U.S. patent application number 16/392543 was filed with the patent office on 2019-12-05 for plant transcriptional regulators.
The applicant listed for this patent is Mendel Biotechnology, Inc.. Invention is credited to Luc Adam, Roger D. Canales, Karen S. Century, Robert A. Creelman, Neal I. Gutterson, Jacqueline E. Heard, Frederick D. Hempel, Cai-Zhong Jiang, Roderick W. Kumimoto, Jeffrey M. Libby, Omaira Pineda, Oliver J. Ratcliffe, Peter P. Repetti, T. Lynne Reuber, Jose Luis Riechmann, James Z Zhang.
Application Number | 20190367565 16/392543 |
Document ID | / |
Family ID | 51062090 |
Filed Date | 2019-12-05 |
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United States Patent
Application |
20190367565 |
Kind Code |
A1 |
Heard; Jacqueline E. ; et
al. |
December 5, 2019 |
PLANT TRANSCRIPTIONAL REGULATORS
Abstract
The invention relates to plant transcription factor
polypeptides, polynucleotides that encode them, homologs from a
variety of plant species, and methods of using the polynucleotides
and polypeptides to produce transgenic plants having improved
tolerance to drought, shade, and low nitrogen conditions, as
compared to wild-type or reference plants.
Inventors: |
Heard; Jacqueline E.;
(Wenham, MA) ; Riechmann; Jose Luis; (Barcelona,
ES) ; Creelman; Robert A.; (Castro Valley, CA)
; Ratcliffe; Oliver J.; (Hayward, CA) ; Canales;
Roger D.; (San Diego, CA) ; Repetti; Peter P.;
(Emeryville, CA) ; Kumimoto; Roderick W.;
(Sacramento, CA) ; Gutterson; Neal I.; (Oakland,
CA) ; Reuber; T. Lynne; (San Mateo, CA) ;
Pineda; Omaira; (Vero Beach, FL) ; Jiang;
Cai-Zhong; (Davis, CA) ; Century; Karen S.;
(Chapel Hill, NC) ; Adam; Luc; (Hayward, CA)
; Zhang; James Z; (Palo Alto, CA) ; Hempel;
Frederick D.; (Sunol, CA) ; Libby; Jeffrey M.;
(Cupertino, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mendel Biotechnology, Inc. |
Hayward |
CA |
US |
|
|
Family ID: |
51062090 |
Appl. No.: |
16/392543 |
Filed: |
April 23, 2019 |
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Application
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Patent Number |
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15821061 |
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10266575 |
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16392543 |
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14229574 |
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15821061 |
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14167768 |
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12705845 |
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14167768 |
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PCT/US04/37584 |
Nov 12, 2004 |
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11435388 |
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10456882 |
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10666642 |
Sep 18, 2003 |
7196245 |
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11981576 |
Oct 30, 2007 |
7888558 |
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10456882 |
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11981576 |
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10714887 |
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12705845 |
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60542928 |
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60527658 |
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60434166 |
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60411837 |
Sep 18, 2002 |
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60465809 |
Apr 24, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8273 20130101; C12N 15/8271 20130101 |
International
Class: |
C07K 14/415 20060101
C07K014/415; C12N 15/82 20060101 C12N015/82 |
Claims
1. A recombinant polynucleotide having a first percent identity,
and the recombinant polynucleotide encodes a polypeptide having a
second percent identity to its full length or to a conserved domain
comprised within the polypeptide; and the polynucleotide is
selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,
81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,
111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,
137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,
163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,
189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,
215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,
241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,
267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,
293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,
319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343,
345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,
371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395,
397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421,
423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447,
449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473,
475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499,
501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525,
527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551,
553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577,
579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603,
605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629,
631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,
657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681,
683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707,
709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733,
735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759,
761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785,
787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,
813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837,
839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863,
865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889,
891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915,
917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,
943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967,
969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993,
995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, and
1017; or the polypeptide is selected from the group consisting of
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,
204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,
230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,
256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,
282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,
308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,
334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,
360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,
386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410,
412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436,
438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,
464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,
490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514,
516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540,
542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566,
568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592,
594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618,
620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644,
646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670,
672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696,
698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722,
724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,
750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774,
776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,
802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826,
828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852,
854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878,
880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904,
906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930,
932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956,
958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982,
984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006,
1008, 1010, 1012, 1014, 1016, 1018, 1019, 1020, 1021, 1022, 1023,
1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034,
1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045,
1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056,
1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067,
1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078,
1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089,
1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100,
1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111,
1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122,
1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133,
1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144,
1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155,
1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166,
1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177,
1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188,
1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199,
1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210,
1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221,
1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232,
1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243,
1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254,
1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265,
1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276,
1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287,
1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298,
1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309,
1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320,
1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331,
1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342,
1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353,
1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364,
1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375,
1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386,
1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397,
1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408,
1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419,
1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430,
1431, 1432, 1433, 1434, 1435, and 1436; and the first percent
identity is selected from the group consisting of: at least 40%, at
least 41%, at least 42%, at least 43%, at least 44%, at least 45%,
at least 46%, at least 47%, at least 48%, at least 49%, at least
50%, at least 51%, at least 52%, at least 53%, at least 54%, at
least 55%, at least 56%, at least 57%, at least 58%, at least 59%,
at least 60%, at least 61%, at least 62%, at least 63%, at least
64%, at least 65%, at least 66%, at least 67%, at least 68%, at
least 69%, at least 70%, at least 71%, at least 72%, at least 73%,
at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, and about 100%; and the
second percent identity is selected from the group consisting of:
at least 50%, at least 51%, at least 52%, at least 53%, at least
54%, at least 55%, at least 56%, at least 57%, at least 58%, at
least 59%, at least 60%, at least 61%, at least 62%, at least 63%,
at least 64%, at least 65%, at least 66%, at least 67%, at least
68%, at least 69%, at least 70%, at least 71%, at least 72%, at
least 73%, at least 74%, at least 75%, at least 76%, at least 77%,
at least 78%, at least 79%, at least 80%, at least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, and about 100%.
2. The recombinant polynucleotide of claim 1, wherein when the
recombinant polynucleotide is introduced into a plant and the
polypeptide is ectopically expressed or overexpressed in the plant,
the plant has an altered trait relative to a control plant that
does not ectopically express or overexpress the recombinant
polynucleotide.
3. The recombinant polynucleotide of claim 2, wherein the altered
trait is selected from the group consisting of: aerial rosettes;
altered inflorescence development; altered architecture; bushier
plant; compact plant; altered branching; altered c/n sensing;
increased tolerance to nitrogen limitation; increased growth on
potassium-free medium; less anthocyanin; altered coloration;
altered floral organ identity and development; altered flower
development; ectopic carpel tissue; altered flower structure;
altered flowering time; altered glucosinolate profile; altered
inflorescence development; altered inflorescence structure; altered
leaf development; altered leaf shape; altered leaf coloration; dark
green color; shiny leaves; upwardly oriented leaves; increased
tocopherol; increased chlorophyll; increased carotenoids; longer
hypocotyls and lack of apical hook in response to ethylene; altered
seed development, ripening, germination; altered seed fatty acid
composition; altered seed oil content; reduction in 16:3 fatty
acids; increased seed oil content; decreased seed oil content;
increased seed protein content; decreased seed protein content;
increased total seed oil and protein content; altered seed shape;
altered shoot development; altered structure of vascular tissues;
altered sugar sensing: greater tolerance to sucrose; more sensitive
to glucose; more tolerant to glucose; reduced cotyledon expansion
in 5% glucose; altered tocopherol composition; reduced trichome
density; increased trichome density; increased trichome size;
reduced trichome density; ectopic trichome formation; glabrous
leaves; increased root hairs; increased tolerance to cold;
increased tolerance to heat; reduced chlorosis in heat; increased
tolerance to salt; increased tolerance to nacl and sucrose;
increased tolerance to sucrose and glucose; increased tolerance to
hyperosmotic stress; better root growth in hyperosmotic stress;
increased seedling vigor in the presence of polyethylene glycol;
better seedling vigor in hyperosmotic stress; better root growth in
hyperosmotic stress; better seedling vigor in 150 mm NaCl; better
seedling vigor in 9.4% sucrose; decreased seedling vigor on high
glucose; increased drought tolerance; constitutive
photomorphogenesis; decreased sensitivity to abscisic acid;
insensitive to aba; downward pedicels; early flowering; early
senescence; late flowering; ectopic trichome formation; increased
trichome number; embryo lethal; enlarged floral organs; short
pedicels; enlarged seedlings; ethylene insensitive when germinated
in the dark on acc; formation of necrotic lesions; leaf and
hypocotyl necrosis; increased seedling vigor in cold; more freezing
tolerant; homeotic transformations; increase in 18:2 fatty acids;
decrease in 18:3 fatty acids; increase in alpha-tocopherol;
increase in chlorophyll a and b; increase in leaf xylose; increase
in M39480; increased anthocyanins; increased leaf fatty acids;
increased leaf fatty acids; increased leaf wax; increased leaf
unsaturated fatty acids; increased lutein content; increased leaf
size; faster development; increased plant size; increased seedling
size; increased seed size; increased seed yield; increased
resistance to Botrytis; increased resistance to Erysiphe; increased
resistance to Fusarium; increased susceptibility to Botrytis;
increased susceptibility to Erysiphe; increased susceptibility to
Fusarium; increased susceptibility to Pseudomonas; increased
resistance to Sclerotinia; increased susceptibility to Sclerotinia;
increased resistance to glyphosate; increased tolerance to
glyphosate; increased sensitivity to ACC; increased shade
tolerance; increased sensitivity to high peg; increased sensitivity
to oxidative stress; long hypocotyls; long petioles; reduced cell
differentiation in meristem; more vascular bundles in stem; pale
green leaves; loss of apical dominance; reduced apical dominance;
reduced branching; reduced fertility; reduced lateral branching;
reduced lignin; reduced petals, sepals and stamens; reduced size;
short pedicels, downward pointing siliques; thicker stem; altered
distribution of vascular bundles; short stamen filaments; seed
color alteration; and smaller and more rounded seeds; increase in
.alpha.-tocopherol; increased chlorophyll a; and increased
chlorophyll b.
4. A transgenic plant transformed with the recombinant
polynucleotide of claim 1.
5. The transgenic plant of claim 4, wherein the recombinant
polynucleotide encodes a polypeptide the expression of which is
regulated by a constitutive, an inducible, or a tissue-enhanced
promoter.
6. A cultured host cell derived from the transgenic plant of claim
4, wherein the cultured host cell comprises the recombinant
polynucleotide.
7. A transgenic seed produced from the transgenic plant of claim 4,
wherein the transgenic seed comprises the recombinant
polynucleotide.
8. A method for producing a transgenic plant having an altered
trait, wherein the method comprises the steps of: (a) providing a
recombinant polynucleotide of claim 1; (b) introducing the
recombinant polynucleotide into a plant; and (c) optionally,
identifying an altered trait in the transgenic plant relative to a
control plant that does not ectopically express or overexpress the
polypeptide; wherein the altered trait is relative to a control
plant that does not ectopically express or overexpress the
recombinant polynucleotide.
9. The method of claim 8, the method steps further comprising: (d)
crossing the transgenic plant with itself or another plant; and (e)
selecting a transgenic seed that develops as a result of said
crossing; and (f) growing a progeny plant from the transgenic seed,
thus producing a transgenic progeny plant having increased the
altered trait.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/821,061, filed Nov. 22, 2017 (now U.S. Pat. No.
10,266,575) which application is a division of U.S. patent
application Ser. No. 14/229,574, filed Mar. 28, 2014 (now U.S. Pat.
No. 9,856,297) which application is a division of U.S. patent
application Ser. No. 14/167,768, filed Jan. 29, 2014 (abandoned)
which application is a division of U.S. patent application Ser. No.
12/705,845, filed Feb. 15, 2010 (now U.S. Pat. No. 8,686,226).
application Ser. No. 12/705,845 is also a continuation-in-part of
U.S. patent application Ser. No. 11/435,388, filed May 15, 2006
(now U.S. Pat. No. 7,663,025), which application is a
continuation-in-part of PCT patent application no.
PCT/US2004/037584, filed Nov. 12, 2004 (expired). PCT/US2004/037584
is a continuation-in-part of U.S. patent application Ser. No.
10/714,887, filed Nov. 13, 2003 (abandoned). PCT/US2004/037584
claims the benefit of U.S. provisional patent application No.
60/542,928, filed Feb. 5, 2004 (expired) and PCT/US2004/037584 also
claims the benefit of U.S. provisional patent application No.
60/527,658, filed Dec. 5, 2003 (expired). application Ser. No.
12/705,845 is also a continuation-in-part of U.S. patent
application Ser. No. 10/714,887, filed Nov. 13, 2003 (abandoned),
which application is a continuation-in-part of U.S. patent
application Ser. No. 10/412,699, filed Apr. 10, 2003 (now U.S. Pat.
No. 7,345,217). application Ser. No. 10/714,887 is also a
continuation-in-part of U.S. patent application Ser. No.
10/456,882, filed Jun. 6, 2003 (abandoned). application Ser. No.
10/714,887 is also a continuation-in-part of U.S. patent
application Ser. No. 10/374,780, filed Feb. 25, 2003 (now U.S. Pat.
No. 7,511,190). application Ser. No. 10/714,887 is also a
continuation-in-part of U.S. patent application Ser. No.
10/666,642, filed Sep. 18, 2003 (now U.S. Pat. No. 7,196,245),
which application claims the benefit of U.S. provisional patent
application No. 60/434,166, filed Dec. 17, 2002 (expired).
application Ser. No. 10/666,642 also claims the benefit of U.S.
provisional patent application No. 60/411,837, filed Sep. 18, 2002
(expired). application Ser. No. 10/666,642 also claims the benefit
of U.S. provisional patent application No. 60/465,809, filed Apr.
24, 2003 (expired). application Ser. No. 12/705,845 is also a
continuation-in-part of U.S. patent application Ser. No.
11/981,576, filed Oct. 30, 2007 (now U.S. Pat. No. 7,888,558), and
U.S. patent application Ser. No. 11/981,576 is a
continuation-in-part of U.S. patent application Ser. No.
10/456,882, filed Jun. 6, 2003 (abandoned). All of these
applications are hereby incorporated by reference in their
entirety.
JOINT RESEARCH AGREEMENT
[0002] The claimed invention, in the field of functional genomics
and the characterization of plant genes for the improvement of
plants, was made by or on behalf of Mendel Biotechnology, Inc. and
Monsanto Company as a result of activities undertaken within the
scope of a joint research agreement in effect on or before the date
the claimed invention was made.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions and methods
for modifying the phenotype of a plant, including altered
carbon/nitrogen balance sensing, improved nitrogen uptake or
assimilation efficiency, improved growth or survival of plants
under conditions of nitrogen limitation, increased tolerance to
drought or other abiotic stress, and/or increased tolerance to
shade.
BACKGROUND OF THE INVENTION
[0004] A plant's traits may be controlled through a number of
cellular processes. One important way to manipulate that control is
through transcription factors--proteins that influence the
expression of a particular gene or sets of genes. Because
transcription factors are key controlling elements of biological
pathways, altering the expression levels of one or more
transcription factors can change entire biological pathways in an
organism. Strategies for manipulating a plant's biochemical,
developmental, or phenotypic characteristics by altering a
transcription factor expression can result in plants and crops with
new and/or improved commercially valuable properties, including
traits that improve yield or survival and yield during periods of
abiotic stress, improve shade tolerance, or alter a plant's sensing
of its carbon/nitrogen balance.
[0005] We have identified numerous polynucleotides encoding
transcription factors, functionally related sequences listed in the
Sequence Listing, and structurally and functionally similar
sequences, developed numerous transgenic plants using these
polynucleotides, and analyzed the plants for their tolerance to
shade, drought stress, and altered carbon-nitrogen balance (C/N)
sensing. In so doing, we have identified important polynucleotide
and polypeptide sequences for producing commercially valuable
plants and crops as well as the methods for making them and using
them. The present invention thus relates to methods and
compositions for producing transgenic plants with improved
tolerance to drought and other abiotic stresses, with altered C/N
sensing, and/or with improved tolerance to shade. This provides
significant value in that the plants may thrive in hostile
environments where low nutrient, light, or water availability
limits or prevents growth of non-transgenic plants. Other aspects
and embodiments of the invention are described below and can be
derived from the teachings of this disclosure as a whole.
SUMMARY OF THE INVENTION
[0006] The present method is directed to recombinant
polynucleotides that confer abiotic stress tolerance in plants when
the expression of any of these recombinant polynucleotides is
altered (e.g., by overexpression). Related sequences that are
encompassed by the invention include nucleotide sequences that
hybridize to the complement of the sequences of the invention under
stringent conditions.
[0007] Related sequences that are also encompassed by the invention
include polypeptide sequences within a given clade or subclade,
that is, sequences that are evolutionarily, functionally and
structurally related. The invention also pertains to a transgenic
plant that comprises a recombinant polynucleotide that encodes a
polypeptide that regulates transcription.
[0008] The invention also includes a transgenic plant that
overexpresses a recombinant polynucleotide comprising a nucleotide
sequence that hybridizes to the complement of any polynucleotide of
the invention under stringent conditions. This transgenic plant has
increased drought, low nitrogen and/or shade tolerance as compared
to a wild-type or non-transformed plant of the same species that
does not overexpress a polypeptide encoded by the recombinant
polynucleotide.
[0009] The invention also encompasses a method for producing a
transgenic plant having increased tolerance to drought, low
nitrogen, and/or shade. These method steps include first providing
an expression vector that contains a nucleotide sequence that
hybridizes to the complement of a polynucleotide of the invention
under stringent hybridization conditions. The expression vector is
then introduced into a plant cell, the plant cell is cultured, from
which a plant is generated. Due to the presence of the expression
vector in the plant, the polypeptide encoded by the nucleotide
sequence is overexpressed. This polypeptide has the property of
regulating drought, low nitrogen, or shade tolerance in a plant,
compared to a control plant that does not overexpress the
polypeptide. After the drought, low nitrogen, or shade-tolerant
transgenic plant is produced, it may be identified by comparing it
with one or more non-transformed plants that do not overexpress the
polypeptide. These method steps may further include selfing or
crossing the abiotic stress-tolerant plant with itself or another
plant, respectively, to produce seed. "Selfing" refers to
self-pollinating, or using pollen from one plant to fertilize the
same plant or another plant in the same line, whereas "crossing"
generally refers to cross pollination with plant from a different
line, such as a non-transformed or wild-type plant, or another
transformed plant from a different transgenic line of plants.
Crossing provides the advantage of being able to produce new
varieties. The resulting seed may then be used to grow a progeny
plant that is transgenic and has increased tolerance to abiotic
stress.
[0010] The invention is also directed to a method for increasing a
plant's tolerance to drought, low nitrogen, or shade. This method
includes first providing a vector that comprises (i) regulatory
elements effective in controlling expression of a polynucleotide
sequence in a target plant, where the regulatory elements flank the
polynucleotide sequence; and (ii) the polynucleotide sequence
itself, which encodes a polypeptide that has the ability to
regulate drought, low nitrogen, or shade tolerance in a plant, as
compared to a control plant of the same species that does not
overexpress the polypeptide. The plant is transformed with the
vector in order to generate a transformed plant with increased
tolerance to drought, low nitrogen, or shade.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0011] The Sequence Listing provides exemplary polynucleotide and
polypeptide sequences of the invention. The traits associated with
the use of the sequences are included in the Examples.
INCORPORATION OF THE SEQUENCE LISTING
[0012] The copy of the Sequence Listing, being submitted
electronically with this patent application, provided under 37 CFR
.sctn. 1.821-1.825, is a read-only memory computer-readable file in
ASCII text format. The Sequence Listing is named
"MDBT008USD2-sequence_listing_replacement.txt", the electronic file
of the Sequence Listing was created on Jul. 27, 2015, and is
2,500,656 bytes in size (or 2,500 kilobytes in size as measured in
MS-WINDOWS). The Sequence Listing is herein incorporated by
reference in its entirety.
FIGURES
[0013] For figures presenting one or more sequences, the SEQ ID NO:
of the sequence(s) is/are provided in parentheses.
[0014] FIG. 1 shows a conservative estimate of phylogenetic
relationships among the orders of flowering plants (modified from
Angiosperm Phylogeny Group (1998) Ann. Missouri Bot. Gard. 84:
1-49). Those plants with a single cotyledon (monocots) are a
monophyletic clade nested within at least two major lineages of
dicots; the eudicots are further divided into rosids and asterids.
Arabidopsis is a rosid eudicot classified within the order
Brassicales; rice is a member of the monocot order Poales. FIG. 1
was adapted from Daly et al. (2001) Plant Physiol. 127:
1328-1333.
[0015] FIG. 2 shows a phylogenic dendogram depicting phylogenetic
relationships of higher plant taxa, including clades containing
tomato and Arabidopsis; adapted from Ku et al. (2000) Proc. Natl.
Acad. Sci. 97: 9121-9126; and Chase et al. (1993) Ann. Missouri
Bot. Gard. 80: 528-580.
[0016] FIG. 3 is a multiple amino acid sequence alignment of
subsequence within the AP2 domain of G47, G2133 and their
orthologs. The first column shows the sequence name followed by the
SEQ ID No. in parentheses. Clade orthologs and paralogs are
indicated by the black bar on the left side of the figure. Of the
sequences examined to date, two valine residues were found that are
present in members of the G47 clade but not outside of the clade
(arrows). Residues that may be used to identify a G47 clade member
are indicated by the residues shown in the boxes in FIG. 3
[0017] FIG. 4 illustrates the relationship of G47 and related
sequences in this phylogenetic tree of the G47 Glade and similar
sequences. The tree building method used was "Neighbor Joining"
with "Systematic Tie-Breaking" and Bootstrapping with 1000
replicates (Uncorrected ("p"), with gaps distributed
proportionally). Full-length polypeptides were used to build the
phylogeny as defined in FIG. 4. The members of the clade shown
within the box are predicted to contain functional homologs of G47.
Abbreviations: At Arabidopsis thaliana; Os Oryza sativa; Zm Zea
mays; Gm Glycine max; Mt Medicago truncatula; Br Brassica rapa; Bo
Brassica oleracea; Ze: Zinnia elegans.
[0018] FIGS. 5A and 5B compare the recovery from a drought
treatment of wild-type controls and two lines of Arabidopsis plants
overexpressing G2133, a paralog of G47. FIGS. 5A and 5B show two
35S::G2133 lines of plants (one line in each figure) in the pot on
the left of each figure and control plants on the right of each
figure. Each pot contained several plants grown under 24 hours
light. All were deprived of water for eight days, and are shown
after re-watering. All of the plants of the G2133 overexpressor
lines recovered, and all of the control plants were either dead or
severely and adversely affected by the drought treatment.
[0019] FIGS. 6A-6C compare a number of homeodomains from the
zinc-finger-homeodomain-type (ZF-HD) proteins related to G2999. The
first column shows the sequence name followed by the SEQ ID No. in
parentheses. _Homeodomains from the ZF-HD type proteins are
distinct from classical types of homeodomains and lie on the
distinct branch of the tree shown in FIG. 7. The relationships
established from this type of alignment of homeodomains were used
to generate the phylogenetic tree shown in FIGS. 7 and 8. Residues
that may be used to identify the G2999 clade are shown in boxes in
FIGS. 6A and 6B.
[0020] FIG. 7 illustrates the relationship of G2999 and related
sequences in this phylogenetic tree of the G2999 clade and similar
sequences comprising ZF-HD-type proteins. The tree building method
used was "Neighbor Joining" with "Systematic Tie-Breaking" and
Bootstrapping with 1000 replicates (Uncorrected ("p"), with gaps
distributed proportionally. All of the sequences shown are members
of the clade and are predicted to be functional homologs of G2999.
Abbreviations: At Arabidopsis thaliana; Os (jap) Oryza sativa
(japonica cultivar group); Os (ind) Oryza sativa (indica cultivar
group); Zm Zea mays; Lj Lotus corniculatus var. japonicus; Bn
Brassica napus; Fb Flaveria bidentis.
[0021] FIG. 8 is a phylogenetic tree (neighbor-joining, 1000
bootstraps) highlighting the relational differences between the
ZF-HD type proteins and the "classical" homeodomain (HD) proteins.
The homeodomains from ZF-HD type proteins lie on a distinct branch
of the tree compared to classical types of homeodomains
(arrow).
[0022] FIGS. 9A-9L represent a multiple amino acid sequence
alignment of G1792 orthologs and paralogs. The first column shows
the sequence name followed by the SEQ ID No. in parentheses. Clade
orthologs and paralogs are indicated by the black bar on the left
side of the figure. Conserved regions of identity are boxed and
bolded while conserved sequences of similarity are boxed with no
bolding. The AP2 conserved domains span alignment coordinates
196-254. The S conserved domain spans alignment coordinates of
301-304. The EDLL conserved domain spans the alignment coordinates
of 393-406 (also see FIG. 10). Abbreviations: At Arabidopsis
thaliana; Os Oryza sativa; Zm Zea mays; Ta Triticum aestivum; Gm
Glycine max; Mt Medicago truncatula.
[0023] FIG. 10 shows a novel conserved domain for the G1792 clade,
herein referred to as the "EDLL domain". The first column shows the
sequence name followed by the SEQ ID No. in parentheses. All clade
members contain a glutamic acid residue at position 3, an aspartic
acid residue at position 8, and a leucine residue at positions 12
and 16. Abbreviations: At Arabidopsis thaliana; Os Oryza sativa; Zm
Zea mays; Ta Triticum aestivum; Gm Glycine max; Mt Medicago
truncatula.
[0024] FIG. 11 illustrates the relationship of G1792 and related
sequences in this phylogenetic tree of the G1792 clade of
transcription factors. The tree building method used was "Neighbor
Joining" with "Systematic Tie-Breaking" and Bootstrapping with 1000
replicates. Only conserved domains were used to build the phylogeny
as defined in FIG. 11. The members of the G1792 clade are shown
within the box. The sequences within the G1792 clade descend from a
common ancestral node (arrow).
[0025] FIG. 12 shows an alignment of G3086, orthologs, and paralog
subsequences. The first column shows the sequence name followed by
the SEQ ID No. in parentheses. The G3086 clade is indicated by the
black bar on the left side of the figure. Residues that may be used
to identify clade members appear in boxes.
[0026] FIG. 13 is a phylogenetic tree of the G3086 clade, including
G3086 and its paralogs and orthologs. Full length, predicted
protein sequences were used to construct a pairwise comparison,
bootstrapped (1000 replicates) neighbor-joining tree, consensus
view. Sequences within the G3086 clade are located within the box.
The sequences within the G3086 clade descend from a common
ancestral node (arrow). Abbreviations: At Arabidopsis thaliana; Os
Oryza sativa; Zm Zea mays; Gm Glycine max.
[0027] FIGS. 14A-14R show a multiple amino acid sequence alignment
of G922 orthologs and paralogs. The first column shows the sequence
name followed by the SEQ ID No. in parentheses. Clade orthologs and
paralogs are indicated by black bar on the left side of the figure.
Residues that appear in boldface represent an acidic, ser/pro-rich
domain that is unique to the G922 clade. Abbreviations: At
Arabidopsis thaliana; Os Oryza sativa; Zm Zea mays; Ta Triticum
aestivum; Gm Glycine max; Le Lycopersicon esculentum; Ps Pisum
sativum.
[0028] FIG. 15 is a phylogenetic tree of the G922 paralogs and
orthologs. Full length, predicted protein sequences were used to
construct a pairwise comparison, bootstrapped (1000 replicates)
neighbor-joining tree, consensus view. Sequences within the G922
clade are located within the box.
[0029] FIG. 16 is a sequence alignment of predicted protein
subsequences within the WRKY domain from G1274 paralogs and
orthologs. The first column shows the sequence name followed by the
SEQ ID No. in parentheses. The sequences within the G1274 clade are
indicated by the black bar to the left of the sequences. Amino acid
residues within the WRKY domain that distinguish the G1274 clade
sequences, and are putatively responsible for conserved
functionality, are indicated within the boxes.
[0030] FIG. 17 represents a phylogenetic tree for the G1274
paralogs and orthologs. Full length, predicted protein sequences
were used to construct a bootstrapped (1000 replicates)
neighbor-joining tree. Gaps and missing data were handled using
pairwise deletion and the distance method used was p-distance.
Sequences within the G1274 clade appear within the box.
[0031] FIGS. 18A-18BB show a multiple sequence alignment of
predicted protein sequences from G2053, and its paralogs and
orthologs. The first column shows the sequence name followed by the
SEQ ID No. in parentheses. The sequences within the G2053 clade are
indicated by the black bar to the left of the alignment. The amino
acid residues in boldface are consensus residues, and those within
the boxes represent conserved, similar residues. Sequences without
a species identifier were found in Arabidopsis.
[0032] FIG. 19 is a phylogenetic tree for the G2053 paralogs and
orthologs. Full length, predicted protein sequences were used to
construct a bootstrapped (1000 replicates) neighbor-joining tree.
Gaps and missing data were handled using pairwise deletion and the
distance method used was p-distance. Sequences within the G2053
Glade appear within the box.
[0033] FIGS. 20A and 20B show the conserved domains making up the
DNA binding domains of G682-like proteins from Arabidopsis,
soybean, rice, and corn. The first column shows the sequence name
followed by the SEQ ID No. in parentheses. G682 and its paralogs
and orthologs are almost entirely composed of a single repeat
MYB-related DNA binding domain that is highly conserved across
plant species. The polypeptide sequences that are representatives
of the G682 subclade are denoted by the vertical bar to the left of
the subsequences. The residues in the boxes in FIG. 20B may be used
to identify G682 subclade members. The residues indicated by the
arrows and in the boxes in FIG. 20B have not been found at
corresponding positions in sequences outside of the G682 subclade.
Prior to this disclosure, no function such as those presented in
Example VIII has been identified for any of the non-Arabidopsis
MYB-related sequences in the G682 subclade.
[0034] FIG. 21 illustrates the relationship of G682 and related
sequences in this phylogenetic tree of the G682 subclade and
similar sequences. This phylogenetic tree of defined conserved
domains of G682 and related polypeptides was constructed with
ClustalW (CLUSTAL W Multiple Sequence Alignment Program version
1.83, 2003) and MEGA2 (http://www.megasoftware.net) software.
ClustalW multiple alignment parameters were as follows:
[0035] Gap Opening Penalty: 10.00
[0036] Gap Extension Penalty: 0.20
[0037] Delay divergent sequences: 30%
[0038] DNA Transitions Weight: 0.50
[0039] Protein weight matrix: Gonnet series
[0040] DNA weight matrix: IUB
[0041] Use negative matrix: OFF
[0042] A FastA formatted alignment was then used to generate a
phylogenetic tree in MEGA2 using the neighbor joining algorithm and
a p-distance model. A test of phylogeny was done via bootstrap with
100 replications and Random Speed set to default. Cut off values of
the bootstrap tree were set to 50%. The G682 subclade of
MYB-related transcription factors, a group of structurally and
functionally related sequences that derive from a single ancestral
node (arrow), appears within the box in FIG. 21. Most of the
members of the subclade within the box have been shown to confer
abiotic stress tolerance and/or altered C/N sensing when the
polypeptides are overexpressed (see Table 13).
[0043] FIG. 22 is a graph representing light quality (percent
transmission vs. wavelength) in the controlled environment plant
growth chamber used for the shade avoidance studies. Because
shading is detected using phytochrome to sense the R:FR ratio in
light, we can mimic the effect of shading by using a filter
designed to prevent only the transmission of red wavelengths. To
determine whether the mechanisms used to sense shading are altered,
we exploit the observation that seedlings of wild-type plants grown
under light deficient in red wavelengths have extended hypocotyls,
indicating a shade avoidance phenotype. Plants overexpressing genes
which produce short hypocotyls under these conditions, and exhibit
a shade tolerance phenotype, would be candidates for further
examination in more rigorous studies (e.g., by looking at
components such as yield under high densities in greenhouse
studies). For the data seen in FIG. 22, a small piece of the filter
was removed and used to determine the percent transmission with a
Beckman DU-650 spectrophotometer. This filter effectively removed
the red region of the visible spectrum yet allowed far-red and blue
to pass through.
[0044] FIG. 23 shows the results of an experiment with 35S::G634
plants versus wild type. Individual seedlings were compared after
being grown under light deficient in red wavelengths (b/FR) and
white light (w). The G634 overexpressors did not exhibit a shade
avoidance phenotype, as indicated by their short hypocotyls
produced under these conditions.
DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0045] The data presented herein represent the results of a screen
of a transcription factor collection to identify genes that can be
applied to reduce yield losses that arise from low nutrient,
drought-related stress, and/or shade avoidance responses.
[0046] We have identified numerous transcription factor genes that
confer improved drought-tolerance relative to wild type plants when
their expression is altered, such as by overexpression or
knocking-out of the gene in transgenic plants. Thus, the present
invention is directed in part to recombinant polynucleotides that
confer drought-related stress tolerance in plants when the
expression of recombinant polynucleotides of the invention is
altered (e.g., by overexpression). In the present studies,
soil-based assays were performed in which transgenic plants are
first deprived of water, evaluated by comparison to control plants,
rewatered, and their recovery also evaluated by comparison to
control plants similarly treated.
[0047] We have also identified numerous transcription factor genes
that confer altered C/N sensing in transgenic Arabidopsis plants.
These experiments were carried out in two phases. A primary screen
was done on seed lots comprised of seed mixed together from each of
two or three independent primary transformants, or on a homozygous
population in the case of the knockout lines. Any lot which showed
a C/N sensing phenotype was subjected to a repeat experiment.
Transgenic lines that exhibited an altered C/N sensing phenotype in
repeat experiments, as compared to control plants, are shown in the
tables and Sequence Listing.
[0048] A secondary screen was then conducted in which either two or
three individual overexpression lines (or a different homozygous
seed lot, in the case of knockout lines) were retested in the
assay. The individual transgenic lines that showed prominent
phenotypes in the second round assay were given an "A" priority
ranking. The set of sequences assigned a "B" priority ranking in
the results table have yet to be confirmed in the secondary screen
or did not show a prominent phenotype.
[0049] We have also identified numerous transcription factor genes
that confer shade tolerance in transgenic Arabidopsis plants. The
principle behind the experiment was as follows: angiosperm plants
have evolved mechanisms to compete with neighboring vegetation for
light. When incident light is filtered or reflected by adjacent
plants, the red wavelengths of the spectrum are removed, resulting
in a fall in the ratio of red to far red light that the plant
perceives. These changes are detected via the phytochrome
photoreceptors and result in extension type growth and accelerated
flowering. Such responses reduce the resources available for
storage and reproduction, which in turn results in poor fruit and
seed development and reduced yield. Given that shade avoidance
responses are often initiated in crops at planting densities where
light availability is not a limiting growth factor, genes that
suppress such effects would offer yield savings.
[0050] In the experiments presented herein, overexpression and
mutant Arabidopsis lines for a transcription factor collection were
grown under light that was deficient in red wavelengths, and was
therefore equivalent to light shaded by vegetation. Transcription
factors were identified that conferred shade tolerance and
prevented the elongated growth that was produced in wild-type
controls under such conditions.
[0051] The present invention relates in part to polynucleotides and
polypeptides, for example, for modifying phenotypes of plants,
particularly those associated with altered C/N sensing, and
improved drought stress and shade tolerance. Throughout this
disclosure, various information sources are referred to and/or are
specifically incorporated. The information sources include
scientific journal articles, patent documents, textbooks, and World
Wide Web browser-inactive page addresses. While the reference to
these information sources clearly indicates that they can be used
by one of skill in the art, each and every one of the information
sources cited herein are specifically incorporated in their
entirety, whether or not a specific mention of "incorporation by
reference" is noted. The contents and teachings of each and every
one of the information sources can be relied on and used to make
and use embodiments of the invention.
[0052] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a plant" includes a plurality of such plants, and a reference
to "a stress" is a reference to one or more stresses and
equivalents thereof known to those skilled in the art, and so
forth.
Definitions
[0053] "Nucleic acid molecule" refers to an oligonucleotide,
polynucleotide or any fragment thereof. It may be DNA or RNA of
genomic or synthetic origin, double-stranded or single-stranded,
and combined with carbohydrate, lipids, protein, or other materials
to perform a particular activity such as transformation or form a
useful composition such as a peptide nucleic acid (PNA).
[0054] "Polynucleotide" is a nucleic acid molecule comprising a
plurality of polymerized nucleotides, for example, at least about
15 or more consecutive polymerized nucleotides. A polynucleotide
may be a nucleic acid, oligonucleotide, nucleotide, or any fragment
thereof. In many instances, a polynucleotide comprises a nucleotide
sequence encoding a polypeptide (or protein) or a domain or
fragment thereof. Additionally, the polynucleotide may comprise a
promoter, an intron, an enhancer region, a polyadenylation site, a
translation initiation site, 5' or 3' untranslated regions, a
reporter gene, a selectable marker, or the like. The polynucleotide
can be single-stranded or double-stranded DNA or RNA. The
polynucleotide optionally comprises modified bases or a modified
backbone. The polynucleotide can be, for example, genomic DNA or
RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a
cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide
can be combined with carbohydrate, lipids, protein, or other
materials to perform a particular activity such as transformation
or form a useful composition such as a peptide nucleic acid (PNA).
The polynucleotide can comprise a sequence in either sense or
antisense orientations. "Oligonucleotide" is substantially
equivalent to the terms amplimer, primer, oligomer, element,
target, and probe and is preferably single-stranded.
[0055] "Gene" or "gene sequence" refers to the partial or complete
coding sequence of a gene, its complement, and its 5' or 3'
untranslated regions. A gene is also a functional unit of
inheritance, and in physical terms is a particular segment or
sequence of nucleotides along a molecule of DNA (or RNA, in the
case of RNA viruses) involved in producing a polypeptide chain. The
latter may be subjected to subsequent processing such as chemical
modification, splicing and folding to obtain a functional protein
or polypeptide. A gene may be isolated, partially isolated, or be
found with an organism's genome. By way of example, a transcription
factor gene encodes a transcription factor polypeptide, which may
be functional or require processing to function as an initiator of
transcription.
[0056] Operationally, genes may be defined by the cis-trans test, a
genetic test that determines whether two mutations occur in the
same gene and that may be used to determine the limits of the
genetically active unit (Rieger et al. (1976) Glossary of Genetics
and Cytogenetics: Classical and Molecular, 4th ed., Springer
Verlag. Berlin). A gene generally includes regions preceding
("leaders"; upstream) and following ("trailers"; downstream) the
coding region. A gene may also include intervening, non-coding
sequences, referred to as "introns", located between individual
coding segments, referred to as "exons". Most genes have an
associated promoter region, a regulatory sequence 5' of the
transcription initiation codon (there are some genes that do not
have an identifiable promoter). The function of a gene may also be
regulated by enhancers, operators, and other regulatory
elements.
[0057] A "recombinant polynucleotide" is a polynucleotide that is
not in its native state, for example, the polynucleotide comprises
a nucleotide sequence not found in nature, or the polynucleotide is
in a context other than that in which it is naturally found, for
example, separated from nucleotide sequences with which it
typically is in proximity in nature, or adjacent (or contiguous
with) nucleotide sequences with which it typically is not in
proximity. For example, the sequence at issue can be cloned into a
vector, or otherwise recombined with one or more additional nucleic
acid.
[0058] An "isolated polynucleotide" is a polynucleotide, whether
naturally occurring or recombinant, that is present outside the
cell in which it is typically found in nature, whether purified or
not. Optionally, an isolated polynucleotide is subject to one or
more enrichment or purification procedures, for example, cell
lysis, extraction, centrifugation, precipitation, or the like.
[0059] A "polypeptide" is an amino acid sequence comprising a
plurality of consecutive polymerized amino acid residues for
example, at least about 15 consecutive polymerized amino acid
residues. In many instances, a polypeptide comprises a polymerized
amino acid residue sequence that is a transcription factor or a
domain or portion or fragment thereof. Additionally, the
polypeptide may comprise: (i) a localization domain; (ii) an
activation domain; (iii) a repression domain; (iv) an
oligomerization domain; or (v) a DNA-binding domain, or the like.
The polypeptide optionally comprises modified amino acid residues,
naturally occurring amino acid residues not encoded by a codon, or
non-naturally occurring amino acid residues.
[0060] "Protein" refers to an amino acid sequence, oligopeptide,
peptide, polypeptide or portions thereof whether naturally
occurring or synthetic.
[0061] "Portion", as used herein, refers to any part of a protein
used for any purpose, but especially for the screening of a library
of molecules that specifically bind to that portion or for the
production of antibodies.
[0062] A "recombinant polypeptide" is a polypeptide produced by
translation of a recombinant polynucleotide. A "synthetic
polypeptide" is a polypeptide created by consecutive polymerization
of isolated amino acid residues using methods well known in the
art. An "isolated polypeptide," whether a naturally occurring or a
recombinant polypeptide, is more enriched in (or out of) a cell
than the polypeptide in its natural state in a wild-type cell, for
example, more than about 5% enriched, or at least 105% relative to
wild type standardized at 100%. Such an enrichment is not the
result of a natural response of a wild-type plant. Alternatively,
or additionally, the isolated polypeptide is separated from other
cellular components with which it is typically associated, for
example, by any of the various protein purification methods
herein.
[0063] "Homology" refers to sequence similarity between a reference
sequence and at least a fragment of a newly sequenced clone insert
or its encoded amino acid sequence. Additionally, the terms
"homology" and "homologous sequence(s)" may refer to one or more
polypeptide sequences that are modified by chemical or enzymatic
means. The homologous sequence may be a sequence modified by
lipids, sugars, peptides, organic or inorganic compounds, by the
use of modified amino acids or the like. Protein modification
techniques are illustrated in Ausubel et al. (eds) Current
Protocols in Molecular Biology, John Wiley & Sons (1998).
[0064] "Identity" or "similarity" refers to sequence similarity
between two polynucleotide sequences or between two polypeptide
sequences, with identity being a more strict comparison. The
phrases "percent identity" and "% identity" refer to the percentage
of sequence similarity found in a comparison of two or more
polynucleotide sequences or two or more polypeptide sequences.
"Sequence similarity" refers to the percent similarity in base pair
sequence (as determined by any suitable method) between two or more
polynucleotide sequences. Two or more sequences can be anywhere
from 0-100% similar, or any integer value therebetween. Identity or
similarity can be determined by comparing a position in each
sequence that may be aligned for purposes of comparison. When a
position in the compared sequence is occupied by the same
nucleotide base or amino acid, then the molecules are identical at
that position. A degree of similarity or identity between
polynucleotide sequences is a function of the number of identical,
matching of corresponding nucleotides at positions shared by the
polynucleotide sequences. A degree of identity of polypeptide
sequences is a function of the number of identical amino acids at
corresponding positions shared by the polypeptide sequences. A
degree of homology or similarity of polypeptide sequences is a
function of the number of amino acids at corresponding positions
shared by the polypeptide sequences.
[0065] With regard to polypeptides, the terms "substantial
identity" or "substantially identical" may refer to sequences of
sufficient similarity and structure to the transcription factors in
the Sequence Listing to produce similar function when expressed or
overexpressed in a plant; in the present invention, this function
is altered C/N sensing or increased tolerance to drought or shade.
Sequences that are at least about 50% identical, and preferably at
least 82% identical, to the instant polypeptide sequences are
considered to have "substantial identity" with the latter.
Sequences having lesser degrees of identity but comparable
biological activity are considered to be equivalents. The structure
required to maintain proper functionality is related to the
tertiary structure of the polypeptide. There are discreet domains
and motifs within a transcription factor that must be present
within the polypeptide to confer function and specificity. These
specific structures are required so that interactive sequences will
be properly oriented to retain the desired activity. "Substantial
identity" may thus also be used with regard to subsequences, for
example, motifs, that are of sufficient structure and similarity,
being at least about 50% identical, and preferably at least 82%
identical, to similar motifs in other related sequences so that
each confers or is required for altered C/N sensing or increased
tolerance to drought or shade.
[0066] The term "amino acid consensus motif" refers to the portion
or subsequence of a polypeptide sequence that is substantially
conserved among the polypeptide transcription factors listed in the
Sequence Listing.
[0067] "Alignment" refers to a number of nucleotide or amino acid
residue sequences aligned by lengthwise comparison so that
components in common (i.e., nucleotide bases or amino acid
residues) may be visually and readily identified. The fraction or
percentage of components in common is related to the homology or
identity between the sequences. Alignments such as those found the
Figures may be used to identify conserved domains and relatedness
within these domains. An alignment may suitably be determined by
means of computer programs known in the art, such as MacVector
(1999) (Accelrys, Inc., San Diego, Calif.).
[0068] A "conserved domain" or "conserved region" as used herein
refers to a region in heterologous polynucleotide or polypeptide
sequences where there is a relatively high degree of sequence
identity between the distinct sequences. AP2 domains are examples
of conserved domains.
[0069] With respect to polynucleotides encoding presently disclosed
transcription factors, a conserved domain is preferably at least 10
base pairs (bp) in length.
[0070] A "conserved domain", with respect to presently disclosed
polypeptides refers to a domain within a transcription factor
family that exhibits a higher degree of sequence homology, such as
at least 70% sequence similarity, including conservative
substitutions, and more preferably at least 79% sequence identity,
and even more preferably at least 81%, or at least about 86%, or at
least about 87%, or at least about 89%, or at least about 91%, or
at least about 95%, or at least about 98% amino acid residue
sequence identity to the conserved domain. Sequences are also
encompassed by the invention that possess or encode conserved
domains that recognizable fall within a given clade of
transcription factor polypeptides and that have comparable
biological activity to the sequences of this invention. A fragment
or domain can be referred to as outside a conserved domain, outside
a consensus sequence, or outside a consensus DNA-binding site that
is known to exist or that exists for a particular transcription
factor class, family, or sub-family. In this case, the fragment or
domain will not include the exact amino acids of a consensus
sequence or consensus DNA-binding site of a transcription factor
class, family or sub-family, or the exact amino acids of a
particular transcription factor consensus sequence or consensus
DNA-binding site. Furthermore, a particular fragment, region, or
domain of a polypeptide, or a polynucleotide encoding a
polypeptide, can be "outside a conserved domain" if all the amino
acids of the fragment, region, or domain fall outside of a defined
conserved domain(s) for a polypeptide or protein. Sequences having
lesser degrees of identity but comparable biological activity are
considered to be equivalents.
[0071] As one of ordinary skill in the art recognizes, conserved
domains may be identified as regions or domains of identity to a
specific consensus sequence (for example, Riechmann et al. (2000)
supra). Thus, by using alignment methods well known in the art, the
conserved domains of the AP2 plant transcription factors may be
determined.
[0072] The conserved domains for a number of the sequences that
confer drought tolerance and altered C/N sensing are found in
Tables 1 and 3, respectively. A comparison of the regions of the
polypeptides in Table 1 or 3 allows one of skill in the art to
identify conserved domains for any of the polypeptides listed or
referred to in this disclosure.
[0073] "Complementary" refers to the natural hydrogen bonding by
base pairing between purines and pyrimidines. For example, the
sequence A-C-G-T (5'->3') forms hydrogen bonds with its
complements A-C-G-T (5'->3') or A-C-G-U (5'->3'). Two
single-stranded molecules may be considered partially
complementary, if only some of the nucleotides bond, or "completely
complementary" if all of the nucleotides bond. The degree of
complementarity between nucleic acid strands affects the efficiency
and strength of hybridization and amplification reactions. "Fully
complementary" refers to the case where bonding occurs between
every base pair and its complement in a pair of sequences, and the
two sequences have the same number of nucleotides.
[0074] The terms "highly stringent" or "highly stringent condition"
refer to conditions that permit hybridization of DNA strands whose
sequences are highly complementary, wherein these same conditions
exclude hybridization of significantly mismatched DNAs.
Polynucleotide sequences capable of hybridizing under stringent
conditions with the polynucleotides of the present invention may
be, for example, variants of the disclosed polynucleotide
sequences, including allelic or splice variants, or sequences that
encode orthologs or paralogs of presently disclosed polypeptides.
Nucleic acid hybridization methods are disclosed in detail by
Kashima et al. (1985) Nature 313:402-404, Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. ("Sambrook"), and by Hames and
Higgins, "Nucleic Acid Hybridisation: A Practical Approach", IRL
Press, Washington, D.C. (1985), which references are incorporated
herein by reference.
[0075] In general, stringency is determined by the temperature,
ionic strength, and concentration of denaturing agents (for
example, formamide) used in a hybridization and washing procedure
(for a more detailed description of establishing and determining
stringency, see below). The degree to which two nucleic acids
hybridize under various conditions of stringency is correlated with
the extent of their similarity. Thus, similar nucleic acid
sequences from a variety of sources, such as within a plant's
genome (as in the case of paralogs) or from another plant (as in
the case of orthologs) that may perform similar functions can be
isolated on the basis of their ability to hybridize with known
transcription factor sequences. Numerous variations are possible in
the conditions and means by which nucleic acid hybridization can be
performed to isolate transcription factor sequences having
similarity to transcription factor sequences known in the art and
are not limited to those explicitly disclosed herein. Such an
approach may be used to isolate polynucleotide sequences having
various degrees of similarity with disclosed transcription factor
sequences, such as, for example, transcription factors having 60%
identity, or more preferably greater than about 70% identity, most
preferably 72% or greater identity with disclosed transcription
factors.
[0076] Regarding the terms "paralog" and "ortholog", homologous
polynucleotide sequences and homologous polypeptide sequences may
be paralogs or orthologs of the claimed polynucleotide or
polypeptide sequence. Orthologs and paralogs are
evolutionarily-related genes that have similar sequence and similar
functions. Orthologs are structurally related genes in different
species that are derived by a speciation event. Paralogs are
structurally related genes within a single species that are derived
by a duplication event. Sequences that are sufficiently similar to
one another will be appreciated by those of skill in the art and
may be based upon percentage identity of the complete sequences,
percentage identity of a conserved domain or sequence within the
complete sequence, percentage similarity to the complete sequence,
percentage similarity to a conserved domain or sequence within the
complete sequence, and/or an arrangement of contiguous nucleotides
or peptides particular to a conserved domain or complete sequence.
Sequences that are sufficiently similar to one another will also
bind in a similar manner to the same DNA binding sites of
transcriptional regulatory elements using methods well known to
those of skill in the art.
[0077] The term "equivalog" describes members of a set of
homologous proteins that are conserved with respect to function
since their last common ancestor. Related proteins are grouped into
equivalog families, and otherwise into protein families with other
hierarchically defined homology types. This definition is provided
at the Institute for Genomic Research (TIGR) world wide web (www)
website, "tigr.org" under the heading "Terms associated with
TIGRFAMs".
[0078] The term "variant", as used herein, may refer to
polynucleotides or polypeptides that differ from the presently
disclosed polynucleotides or polypeptides, respectively, in
sequence from each other, and as set forth below.
[0079] With regard to polynucleotide variants, differences between
presently disclosed polynucleotides and polynucleotide variants are
limited so that the nucleotide sequences of the former and the
latter are closely similar overall and, in many regions, identical.
Due to the degeneracy of the genetic code, differences between the
former and latter nucleotide sequences may be silent (i.e., the
amino acids encoded by the polynucleotide are the same, and the
variant polynucleotide sequence encodes the same amino acid
sequence as the presently disclosed polynucleotide. Variant
nucleotide sequences may encode different amino acid sequences, in
which case such nucleotide differences will result in amino acid
substitutions, additions, deletions, insertions, truncations or
fusions with respect to the similar disclosed polynucleotide
sequences. These variations may result in polynucleotide variants
encoding polypeptides that share at least one functional
characteristic. The degeneracy of the genetic code also dictates
that many different variant polynucleotides can encode identical
and/or substantially similar polypeptides in addition to those
sequences illustrated in the Sequence Listing.
[0080] Presently disclosed polypeptide sequences and similar
polypeptide variants may differ in amino acid sequence by one or
more substitutions, additions, deletions, fusions and truncations,
which may be present in any combination. These differences may
produce silent changes and result in a functionally equivalent
transcription factor. Thus, it will be readily appreciated by those
of skill in the art, that any of a variety of polynucleotide
sequences is capable of encoding the transcription factors and
transcription factor homolog polypeptides of the invention. A
polypeptide sequence variant may have "conservative" changes,
wherein a substituted amino acid has similar structural or chemical
properties. Deliberate amino acid substitutions may thus be made on
the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as a substantial amount of the functional or
biological activity of the transcription factor is retained. For
example, negatively charged amino acids may include aspartic acid
and glutamic acid, positively charged amino acids may include
lysine and arginine, and amino acids with uncharged polar head
groups having similar hydrophilicity values may include leucine,
isoleucine, and valine; glycine and alanine; asparagine and
glutamine; serine and threonine; and phenylalanine and tyrosine
(for more detail on conservative substitutions, see Table 6). More
rarely, a variant may have "non-conservative" changes, for example,
replacement of a glycine with a tryptophan. Similar minor
variations may also include amino acid deletions or insertions, or
both. Related polypeptides may comprise, for example, additions
and/or deletions of one or more N-linked or O-linked glycosylation
sites, or an addition and/or a deletion of one or more cysteine
residues. Guidance in determining which and how many amino acid
residues may be substituted, inserted or deleted without abolishing
functional or biological activity may be found using computer
programs well known in the art, for example, DNASTAR software (U.S.
Pat. No. 5,840,544).
[0081] Also within the scope of the invention is a variant of a
transcription factor nucleic acid listed in the Sequence Listing,
that is, one having a sequence that differs from the one of the
polynucleotide sequences in the Sequence Listing, or a
complementary sequence, that encodes a functionally equivalent
polypeptide (i.e., a polypeptide having some degree of equivalent
or similar biological activity) but differs in sequence from the
sequence in the Sequence Listing, due to degeneracy in the genetic
code. Included within this definition are polymorphisms that may or
may not be readily detectable using a particular oligonucleotide
probe of the polynucleotide encoding polypeptide, and improper or
unexpected hybridization to allelic variants, with a locus other
than the normal chromosomal locus for the polynucleotide sequence
encoding polypeptide.
[0082] "Allelic variant" or "polynucleotide allelic variant" refers
to any of two or more alternative forms of a gene occupying the
same chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations may be "silent" or may encode
polypeptides having altered amino acid sequence. "Allelic variant"
and "polypeptide allelic variant" may also be used with respect to
polypeptides, and in this case the term refer to a polypeptide
encoded by an allelic variant of a gene.
[0083] "Splice variant" or "polynucleotide splice variant" as used
herein refers to alternative forms of RNA transcribed from a gene.
Splice variation naturally occurs as a result of alternative sites
being spliced within a single transcribed RNA molecule or between
separately transcribed RNA molecules, and may result in several
different forms of mRNA transcribed from the same gene. Thus,
splice variants may encode polypeptides having different amino acid
sequences, which may or may not have similar functions in the
organism. "Splice variant" or "polypeptide splice variant" may also
refer to a polypeptide encoded by a splice variant of a transcribed
mRNA.
[0084] As used herein, "polynucleotide variants" may also refer to
polynucleotide sequences that encode paralogs and orthologs of the
presently disclosed polypeptide sequences. "Polypeptide variants"
may refer to polypeptide sequences that are paralogs and orthologs
of the presently disclosed polypeptide sequences.
[0085] "Ligand" refers to any molecule, agent, or compound that
will bind specifically to a complementary site on a nucleic acid
molecule or protein. Such ligands stabilize or modulate the
activity of nucleic acid molecules or proteins of the invention and
may be composed of at least one of the following: inorganic and
organic substances including nucleic acids, proteins,
carbohydrates, fats, and lipids.
[0086] "Modulates" refers to a change in activity (biological,
chemical, or immunological) or lifespan resulting from specific
binding between a molecule and either a nucleic acid molecule or a
protein.
[0087] The term "plant" includes whole plants, shoot vegetative
organs/structures (for example, leaves, stems and tubers), roots,
flowers and floral organs/structures (for example, bracts, sepals,
petals, stamens, carpels, anthers and ovules), seed (including
embryo, endosperm, and seed coat) and fruit (the mature ovary),
plant tissue (for example, vascular tissue, ground tissue, and the
like) and cells (for example, guard cells, egg cells, and the
like), and progeny of same. The class of plants that can be used in
the method of the invention is generally as broad as the class of
higher and lower plants amenable to transformation techniques,
including angiosperms (monocotyledonous and dicotyledonous plants),
gymnosperms, ferns, horsetails, psilophytes, lycophytes,
bryophytes, and multicellular algae (as shown, for example, in FIG.
1, adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333,
and in FIG. 2, adapted from Ku et al. (2000) Proc. Natl. Acad. Sci.
97: 9121-9126; and in Tudge (2000) in The Variety of Life, Oxford
University Press, New York, N.Y., pp. 547-606).
[0088] A "transgenic plant" refers to a plant that contains genetic
material not found in a wild-type plant of the same species,
variety or cultivar. The genetic material may include a transgene,
an insertional mutagenesis event (such as by transposon or T-DNA
insertional mutagenesis), an activation tagging sequence, a mutated
sequence, a homologous recombination event or a sequence modified
by chimeraplasty. Typically, the foreign genetic material has been
introduced into the plant by human manipulation, but any method can
be used as one of skill in the art recognizes.
[0089] A transgenic plant may contain an expression vector or
cassette. The expression cassette typically comprises a
polypeptide-encoding sequence operably linked (i.e., under
regulatory control of) to appropriate inducible or constitutive
regulatory sequences that allow for the expression of polypeptide.
The expression cassette can be introduced into a plant by
transformation or by breeding after transformation of a parent
plant. A plant refers to a whole plant as well as to a plant part,
such as seed, fruit, leaf, or root, plant tissue, plant cells or
any other plant material, for example, a plant explant, as well as
to progeny thereof, and to in vitro systems that mimic biochemical
or cellular components or processes in a cell.
[0090] "Wild type" or "wild-type", as used herein, refers to a
plant cell, seed, plant component, plant tissue, plant organ or
whole plant that has not been genetically modified or treated in an
experimental sense. Wild-type cells, seed, components, tissue,
organs or whole plants may be used as controls to compare levels of
expression and the extent and nature of trait modification with
cells, tissue or plants of the same species in which a
transcription factor expression is altered, for example, in that it
has been knocked out, overexpressed, or ectopically expressed.
[0091] A "control plant" as used in the present invention refers to
a plant cell, seed, plant component, plant tissue, plant organ or
whole plant used to compare against transgenic or genetically
modified plant for the purpose of identifying an enhanced phenotype
in the transgenic or genetically modified plant. A control plant
may in some cases be a transgenic plant line that comprises an
empty vector or marker gene, but does not contain the recombinant
polynucleotide of the present invention that is expressed in the
transgenic or genetically modified plant being evaluated. In
general, a control plant is a plant of the same line or variety as
the transgenic or genetically modified plant being tested. A
suitable control plant would include a genetically unaltered or
non-transgenic plant of the parental line used to generate a
transgenic plant herein.
[0092] "Fragment", with respect to a polynucleotide, refers to a
clone or any part of a polynucleotide molecule that retains a
usable, functional characteristic. Useful fragments include
oligonucleotides and polynucleotides that may be used in
hybridization or amplification technologies or in the regulation of
replication, transcription or translation. A "polynucleotide
fragment" refers to any subsequence of a polynucleotide, typically,
of at least about nine consecutive nucleotides, preferably at least
about 30 nucleotides, more preferably at least about 50
nucleotides, of any of the sequences provided herein. Exemplary
polynucleotide fragments are the first sixty consecutive
nucleotides of the transcription factor polynucleotides listed in
the Sequence Listing. Exemplary fragments include fragments
comprising a region that encodes a conserved domain (for example,
an AP2 domain) of a transcription factor.
[0093] Fragments may also include subsequences of polypeptides and
protein molecules, or a subsequence of the polypeptide. Fragments
may have uses in that they may have antigenic potential. In some
cases, the fragment or domain is a subsequence of the polypeptide
which performs at least one biological function of the intact
polypeptide in substantially the same manner, or to a similar
extent, as does the intact polypeptide. For example, a polypeptide
fragment can comprise a recognizable structural motif or functional
domain such as a DNA-binding site or domain that binds to a DNA
promoter region, an activation domain, or a domain for
protein-protein interactions, and may initiate transcription.
Fragments can vary in size from as few as 3 amino acid residues to
the full length of the intact polypeptide, but are preferably at
least about 30 amino acid residues in length and more preferably at
least about 60 amino acid residues in length. Exemplary polypeptide
fragments are the first twenty consecutive amino acids of the
transcription factor polypeptides listed in the Sequence Listing.
Exemplary fragments also include fragments that comprise an AP2
domain of a transcription factor, for example, amino acid residues
10-77 of G2133 (SEQ ID NO: 12), as noted in Table 1.
[0094] The invention also encompasses production of DNA sequences
that encode transcription factors and transcription factor
derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any
of the many available expression vectors and cell systems using
reagents well known in the art. Moreover, synthetic chemistry may
be used to introduce mutations into a sequence encoding
transcription factors or any fragment thereof.
[0095] "Derivative" refers to the chemical modification of a
nucleic acid molecule or amino acid sequence. Chemical
modifications can include replacement of hydrogen by an alkyl,
acyl, or amino group or glycosylation, pegylation, or any similar
process that retains or enhances biological activity or lifespan of
the molecule or sequence.
[0096] A "trait" refers to a physiological, morphological,
biochemical, or physical characteristic of a plant or particular
plant material or cell. In some instances, this characteristic is
visible to the human eye, such as seed or plant size, or can be
measured by biochemical techniques, such as detecting the protein,
starch, or oil content of seed or leaves, or by observation of a
metabolic or physiological process, for example, by measuring
tolerance to water deprivation or particular salt or sugar
concentrations, or by the observation of the expression level of a
gene or genes, for example, by employing Northern analysis, RT-PCR,
microarray gene expression assays, or reporter gene expression
systems, or by agricultural observations such as drought stress
tolerance or yield. Any technique can be used to measure the amount
of, comparative level of, or difference in any selected chemical
compound or macromolecule in the transgenic plants, however.
[0097] "Trait modification" refers to a detectable difference in a
characteristic in a plant ectopically expressing a polynucleotide
or polypeptide of the present invention relative to a plant not
doing so, such as a wild-type plant. In some cases, the trait
modification can be evaluated quantitatively. For example, the
trait modification can entail at least about a 2% increase or
decrease in an observed trait, or an even greater difference, as
compared with a wild-type or control plant. It is known that there
can be a natural variation in the modified trait. Therefore, the
trait modification observed entails a change of the normal
distribution and magnitude of the trait in the plants compared with
the distribution and magnitude observed in wild-type plants.
[0098] The term "transcript profile" refers to the expression
levels of a set of genes in a cell in a particular state,
particularly by comparison with the expression levels of that same
set of genes in a cell of the same type in a reference state. For
example, the transcript profile of a particular transcription
factor in a suspension cell is the expression levels of a set of
genes in a cell repressing or overexpressing that transcription
factor compared with the expression levels of that same set of
genes in a suspension cell that has normal levels of that
transcription factor. The transcript profile can be presented as a
list of those genes whose expression level is significantly
different between the two treatments, and the difference ratios.
Differences and similarities between expression levels may also be
evaluated and calculated using statistical and clustering
methods.
[0099] "Ectopic expression or altered expression" in reference to a
polynucleotide indicates that the pattern of expression in, for
example, a transgenic plant or plant tissue, is different from the
expression pattern in a wild-type plant or a reference plant of the
same species. The pattern of expression may also be compared with a
reference expression pattern in a wild-type plant of the same
species. For example, the polynucleotide or polypeptide is
expressed in a cell or tissue type other than a cell or tissue type
in which the sequence is expressed in the wild-type plant, or by
expression at a time other than at the time the sequence is
expressed in the wild-type plant, or by a response to different
inducible agents, such as hormones or environmental signals, or at
different expression levels (either higher or lower) compared with
those found in a wild-type plant. The term also refers to altered
expression patterns that are produced by lowering the levels of
expression to below the detection level or completely abolishing
expression. The resulting expression pattern can be transient or
stable, constitutive or inducible. In reference to a polypeptide,
the term "ectopic expression or altered expression" further may
relate to altered activity levels resulting from the interactions
of the polypeptides with exogenous or endogenous modulators or from
interactions with factors or as a result of the chemical
modification of the polypeptides.
[0100] The term "overexpression" as used herein refers to a greater
expression level of a gene in a plant, plant cell or plant tissue,
compared to expression in a wild-type plant, cell or tissue, at any
developmental or temporal stage for the gene. Overexpression can
occur when, for example, the genes encoding one or more
transcription factors are under the control of a strong promoter
described herein (for example, the cauliflower mosaic virus 35S
transcription initiation region), or overexpression can be induced
when an appropriate environmental signal is present. Overexpression
may occur throughout a plant or in specific tissues of the plant,
depending on the promoter used, as described below.
[0101] Overexpression may take place in plant cells normally
lacking expression of polypeptides functionally equivalent or
identical to the present transcription factors. Overexpression may
also occur in plant cells where endogenous expression of the
present transcription factors or functionally equivalent molecules
normally occurs, but such normal expression is at a lower level.
Overexpression thus results in a greater than normal production, or
"overproduction" of the transcription factor in the plant, cell or
tissue.
[0102] The term "transcription regulating region" refers to a DNA
regulatory sequence that regulates expression of one or more genes
in a plant when a transcription factor having one or more specific
binding domains binds to the DNA regulatory sequence. Transcription
factors of the present invention may possess, for example, an AP2
domain, in which case the AP2 domain of the transcription factor
binds to a transcription regulating region, such as AtERF1, which
binds to the motif AGCCGCC (the "GCC box") that are present in
promoters of genes such as PDF1.2. The transcription factors of the
invention also comprise an amino acid subsequence that forms a
transcription activation domain that regulates expression of one or
more abiotic stress tolerance genes in a plant when the
transcription factor binds to the regulating region.
[0103] A "sample" with respect to a material containing nucleic
acid molecules may comprise a bodily fluid; an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; genomic
DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; a forensic sample; and the like. In this
context "substrate" refers to any rigid or semi-rigid support to
which nucleic acid molecules or proteins are bound and includes
membranes, filters, chips, slides, wafers, fibers, magnetic or
nonmagnetic beads, gels, capillaries or other tubing, plates,
polymers, and microparticles with a variety of surface forms
including wells, trenches, pins, channels and pores. A substrate
may also refer to a reactant in a chemical or biological reaction,
or a substance acted upon (for example, by an enzyme).
Transcription Factors Modify Expression of Endogenous Genes
[0104] A transcription factor may include, but is not limited to,
any polypeptide that can activate or repress transcription of a
single gene or a number of genes. As one of ordinary skill in the
art recognizes, transcription factors can be identified by the
presence of a region or domain of structural similarity or identity
to a specific consensus sequence or the presence of a specific
consensus DNA-binding site or DNA-binding site motif (for example,
in Riechmann et al. (2000) Science 290: 2105-2110).
[0105] Generally, the transcription factors encoded by the present
sequences are involved in cell differentiation and proliferation
and the regulation of growth. Accordingly, one skilled in the art
would recognize that by expressing the present sequences in a
plant, one may change the expression of autologous genes or induce
the expression of introduced genes. By affecting the expression of
similar autologous sequences in a plant that have the biological
activity of the present sequences, or by introducing the present
sequences into a plant, one may alter a plant's phenotype to one
with improved traits related to drought stress, shade tolerance or
C/N sensing. The sequences of the invention may also be used to
transform a plant and introduce desirable traits not found in the
wild-type cultivar or strain. Plants may then be selected for those
that produce the most desirable degree of over- or under-expression
of target genes of interest and coincident trait improvement.
[0106] The sequences of the present invention may be from any
species, particularly plant species, in a naturally-occurring form
or from any source whether natural, synthetic, semi-synthetic or
recombinant. The sequences of the invention may also include
fragments of the present amino acid sequences. Where "amino acid
sequence" is recited to refer to an amino acid sequence of a
naturally occurring protein molecule, "amino acid sequence" and
like terms are not meant to limit the amino acid sequence to the
complete native amino acid sequence associated with the recited
protein molecule.
[0107] In addition to methods for modifying a plant phenotype by
employing one or more polynucleotides and polypeptides of the
invention described herein, the polynucleotides and polypeptides of
the invention have a variety of additional uses. These uses include
their use in the recombinant production (i.e., expression) of
proteins; as regulators of plant gene expression, as diagnostic
probes for the presence of complementary or partially complementary
nucleic acids (including for detection of natural coding nucleic
acids); as substrates for further reactions, for example, mutation
reactions, PCR reactions, or the like; as substrates for cloning
for example, including digestion or ligation reactions; and for
identifying exogenous or endogenous modulators of the transcription
factors. In many instances, a polynucleotide comprises a nucleotide
sequence encoding a polypeptide (or protein) or a domain or
fragment thereof. Additionally, the polynucleotide may comprise a
promoter, an intron, an enhancer region, a polyadenylation site, a
translation initiation site, 5' or 3' untranslated regions, a
reporter gene, a selectable marker, or the like. The polynucleotide
can be single-stranded or double-stranded DNA or RNA. The
polynucleotide optionally comprises modified bases or a modified
backbone. The polynucleotide can be, for example, genomic DNA or
RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a
cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide
can comprise a sequence in either sense or antisense
orientations.
[0108] Expression of genes that encode transcription factors that
modify expression of endogenous genes, polynucleotides, and
proteins are well known in the art. In addition, transgenic plants
comprising isolated polynucleotides encoding transcription factors
may also modify expression of endogenous genes, polynucleotides,
and proteins. Examples include Peng et al. (1997) Genes Development
11: 3194-3205, and Peng et al. (1999) Nature, 400: 256-261). In
addition, many others have demonstrated that an Arabidopsis
transcription factor expressed in an exogenous plant species
elicits the same or very similar phenotypic response (for example,
in Fu et al. (2001) Plant Cell 13: 1791-1802; Nandi et al. (2000)
Curr. Biol. 10: 215-218; Coupland (1995) Nature 377: 482-483; and
Weigel and Nilsson (1995) Nature 377: 482-500).
[0109] In another example, Mandel et al. (1992) Cell 71-133-143,
and Suzuki et al. (2001) Plant J. 28: 409-418 teach that a
transcription factor expressed in another plant species elicits the
same or very similar phenotypic response of the endogenous
sequence, as often predicted in earlier studies of Arabidopsis
transcription factors in Arabidopsis (Mandel et al. (1992) supra;
and Suzuki et al. (2001) supra). Other examples include Muller et
al. (2001) Plant J. 28: 169-179; Kim et al. (2001) Plant J. 25:
247-259; Kyozuka and Shimamoto (2002) Plant Cell Physiol. 43:
130-135; Boss and Thomas (2002) Nature, 416: 847-850; He et al.
(2000) Transgenic Res. 9: 223-227; and Robson et al. (2001) Plant
J. 28: 619-631.
[0110] In yet another example, Gilmour et al. (1998) Plant J. 16:
433-442, teach an Arabidopsis AP2 transcription factor, CBF1,
which, when overexpressed in transgenic plants, increases plant
freezing tolerance. Jaglo et al. (2001) Plant Physiol. 127:
910-917, further identified sequences in Brassica napus which
encode CBF-like genes and that transcripts for these genes
accumulated rapidly in response to low temperature. Transcripts
encoding CBF-like proteins were also found to accumulate rapidly in
response to low temperature in wheat, as well as in tomato. An
alignment of the CBF proteins from Arabidopsis, B. napus, wheat,
rye, and tomato revealed the presence of conserved consecutive
amino acid residues, PKK/RPAGRxKFxETRHP (SEQ ID NO: 1260) and DSAWR
(SEQ ID NO: 1261), that bracket the AP2/EREBP DNA binding domains
of the proteins and distinguish them from other members of the
AP2/EREBP protein family (Jaglo et al. (2001) supra).
[0111] Transcription factors mediate cellular responses and control
traits through altered expression of genes containing cis-acting
nucleotide sequences that are targets of the introduced
transcription factor. It is well appreciated in the art that the
effect of a transcription factor on cellular responses or a
cellular trait is determined by the particular genes whose
expression is either directly or indirectly (for example, by a
cascade of transcription factor binding events and transcriptional
changes) altered by transcription factor binding. In a global
analysis of transcription comparing a standard condition with one
in which a transcription factor is overexpressed, the resulting
transcript profile associated with transcription factor
overexpression is related to the trait or cellular process
controlled by that transcription factor. For example, the PAP2 gene
(and other genes in the MYB family) have been shown to control
anthocyanin biosynthesis through regulation of the expression of
genes known to be involved in the anthocyanin biosynthetic pathway
(Bruce et al. (2000) Plant Cell, 12: 65-79; Borevitz et al. (2000)
Plant Cell 12: 2383-93). Further, global transcript profiles have
been used successfully as diagnostic tools for specific cellular
states (for example, cancerous vs. non-cancerous; Bhattacharjee et
al. (2001) Proc Natl. Acad. Sci., USA, 98: 13790-13795; Xu et al.
(2001) Proc. Natl. Acad. Sci., USA, 98: 15089-15094). Consequently,
it is evident to one skilled in the art that similarity of
transcript profile upon overexpression of different transcription
factors would indicate similarity of transcription factor
function.
Polypeptides and Polynucleotides of the Invention
[0112] The present invention provides, among other things,
transcription factors (TFs), and transcription factor homolog
polypeptides, and isolated or recombinant polynucleotides encoding
the polypeptides, or novel sequence variant polypeptides or
polynucleotides encoding novel variants of transcription factors
derived from the specific sequences provided here.
[0113] The polynucleotides of the invention can be or were
ectopically expressed in overexpressor plant cells and the changes
in the expression levels of a number of genes, polynucleotides,
and/or proteins of the plant cells observed. Therefore, the
polynucleotides and polypeptides can be employed to change
expression levels of a genes, polynucleotides, and/or proteins of
plants or plant cells. These polypeptides and polynucleotides may
be employed to modify a plant's characteristics, particularly
drought tolerance, shade tolerance, and/or C/N sensing. The
polynucleotides of the invention can be or were ectopically
expressed in overexpressor or knockout plants and the changes in
the characteristic(s) or trait(s) of the plants observed.
Therefore, the polynucleotides and polypeptides can be employed to
improve the characteristics of plants. The polypeptide sequences of
the sequence listing have been shown to confer increased drought or
shade tolerance or altered C/N sensing when these polypeptides are
overexpressed in Arabidopsis plants. These polynucleotides have
been shown to have a strong association with these traits, in that
plants that overexpress these sequences are more tolerant to
drought, shade, or have altered C/N sensing, respectively. The
invention also encompasses a complement of the polynucleotides. The
polynucleotides are also useful for screening libraries of
molecules or compounds for specific binding and for creating
transgenic plants having improved traits. Altering the expression
levels of equivalogs of these sequences, including paralogs and
orthologs in the Sequence Listing, and other orthologs that are
structurally and sequentially similar to the former orthologs, has
been shown and is expected to confer similar phenotypes, including
altered C/N sensing, drought and/or shade tolerance in plants.
[0114] In some cases, exemplary polynucleotides encoding the
polypeptides of the invention were identified in the Arabidopsis
thaliana GenBank database using publicly available sequence
analysis programs and parameters. Sequences initially identified
were then further characterized to identify sequences comprising
specified sequence strings corresponding to sequence motifs present
in families of known transcription factors. In addition, further
exemplary polynucleotides encoding the polypeptides of the
invention were identified in the plant GenBank database using
publicly available sequence analysis programs and parameters.
Sequences initially identified were then further characterized to
identify sequences comprising specified sequence strings
corresponding to sequence motifs present in families of known
transcription factors. Polynucleotide sequences meeting such
criteria were confirmed as transcription factors.
[0115] Additional polynucleotides of the invention were identified
by screening Arabidopsis thaliana and/or other plant cDNA libraries
with probes corresponding to known transcription factors under low
stringency hybridization conditions. Additional sequences,
including full length coding sequences were subsequently recovered
by the rapid amplification of cDNA ends (RACE) procedure, using a
commercially available kit according to the manufacturer's
instructions. Where necessary, multiple rounds of RACE are
performed to isolate 5' and 3' ends. The full-length cDNA was then
recovered by a routine end-to-end polymerase chain reaction (PCR)
using primers specific to the isolated 5' and 3' ends. Exemplary
sequences are provided in the Sequence Listing.
[0116] The polynucleotides are particularly useful when they are
hybridizable array elements in a microarray. Such a microarray can
be employed to monitor the expression of genes that are
differentially expressed in response to limited light, drought,
other osmotic stresses, or low nitrogen availability. The
microarray can be used in large scale genetic or gene expression
analysis of a large number of polynucleotides; or in the diagnosis
of, for example, drought stress before phenotypic symptoms are
evident. Furthermore, the microarray can be employed to investigate
cellular responses, such as cell proliferation, transformation, and
the like.
[0117] When the polynucleotides of the invention may also be used
as hybridizable array elements in a microarray, the array elements
are organized in an ordered fashion so that each element is present
at a specified location on the substrate. Because the array
elements are at specified locations on the substrate, the
hybridization patterns and intensities (which together create a
unique expression profile) can be interpreted in terms of
expression levels of particular genes and can be correlated with a
particular stress, pathology, or treatment.
[0118] The invention also entails an agronomic composition
comprising a polynucleotide of the invention in conjunction with a
suitable carrier and a method for altering a plant's trait using
the composition.
[0119] Examples of specific polynucleotide and polypeptides of the
invention, and equivalog sequences, along with descriptions of the
gene families that comprise these polynucleotides and polypeptides,
are provided below.
[0120] Examples of specific polynucleotide and polypeptides of the
invention, and equivalog sequences, are provided below.
[0121] Polypeptide sequences of the sequence listing, including,
for example, Arabidopsis sequences G2133, G1274, G922, G2999,
G3086, G354, G1792, G2053, G975, G1069, G916, G1820, G2701, G47,
G2854, G2789, G634, G175, G2839, G1452, G3083, G489, G303, G2992,
and G682 (SEQ ID NOs: 12, 6, 4, 14, 16, 228, 8, 10, 238, 240, 236,
244, 246, 2, 252, 248, 232, 224, 250, 242, 254, 230, 226, 50 and
234, respectively) have been shown to confer increased drought
tolerance when expression of these polypeptides is altered in
Arabidopsis plants. These polynucleotides have been shown to have a
strong association with drought stress tolerance, in that plants
that overexpress these sequences are more tolerant to drought.
Exemplary sequences of the invention include G2133, G47, and
structurally and functionally-related sequences found in the G47
clade of transcription factor polypeptides (examples of which may
be found in FIGS. 3 and 4).
[0122] A number of the polypeptide sequences of the sequence
listing, including, for example, G682, G226, G1816, G2718, G24,
G154, G384, G486, G545, G760, G773, G937, G971, G988, G989, G1069,
G1090, G1322, G1587, G1666, G1700, G1818, G1868, G1888, G2117,
G2131, G2520, G2522, G2789, G8, G27, G156, G161, G168, G183, G189,
G200, G234, G237, G275, G326, G347, G427, G505, G590, G602, G618,
G635, G643, G653, G657, G837, G866, G872, G904, G912, G932, G958,
G964, G975, G979, G1049, G1246, G1255, G1266, G1331, G1332, G1494,
G1535, G1649, G1750, G1773, G1835, G1930, G2053, G2057, G2133,
G2144, G2145, G2295, G2512, G2531, G2535, G2590, and G2719 (SEQ ID
NOs: 234, 286, 312, 324, 420, 422, 424, 294, 426, 428, 430, 432,
434, 436, 438, 240, 440, 442, 444, 446, 448, 450, 452, 454, 456,
458, 460, 462, 248, 464, 466, 468, 470, 472, 474, 476, 478, 480,
482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506,
508, 510, 512, 514, 516, 518, 520, 522, 238, 524, 526, 528, 530,
532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 10, 552, 12, 554,
556, 558, 560, 562, 564, 566, and 568, respectively) have been
shown to confer altered C/N sensing when expression of these
polypeptides is altered in Arabidopsis plants. A number of these
polynucleotides have also been shown to confer increased tolerance
to low nutrient (e.g., nitrogen-limited) environments and other
abiotic stress tolerances such as drought, heat, and cold.
Exemplary sequences of the invention include G682 and structurally
and functionally-related sequences found in the G682 subclade
(examples may be found in FIGS. 20A, 20B and 21).
[0123] A number of the polypeptide sequences of the sequence
listing, including, for example, G634, G1048, G1100, G1412, G2505,
G1796, G1995, G2467, G2550, G2640, G2686, and G2789 (SEQ ID NOs:
232, 808, 810, 658, 818, 812, 814, 816, 820, 822, 824 and 248,
respectively) have also been shown to confer increased shade
tolerance when expression of these polypeptides is altered in
Arabidopsis plants. Equivalogs of these sequences, including
paralogs and orthologs in the Sequence Listing, and other orthologs
that are structurally and sequentially similar to the former
orthologs, are expected to confer increased shade tolerance in
plants when their expression is altered. Exemplary sequences of the
invention include G634 and structurally and functionally-related
sequences found in the 6634 clade (examples of which may be found
in Table 8).
[0124] The invention also encompasses the complements of these
polynucleotides. The polynucleotides are also useful for screening
libraries of molecules or compounds for specific binding and for
creating transgenic plants having altered C/N sensing or increased
abiotic stress or shade tolerance. Equivalogs of these sequences,
including paralogs and orthologs in the Sequence Listing, and other
orthologs that are structurally and sequentially similar to the
former orthologs, are expected to confer altered C/N sensing and/or
abiotic stress tolerance in plants when their expression is
altered.
[0125] The AP2 Family, Including the G47/G2133 and G1792
Clades.
[0126] AP2 (APETALA2) and EREBPs (Ethylene-Responsive Element
Binding Proteins) are the prototypic members of a family of
transcription factors unique to plants, whose distinguishing
characteristic is that they contain the so-called AP2 DNA-binding
domain (Riechmann and Meyerowitz (1998) Biol. Chem. 379: 633-646).
The AP2 domain was first recognized as a repeated motif within the
Arabidopsis thaliana AP2 protein (Jofuku et al. (1994) Plant Cell
6: 1211-1225). Shortly afterwards, four DNA-binding proteins from
tobacco were identified that interact with a sequence that is
essential for the responsiveness of some promoters to the plant
hormone ethylene, and were designated as ethylene-responsive
element binding proteins (EREBPs; Ohme-Takagi et al. (1995) Plant
Cell 7: 173-182). The DNA-binding domain of EREBP-2 was mapped to a
region that was common to all four proteins (Ohme-Takagi et al
(1995) supra), and that was found to be closely related to the AP2
domain (Weigel (1995) Plant Cell 7: 388-389) but that did not bear
sequence similarity to previously known DNA-binding motifs.
[0127] AP2/EREBP genes form a large family, with many members known
in several plant species (Okamuro et al. (1997) Proc. Natl. Acad.
Sci. USA 94: 7076-7081; Riechmann and Meyerowitz (1998) supra). The
number of AP2/EREBP genes in the Arabidopsis thaliana genome is
approximately 145 (Riechmann et al. (2000) Science 290: 2105-2110).
The APETALA2 class is characterized by the presence of two AP2 DNA
binding domains, and contains 14 genes. The AP2/ERF is the largest
subfamily, and includes 125 genes which are involved in abiotic
(DREB subgroup) and biotic (ERF subgroup) stress responses and the
RAV subgroup includes 6 genes which all have a B3 DNA binding
domain in addition to the AP2 DNA binding domain (Kagaya et al.
(1999) Nucleic Acids Res. 27: 470-478).
[0128] Arabidopsis AP2 is involved in the specification of sepal
and petal identity through its activity as a homeotic gene that
forms part of the combinatorial genetic mechanism of floral organ
identity determination and it is also required for normal ovule and
seed development (Bowman et al. (1991) Development 112: 1-20;
Jofuku et al. (1994) supra). Arabidopsis ANT is required for ovule
development and it also plays a role in floral organ growth
(Elliott et al. (1996) Plant Cell 8: 155-168; Klucher et al. (1996)
Plant Cell 8: 137-153). Finally, maize G115 regulates leaf
epidermal cell identity (Moose et al. (1996) Genes Dev. 10:
3018-3027).
[0129] The attack of a plant by a pathogen may induce defense
responses that lead to resistance to the invasion, and these
responses are associated with transcriptional activation of
defense-related genes, among them those encoding
pathogenesis-related (PR) proteins. The involvement of EREBP-like
genes in controlling the plant defense response is based on the
observation that many PR gene promoters contain a short cis-acting
element that mediates their responsiveness to ethylene (ethylene
appears to be one of several signal molecules controlling the
activation of defense responses). Tobacco EREBP-1, -2, -3, and -4,
and tomato Pti4, Pti5 and Pti6 proteins have been shown to
recognize such cis-acting elements (Ohme-Takagi (1995) supra; Zhou
et al. (1997) EMBO J. 16: 3207-3218). In addition, Pti4, Pti5, and
Pti6 proteins have been shown to directly interact with Pto, a
protein kinase that confers resistance against Pseudomonas syringae
pv tomato (Zhou et al. (1997) supra). Plants are also challenged by
adverse environmental conditions like cold or drought, and
EREBP-like proteins appear to be involved in the responses to these
abiotic stresses as well. COR (for cold-regulated) gene expression
is induced during cold acclimation, the process by which plants
increase their resistance to freezing in response to low unfreezing
temperatures. The Arabidopsis EREBP-like gene CBF1 (Stockinger et
al. (1997) Proc. Natl. Acad. Sci. USA 94: 1035-1040) is a regulator
of the cold acclimation response, because ectopic expression of
CBF1 in Arabidopsis transgenic plants induced COR gene expression
in the absence of a cold stimulus, and the plant freezing tolerance
was increased (Jaglo-Ottosen et al. (1998) Science 280: 104-106).
Finally, another Arabidopsis EREBP-like gene, ABI4, is involved in
ABA signal transduction, because abi4 mutants are insensitive to
ABA (ABA is a plant hormone that regulates many agronomically
important aspects of plant development; Finkelstein et al. (1998)
Plant Cell 10: 1043-1054).
[0130] Of the sequences examined to date, two valine residues were
found that are present in members of the G47 clade but not outside
of the clade (indicated by the arrows in FIG. 3). All members of
the clade examined thus far have the subsequence:
[0131] V-(X).sub.17-A-A-V-A-H-D-X-A (SEQ ID NO: 1262), where X is
any amino acid and the identified residues are indicated by the
residues shown in the boxes in FIG. 3.
[0132] The SCR Family, Including the G922 Clade.
[0133] The SCARECROW gene, which regulates an asymmetric cell
division essential for proper radial organization of root cell
layers, was isolated from Arabidopsis thaliana by screening a
genomic library with sequences flanking a T-DNA insertion causing a
"scarecrow" mutation (Di Laurenzio et al. (1996) Cell 86, 423-433).
The gene product was tentatively described as a transcription
factor based on the presence of homopolymeric stretches of several
amino acids, the presence of a basic domain similar to that of the
basic-leucine zipper family of transcription factors, and the
presence of leucine heptad repeats. The presence of several
Arabidopsis ESTs with gene products homologous to the SCARECROW
gene were noted. The ability of the SCARECROW gene to complement
the scarecrow mutation was also demonstrated (Malamy et al. (1997)
Plant J. 12, 957-963).
[0134] More recently, the SCARECROW homologue RGA, which encodes a
negative regulator of the gibberellin signal transduction pathway,
was isolated from Arabidopsis by genomic subtraction (Silverstone
et al. (1998) Plant Cell 10, 155-169). The RGA gene was shown to be
expressed in many different tissues and the RGA protein was shown
to be localized to the nucleus. The same gene was isolated by
Truong (Truong et al. (1997) FEBS Lett. 410: 213-218) by
identifying cDNA clones which complement a yeast nitrogen
metabolism mutant, suggesting that RGA may be involved in
regulating diverse metabolic processes. Another SCARECROW homologue
designated GAI, which also is involved in gibberellin signaling
processes, has been isolated by Peng (Peng et al. (1997) Genes Dev.
11, 3194-3205). Interestingly, GAI is the gene that initiated the
Green Revolution. Peng et al. (Peng et al. (1999) Nature 6741,
256-261) have recently shown that maize GAI orthologs, when
mutated, result in plants that are shorter, have increased seed
yield, and are more resistant to damage by rain and wind than wild
type plants. Based on the inclusion of the GAI, RGA and SCR genes
in this family, it has also been referred to as the GRAS family
(Pysh et al. (1999) Plant J 18, 111-19).
[0135] The scarecrow gene family has 32 members in the Arabidopsis
genome.
[0136] The WRKY Family, Including the G1274 Clade.
[0137] The WRKY family of transcription factors is thus far only
found in plants. It is primarily characterized by a 60 amino acid
conserved DNA binding domain and a zinc finger domain. The family
is divided into groups based on whether the protein has two or only
one WRKY domain (Groups I and II, respectively), and further
subdivided based on a unique variation of the zing finger motif
(Group III) as described by Eulgem (Eulgem et al. (2000) Trends
Plant Science 5:199-206). G1274 (polynucleotide SEQ ID NO: 5 and
polypeptide SEQ ID NO: 6) belongs to the so-called Group II class
of WRKY proteins, which can be further subdivided into 5 groups
(a-e) based on conserved structural features outside of the WRKY
domain. G1274 is a member of the IIc subgroup.
[0138] The phylogenetic tree in FIG. 17 uses other closely related
members of the WRKY Group IIc family as a natural out-group to the
G1274 clade. Using either the full protein, or WRKY domain, the
potentially orthologous sequences shown on the tree appear most
closely related to the G1274 paralog clade. FIG. 16 indicates amino
acids within the WRKY domain that differentiate the G1274 clade
from the out-group. Notable for the G1274 clade are the conserved K
at position 264, the N at position 275, the S at position 280, and
the F/Y at position 299 (indicated by arrows in FIG. 16). These
residues are potentially responsible for the conserved
structure/function of this clade with regard to drought tolerance.
The G1274 domain may thus be distinguished by the subsequence:
TABLE-US-00001 (SEQ ID NO: 1263)
RR-K-Y-G-K-K-(X).sub.8-R-N-Y-(X).sub.2-C-S-(X).sub.5-V-K-K-X-V-X-
R-(X).sub.6-Y/F-V .
[0139] Amino acid residues within the WRKY domain that distinguish
the G1274 clade sequences, and are putatively responsible for
conserved functionality, are indicated within the boxes in FIG.
16.
[0140] Based on full-length protein sequence, G1758 appears firmly
in the G1274 clade. However, FIG. 16 shows that, within the WRKY
domain, G1758 is intermediate between the out-group and the claimed
sequences. These amino acid differences may represent specific
changes that retain drought tolerance function, or possibly more
finely delineate the key residues required for function.
[0141] The NAC Family, Including the G2053 Clade.
[0142] The NAC family is a group of transcription factors that
share a highly conserved N-terminal domain of about 150 amino
acids, designated the NAC domain (NAC stands for Petunia, N A M,
and Arabidopsis, ATAF1, ATAF2 and CUC2). This is believed to be a
novel domain that is present in both monocot and dicot plants but
is absent from yeast and animal proteins. One hundred and twelve
members of the NAC family have been identified in the Arabidopsis
genome. The NAC class of proteins can be divided into at least two
sub-families on the basis of amino acid sequence similarities
within the NAC domain. One sub-family is built around the NAM and
CUC2 (cup-shaped cotyledon) proteins whilst the other sub-family
contains factors with a NAC domain similar to those of ATAF1 and
ATAF2.
[0143] Thus far, little is known about the function of different
NAC family members. This is surprising given that there are 113
members in Arabidopsis. However, NAM, CUC1 and CUC2 are thought to
have vital roles in the regulation of embryo and flower
development. In Petunia, nam mutant embryos fail to develop a shoot
apical meristem (SAM) and have fused cotyledons. These mutants
sometimes generate escape shoots that produce defective flowers
with extra petals and fused organs. In Arabidopsis, the cuc1 and
cuc2 mutations have somewhat similar effects, causing defects in
SAM formation and the separation of cotyledons, sepals and
stamens.
[0144] Although nam and cuc mutants exhibit comparable defects
during embryogenesis, the penetrance of these phenotypes is much
lower in cuc mutants. Functional redundancy of the CUC genes in
Arabidopsis may explain this observation. In terms of the flower
phenotype there are notable differences between nam and cuc
mutants. Flowers of cuc mutants do not contain additional organs
and the formation of sepals and stamens is most strongly affected.
In nam mutants, by contrast, the flowers do carry additional organs
and petal formation is more markedly affected than that of other
floral organs. These apparent differences might be explained in two
ways: the NAM and CUC proteins have been recruited into different
roles in development of Arabidopsis and Petunia flowers.
Alternatively, the proteins could share a common function between
the two species, with the different mutant floral phenotypes
arising from variations in the way other genes (that participate in
the same developmental processes) are affected by defects in NAM or
CUC.
[0145] A further gene from this family, NAP (NAC-like activated by
AP3/PI) is also involved in flower development and is thought to
influence the transition between cell division and cell expansion
in stamens and petals. Overall, then, the NAC proteins mainly
appear to regulate developmental processes.
[0146] The ZF-HD Family, Including the G2999 Clade.
[0147] Since their discovery in 1983, the homeobox genes (the name
of which derives from the homeotic mutations that affect Drosophila
development) have been found in all eukaryotes examined, including
yeast, plants, and animals (McGinnis et al. (1984) Nature 308:
428-433; McGinnis et al. (1984) Cell 37: 403-408; Scott et al.
(1984) Proc. Natl. Acad. Sci. U.S.A. 81: 4115-4119; Scott et al.
(1989) Biochim. Biophys. Acta. 989, 25-48; Shepherd et al. (1984)
Nature 310: 70-71; Gehring et al. (1987) Science 236: 1245-1252;
Vollbrecht et al. (1991) Nature 350: 241-243; Ruberti et al. (1991)
EMBO J. 10: 1787-1791; and Schena and Davis (1992) Genes. Dev. 7,
367-379. The homeobox (HB) is a conserved DNA stretch that encodes
an approximate 61 amino acid region termed the homeodomain (HD). It
is well demonstrated that homeodomain proteins are transcription
factors, and that the homeodomain is responsible for sequence
specific recognition and binding of DNA (Affolter et al. (1990)
Curr Opin Cell Biol. 2: 485-495; Hayashi and Scott (1990) Cell 63:
883-894, and references therein). Genetic and structural analysis
indicate that the homeodomain operates by fitting the most
conserved of three .alpha.-helices, helix 3, directly into the
major groove of the DNA (Hanes and Brent (1989) Cell 57: 1275-1283;
Hanes and Brent (1991) Science 251: 426-430; Kissinger et al.
(1990) Cell 63: 579-590; and Wolberger et al. (1991) Cell 67:
517-528). A general review on the homeobox genes is provided by
Duboule, D. (1994). Guidebook to the Homeobox Genes. Oxford, Oxford
University Press.
[0148] Homeobox genes play many important roles in the
developmental processes of multicellular animals. In Drosophila,
for example, a variety of these genes have functions in embryo
development. Initially, they act maternally to establish
anterior-posterior polarity. Later, homeobox genes are known to
regulate the segmentation process, dorso-ventral differentiation,
and control cell fate determination in the eye and nervous system
(Scott et al. (1989) supra).
[0149] A large number of homeodomain proteins have now been
identified in a range of higher plants (Burglin (1997) Nucleic
Acids Res. 25: 4173-4180; Burglin (1998) Dev. Genes Evol. 208:
113-116), which are herein defined as the containing the
`classical` type of homeodomain (FIGS. 6A-6C). These exhibit many
differences to animal homeodomain proteins outside the conserved
domain, but all contain the signature WFXNX[RK] (SEQ ID NO: 1264;
X=any amino acid, [RK] indicates either an R or K residue at this
position) within the third helix. Data from the Genome Initiative
indicate that there are around 90 Arabidopsis classical homeobox
genes. These are now being implicated in the control of a wide
range of different processes. In many cases, plant homeodomains are
found in proteins in combination with additional regulatory motifs
such as leucine zippers. Classical plant homeodomain proteins can
be broadly categorized into the following different classes based
on homologies within the family, and the presence of other types of
domain: KNOX class I, KNOX class II, HD-BEL1, HD-ZIP class I,
HD-ZIP class II, HD-ZIP class III, HD-ZIP class IV (GL2 like), PHD
finger type, and WUSCHEL-like (Freeling and Hake (1985); Genetics
111: 617-634 Vollbrecht et al. (1991) supra; Schindler et al.
(1993) Plant J. 4:137-150; Sessa et al. (1994)). In: Puigdomenech
P, Coruzzi G, (eds) Molecular genetic analysis of plant development
and metabolism, pp. 411-426. Springer Verlag, Berlin; Kerstetter et
al. (1994) Plant Cell 6: 1877-1887; Kerstetter et al. (1997)
Development 124: 3045-3054; Burglin (1997) supra; Burglin (1998)
supra; Schoof et al. (2000) Cell 100: 635-644).
[0150] Recently a novel class of proteins was discovered that
contain a domain similar to the classical homeodomain, in
combination with N-terminal zinc finger motifs, by Windhovel
(Windhovel et al. (2001) Plant Mol. Biol. 45: 201-214), while
studying the regulatory mechanisms responsible for the mesophyll
specific expression of the C4 phosphoenolpyruvate gene of Flavaria
trinervia. Using a yeast one-hybrid screen, these workers recovered
five cDNA clones, which encoded proteins that were capable of
specifically binding the promoter of the Flavaria C4
phosphoenolpyruvate gene, but not the promoter of a Flavaria C3
phosphoenolpyruvate gene. One-hybrid experiments and in vitro DNA
binding studies were then used to confirm that these proteins
specifically interact with the proximal region of the C4
phosphoenolpyruvate gene. Four of five clones [FtHB1 (GenBank
accession Y18577), FbHB2 (GenBank accession Y18579), FbHB3 (GenBank
accession Y18580), and FbHB4 (GenBank accession Y18581), (the fifth
clone encoded a histone)] all encoded a novel type of protein that
contained two types of highly conserved domains. At the C-termini,
a region was apparent that had many of the features of a
homeodomain, whereas at the N-termini, two putative zinc finger
motifs were present. Yeast two-hybrid experiments were used to show
that the zinc finger motifs are sufficient to confer homo and
hetero-dimerization between the proteins, and mutagenesis
experiments demonstrated that conserved cysteine residues within
the motifs are essential for such dimerization. Given the presence
of the potential homeodomain and zinc fingers, Windhovel (Windhovel
et al. (2001) supra) named this new class of proteins as the ZF-HD
group.
[0151] That four proteins of this type were identified in the above
studies suggested that the family might have a specific role in
establishing expression of the C4 phosphoenolpyruvate gene within
mesophyll cells. However, database searches revealed that proteins
of this class are also present in C3 species, indicating that they
likely have additional roles outside of C4 photosynthesis
(Windhovel et al. (2001) supra). In particular, the Arabidopsis
genome encodes fourteen proteins of this type, but the functional
analysis of these proteins has yet to be publicly reported.
[0152] Secondary structure analyses performed by Windhovel
(Windhovel et al. (2001) supra) indicated that the putative
homeodomains of the ZF-HD proteins contain three .alpha.-helices
similar to those recognized in the classes of homeodomain already
found in plants (Duboule (1994) supra). Interestingly, though, if
full-length proteins of the ZF-HD group are blasted against
databases, they do not preferentially align with the known classes
of plant homeodomain proteins. Furthermore, a phylogenetic tree
based on comparing the classical versus ZF-HD type homeodomains
reveal that the latter occupy a distinct node of the tree (FIG.
8).
[0153] A careful examination of the ZF-HD proteins reveals a
particular striking difference to the classical plant homeodomain.
All of the 90 or so previously recognized plant homeodomain
proteins contain the signature WFXNX[RK] (SEQ ID NO: 1264; X=any
amino acid) within the third helix. However, the ZF-HD proteins all
lack the invariant F residue in this motif and generally contain an
M in its place. This structural distinction, combined with the
presence of ZF motifs in other regions of the protein, could confer
functional properties on ZF-HD proteins that are different to those
found in other HD containing proteins.
[0154] Residues that may be used to identify the G2999 clade are
shown in boxes in FIGS. 6A and 6B. As shown in FIGS. 6A and 6B, a
number of amino acid residues may be used to identify G2999 clade
members. Of the G2999 clade members examined to date, each is a
ZF-HD polypeptide and comprises the consensus subsequence:
K-(X).sub.17-W-(X).sub.13-15-C-(X).sub.12-W-(X).sub.2-N-N/H-K (SEQ
ID NO: 1265, 1266 or 1267), where X is any amino acid.
[0155] The HLH/MYC Family, Including the G3086 Clade.
[0156] The bHLH protein family is a group of transcription factors
found in mammals and plants. The typical feature of this family of
transcription factors is that they share a highly conserved
approximately 50 amino acid DNA-binding domain. This domain
consists of a basic region of 14 amino acids followed by a first
helix, a loop region of seven amino acids and a second helix
(Littlewood et al. (1994) Prot. Profile 1: 639-709). In plants,
members of this family also share, besides the bHLH domain, a
highly conserved 200 amino acid N-terminal domain. Functional
analysis revealed that small deletions in the N-terminal domain
inactivate the B protein, a member of bHLH protein family, in Z.
mays (Goff et al. (1992) Genes Dev. 6: 864-875). It has also been
shown that the N-terminal domain can interact with one of other
transcription factors (Myb proteins) to regulate anthocyanin
biosynthesis in Z. mays (Goff et al. (1992) supra).
[0157] In mammalian systems, members of this family have been shown
to control development and differentiation of a variety of cell
types. The bHLH proteins play essential roles in neurogenesis or
neural development, and myogenesis (Littlewood et al. (1994)
supra).
[0158] Plant bHLH proteins have been shown to play an important
role in the regulation of anthocyanin biosynthesis, in the control
of trichome development, in phytochrome signaling transduction
pathway, and in the regulation of dehydration- and ABA-inducible
gene expression. It has suggested that the R locus of maize is
responsible for determining the temporal and spatial pattern of
anthocyanin pigmentation in the plant. The R gene family consists
of B, S, and Lc genes, which encode a transcription factor of the
basic helix-loop-helix class (Goff et al. (1992) supra, Ludwig
(1990) Cell 62: 849-851). A gene encoding a basic helix-loop-helix
protein has been cloned as a phytochrome-interacting factor in a
genetic screen for T-DNA-tagged Arabidopsis mutants as well as in a
yeast two-hybrid screen. The protein functions as a
positively-acting signaling intermediate (Halliday et al. (1999)
Proc. Natl. Acad. Sci. USA. 96:5832-5837, Ni et al. (1998) Cell 95:
657-667). A new mutant, hfr1 (long hypocotyl in far-red) has been
isolated from Quail's lab. The hfr1 mutant exhibits a reduction in
seedling responsiveness specifically to continuous far-red light
(FRc), thereby suggesting a locus likely to be involved in
phytochrome A (phyA) signal transduction. HFR1 encodes a nuclear
protein with strong similarity to the bHLH family of DNA-binding
proteins but with an atypical basic region. In contrast to PIF3, a
related bHLH protein previously shown to bind phyB, HFR1 did not
bind either phyA or B. However, HFR1 did bind PIF3, suggesting
heterodimerization, and both the HFR1/PIF3 complex and PIF3
homodimer bound preferentially to the Pfr form of both
phytochromes. Thus, HFR1 may function to modulate phyA signaling
via heterodimerization with PIF3. HFR1 mRNA is 30-fold more
abundant in FRc than in continuous red light, suggesting a
potential mechanistic basis for the specificity of HFR1 to phyA
signaling.
[0159] The rd22BP1 protein of Arabidopsis has a typical DNA-binding
domain of a basic region helix-loop-helix motif. It has been shown
that transcription of the rd22BP1 gene is induced by dehydration
stress and phytohormone ABA treatment, and its induction precedes
that of rd22, a dehydration-responsive gene (Abe et al. (1997)
Plant Cell 9: 1859-1868).
[0160] Plant bHLH proteins may also play a crucial role in the
process of nitrogen fixation, probably not acting as a
transcription factor. A protein with a helix-loop-helix motif was
identified as a symbiotic ammonium transport protein by functional
complementation of the yeast NH.sub.4+ transport mutant with a
soybean nodule cDNA (Kaiser et al. (1998) Science 1998 281:
1202-1206). Using similar complementation approach of the yeast
fet3fet4 mutant strain, an iron transport protein was isolated from
an iron-deficient maize root cDNA expression library. The protein
had 44% identity with an Arabidopsis bHLH-like protein RAP1 that
binds the G-box sequence via a basic region helix-loop-helix
(Loulergue (1998) Gene 225:47-57).
[0161] Another bHLH gene has been recently identified as ind1
(Liljegren et al. (2000) in 11th International Conference on
Arabidopsis Research, Madison, Wis.; TAIR Accession Publication No.
1547039). They found that fruit from a knockout mutant do not show
dehiscence zone differentiation. In addition, their results suggest
that ind1 may mediate cell differentiation during Arabidopsis fruit
development. A cytokinin-repressed gene CRR12 with a basic
region/helix-loop-helix motif was identified from a cucumber
cotyledon cDNA library. It was found that the level of CRR12
transcripts decreased in response to either cytokinins or light in
etiolated cotyledons. The mRNA was low in cotyledons and leaves of
light-grown plants, but it increased during dark incubation.
[0162] As shown in FIG. 12, a number of amino acid residues may be
used to identify G3086 clade members. Of the G3086 clade members
examined to date, each of their sequences comprise the consensus
subsequence:
K-(X).sub.5-H-X-R-S-I-A-X-R-X-R-R-T-R/K-I-(X).sub.6-L-(X).sub.2-L-X-P-(X)-
.sub.2-D-K-Q-T-(X).sub.4-5-M-(X).sub.8-K-X-L-Q (SEQ ID NOs: 1268 or
1269), where X is any amino acid.
[0163] Table 1 shows exemplary sequences of the invention that, in
many cases, confer drought tolerance when overexpressed. The
polypeptides are identified by polypeptide SEQ ID NO and Identifier
(for example, Gene ID (GID) No., accession number or other name),
presented in order of similarity to the first Arabidopsis sequence
listed for each set, and includes the conserved domains of the
polypeptide in amino acid coordinates, the respective domain
sequences, and the extent of identity in percentage terms to the
first Arabidopsis sequence listed for each set.
TABLE-US-00002 TABLE 1 Gene families and binding domains for
exemplary sequences conferring drought tolerance, including
paralogs and orthologs Conserved Conserved Domains in Domains in
SEQ ID SEQ Polypeptide Polypeptide NO: of % ID in ID Amino Acid
Base Conserved conserved conserved NO: GID Species Coordinates
Coordinates Domain Sequence domain domain % to G2133 12 G2133
Arabidopsis AP2: 10-77 AP2: 53-256 DQSKYKGIRRRKWGKW 1270 100%
thaliana VSEIRVPGTRQRLWLGSF STAEGAAVAHDVAFYCL HRPSSLDDESFNFPHLL 94
G3646 Brassica AP2: 10-77 AP2: 203-406 HQAKYKGIRRRKWGKW 1271 91%
oleracea VSEIRVPATRERLWLGSF STAEGAAVAHDVAFYCL HRPSSLDNEAFNFPHLL 92
G3645 Brassica rapa AP2: 10-75 AP2: 40-237 TQSKYKGIRRRKWGKW 1272
89% subsp. VSEIRVPGTRDRLWLGSF Pekinensis STAEGAAVAHDVAFYCL
HQPNSLESLNFPHLL 2 G47 Arabidopsis AP2: 10-75 AP2: 65-262
SQSKYKGIRRRKWGKWV 1273 88% thaliana SEIRVPGTRDRLWLGSFS
TAEGAAVAHDVAFFCLH QPDSLESLNFPHLL 88 G3643 Glycine max AP2: 13-78
AP2: 101-298 TNNKLKGVRRRKWGKW 1274 69% VSEIRVPGTQERLWLGTY
ATPEAAAVAHDVAVYCL SRPSSLDKLNFPETL 96 G3647 Zinnia AP2: 13-78 AP2:
53-250 SQKTYKGVRCRRWGKW 1275 63% elegans VSEIRVPGSRERLWLGTY
STPEGAAVAHDVASYCL KGNTSFHKLNIPSML 90 G3644 Oryza sativa AP2: 52-122
AP2: 154-366 ERCRYRGVRRRRWGKW 1276 54% (japonica VSEIRVPGTRERLWLGSY
cultivar- ATPEAAAVAHDTAVYFL group) RGGAGDGGGGGATLNFP ERA 98 G3649
Oryza sativa AP2: 15-87 AP2: 43-261 EMMRYRGVRRRRWGK 1277 53%
(japonica WVSEIRVPGTRERLWLGS cultivar- YATAEAAAVAHDAAVC group)
LLRLGGGRRAAAGGGGG LNFPARA 100 G3651 Oryza sativa AP2: 60-130 AP2:
178-390 ERCRYRGVRRRRWGKW 1278 52% (japonica VSEIRVPGTRERLWLGSY
cultivar- ATPEAAAVAHDTAVYFL group) RGGAGDGGGGGATAQLP GAR %ID to
G922 4 G922 Arabidopsis 1st SCR: 134- 1st SCR: 400-
RRLFFEMFPILKVSYLLT 1279 100% thaliana 199 597 NRAILEAMEGEKMVHVI
DLDASEPAQWLALLQAF NSRPEGPPHLRITG 4 G922 Arabidopsis 2nd SCR: 2nd
SCR: 994- FLNAIWGLSPKVMVVTE 1280 100% thaliana 332-401 1203
QDSDHNGSTLMERLLESL YTYAALFDCLETKVPRTS QDRIKVEKMLFGEEIKN 4 G922
Arabidopsis 3rd SCR: 405- 3rd SCR: 1213- CEGEERRERHEKLEKWS 1281
100% thaliana 478 1434 QRIDLAGFGNVPLSYYA MLQARRLLQGCGFDGYR
IKEESGCAVICWQDRPLY SVSAW 220 G3824 Lycopersicon 1st SCR: 42- 1st
SCR: 134- RKMFFEIFPFLKVAFVVT 1282 69% esculentum 107 331
NQAIIEAMEGEKMVHIVD LNAAEPLQWRALLQDLS ARPEGPPHLRITG 220 G3824
Lycopersicon 2nd SCR: 2nd SCR: 713- FLNALWGLSPKVMVVTE 1283 78%
esculentum 235-304 922 QDANHNGTTLMERLSES LHFYAALFDCLESTLPRT
SLERLKVEKMLLGEEIRN 220 G3824 Lycopersicon 3rd SCR: 308- 3rd SCR:
932- CEGIERKERHEKLEKWFQ 1284 77% esculentum 381 1153
RFDTSGFGNVPLSYYAM LQARRLLQSYSCEGYKIK EDNGCVVICWQDRPLFS VSSW 212
G3810 Glycine max 1st SCR: 106- 1st SCR: 316- QKLFFELFPFLKVAFVLT
1285 68% 171 513 NQAIIEAMEGEKVIHIIDL NAAEAAQWIALLRVLSA HPEGPPHLRITG
212 G3810 Glycine max 2nd SCR: 2nd SCR: 913- FLNALWGLSPKVMVVTE 1286
80% 305-374 1122 QDCNHNGPTLMDRLLEA LYSYAALFDCLESTVSRT
SLERLRVEKMLFGEEIKN 212 G3810 Glycine max 3rd SCR: 378- 3rd SCR:
1132- CEGSERKERHEKLEKWF 1287 71% 451 1353 QRFDLAGFGNVPLSYFG
MVQARRFLQSYGCEGYR MRDENGCVLICWEDRPM YSISAW 214 G3811 Glycine max
1st SCR: 103- 1st SCR: 361- QKLFFELLPFLKESYILTN 1288 68% 168 558
QAIVEAMEGEKMVHIVD LYGAGPAQWISLLQVLS ARPEGPPHLRITG 214 G3811 Glycine
max 2nd SCR: 2nd SCR: 940- FLNALWGLSPKVMVVTE 1289 74% 296-365 1149
QDFNHNCLTMMERLAEA LFSYAAYFDCLESTVSRA SMDRLKLEKMLFGEEIK N 214 G3811
Glycine max 3rd SCR: 369- 3rd SCR: 1159- CEGCERKERHEKMDRWI 1290 60%
442 1380 QRLDLSGFANVPISYYGM LQGRRFLQTYGCEGYKM REECGRVMICWQERSLFS
ITAW 218 G3814 Oryza sativa 1st SCR: 123- 1st SCR: 367-
RRHMFDVLPFLKLAYLT 1291 60% (japonica 190 570 TNHAILEAMEGERFVHV
cultivar- VDFSGPAANPVQWIALF group) HAFRGRREGPPHLRITA 218 G3814
Oryza sativa 2nd SCR: 2nd SCR: 994- FLSAVRSLSPKIMVMTEQ 1292 48%
(japonica 332-400 1200 EANHNGGAFQERFDEAL cultivar-
NYYASLFDCLQRSAAAA group) AERARVERVLLGEEIRG 218 G3814 Oryza sativa
3rd SCR: 404- 3rd SCR: 1210- CEGAERVERHERARQWA 1293 46% (japonica
480 1440 ARMEAAGMERVGLSYSG cultivar- AMEARKLLQSCGWAGP group)
YEVRHDAGGHGFFFCWH KRPLYAVTAW 216 G3813 Oryza sativa 1st SCR: 129-
1st SCR: 385- RRHFLDLCPFLRLAGAAA 1294 53% (japonica 194 582
NQSILEAMESEKIVHVIDL cultivar- GGADATQWLELLHLLAA group) RPEGPPHLRLTS
216 G3813 Oryza sativa 2nd SCR: 2nd SCR: 868- FLGALWGLSPKVMVVAE
1295 61% (japonica 290-359 1077 QEASHNAAGLTERFVEA cultivar-
LNYYAALFDCLEVGAAR group) GSVERARVERWLLGEEIK N 216 G3813 Oryza
sativa 3rd SCR: 363- 3rd SCR: 1087- CDGGERRERHERLERWA 1296 64%
(japonica 436 1308 RRLEGAGFGRVPLSYYA cultivar- LLQARRVAQGLGCDGFK
group) VREEKGNFFLCWQDRAL FSVSAW 222 G3827 Oryza sativa 2nd SCR: 2nd
SCR: 676- DVESLRGLSLKVMVVTE 1297 55% (japonica 226-295 885
QEVSHNAAGLTERFVEA cultivar- LNYYAALFDCLEVGGAR group)
GSVERTRVERWLLGEEIK N 222 G3827 Oryza sativa 3rd SCR: 299- 3rd SCR:
895- CDGGERRERHERLEGAG 1298 60% (japonica 365 1095
FGRVPLSYYALLQARRV cultivar- AQGLGCDGFKVREEKGN group)
FFLCWQDRALFSVSAW %ID to G1274 6 G1274 Arabidopsis WRKY: 110- WRKY:
328- DDGFKWRKYGKKSVKN 1299 100% thaliana 166 498 NINKRNYYKCSSEGCSVK
KRVERDGDDAAYVITTY EGVHNH 140 G3724 Glycine max WRKY:107- WRKY: 390-
DDGYKWRKYGKKSVKS 1300 84% 163 560 SPNLRNYYKCSSGGCSV
KKRVERDRDDYSYVITT YEGVHNH 148 G3728 Zea mays WRKY: 108- WRKY: 1075-
DDGFKWRKYGKKAVKN 1301 82% 164 1245 SPNPRNYYRCSSEGCGVK
KRVERDRDDPRYVITTY DGVHNH 206 G3802 Sorghum WRKY: 110- WRKY: 386-
DDGFKWRKYGKKAVKN 1302 82% bicolor 166 556 SPNPRNYYRCSSEGCGVK
KRVERDRDDPRYVITTY DGVHNH 210 G3804 Zea mays WRKY: 108- WRKY: 438-
DDGFKWRKYGKKAVKN 1303 82% 164 608 SPNPRNYYRCSSEGCGVK
KRVERDRDDPRYVITTY DGVHNH 146 G3727 Zea mays WRKY: 102- WRKY: 391-
DDGFKWRKYGKKAVKS 1304 80% 158 561 SPNPRNYYRCSSEGCGVK
KRVERDRDDPRYVITTY DGVHNH 154 G3731 Lycopersicon WRKY: 95- WRKY:
297- DDGFKCRKYGKKMVKN 1305 80% esculentum 151 467 NPNPRNYYKCSSGGCNV
KKRVERDNKDSSYVITTY EGIHNH 156 G3732 Solanum WRKY: 95- WRKY: 309-
DDGFKWRKYGKKMVKN 1306 80% tuberosum 151 479 SSNPRNYYKCSSGGCNV
KKRVERDNEDSSYVITTY EGIHNH 158 G3733 Hordeum WRKY: 131- WRKY: 641-
DDGYKWRKYGKKSVKN 1307 80% vulgare 187 811 SPNPRNYYRCSTEGCSVK
KRVERDRDDPAYVVTTY EGTHSH 204 G3797 Lactuca WRKY: 118- WRKY: 363-
DDGFKWRKYGKKMVKN 1308 80% sativa 174 533 SPNPRNYYRCSAAGCSV
KKRVERDVEDARYVITT YEGIHNH 208 G3803 Glycine max WRKY: 111- WRKY:
367- DDGYKWRKYGKKTVKN 1309 80% 167 537 NPNPRNYYKCSGEGCNV
KKRVERDRDDSNYVLTT YDGVHNH 132 G3720 Zea mays WRKY: 135- WRKY: 403-
DDGYKWRKYGKKSVKN 1310 78% 191 573 SPNPRNYYRCSTEGCNV
KKRVERDKDDPSYVVTT YEGMHNH
134 G3721 Oryza sativa WRKY: 96- WRKY: 342- DDGFKWRKYGKKAVKN 1311
78% (japonica 152 512 SPNPRNYYRCSTEGCNV cultivar- KKRVERDREDHRYVITT
group) YDGVHNH 136 G3722 Zea mays WRKY: 129- WRKY: 430-
DDGYKWRKYGKKSVKN 1312 78% 185 600 SPNPRNYYRCSTEGCNV
KKRVERDRDDPRYVVTM YEGVHNH 144 G3726 Oryza sativa WRKY: 135- WRKY:
459- DDGYKWRKYGKKSVKN 1313 78% (japonica 191 629 SPNPRNYYRCSTEGCNV
cultivar- KKRVERDKDDPSYVVTT group) YEGTHNH 202 G3795 Capsicum WRKY:
95- WRKY: 302- DDGYKWRKYGKKMVK 1314 78% annuum 151 472
NSPNPRNYYRCSVEGCPV KKRVERDKEDSRYVITTY EGVHNH 30 G1275 Arabidopsis
WRKY: 113- WRKY: 394- DDGFKWRKYGKKMVKN 1315 77% thaliana 169 564
SPHPRNYYKCSVDGCPV KKRVERDRDDPSFVITTY EGSHNH 138 G3723 Glycine max
WRKY: 113- WRKY: 715- DDGYKWRKYGKKTVKS 1316 77% 169 885
SPNPRNYYKCSGEGCDV KKRVERDRDDSNYVLTT YDGVHNH 152 G3730 Oryza sativa
WRKY: 107- WRKY: 385- DDGFKWRKYGKKAVKS 1317 77% (japonica 163 555
SPNPRNYYRCSAAGCGV cultivar- KKRVERDGDDPRYVVTT group) YDGVHNH 130
G3719 Zea mays WRKY: 91- WRKY: 428- DDGFKWRKYGKKAVKS 1318 75% 147
598 SPNPRNYYRCSTEGSGVK KRVERDSDDPRYVVTTY DGVHNH 142 G3725 Oryza
sativa WRKY: 158- WRKY: 688- DDGYKWRKYGKKSVKN 1319 75% (japonica
214 858 SPNPRNYYRCSTEGCNV cultivar- KKRVERDKNDPRYVVT group)
MYEGIHNH 150 G3729 Oryza sativa WRKY: 137- WRKY: 452-
DDGYRWRKYGKKMVKN 1320 75% (japonica 193 622 SPNPRNYYRCSSEGCRVK
cultivar- KRVERARDDARFVVTTY group) DGVHNH 32 G1758 Arabidopsis
WRKY: 109- WRKY: 393- DDGYKWRKYGKKPITGS 1321 57% thaliana 165 563
PFPRHYHKCSSPDCNVKK KIERDTNNPDYILTTYEG RHNH %ID to G1792 8 G1792
Arabidopsis AP2: 16-80 AP2: 122-316 KQARFRGVRRRPWGKFA 1322 100%
thaliana AEIRDPSRNGARLWLGTF ETAEEAARAYDRAAFNL RGHLAILNFPNEY 86
G3520 Glycine max AP2: 14-78 AP2: 50-244 EEPRYRGVRRRPWGKFA 1323 80%
AEIRDPARHGARVWLGT FLTAEEAARAYDRAAYE MRGALAVLNFPNEY 82 G3518 Glycine
max AP2: 13-77 AP2: 134-328 VEVRYRGIRRRPWGKFA 1324 76%
AEIRDPTRKGTRIWLGTF DTAEQAARAYDAAAFHF RGHRAILNFPNEY 84 G3519 Glycine
max AP2: 13-77 AP2: 93-287 CEVRYRGIRRRPWGKFA 1325 76%
AEIRDPTRKGTRIWLGTF DTAEQAARAYDAAAFHF RGHRAILNFPNEY 160 G3735
Medicago AP2: 23-87 AP2: 148-342 DQIKYRGIRRRPWGKFA 1326 76%
truncatula AEIRDPTRKGTRIWLGTF DTAEQAARAYDAAAFHF RGHRAILNFPNEY 34
G1791 Arabidopsis AP2: 10-74 AP2: 63-257 NEMKYRGVRKRPWGKY 1327 72%
thaliana AAEIRDSARHGARVWLG TFNTAEDAARAYDRAAF GMRGQRAILNFPHEY 70
G3380 Oryza sativa AP2: 18-82 AP2: 138-332 ETTKYRGVRRRPSGKFA 1328
72% (japonica AEIRDSSRQSVRVWLGTF cultivar- DTAEEAARAYDRAAYA group)
MRGHLAVLNFPAEA 74 G3383 Oryza sativa AP2: 9-73 AP2: 25-219
TATKYRGVRRRPWGKFA 1329 72% (japonica AEIRDPERGGARVWLGT cultivar-
FDTAEEAARAYDRAAYA group) QRGAAAVLNFPAAA 18 G30 Arabidopsis AP2:
16-80 AP2: 86-280 EQGKYRGVRRRPWGKY 1330 70% thaliana
AAEIRDSRKHGERVWLG TFDTAEDAARAYDRAAY SMRGKAAILNFPHEY 72 G3381 Oryza
sativa AP2: 14-78 AP2: 122-316 LVAKYRGVRRRPWGKFA 1331 70% (japonica
AEIRDSSRHGVRVWLGTF cultivar- DTAEEAARAYDRSAYSM group) RGANAVLNFPADA
76 G3515 Oryza sativa AP2: 11-75 AP2: 53-247 SSSSYRGVRKRPWGKFA 1332
70% (japonica AEIRDPERGGARVWLGT cultivar- FDTAEEAARAYDRAAFA group)
MKGATAMLNFPGDH 78 G3516 Zea mays AP2: 6-70 AP2: 16-210
KEGKYRGVRKRPWGKF 1333 70% AAEIRDPERGGSRVWLG TFDTAEEAARAYDRAAF
AMKGATAVLNFPASG 164 G3737 Oryza sativa AP2: 8-72 AP2: 233-427
AASKYRGVRRRPWGKFA 1334 70% (japonica AEIRDPERGGSRVWLGTF cultivar-
DTAEEAARAYDRAAFAM group) KGAMAVLNFPGRT 36 G1795 Arabidopsis AP2:
11-75 AP2: 57-251 EHGKYRGVRRRPWGKY 1335 69% thaliana
AAEIRDSRKHGERVWLG TFDTAEEAARAYDQAAY SMRGQAAILNFPHEY 200 G3794 Zea
mays AP2: 6-70 AP2: 135-329 EPTKYRGVRRRPSGKFA 1336 69%
AEIRDSSRQSVRMVVLGTF DTAEEAARAYDRAAYA MRGQIAVLNFPAEA 80 G3517 Zea
mays AP2: 13-77 AP2: 76-270 EPTKYRGVRRRPWGKYA 1337 67%
AEIRDSSRHGVRIWLGTF DTAEEAARAYDRSANSM RGANAVLNFPEDA 162 G3736
Triticum AP2: 12-76 AP2: 163-357 EPTKYRGVRRRPWGKFA 1338 67%
aestivum AEIRDSSRHGVRMWLGT FDTAEEAAAAYDRSAYS MRGRNAVLNFPDRA 166
G3739 Zea mays AP2: 13-77 AP2: 211-405 EPTKYRGVRRRPWGKYA 1339 67%
AEIRDSSRHGVRIWLGTF DTAEEAARAYDRSAYSM RGANAVLNFPEDA %ID to G2053 10
G2053 Arabidopsis NAC: 6-152 NAC: 16-456 GLRFRPTDKEIVVDYLRP 1340
100% thaliana KNSDRDTSHVDRVISTVT IRSFDPWELPCQSRIKLKD
ESWCFFSPKENKYGRGD QQIRKTKSGYWKITGKPK PILRNRQEIGEKKVLMFY
MSKELGGSKSDWVMHE YHAFSPTQMMMTYTICK VMFKGD 20 G515 Arabidopsis NAC:
6-149 NAC: 93-524 GLRFCPTDEEIVVDYLWP 1341 78% thaliana
KNSDRDTSHVDRFINTVP VCRLDPWELPCQSRIKLK DVAWCFFRPKENKYGRG
DQQMRKTKSGFWKSTGR PKPIMRNRQQIGEKKILM FYTSKESKSDWVIHEYHG
FSHNQMMMTYTLCKVM FNGG 24 G517 Arabidopsis NAC: 6-153 NAC: 16-459
GFRFRPNDEEIVDHYLRP 1342 62% thaliana KNLDSDTSHVDEVISTVD
ICSFEPWDLPSKSMIKSRD GVWYFFSVKEMKYNRG DQQRRRTNSGFWKKTGK
TMTVMRKRGNREKIGEK RVLVFKNRDGSKTDWV MHEYHATSLFPNQMMTY TVCKVEFKGE 22
G516 Arabidopsis NAC: 6-141 NAC: 16-423 GFRFRPTDGEIVDIYLRPK 1343
55% thaliana NLESNTSHVDEVISTVDIC SFDPWDLPSHSRMKTRD QVWYFFGRKENKYGKG
DRQIRKTKSGFWKKTGV TMDIMRKTGDREKIGEK RVLVFKNHGGSKSDWA
MHEYHATFSSPNQGE %ID to G2999 14 G2999 Arabidopsis ZF: 80-133 ZF:
280-441 ARYRECQKNHAASSGGH 1344 100% thaliana VVDGCGEFMSSGEEGTV
ESLLCAACDCHRSFERKE ID 14 G2999 Arabidopsis HB: 198-261 HB: 634-825
KKRFRTKFNEEQKEKMM 1345 100% thaliana EFAEKIGWRMTKLEDDE
VNRFCREIKVKRQVFKV WMHNNKQAAKKKD 62 G2998 Arabidopsis ZF: 74-127 ZF:
220-381 VRYRECLKNHAASVGGS 1346 79% thaliana VHDGCGEFMPSGEEGTIE
ALRCAACDCHRNFERKE MD 62 G2998 Arabidopsis HB: 240-303 HB: 718-909
KKRFRTKFTTDQKERMM 1347 78% thaliana DFAEKLGWRMNKQDEE
ELKRFCGEIGVKRQVFKV WMHNNKNNAKKPP 64 G3000 Arabidopsis ZF: 58-111
ZF: 318-479 AKYRECQKNHAASTGGH 1348 77% thaliana VVDGCCEFMAGGEEGTL
GALKCAACNCHRSFHRK EVY 64 G3000 Arabidopsis HB: 181-244 HB: 687-878
KKRVRTKINEEQKEKMK 1349 65% thaliana EFAERLGWRMQKKDEEE
IDKFCRMVNLRRQVFKV WMHNNKQAMKRNN 106 G3670 Lotus ZF: 62-115 ZF:
184-345 VRYRECQKNHAVSEGGH 1350 74% corniculatus AVDGCCEFMAAGDEGTL
var. EAVICAACNCHRNFHRK japonicus EID 106 G3670 Lotus HB: 207-270
HB: 619-810 KKRYRTKFTPEQKEKML 1351 57% corniculatus
AFAEELGWRIQKHQEAA var. VEQFCAETCVRRNVLKV japonicus WMHNNKNTLGKKP
110 G3674 Oryza sativa ZF: 61-114 ZF: 274-435 ARYRECLKNHAVGIGGH
1352 72% (indica AVDGCGEFMASGEEGSI cultivar- DALRCAACGCHRNFHRK
group) ESE 110 G3674 Oryza sativa HB: 226-289 HB: 769-960
KKRFRTKFTQEQKDKML 1353 59% (indica AFAERLGWRIQKHDEAA cultivar-
VQQFCEEVCVKRHVLKV group) WMHNNKHTLGKKA 102 G3663 Lotus ZF: 88-141
ZF: 262-423 IRYRECLRNHAARLGSHV 1354 70% corniculatus
TDGCGEFMPNGEQGTPE var. SLICAACECHRNFERKEA japonicus Q 102 G3663
Lotus HB: 219-282 HB: 655-846 KKRFRTKFTQQQKDRM 1355 64%
corniculatus MEFAEKLGWKIQKQDEE var. EVKQFCSHVGVKRQAFK
japonicus VWMHNSKQAMKKKQ 108 G3671 Oryza sativa ZF: 40-93 ZF:
233-394 GRYRECLKNHAVGIGGH 1356 70% (japonica AVDGCGEFMAAGEEGTI
cultivar- DALRCAACNCHRNFHRK group) ESE 108 G3671 Oryza sativa HB:
200-263 HB: 713-904 KKRFRTKFTQEQKDKML 1357 59% (japonica
AFAERVGWRIQKHDEAA cultivar- VQQFCDEVGVKRHVLKV group) WMHNNKHTLGKKL
60 G2997 Arabidopsis ZF: 47-100 ZF: 263-424 IRYRECLKNHAVNIGGHA 1358
68% thaliana VDGCCEFMPSGEDGTLD ALKCAACGCHRNFHRKE TE 60 G2997
Arabidopsis HB: 157-220 HB: 593-784 TKRFRTKFTAEQKEKML 1359 59%
thaliana AFAERLGWRIQKHDDVA VEQFCAETGVRRQVLKI WMHNNKNSLGKKP 116
G3683 Oryza sativa ZF: 72-125 ZF: 214-375 ARYRECLKNHAAAIGGS 1360
68% (japonica ATDGCGEFMPGGEEGSL cultivar- DALRCSACGCHRNFHRK group)
ELD 116 G3683 Oryza sativa HB: 193-256 HB: 577-768
RKRERTKFTAEQKARML 1361 59% (japonica GFAEEVGWRLQKLEDAV cultivar-
VQRFCQEVGVKRRVLKV group) WMHNNKHTLARRH 112 G3675 Brassica ZF:
49-102 ZF: 201-362 VRYRECLKNHAVNIGGH 1362 66% napus
AVDGCCEFMPSGEDGSL DALKCAACGCHRNFHRK ETE 112 G3675 Brassica HB:
162-225 HB: 540-731 AKRFRTKFTAEQKDKML 1363 56% napus
AFAERLGWRIQKHDDAA VEQFCAETGVRRQVLKI WMHNNKNSLGRKP 122 G3690 Oryza
sativa ZF: 161-213 ZF: 481-639 WRYRECLKNHAARMGA 1364 66% (japonica
HVLDGCGEFMSSPGDGA cultivar- AALACAACGCHRSFHRR group) EPA 122 G3690
Oryza sativa HB: 318-381 HB: 952-1143 KKRERTKFTAEQKERIVIR 1365 56%
(japonica EFAHRVGWRIHKPDAAA cultivar- VDAFCAQVGVSRRVLKV group)
WMHNNKHLAKTPP 104 G3668 Flaveria ZF: 42-95 ZF: 410-571
YRYKECLKNHAVGIGGQ 1366 64% bidentis AVDGCGEFMAAGDEGTL
DALKCAACNCHRNFHRK EVE 104 G3668 Flaveria HB: 174-237 HB: 806-997
KKRFRTKFTQDQKDRML 1367 54% bidentis AFSEALGWRIQKHDEAA
VQQFCNETGVKRHVLKV WMHNNKHTIGKKP 58 G2996 Arabidopsis ZF: 73-126 ZF:
241-402 FRFRECLKNQAVNIGGH 1368 64% thaliana AVDGCGEFMPAGIEGTID
ALKCAACGCHRNFHRKE LP 58 G2996 Arabidopsis HB: 191-254 HB: 595-786
RKRHRTKFTAEQKERML 1369 53% thaliana ALAERIGWRIQRQDDEVI
QRFCQETGVPRQVLKVW LHNNKHTLGKSP 54 G2994 Arabidopsis ZF: 88-141 ZF:
329-490 IKYKECLKNHAAAMGGN 1370 62% thaliana ATDGCGEFMPSGEDGSIE
ALTCSACNCHRNFHRKE VE 54 G2994 Arabidopsis HB: 218-281 HB: 719-910
KKRFRTKFTPEQKEKMLS 1371 65% thaliana FAEKVGWKIQRQEDCVV
QRFCEEIGVKRRVLKVW MHNNKIHFSKKN 120 G3686 Oryza sativa ZF: 38-88 ZF:
112-264 CRYHECLRNHAAASGGH 1372 62% (indica VVDGCGEFMPASFEEPL
cultivar- ACAACGCHRSFHRRDPS group) 120 G3686 Oryza sativa HB:
159-222 HB: 475-666 RRRSRTTFTREQKEQML 1373 50% (indica
AFAERVGWRIQRQEEAT cultivar- VEHFCAQVGVRRQALKV group) WMHNNKHSFKQKQ
52 G2993 Arabidopsis ZF: 85-138 ZF: 442-603 IKYKECLKNHAATMGGN 1374
61% thaliana AIDGCGEFMPSGEEGSIE ALTCSVCNCHRNFHRRE IL 52 G2993
Arabidopsis HB: 222-285 HB: 853-1044 KKRFRTKFTQEQKEKMIS 1375 57%
thaliana FAERVGWKIQRQEESVV QQLCQEIGIRRRVLKVW MHNNKQNLSKKS 48 G2991
Arabidopsis ZF: 54-109 ZF: 218-385 ATYKECLKNHAAGIGGH 1376 60%
thaliana ALDGCGEFMPSPSFNSND PASLTCAACGCHRNFHR REED 48 G2991
Arabidopsis HB: 179-242 HB: 593-784 RKRFRTKFSQYQKEKMF 1377 59%
thaliana EFSERVGWRMPKADDVV VKEFCREIGVDKSVFKV WMHNNKISGRSGA 114
G3680 Zea mays ZF: 34-89 ZF: 223-390 PLYRECLKNHAASLGGH 1378 60%
AVDGCGEFMPSPGANPA DPTSLKCAACGCHRNFH RRTLE 114 G3680 Zea mays HB:
222-285 HB: 787-978 RKRFRTKFTAEQKQRMQ 1379 50% ELSERLGWRLQKRDEAIV
DEWCRDIGVGKGVFKV WMHNNKHNFLGGH 118 G3685 Oryza sativa ZF: 43-95 ZF:
216-374 VRYHECLRNHAAAMGG 1380 59% (japonica HVVDGCREFMPMPGDA
cultivar- ADALKCAACGCHRSFHR group) KDDG 118 G3685 Oryza sativa HB:
172-235 HB: 603-794 RKRFRTKFTPEQKEQML 1381 56% (japonica
AFAERVGWRMQKQDEA cultivar- LVEQFCAQVGVRRQVFK group) VWMHNNKSSIGSSS
44 G2989 Arabidopsis ZF: 50-105 ZF: 208-375 VTYKECLKNHAAAIGGH 1382
58% thaliana ALDGCGEFMPSPSSTPSD PTSLKCAACGCHRNFHR RETD 44 G2989
Arabidopsis HB: 192-255 HB: 634-825 RKRFRTKFSSNQKEKMH 1383 59%
thaliana EFADRIGWKIQKRDEDEV RDFCREIGVDKGVLKVW MHNNKNSFKFSG 46 G2990
Arabidopsis ZF: 54-109 ZF: 206-373 FTYKECLKNHAAALGGH 1384 57%
thaliana ALDGCGEFMPSPSSISSDP TSLKCAACGCHRNFHRR DPD 46 G2990
Arabidopsis HB: 200-263 HB: 644-835 RKRFRTKFSQFQKEKMH 1385 57%
thaliana EFAERVGWKMQKRDED DVRDFCRQIGVDKSVLK VWMHNNKNTFNRRD 66 G3001
Arabidopsis ZF: 62-113 ZF: 222-377 PHYYECRKNHAADIGTT 1386 57%
thaliana AYDGCGEFVSSTGEEDSL NCAACGCHRNFHREELI 66 G3001 Arabidopsis
HB: 179-242 HB: 573-764 VKRLKTKFTAEQIEKMR 1387 42% thaliana
DYAEKLRWKVRPERQEE VEEFCVEIGVNRKNFRIW MNNHKDKIIIDE 50 G2992
Arabidopsis ZF: 29-84 ZF: 85-252 VCYKECLKNHAANLGGH 1388 55%
thaliana ALDGCGEFMPSPTATSTD PSSLRCAACGCHRNFHRR DPS 50 G2992
Arabidopsis HB: 156-219 HB: 466-657 RKRTRTKFTPEQKIKMRA 1389 48%
thaliana FAEKAGWKINGCDEKSV REFCNEVGIERGVLKVW MHNNKYSLLNGK 128 G3695
Oryza sativa ZF: 22-71 ZF: 64-213 GKYKECMRNHAAAMGG 1390 51%
(japonica QAFDGCGEYMPASPDSL cultivar- KCAACGCHRSFHRRAAA group) 128
G3695 Oryza sativa HB: 164-227 HB: 490-681 RKRERTKFTPEQKERMRE 1391
57% (japonica FAEKQGWRINRNDDGAL cultivar- DRFCVEIGVKRHVLKVW group)
MHNHKNQLASSP 56 G2995 Arabidopsis ZF: 3-58 ZF: 143-310
VLYNECLKNHAVSLGGH 1392 50% thaliana ALDGCGEFTPKSTTILTDP
PSLRCDACGCHRNFHRRS PS 56 G2995 Arabidopsis HB: 115-178 HB: 479-670
KKHKRTKFTAEQKVKMR 1393 45% thaliana GFAERAGWKINGWDEK
WVREFCSEVGIERKVLK VWIHNNKYFNNGRS 124 G3692 Oryza sativa ZF: 10-61
ZF: 28-183 EVYRECMRNHAAKLGTY 1394 48% (japonica ANDGCCEYTPDDGHPAG
cultivar- LLCAACGCHRNFHRKDF group) L 124 G3692 Oryza sativa HB:
119-188 HB: 355-564 RRRTRTKFTEEQKARML 1395 58% (japonica
RFAERLGWRMPKREPGR cultivar- APGDDEVARFCREIGVNR group)
QVFKVWMHNHKAGGGG GG 126 G3694 Oryza sativa ZF: 1-40 ZF: 1-120
MGAHVLDGCGEFMSSPG 1396 48% (japonica DGAAALACAACGCHRSF cultivar-
HRREPA group) 126 G3694 Oryza sativa HB: 145-208 HB: 433-624
KKRERTKFTAEQKERMR 1397 56% (japonica EFAHRVGWRIHKPDAAA cultivar-
VDAFCAQVGVSRRVLKV group) WMHNNKLLAKTPP 68 G3002 Arabidopsis ZF:
5-53 ZF: 81-227 CVYRECMRNHAAKLGSY 1398 42% thaliana
AIDGCREYSQPSTGDLCV ACGCHRSYHRRIDV 68 G3002 Arabidopsis HB: 106-168
HB: 384-572 QRRRKSKFTAEQREAMK 1399 35% thaliana DYAAKLGWTLKDKRAL
REEIRVECEGIGVTRYHE KTWVNNNKKFYH %ID to G3086 16 G3086 Arabidopsis
HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1400 100% thaliana 307-365
1059-1235 KISERMRKLQDLVPNMD TQTNTADMLDLAVQYIK DLQEQVK 188 G3767
Glycine max HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1401 93% 146-204
436-612 KISERMRKLQDLVPNMD KQTNTADMLDLAVDYIK DLQKQVQ 190 G3768
Glycine max HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1402 93% 190-248
568-744 KISERMRKLQDLVPNMD KQTNTADMLDLAVDYIK DLQKQVQ 192 G3769
Glycine max HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1403 93% 240-298
718-894 KISERMRKLQDLVPNMD
KQTNTADMLDLAVEYIK DLQNQVQ 174 G3744 Oryza sativa HLH/MYC: HLH/MYC:
KRGCATHPRSIAERVRRT 1404 89% (japonica 71-129 211-387
RISERIRKLQELVPNMDK cultivar- QTNTADMLDLAVDYIKD group) LQKQVK 178
G3755 Zea mays HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1405 89% 97-155
289-465 KISERIRKLQELVPNMDK QTNTSDMLDLAVDYIKD LQKQVK 26 G592
Arabidopsis HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1406 88% thaliana
282-340 964-1140 RISERMRKLQELVPNMD KQTNTSDMLDLAVDYIK DLQRQYK 186
G3766 Glycine max HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1407 88%
35-93 103-279 RISERMRKLQELVPHMD KQTNTADMLDLAVEYIK DLQKQFK 172 G3742
Oryza sativa HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1408 86%
(japonica 199-257 595-771 RISERIRKLQELVPNMEK cultivar-
QTNTADMLDLAVDYIKE group) LQKQVK 198 G3782 Pinus taeda HLH/MYC:
HLH/MYC: KRGCATHPRSIAERVRRT 1409 80% 471-530 1411-1590
RISERMRKLQELVPNSDK QTVNIADMLDEAVEYVK SLQKQVQ 176 G3746 Oryza sativa
HLH/MYC: HLH/MYC: KRGCATHPRSIAERERRT 1410 79% (japonica 312-370
934-1110 RISKRLKKLQDLVPNMD cultivar- KQTNTSDMLDIAVTYIKE group)
LQGQVE 184 G3765 Glycine max HLH/MYC: HLH/MYC: KRGFATHPRSIAERVRRT
1411 79% 147-205 439-615 RISERIRKLQELVPTMDK QTSTAEMLDLALDYIKDL
QKQFK 194 G3771 Glycine max HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT
1412 79% 84-142 250-426 RISDRIRKLQELVPNMDK QTNTADMLDEAVAYVKF LQKQIE
28 G1134 Arabidopsis HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1413 77%
thaliana 187-245 619-795 RISDRIRKLQELVPNMDK QTNTADMLEEAVEYVKV
LQRQIQ 168 G3740 Oryza sativa HLH/MYC: HLH/MYC: KRGCATHPRSIAERERRT
1414 77% (japonica 141-199 421-597 RISEKLRKLQELVPNMDK cultivar-
QTSTADMLDLAVEHIKG group) LQSQLQ 180 G3763 Glycine max HLH/MYC:
HLH/MYC: KRGFATHPRSIAERERRT 1415 77% 161-219 481-657
RISARIKKLQDLFPKSDK QTSTADMLDLAVEYIKD LQKQVK 182 G3764 Glycine max
HLH/MYC: HLH/MYC: KRGFATHPRSIAERVRRT 1416 77% 370-428 1108-1284
RISERIKKLQDLFPKSEKQ TSTADMLDLAVEYIKDL QQKVK 196 G3772 Glycine max
HLH/MYC: HLH/MYC: KRGCATHPRSIAERERRT 1417 77% 211-269 631-807
RISGKLKKLQDLVPNMD KQTSYADMLDLAVQHIK GLQTQVQ 40 G2555 Arabidopsis
HLH/MYC: HLH/MYC: KRGCATHPRSIAERVRRT 1418 76% thaliana 184-242
726-902 RISDRIRRLQELVPNMDK QTNTADMLEEAVEYVKA LQSQIQ 170 G3741 Oryza
sativa HLH/MYC: HLH/MYC: KRGCATHPRSIAERERRT 1419 76% (japonica
288-346 862-1038 RISEKLRKLQALVPNMD cultivar- KQTSTSDMLDLAVDHIK
group) GLQSQLQ 38 G2149 Arabidopsis HLH/MYC: HLH/MYC:
KRGCATHPRSIAERERRT 1420 74% thaliana 286-344 927-1103
RISGKLKKLQDLVPNMD KQTSYSDMLDLAVQHIK GLQHQLQ 42 G2766 Arabidopsis
HLH/MYC: HLH/MYC: KRGFATHPRSIAERERRT 1421 72% thaliana 234-292
778-954 RISGKLKKLQELVPNMD KQTSYADMLDLAVEHIK GLQHQVE
The MYB-Related Family, Including the G682 Subclade
[0164] MYB transcription factors are found in both plants and
animals. The MYB-related class of transcription factors is a
heterogeneous group of 54 proteins that are connected to one
another through their evolutionary relationship with proteins
containing a MYB DNA binding motif. MYB proteins share a signature
DNA-binding domain of approximately 50 amino acids that contains a
series of highly conserved residues with a characteristic spacing.
Critical in the formation of the tertiary structure of the
conserved MYB motif is a series of consistently spaced tryptophan
residues. Animal MYBs contain three repeats of the MYB domain: R1,
R2, and R3. Plant MYBs usually contain two imperfect MYB repeats
near their amino termini: R2 and R3 (136 in Arabidopsis genome)
although there is a small subgroup of three repeat (R1R2R3) MYBs
similar to those found in animals, numbering approximately five in
the Arabidopsis genome. Each MYB repeat has the potential to form
three alpha-helical segments, resembling a helix-turn-helix
structure. Repeats R2 and R3 are responsible for the
sequence-specific DNA-binding of MYB proteins (Howe et al. (1990)
EMBO J. 9: 161-169). Once bound, MYB proteins function to
facilitate transcriptional activation or repression, and this
sometimes involves interaction with a protein partner (Goff et al.
(1992) Genes Dev. 6: 864-875).
[0165] G682 is a member of the MYB-related family of transcription
factors. There appear to be 48 Myb-related genes in Arabidopsis.
The Myb-related genes are similar to the classic plant Myb(R1)R2R3
genes in that they share a signature DNA-binding domain sequence of
approximately 45 amino acids that contains a series of highly
conserved residues with a characteristic spacing. Unlike the
Myb(R1)R2R3 genes, which generally contain two or three Myb
repeats, the majority of the Myb-related genes contain only one
complete Myb domain. There are several Myb-related genes that have
two repeat domains, however the spacing between the domains is
greater than that seen in the Myb(R1)R2R3 family and the sequence
of each domain bears a much higher similarity to the genes in the
Myb-related family than the Myb(R1)R2R3 family. The G682 coding
sequence corresponds to At4G01060, annotated by the Arabidopsis
Genome initiative. This gene is one of a five-member clade of
related proteins that range in size from 75 to 112 amino acids.
These proteins contain a single MYB repeat. Two well characterized
transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1/G214) and
LATE ELONGATED HYPOCOTYL (LHY/G680) are among the other MYB-related
proteins that contain single MYB repeats (Wang et al. (1997) Plant
Cell. 9: 491507; Schaffer et al. (1998) Cell 93: 1219-1229).
[0166] All members of the G682 subclade were found to have
epidermal cell type alterations when overexpressed in Arabidopsis;
for instance, so far all characterized members of the clade show
increased numbers of root hairs compared to wild type plants, as
well as a reduction in trichome number. In addition, overexpression
lines for all members of the clade showed a reduction in
anthocyanin accumulation in response to stress, and enhanced
tolerance to abiotic stress. In the case of 35S::G682 transgenic
lines, an enhanced tolerance to high heat conditions was observed.
Heat can cause osmotic stress; it is therefore consistent that
these transgenic lines were also more tolerant to drought stress in
a soil-based assay. Table 2 summarizes the data for a variety of
abiotic stresses with G682 and its clade members. Another common
feature of all of the members of this clade that have thus far been
examined (constituting a majority of the sequences appearing in the
box in FIG. 21) is that they enhance a plant's performance in
nitrogen limiting conditions, as evidenced by altered C/N sensing
and/or germination assays in low nitrogen environments.
[0167] The difference in the phenotypic responses of the
overexpression lines suggests that each of these genes could have
slightly different but related functions in the plant. One of the
G682 subclade members, G1816 (TRIPTYCHON, TRY), is only partially
redundant with CAPRICE (CPC; Schellmann et al. (2002) EMBO J. 21:
5036-5046). No genetic data has been reported for G682, G226, or
G2718 in the literature.
[0168] Epidermal cell fate specification in the root, the
hypocotyl, the leaf and the seed coat involves similar set of genes
that presumably function in mechanistically similar ways in the
various epidermal cell types. The signals that specify epidermal
cell fate in different parts of the plant must therefore feed into
a common signal transduction cascade. Such a cascade, consisting of
members of the same gene family (that have evolved from gene
duplication of common ancestors) must have adopted new and
different functions to variable degrees, in different regions of
the plant.
[0169] Table 2 compiles a list of genes that have been implicated
in root hair and trichome cell specification through genetic and
biochemical characterization in Arabidopsis from the public
literature as well as from our own discoveries.
TABLE-US-00003 TABLE 2 Genes implicated in root hair and trichome
cell specification Gene Name CAPRICE TRY GL3 GL1 WER GL2 TTG1 (CPC)
(G1816) Gene bHLH/MYC MYB- MYB- HD n/a MYB-related MYB-related
Family (R1)R2R3 (R1)R2R3 Loss-of- None Glabrous All cell files
Ectopic hairs, All cell files No root hairs, wild-type Function
detected are hairs glabrous are hairs, ectopic roots, ectopic
glabrous trichomes trichomes Gain-of- Ectopic Ectopic Wild-type
Wild-type Wild-type Ectopic root Ectopic root Function trichomes
trichomes hairs, glabrous hairs, glabrous Site of Leaf Leaf Root
Leaf Leaf Leaf Leaf Activity Epidermis Epidermis Epidermis
epidermis, epidermis, epidermis and epidermis and root epidermis
root epidermis root epidermis root epidermis and seed coat and seed
coat Reference Payne et al. (Di Cristina Lee and Masucci et al.,
Galway et al. Wada et al. Schellmann (2000) et al. (1996)
Schiefelbein (1994), (1994) (1997) et al. (2002) (1999) DiCristina
et al. (1996) References: Di Cristina et al. (1996) Plant J. 10:
393-402 Galway et al (1994) Dev. Biol. 166,: 740-754 Lee and
Schiefelbein (1999) Cell 99: 473-483 Masucci et al. (1994) Plant
Physiol. 106: 1335-1346 Payne et al. (2000) Genetics 156: 1349-1362
Schellmann et al. (2002) EMBO J. 21: 5036-5046 Wada et al. (1997)
Science 277: 1113-1116
[0170] In recently proposed genetic models to explain trichome and
root hair cell specification, a theoretical model of lateral
inhibition first put forth by Wigglesworth (1940) J. Exp. Biol. 17:
180-200) was used (Schellmann et al. (2002) supra; Lee and
Schiefelbein (2002) Plant Cell 14: 611-618). Lateral inhibition is
a process whereby a cell that has taken a certain fate prevents its
neighbors from taking that same fate. The mechanism of lateral
inhibition involves diffusible activators and repressors. The
activator complex stimulates its own expression as well as that of
the repressor. The repressor then moves across cell boundaries to
suppress the activator complex found in neighboring cells. Since it
is conceivable that both activator and repressor are capable of
diffusion across cell boundaries, in this model it is proposed that
the repressor is slightly smaller and therefore diffuses more
quickly resulting in the overall suppression of the activator in
neighboring cells (Schellmann et al. (2002) supra). In other words,
in cells where the proteins are initially being produced, the
scales are still tipped in the direction of the activator and in
the neighboring cells the scales are tipped in the direction of the
repressor.
[0171] In leaf epidermal tissue, the default program is the
formation of a trichome cell fate through the activity of the
homeobox transcription factor, GLABRA2 (GL2). GL2 is known to be
induced by the proposed "activator complex" that is composed of
GL1, a MYB-related protein, TTG1 a WD-40 repeat containing protein,
and GL3, a bHLH transcription factor. The formation of this complex
is supported by genetic data as well as by biochemical data. Yeast
2-hybrid data shows that GL3 interacts with both TTG1 and GL1
(Payne et al. (2000) supra). A non-trichome cell fate, on the other
hand, is specified in neighboring cells through the combined
activity of two repressors, TRY (G1816) and CPC. TRY and CPC are
paralogs and most likely function in a very similar manner.
However, based on the different phenotypes of try and cpc mutants
with respect to trichome initiation, and the additive phenotype of
the double mutant, Schellmann et al. (Schellmann et al. (2002)
supra) concluded that their function was slightly different, and
proposed that CPC and TRY might interact with different proteins in
the "activator complex". This might explain the differences in the
phenotypes observed in the mutants.
[0172] In the lateral inhibition model described above for trichome
cell specification, GL1, TTG1 and GL3 function in a regulatory
feedback loop, enhancing their own expression. A complex composed
of those three proteins, activates GL2 that then functions in
promoting trichome cell fate. The GL1/TTG/GL3 complex also serves
to activate the repressors CPC and TRY that then results in the
prevention of trichome formation in neighboring cells.
[0173] Similarly in the root epidermis, but with reverse logic, the
"activator complex" promotes a non-hair cell fate. In neighboring
cells where the repressor activity accumulates to a greater degree,
a hair cell fate is determined. Involvement of CPC in a lateral
inhibition model in root hair cell specification was supported by a
series of genetic experiments recently described (Lee and
Schiefelbein (2002) supra). The proposed "activator" that is
important for the specification of a non-root hair cell fate is
thought to be composed of WER (MYB-related transcription factor and
paralog to GL1), TTG1 and a bHLH transcription factor that has yet
to be identified. (The maize bHLH transcription factor, R, was
capable of suppressing the ttg1 root hair phenotype suggesting that
a similar bHLH is involved in this process). Genetic data supports
the model that proposes that the activator complex activates the
homeodomain transcription factor GL2 (a positive regulator of
atrichoblast [non-hair] cell fate in the root). The repressor
proteins in this model are, again, postulated to be CPC and TRY
(G1816). Consistent with this model, Lee and Schiefelbein (Lee and
Schiefelbein (2002) supra) have shown that CPC inhibits the
expression of WER, GL2 and itself. They have also shown that WER
activates GL2 and CPC.
[0174] Candidate genes for the bHLH component of the "activator
complex" in root hair development are G1666 (TT8) and G581. Both
genes are similar in sequence to the maize R-gene and we found that
both had seed anthocyanin phenotypes when overexpressed.
Anthocyanin production is consistent with genes that potentially
have maize R-like activity.
[0175] The fact that all of the G682 subclade members have slightly
different phenotypes suggests that the genes do not have completely
overlapping or redundant functions in the plant. The low nitrogen
and other abiotic stress tolerance phenotypes in these lines may be
related to the increase in root hairs on the root epidermis.
Increasing root hair density could provide an increase in
absorptive surface area and an increase in nitrate transporters
that are normally found there. Alternatively, ectopic expression of
these transcription factors may affect stomate formation as has
been reported for wer, ttg1 and g12 mutations (Hung et al. (1998)
Plant Physiol. 117: 73-84; Berger et al. (1998) Dev. Biol. 194:
226-234; Lee and Schiefelbein (1999) supra). Such alterations in
stomate production could alter plant water status. Interestingly,
our data also indicated that G1816 (TRY) overexpression lines had a
glucose sugar sensing phenotype. Several sugar-sensing mutants have
turned out to be allelic to ABA and ethylene mutants. This
potentially implicates G1816 in hormone signaling.
[0176] Because the G682 subclade members are short proteins that
are comprised of almost exclusively a DNA binding motif, it is
possible that they function as repressors. Repression could occur
at the level of DNA binding through competition with other factors
at target promoters. Repression through protein-protein
interactions, though, cannot be excluded. An alternative model is
that the G682 subclade members function by activating a second
pathway that has not yet been identified.
[0177] The residues in the boxes in FIG. 20B may be used to
identify G682 subclade members. Of the sequences examined to date,
a valine (corresponding to position 50 of G682 and a glutamate
residue (at a position corresponding to position 70 of G682) were
found that are present in members of the G682 subclade (these
residues may be found in the boxes and below the arrows in FIG.
20B) but not outside of the subclade. All members of the clade
examined thus far have the subsequence:
[0178]
E-(X).sub.9-L-V-G-(X).sub.2-W-(X).sub.2-I-A-G-R-(X).sub.2-G-R-(X).s-
ub.5-E-(X).sub.2-W (SEQ ID NO: 1422), where X is any amino
acid.
[0179] Table 3 shows the G682 subclade polypeptides identified by
polypeptide SEQ ID NO and Identifier (e.g., Gene ID (GID) No.,
accession number or other name), and includes the species from
which each sequence was derived, the coordinates of the MYB-related
domains in polypeptide amino acid coordinates and polynucleotide
base coordinates, the respective domain sequences, and the extent
of identity in percentage terms to the MYB-related domain of G682.
It is of interest to note that a number of non-Arabidopsis monocot
and dicot sequences are more similar to 6682 than a number of the
Arabidopsis paralogs that are functionally similar to G682.
TABLE-US-00004 TABLE 3 Gene families and binding domains for
exemplary sequences altering C/N sensing, including paralogs and
orthologs Polypeptide Polypeptide SEQ ID % ID to Amino Acid Base
NO: of MYB- Coordinates Coordinates Myb- related SEQ of the MYB- of
the MYB- related Domain ID Iden- related related MYB-related Domain
of NO: tifier Species Domain Domain Domain Sequence Sequence G682
234 G682 Arabidopsis 33-77 99-233 VNMSQEEEDLVSRMH 1423 100%
thaliana KLVGDRWELIAGRIPG RTAGEIERFWVMKN 324 G2718 Arabidopsis
32-76 94-228 IAMAQEEEDLICRMYK 1424 80% thaliana LVGERWDLIAGRIPGRT
AEEIERFWVMKN 328 G3393 Oryza sativa 31-75 172-306 VHFTEEEEDLVFRMHR
1425 71% (japonica LVGNRWELIAGRIPGRT cultivar- AKEVEMFWAVKH group)
326 G3392 Oryza sativa 32-76 143-277 VHFTEEEEDIVFRMHRL 1426 68%
(japonica VGNRWELIAGRIPGRT cultivar- AEEVEKFWAIKH group) 360 G3431
Zea mays 31-75 94-228 VDFTEAEEDLVSRMHR 1427 68% LVGNRWEIIAGRIPGRT
AEEVEMFWSKKY 370 G3444 Zea mays 31-75 104-238 VDFTEAEEDLVSRMHR 1428
68% LVGNRWEIIAGRIPGRT AEEVEMFWSKKY 382 G3450 Glycine max 20-64
83-217 IHMSEQEEDLIRRMYK 1429 68% LVGDKWNLIAGRIPGR KAEEIERFWIMRH 312
G1816 Arabidopsis 30-74 88-222 INMIEQEEDLIFRMYRL 1430 64% thaliana
VGDRWDLIAGRVPGRQ PEEIERYWIMRN 286 G226 Arabidopsis 38-82 121-255
ISMTEQEEDLISRMYRL 1431 62% thaliana VGNRWDLIAGRVVGR KANEIERYWIMRN
380 G3449 Glycine max 26-70 95-229 VEFSEDEETLIIRMYKL 1432 62%
VGERWSLIAGRIPGRTA EEIEKYWTSRF 378 G3448 Glycine max 26-70 96-230
VEFSEDEETLIIRMYKL 1433 60% VGERWSIIAGRIPGRTA EEIEKYWTSRF 372 G3445
Glycine max 25-69 89-223 VEFSEAEEILIAMVYNL 1434 55%
VGERWSLIAGRIPGRTA EEIEKYWTSRF 374 G3446 Glycine max 26-70 92-226
VEFSEAEEILIAMVYNL 1435 55% VGERWSLIAGRIPGRTA EEIEKYWTSRF 376 G3447
Glycine max 26-70 85-219 VEFSEAEEILIAMVYNL 1436 55%
VGERWSLIAGRIPGRTA EEIEKYWTSRF
[0180] Table 4 shows polypeptides of the invention identified by
SEQ ID NO; Identifier (for example, Gene ID (GID) No); the
transcription factor family to which the polypeptide belongs, and
conserved domains of the polypeptide. The first column shows the
polypeptide SEQ ID NO; the third column shows the transcription
factor family to which the polynucleotide belongs; and the fourth
column shows the amino acid residue positions of the conserved
domain in amino acid (AA) coordinates.
TABLE-US-00005 TABLE 4 Gene families and conserved domains Poly-
peptide Conserved Domains SEQ ID in Amino NO: Identifier Family
Acid Coordinates 224 G175 WRKY 178-234, 372-428 226 G303 HLH/MYC
92-161 228 G354 Z-C2H2 42-62, 88-109 230 G489 CAAT 57-156 232 G634
TH 62-147, 189-245 234 G682 MYB-related 27-63 236 G916 WRKY 293-349
238 G975 AP2 4-71 240 G1069 AT-hook 67-75, 76-218 242 G1452 NAC
55-196 244 G1820 CAAT 70-133 246 G2701 MYB-related 33-81, 129-183
248 G2789 AT-hook 59-67, 68-208 250 G2839 Z-C2H2 34-60, 85-113 252
G2854 ACBF-like 110-250 254 G3083 bZIP-ZW2 75-105, 188-215 256 G184
WRKY 295-352 258 G186 WRKY 312-369 260 G353 Z-C2H2 41-61, 84-104
262 G512 NAC 24-166 264 G596 AT-hook 89-96 266 G714 CAAT 58-148 268
G877 WRKY 272-328, 487-603 270 G1357 NAC 17-158 272 G1387 AP2 4-71
274 G1634 MYB-related 129-180 276 G1889 Z-C2H2 80-100 278 G1940
ACBF-like 156-228 280 G1974 Z-C2H2 32-60, 72-116 282 G2153 AT-hook
75-94, 162-206 284 G2583 AP2 4-71 286 G226 MYB-related 28-78 288
G481 CAAT 20-109 290 G482 CAAT 25-116 292 G485 CAAT 21-116 294 G486
CAAT 5-66 296 G1067 AT-hook 86-92, 94-247 298 G1070 AT-hook 98-120
300 G1073 AT-hook 34-42, 43-187 302 G1075 AT-hook 78-85 304 G1076
AT-hook 82-89 306 G1248 CAAT 46-155 308 G1364 CAAT 29-118 310 G1781
CAAT 35-130 312 G1816 MYB-related 31-81 314 G1945 AT-hook 49-71 316
G2155 AT-hook 18-38 318 G2156 AT-hook 72-78, 80-232 320 G2345 CAAT
26-152 322 G2657 AT-hook 116-129 324 G2718 MYB-related 21-76 326
G3392 MYB-related 21-72 328 G3393 MYB-related 20-71 330 G3394 CAAT
37-126 332 G3395 CAAT 19-108 334 G3396 CAAT 21-110 338 G3397 CAAT
23-112 338 G3398 CAAT 21-110 340 G3399 AT-hook 99-107, 108-253 342
G3400 AT-hook 83-89, 91-237 344 G3401 AT-hook 35-41, 43-186 346
G3403 AT-hook 58-64, 66-207 348 G3404 AT-hook 111-117,119-263 350
G3405 AT-hook 97-103, 105-248 352 G3406 AT-hook 82-88, 90-232 354
G3407 AT-hook 63-71, 72-220 356 G3408 AT-hook 83-89, 91-247 358
G3429 CAAT 35-124 360 G3431 MYB-related 20-71 362 G3434 CAAT 18-107
364 G3435 CAAT 22-111 366 G3436 CAAT 20-109 368 G3437 CAAT 54-143
370 G3444 MYB-related 20-71 372 G3445 MYB-related 15-65 374 G3446
MYB-related 16-66 376 G3447 MYB-related 16-66 378 G3448 MYB-related
15-66 380 G3449 MYB-related 15-66 382 G3450 MYB-related 9-60 384
G3456 AT-hook 44-52, 53-195 386 G3458 AT-hook 56-62, 64-207 388
G3459 AT-hook 77-85, 86-228 390 G3460 AT-hook 74-82, 83-225 392
G3462 AT-hook 82-88, 90-237 394 G3470 CAAT 27-116 396 G3471 CAAT
26-115 398 G3472 CAAT 25-114 400 G3473 CAAT 23-113 402 G3474 CAAT
25-114 404 G3475 CAAT 23-112 406 G3476 CAAT 26-115 408 G3477 CAAT
27-116 410 G3478 CAAT 23-112 412 G3556 AT-hook 45-51, 53-196 414
G3835 CAAT 4-92 416 G3836 CAAT 34-122 418 G3837 CAAT 35-123 420 G24
AP2 25-92 422 G154 MADS 2-57 424 G384 HB 14-77 294 G486 CAAT 5-66
426 G545 Z-C2H2 82-102, 136-154 428 G760 NAC 12-156 430 G773 NAC
17-159 432 G937 GARP 197-246 434 G971 AP2 120-186 436 G988 SCR
146-217, 278-366, 370-444 438 G989 SCR 121-186, 238-327, 326-399
240 G1069 AT-hook 67-74 440 G1090 AP2 17-84 442 G1322 MYB-(R1)R2R3
26-30 444 G1587 HB 61-121 446 G1666 HLH/MYC 353-420 448 G1700
RING/C3H2C3 93-134 450 G1818 CAAT 36-113 452 G1868 GRF-like 164-270
454 G1888 Z-CO-like 5-50 456 G2117 bZIP 46-106 458 G2131 AP2
50-121, 146-217 460 G2520 HLH/MYC 135-206 462 G2522 AT-hook
At-hooks: 101-109 & 134-142 2nd domain: 143-291 248 G2789
AT-hook 53-73, 121-165 464 G8 AP2 151-217,243-293 466 G27 AP2
37-104 468 G156 MADS 2-57 470 G161 MADS 6-62 472 G168 MADS 1-57 474
G183 WRKY 307-368 476 G189 WRKY 240-297 478 G200 MYB-(R1)R2R3
12-116 480 G234 MYB-(R1)R2R3 14-115 482 G237 MYB-(R1)R2R3 11-113
484 G275 AKR 308-813 486 G326 Z-CO-like 11-94, 354-400 488 G347
Z-LSDlike 9-39, 50-70, 80-127 490 G427 HB 307-370 492 G505 NAC
20-170 494 G590 HLH/MYC 202-254 496 G602 DBP 110-162 498 G618 TEO
32-89 500 G635 TH 239-323 502 G643 TH 47-85 504 G653 Z-LIM 10-61,
109-160 506 G657 MYB-(R1)R2R3 35-187 508 G837 AKR 250-754 510 G866
WRKY 43-300 512 G872 AP2 18-84 514 G904 RING/C3H2C3 117-158 516
G912 AP2 51-118 518 G932 MYB-(R1)R2R3 14-118 520 G958 NAC 7-156 522
G964 HB 126-186 238 G975 AP2 4-71 524 G979 AP2 63-139, 165-233 526
G1049 bZIP 77-132 528 G1246 MYB-(R1)R2R3 27-139 530 G1255 Z-CO-like
18-56 532 G1266 AP2 79-147 534 G1331 MYB-(R1)R2R3 8-109 536 G1332
MYB-(R1)R2R3 13-116 538 G1494 HLH/MYC 261-311 540 G1535 HB 109-169
542 G1649 HLH/MYC 225-295 544 G1750 AP2 115-182 546 G1773
RING/C3HC4 139-184 548 G1835 GATA/Zn 224-296 550 G1930 AP2 59-124,
179-273 10 G2053 NAC 6-152 552 G2057 TEO 46-103 12 G2133 AP2 10-77
554 G2144 HLH/MYC 203-283 556 G2145 HLH/MYC 166-243 558 G2295 MADS
1-57 560 G2512 AP2 79-147 562 G2531 NAC 52-212 564 G2535 NAC 11-114
566 G2590 MADS 2-57 568 G2719 MYB-(R1)R2R3 56-154 570 G9 AP2
62-127, 184-277 572 G12 AP2 27-94 574 G40 AP2 45-112 576 G41 AP2
39-106 578 G42 AP2 48-115 2 G47 AP2 10-75 580 G170 MADS 2-57 582
G216 MYB-(R1)R2R3 49-151 584 G221 MYB-(R1)R2R3 21-125 586 G232
MYB-(R1)R2R3 14-115 588 G249 MYB-(R1)R2R3 19-116 590 G256
MYB-(R1)R2R3 13-115 592 G350 Z-C2H2 91-113, 150-170 594 G351 Z-C2H2
77-97, 118-140 596 G385 HB 60-123 598 G389 HB 84-147 600 G398 HB
128-191 602 G399 HB 126-186 604 G425 HB 305-365 606 G426 HB 346-406
608 G440 AP2 122-189 610 G441 AP2 40-107 20 G515 NAC 6-149 22 G516
NAC 6-141 24 G517 NAC 6-153 612 G518 NAC 7-153 614 G572 bZIP
120-186 264 G596 AT-hook 89-96 616 G654 Z-LIM 10-61, 108-159 618
G666 MYB-(R1)R2R3 14-118 620 G668 MYB-(R1)R2R3 13-113 622 G759 NAC
17-159 624 G789 HLH/MYC 253-313 626 G829 AKR 250-754 628 G864 AP2
119-186 630 G867 AP2 59-124, 184-276 632 G883 WRKY 245-302 634 G914
AP2 106-162, 198-238 636 G957 NAC 12-182 638 G961 NAC 12-180 640
G993 AP2 69-134, 191-290 642 G1011 MADS 2-57 644 G1065 DBP 101-210
646 G1071 AT-hook 98-111, 132-138, 140-286 648 G1277 AP2 18-85 650
G1309 MYB-(R1)R2R3 9-114 652 G1337 Z-CO-like 9-75 654 G1379 AP2
18-85 656 G1386 AP2 42-109 272 G1387 AP2 4-71 658 G1412 NAC 13-162
660 G1439 GRF-like 133-239 662 G1482 Z-CO-like 5-63 664 G1484
Z-CO-like 16-39 666 G1588 HB 66-124 668 G1752 AP2 83-151 670 G1836
CAAT 30-164 672 G1942 HLH/MYC 178-270 674 G2065 MADS 1-57 676 G2106
AP2 56-139, 165-233 678 G2107 AP2 27-94
680 G2148 HLH/MYC 130-268 282 G2153 AT-hook 75-94, 162-206 682
G2180 NAC 7-156 684 G2513 AP2 27-94 686 G2545 HB 215-278 688 G2576
AP2 9-75 284 G2583 AP2 4-71 690 G3041 NAC 8-136 692 G3362 AP2
41-108 694 G3364 AP2 51-114 696 G3365 AP2 41-108 698 G3366 AP2
53-117 700 G3367 AP2 51-114 702 G3368 AP2 51-120 704 G3369 AP2
107-170 706 G3370 AP2 29-99 708 G3371 AP2 36-102 710 G3372 AP2
30-95 712 G3373 AP2 43-109 714 G3374 AP2 51-118 716 G3375 AP2
49-113 718 G3376 AP2 51-115 720 G3377 AP2 41-107 722 G3378 AP2
83-154 724 G3379 AP2 47-119 726 G3384 MYB-(R1)R2R3 14-118 728 G3385
MYB-(R1)R2R3 14-118 730 G3386 MYB-(R1)R2R3 14-118 732 G3388 AP2
66-129, 181-274 734 G3389 AP2 64-129, 177-266 736 G3390 AP2 66-131,
192-294 738 G3391 AP2 79-148,215-300 344 G3401 AT-hook 35-41,
43-186 346 G3403 AT-hook 58-64, 66-207 740 G3432 AP2 75-140,
212-299 742 G3433 AP2 80-151,210-291 744 G3438 AP2 50-116 746 G3439
AP2 57-126 748 G3440 AP2 49-116 750 G3441 AP2 55-120 752 G3442 AP2
61-127 754 G3451 AP2 80-141, 209-308 756 G3452 AP2 51-116, 171-266
758 G3453 AP2 57-122, 177-272 760 G3454 AP2 74-141, 203-302 384
G3456 AT-hook 44-50, 52-195 392 G3462 AT-hook 82-88, 90-237 762
G3463 AP2 60-125 764 G3464 AP2 50-114 766 G3465 AP2 61-125 768
G3466 AP2 63-127 770 G3467 AP2 60-123 772 G3468 AP2 63-128 774
G3469 AP2 16-79 776 G3497 AP2 51-114 778 G3498 AP2 50-114 780 G3499
AP2 46-111 782 G3500 MYB-(R1)R2R3 14-118 784 G3501 MYB-(R1)R2R3
14-118 786 G3502 MYB-(R1)R2R3 14-119 788 G3537 MYB-(R1)R2R3 14-118
790 G3538 MYB-(R1)R2R3 13-117 792 G3539 MYB-(R1)R2R3 14-118 794
G3540 MYB-(R1)R2R3 14-118 796 G3541 MYB-(R1)R2R3 14-118 412 G3556
AT-hook 45-51, 53-196 88 G3643 AP2 13-78 90 G3644 AP2 52-122 92
G3645 AP2 10-75 94 G3646 AP2 10-77 96 G3647 AP2 13-78 98 G3649 AP2
15-87 100 G3651 AP2 60-130 798 G3652 AP2 13-78 800 G3653 AP2 41-107
802 G3654 AP2 9-76 804 G3655 AP2 31-96 806 G3656 AP2 23-86 232 G634
TH 62-147, 189-245, 808 G1048 bZIP 138-190 810 G1100 RING/C3H2C3
96-137 658 G1412 NAC 13-162 812 G1796 AP2 54-121 814 G1995 Z-C2H2
93-113 816 G2467 HS 28-119 818 G2505 NAC 9-137 820 G2550 HB 345-408
822 G2640 SRS 146-189 824 G2686 WRKY 122-173 248 G2789 AT-hook
53-73, 121-165, 420 G24 AP2 25-92 826 G38 AP2 76-143 828 G44 AP2
85-154 830 G230 MYB-(R1)R2R3 13-114 480 G234 MYB-(R1)R2R3 14-115
832 G261 HS 15-106 834 G271 AKR 41-106, 325-363, 226 G303 HLH/MYC
92-161 836 G359 Z-C2H2 49-69 838 G377 RING/C3H2C3 85-128 840 G388
HB 98-158 842 G435 HB 4-67 844 G442 AP2 66-138 846 G468 IAA 86-102,
141-171, 848 G571 bZIP 160-220, 441-452, 850 G652 Z-CLDSH 28-49,
137-151, 182-196, 852 G664 MYB-(R1)R2R3 14-116 854 G772 NAC 27-176
856 G798 Z-Dof 19-47 858 G818 HS 71-162 434 G971 AP2 120-186 860
G974 AP2 80-147 436 G988 SCR 146-217, 278-366, 370-444, 862 G1062
HLH/MYC 308-359 240 G1069 AT-hook 67-74 864 G1129 HLH/MYC 171-244
866 G1137 HLH/MYC 264-314 868 G1425 NAC 20-173 870 G1517 RING/C3HC4
312-349 872 G1655 HLH/MYC 134-192 874 G1743 RING/C3H2C3 94-136 876
G1789 MYB-related 12-62 878 G1806 bZIP 165-225 880 G1911
MYB-related 12-62 882 G2011 HS 55-146 316 G2155 AT-hook 18-38 884
G2215 bZIP-NIN 150-246 886 G2452 MYB-related 28-79, 146-194, 888
G2455 YABBY 10-48, 107-154, 890 G2510 AP2 42-109 892 G2515 MADS
1-57 894 G2571 AP2 133-200 896 G2702 MYB-(R1)R2R3 31-131 898 G2763
HLH/MYC 140-210 900 G2774 HLH/MYC 157-227 892 G2888 Z-C2H2 41-61,
120-140, 904 G2958 IAA 88-104, 143-172, 906 G5 AP2 149-216 572 G12
AP2 27-94 908 G197 MYB-(R1)R2R3 14-116 910 G207 MYB-(R1)R2R3 6-106
912 G227 MYB-(R1)R2R3 13-112 586 G232 MYB-(R1)R2R3 14-115 914 G242
MYB-(R1)R2R3 6-105 916 G255 MYB-(R1)R2R3 14-116 918 G265 HS 13-104
920 G361 Z-C2H2 43-63 922 G362 Z-C2H2 62-82 924 G370 Z-C2H2 97-117
926 G504 NAC 16-178 928 G554 bZIP 82-142 930 G555 bZIP 38-110 932
G556 bZIP 83-143 934 G558 bZIP 45-105 936 G578 bZIP 36-96 264 G596
AT-hook 89-96 938 G629 bZIP 92-152 622 G759 NAC 17-159 430 G773 NAC
17-159 940 G776 NAC 27-175 942 G812 HS 29-120 634 G914 AP2 106-162,
198-238, 944 G997 MYB-related 9-59 946 G1133 HLH/MYC 256-326 948
G1141 AP2 75-142 950 G1198 bZIP 173-223 648 G1277 AP2 18-85 952
G1335 Z-CLDSH 24-43, 131-144, 185-203, 654 G1379 AP2 18-85 954
G1454 NAC 9-178 956 G1664 HLH/MYC 258-328 958 G1897 Z-Dof 34-62 314
G1945 AT-hook 49-71 960 G1991 Z-C2H2 6-26, 175-195, 224-226, 282
G2153 AT-hook 75-94, 162-206, 962 G2216 bZIP-NIN 90-139 964 G2546
HB 349-413 966 G2586 WRKY 103-160 968 G2587 WRKY 108-165 970 G2635
NAC 8-161 972 G2639 SRS 114-167 974 G2642 SRS 54-97 976 G2721
MYB-related 10-60 978 G2826 Z-C2H2 75-95 980 G2838 Z-C2H2 57-77 982
G2866 IAA 84-100, 139-168, 344 G3401 AT-hook 35-41, 43-186, 346
G3403 AT-hook 58-64, 66-207, 348 G3408 AT-hook 83-89, 91-247, 384
G3456 AT-hook 44-50, 52-195, 392 G3462 AT-hook 82-88, 90-237, 984
G3503 MYB-(R1)R2R3 14-116 986 G3504 MYB-(R1)R2R3 14-116 988 G3505
MYB-(R1)R2R3 14-116 990 G3506 MYB-(R1)R2R3 14-116 992 G3507
MYB-(R1)R2R3 14-116 994 G3508 MYB-(R1)R2R3 14-116 996 G3509
MYB-(R1)R2R3 14-116 998 G3527 MYB-(R1)R2R3 13-117 1000 G3528
MYB-(R1)R2R3 13-117 1002 G3529 MYB-(R1)R2R3 14-116 1004 G3531
MYB-(R1)R2R3 14-116 1006 G3532 MYB-(R1)R2R3 14-116 1008 G3533
MYB-(R1)R2R3 14-116 1010 G3534 MYB-(R1)R2R3 14-116 412 G3556
AT-hook 45-51, 53-196, 806 G3656 AP2 23-86 1012 G3809 NAC
25-236
Producing Polypeptides
[0181] The polynucleotides of the invention include sequences that
encode transcription factors and transcription factor homolog
polypeptides and sequences complementary thereto, as well as unique
fragments of coding sequence, or sequence complementary thereto.
Such polynucleotides can be, for example, DNA or RNA, the latter
including mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic
DNA, oligonucleotides, etc. The polynucleotides are either
double-stranded or single-stranded, and include either, or both
sense (i.e., coding) sequences and antisense (i.e., non-coding,
complementary) sequences. The polynucleotides include the coding
sequence of a transcription factor, or transcription factor homolog
polypeptide, in isolation, in combination with additional coding
sequences (for example, a purification tag, a localization signal,
as a fusion-protein, as a pre-protein, or the like), in combination
with non-coding sequences (for example, introns or inteins,
regulatory elements such as promoters, enhancers, terminators, and
the like), and/or in a vector or host environment in which the
polynucleotide encoding a transcription factor or transcription
factor homolog polypeptide is an endogenous or exogenous gene.
[0182] A variety of methods exist for producing the polynucleotides
of the invention. Procedures for identifying and isolating DNA
clones are well known to those of skill in the art, and are
described in, for example, Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology, vol. 152 Academic Press,
Inc., San Diego, Calif. ("Berger"); Sambrook et al. Molecular
Cloning--A Laboratory Manual (2nd ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and
Current Protocols in Molecular Biology, Ausubel et al. eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2000) ("Ausubel").
[0183] Alternatively, polynucleotides of the invention, can be
produced by a variety of in vitro amplification methods adapted to
the present invention by appropriate selection of specific or
degenerate primers. Examples of protocols sufficient to direct
persons of skill through in vitro amplification methods, including
the polymerase chain reaction (PCR) the ligase chain reaction
(LCR), Q.beta.-replicase amplification and other RNA polymerase
mediated techniques (for example, NASBA), for example, for the
production of the homologous nucleic acids of the invention are
found in Berger (supra), Sambrook (supra), and Ausubel (supra), as
well as Mullis et al. (1987) PCR Protocols A Guide to Methods and
Applications (Innis et al. eds) Academic Press Inc. San Diego,
Calif. (1990) (Innis). Improved methods for cloning in vitro
amplified nucleic acids are described in Wallace et al. U.S. Pat.
No. 5,426,039. Improved methods for amplifying large nucleic acids
by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685
and the references cited therein, in which PCR amplicons of up to
40 kb are generated. One of skill will appreciate that essentially
any RNA can be converted into a double-stranded DNA suitable for
restriction digestion, PCR expansion and sequencing using reverse
transcriptase and a polymerase (for example, in Ausubel, Sambrook
and Berger, all supra).
[0184] Alternatively, polynucleotides and oligonucleotides of the
invention can be assembled from fragments produced by solid-phase
synthesis methods. Typically, fragments of up to approximately 100
bases are individually synthesized and then enzymatically or
chemically ligated to produce a desired sequence, for example, a
polynucleotide encoding all or part of a transcription factor. For
example, chemical synthesis using the phosphoramidite method is
described, for example, by Beaucage et al. (1981) Tetrahedron
Letters 22: 1859-1869; and Matthes et al. (1984) EMBO J. 3:
801-805. According to such methods, oligonucleotides are
synthesized, purified, annealed to their complementary strand,
ligated and then optionally cloned into suitable vectors. And if so
desired, the polynucleotides and polypeptides of the invention can
be custom ordered from any of a number of commercial suppliers.
Homologous Sequences
[0185] Sequences homologous, i.e., that share significant sequence
identity or similarity, to those provided in the Sequence Listing,
derived from Arabidopsis thaliana or from other plants of choice,
are also an aspect of the invention. Homologous sequences can be
derived from any plant including monocots and dicots and in
particular agriculturally important plant species, including but
not limited to, crops such as soybean, wheat, corn (maize), potato,
cotton, rice, rape, oilseed rape (including canola), sunflower,
alfalfa, clover, sugarcane, and turf; or fruits and vegetables,
such as banana, blackberry, blueberry, strawberry, and raspberry,
cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant,
grapes, honeydew, lettuce, mango, melon, onion, papaya, peas,
peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco,
tomato, tomatillo, watermelon, rosaceous fruits (such as apple,
peach, pear, cherry and plum) and vegetable brassicas (such as
broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi).
Other crops, including fruits and vegetables, whose phenotype can
be changed and which comprise homologous sequences include barley;
rye; millet; sorghum; currant; avocado; citrus fruits such as
oranges, lemons, grapefruit and tangerines, artichoke, cherries;
nuts such as the walnut and peanut; endive; leek; roots such as
arrowroot, beet, cassava, turnip, radish, yam, and sweet potato;
and beans. The homologous sequences may also be derived from woody
species, such as pine, poplar and eucalyptus, or mint or other
labiates. In addition, homologous sequences may be derived from
plants that are evolutionarily related to crop plants, but which
may not have yet been used as crop plants. Examples include deadly
nightshade (Atropa belladona), related to tomato; jimson weed
(Datura strommium), related to peyote; and teosinte (Zea species),
related to corn (maize).
Orthologs and Paralogs
[0186] Homologous sequences as described above can comprise
orthologous or paralogous sequences. Several different methods are
known by those of skill in the art for identifying and defining
these functionally homologous sequences. Three general methods for
defining orthologs and paralogs are described; an ortholog, paralog
or homolog may be identified by one or more of the methods
described below.
[0187] Within a single plant species, gene duplication may cause
two copies of a particular gene, giving rise to two or more genes
with similar sequence and often similar function known as paralogs.
A paralog is therefore a similar gene formed by duplication within
the same species. Paralogs typically cluster together or in the
same Glade (a group of similar genes) when a gene family phylogeny
is analyzed using programs such as CLUSTAL (Thompson et al. (1994)
Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods
Enzymol. 266: 383-402). Groups of similar genes can also be
identified with pair-wise BLAST analysis (Feng and Doolittle (1987)
J. Mol. Evol. 25: 351-360). For example, a clade of very similar
MADS domain transcription factors from Arabidopsis all share a
common function in flowering time (Ratcliffe et al. (2001) Plant
Physiol. 126: 122-132), and a group of very similar AP2 domain
transcription factors from Arabidopsis are involved in tolerance of
plants to freezing (Gilmour et al. (1998) Plant J. 16: 433-442).
Analysis of groups of similar genes with similar function that fall
within one clade can yield sub-sequences that are particular to the
clade. These sub-sequences, known as consensus sequences, can not
only be used to define the sequences within each clade, but define
the functions of these genes; genes within a clade may contain
paralogous sequences, or orthologous sequences that share the same
function (for example, in Mount (2001), in Bioinformatics: Sequence
and Genome Analysis Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., page 543).
[0188] Speciation, the production of new species from a parental
species, can also give rise to two or more genes with similar
sequence and similar function. These genes, termed orthologs, often
have an identical function within their host plants and are often
interchangeable between species without losing function. Because
plants have common ancestors, many genes in any plant species will
have a corresponding orthologous gene in another plant species.
Once a phylogenic tree for a gene family of one species has been
constructed using a program such as CLUSTAL (Thompson et al. (1994)
Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) supra)
potential orthologous sequences can be placed into the phylogenetic
tree and their relationship to genes from the species of interest
can be determined. Orthologous sequences can also be identified by
a reciprocal BLAST strategy. Once an orthologous sequence has been
identified, the function of the ortholog can be deduced from the
identified function of the reference sequence.
[0189] Transcription factor gene sequences are conserved across
diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75:
519-530; Lin et al. (1991) Nature 353: 569-571; Sadowski et al.
(1988) Nature 335: 563-564). Plants are no exception to this
observation; diverse plant species possess transcription factors
that have similar sequences and functions.
[0190] Orthologous genes from different organisms have highly
conserved functions, and very often essentially identical functions
(Lee et al. (2002) Genome Res. 12: 493-502; Remm et al. (2001) J.
Mol. Biol. 314: 1041-1052). Paralogous genes, which have diverged
through gene duplication, may retain similar functions of the
encoded proteins. In such cases, paralogs can be used
interchangeably with respect to certain embodiments of the instant
invention (for example, transgenic expression of a coding
sequence). An example of such highly related paralogs is the CBF
family, with four well-defined members in Arabidopsis (CBF 1, CBF2,
CBF3 and CBF4) and at least one ortholog in Brassica napus, all of
which control pathways involved in both freezing and drought stress
(Gilmour et al. (1998) Plant J. 16: 433-442; Jaglo et al. (1998)
Plant Physiol. 127: 910-917).
[0191] The following references represent a small sampling of the
many studies that demonstrate that conserved transcription factor
genes from diverse species are likely to function similarly (i.e.,
regulate similar target sequences and control the same traits), and
that transcription factors may be transformed into diverse species
to confer or improve traits. [0192] (1) Distinct Arabidopsis
transcription factors, including G28 (U.S. Pat. No. 6,664,446),
G482 (US Patent Application 20040045049; SEQ ID NO: 290 in the
present Sequence Listing), G867 (US Patent Application 20040098764;
SEQ ID NO: 630 in the present Sequence Listing), and G1073 (US
Patent Application 20040128712; SEQ ID NO: 300 in the present
Sequence Listing), have been shown to confer abiotic stress
tolerance when the sequences are overexpressed. The polypeptides
sequences belong to distinct clades of transcription factor
polypeptides that include members from diverse species. In each
case, a significant number of sequences derived from both dicots
and monocots have been shown to confer tolerance to various abiotic
stresses when the sequences were overexpressed. [0193] (2) The
Arabidopsis NPR1 gene regulates systemic acquired resistance (SAR)
(Cao et al. (1997) Cell 88: 57-63); over-expression of NPR1 leads
to enhanced resistance in Arabidopsis. When either Arabidopsis NPR1
or the rice NPR1 ortholog was overexpressed in rice (which, as a
monocot, is diverse from Arabidopsis), challenge with the rice
bacterial blight pathogen Xanthomonas oryzae pv. Oryzae, the
transgenic plants displayed enhanced resistance (Chern et al.
(2001) Plant J. 27: 101-113). NPR1 acts through activation of
expression of transcription factor genes, such as TGA2 (Fan and
Dong (2002) Plant Cell 14: 1377-1389).
[0194] (3) E2F genes are involved in transcription of plant genes
for proliferating cell nuclear antigen (PCNA). Plant E2Fs share a
high degree of similarity in amino acid sequence between monocots
and dicots, and are even similar to the conserved domains of the
animal E2Fs. Such conservation indicates a functional similarity
between plant and animal E2Fs. E2F transcription factors that
regulate meristem development act through common cis-elements, and
regulate related (PCNA) genes (Kosugi and Ohashi, (2002) Plant J.
29: 45-59). [0195] (4) The ABI5 gene (ABA insensitive 5) encodes a
basic leucine zipper factor required for ABA response in the seed
and vegetative tissues. Co-transformation experiments with ABI5
cDNA constructs in rice protoplasts resulted in specific
transactivation of the ABA-inducible wheat, Arabidopsis, bean, and
barley promoters. These results demonstrate that sequentially
similar ABI5 transcription factors are key targets of a conserved
ABA signaling pathway in diverse plants. (Gampala et al. (2001) J.
Biol. Chem. 277: 1689-1694). [0196] (5) Sequences of three
Arabidopsis GAMYB-like genes were obtained on the basis of sequence
similarity to GAMYB genes from barley, rice, and L. temulentum.
These three Arabidopsis genes were determined to encode
transcription factors (AtMYB33, AtMYB65, and AtMYB101) and could
substitute for a barley GAMYB and control .alpha.-amylase
expression (Gocal et al. (2001) Plant Physiol. 127: 1682-1693).
[0197] (6) The floral control gene LEAFY from Arabidopsis can
dramatically accelerate flowering in numerous dictoyledonous
plants. Constitutive expression of Arabidopsis LEAFY also caused
early flowering in transgenic rice (a monocot), with a heading date
that was 26-34 days earlier than that of wild-type plants. These
observations indicate that floral regulatory genes from Arabidopsis
are useful tools for heading date improvement in cereal crops (He
et al. (2000) Transgenic Res. 9: 223-227). [0198] (7) Bioactive
gibberellins (GAs) are essential endogenous regulators of plant
growth. GA signaling tends to be conserved across the plant
kingdom. GA signaling is mediated via GAI, a nuclear member of the
GRAS family of plant transcription factors. Arabidopsis GAI has
been shown to function in rice to inhibit gibberellin response
pathways (Fu et al. (2001) Plant Cell 13: 1791-1802). [0199] (8)
The Arabidopsis gene SUPERMAN (SUP), encodes a putative
transcription factor that maintains the boundary between stamens
and carpels. By over-expressing Arabidopsis SUP in rice, the effect
of the gene's presence on whorl boundaries was shown to be
conserved. This demonstrated that SUP is a conserved regulator of
floral whorl boundaries and affects cell proliferation (Nandi et
al. (2000) Curr. Biol. 10: 215-218). [0200] (9) Maize, petunia and
Arabidopsis myb transcription factors that regulate flavonoid
biosynthesis are genetically similar and affect the same trait in
their native species; therefore, sequence and function of these myb
transcription factors correlate with each other in these diverse
species (Borevitz et al. (2000) Plant Cell 12: 2383-2394). [0201]
(10) Wheat reduced height-1 (Rht-B1/Rht-D1) and maize dwarf-8 (d8)
genes are orthologs of the Arabidopsis gibberellin insensitive
(GAI) gene. Both of these genes have been used to produce dwarf
grain varieties that have improved grain yield. These genes encode
proteins that resemble nuclear transcription factors and contain an
SH2-like domain, indicating that phosphotyrosine may participate in
gibberellin signaling. Transgenic rice plants containing a mutant
GAI allele from Arabidopsis have been shown to produce reduced
responses to gibberellin and are dwarfed, indicating that mutant
GAI orthologs could be used to increase yield in a wide range of
crop species (Peng et al. (1999) Nature 400: 256-261).
[0202] Transcription factors that are homologous to the listed
sequences will typically share at least about 70% amino acid
sequence identity in their conserved domain. More closely related
transcription factors can share at least about 79% or about 90% or
about 95% or about 98% or more sequence identity with the listed
sequences, or with the listed sequences but excluding or outside a
known consensus sequence or consensus DNA-binding site, or with the
listed sequences excluding one or all conserved domains. Factors
that are most closely related to the listed sequences share, for
example, at least about 85%, about 90% or about 95% or more %
sequence identity to the listed sequences, or to the listed
sequences but excluding or outside a known consensus sequence or
consensus DNA-binding site or outside one or all conserved domain.
At the nucleotide level, the sequences will typically share at
least about 40% nucleotide sequence identity, preferably at least
about 50%, about 60%, about 70% or about 80% sequence identity, and
more preferably about 85%, about 90%, about 95% or about 97% or
more sequence identity to one or more of the listed sequences, or
to a listed sequence but excluding or outside a known consensus
sequence or consensus DNA-binding site, or outside one or all
conserved domain. The degeneracy of the genetic code enables major
variations in the nucleotide sequence of a polynucleotide while
maintaining the amino acid sequence of the encoded protein. AP2
domains within the AP2 transcription factor family may exhibit a
higher degree of sequence homology, such as at least 70% amino acid
sequence identity including conservative substitutions, and
preferably at least 80% sequence identity, and more preferably at
least 85%, or at least about 86%, or at least about 87%, or at
least about 88%, or at least about 90%, or at least about 95%, or
at least about 98% sequence identity. Transcription factors that
are homologous to the listed sequences should share at least 30%,
or at least about 60%, or at least about 75%, or at least about
80%, or at least about 90%, or at least about 95% amino acid
sequence identity over the entire length of the polypeptide or the
homolog.
[0203] Percent identity can be determined electronically, for
example, by using the MEGALIGN program (DNASTAR, Inc. Madison,
Wis.). The MEGALIGN program can create alignments between two or
more sequences according to different methods, for example, the
clustal method (for example, in Higgins and Sharp (1988) Gene 73:
237-244). The clustal algorithm groups sequences into clusters by
examining the distances between all pairs. The clusters are aligned
pairwise and then in groups. Other alignment algorithms or programs
may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST,
and which may be used to calculate percent similarity. These are
available as a part of the GCG sequence analysis package
(University of Wisconsin, Madison, Wis.), and can be used with or
without default settings. ENTREZ is available through the National
Center for Biotechnology Information. In one embodiment, the
percent identity of two sequences can be determined by the GCG
program with a gap weight of 1, for example, each amino acid gap is
weighted as if it were a single amino acid or nucleotide mismatch
between the two sequences (U.S. Pat. No. 6,262,333).
[0204] Other techniques for alignment are described in Methods in
Enzymology, vol. 266, Computer Methods for Macromolecular Sequence
Analysis (1996), ed. Doolittle, Academic Press, Inc., San Diego,
Calif., USA. Preferably, an alignment program that permits gaps in
the sequence is utilized to align the sequences. The Smith-Waterman
is one type of algorithm that permits gaps in sequence alignments
(Shpaer (1997) Methods Mol. Biol. 70: 173-187). Also, the GAP
program using the Needleman and Wunsch alignment method can be
utilized to align sequences. An alternative search strategy uses
MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a
Smith-Waterman algorithm to score sequences on a massively parallel
computer. This approach improves ability to pick up distantly
related matches, and is especially tolerant of small gaps and
nucleotide sequence errors. Nucleic acid-encoded amino acid
sequences can be used to search both protein and DNA databases.
[0205] The percentage similarity between two polypeptide sequences,
for example, sequence A and sequence B, is calculated by dividing
the length of sequence A, minus the number of gap residues in
sequence A, minus the number of gap residues in sequence B, into
the sum of the residue matches between sequence A and sequence B,
times one hundred. Gaps of low or of no similarity between the two
amino acid sequences are not included in determining percentage
similarity. Percent identity between polynucleotide sequences can
also be counted or calculated by other methods known in the art,
for example, the Jotun Hein method (for example, in Hein (1990)
Methods Enzymol. 183: 626-645). Identity between sequences can also
be determined by other methods known in the art, for example, by
varying hybridization conditions (US Patent Application No.
20010010913).
[0206] Thus, the invention provides methods for identifying a
sequence similar or paralogous or orthologous or homologous to one
or more polynucleotides as noted herein, or one or more target
polypeptides encoded by the polynucleotides, or otherwise noted
herein and may include linking or associating a given plant
phenotype or gene function with a sequence. In the methods, a
sequence database is provided (locally or across an internet or
intranet) and a query is made against the sequence database using
the relevant sequences herein and associated plant phenotypes or
gene functions.
[0207] In addition, one or more polynucleotide sequences or one or
more polypeptides encoded by the polynucleotide sequences may be
used to search against a BLOCKS (Bairoch et al. (1997) Nucleic
Acids Res. 25: 217-221), PFAM, and other databases which contain
previously identified and annotated motifs, sequences and gene
functions. Methods that search for primary sequence patterns with
secondary structure gap penalties (Smith et al. (1992) Protein
Engineering 5: 35-51) as well as algorithms such as Basic Local
Alignment Search Tool (BLAST; Altschul (1993) J. Mol. Evol. 36:
290-300; Altschul et al. (1990) J. Mol. Biol. 215: 403-410), BLOCKS
(Henikoff and Henikoff (1991) Nucleic Acids Res. 19: 6565-6572),
Hidden Markov Models (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6:
361-365; Sonnhammer et al. (1997) Proteins 28: 405-420), and the
like, can be used to manipulate and analyze polynucleotide and
polypeptide sequences encoded by polynucleotides. These databases,
algorithms and other methods are well known in the art and are
described in Ausubel et al. (1997) Short Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., unit 7.7) and in
Meyers (1995) Molecular Biology and Biotechnology, Wiley VCH, New
York, N.Y., p 856-853).
[0208] A further method for identifying or confirming that specific
homologous sequences control the same function is by comparison of
the transcript profile(s) obtained upon overexpression or knockout
of two or more related transcription factors. Since transcript
profiles are diagnostic for specific cellular states, one skilled
in the art will appreciate that genes that have a highly similar
transcript profile (for example, with greater than 50% regulated
transcripts in common, more preferably with greater than 70%
regulated transcripts in common, most preferably with greater than
90% regulated transcripts in common) will have highly similar
functions. Fowler et al. (2002) Plant Cell 14: 1675-1679) have
shown that three paralogous AP2 family genes (CBF1, CBF2 and CBF3),
each of which is induced upon cold treatment, and each of which can
condition improved freezing tolerance, have highly similar
transcript profiles. Once a transcription factor has been shown to
provide a specific function, its transcript profile becomes a
diagnostic tool to determine whether putative paralogs or orthologs
have the same function.
[0209] Furthermore, methods using manual alignment of sequences
similar or homologous to one or more polynucleotide sequences or
one or more polypeptides encoded by the polynucleotide sequences
may be used to identify regions of similarity and AP2 binding
domains. Such manual methods are well-known of those of skill in
the art and can include, for example, comparisons of tertiary
structure between a polypeptide sequence encoded by a
polynucleotide that comprises a known function with a polypeptide
sequence encoded by a polynucleotide sequence which has a function
not yet determined. Such examples of tertiary structure may
comprise predicted .alpha.-helices, .beta.-sheets, amphipathic
helices, leucine zipper motifs, zinc finger motifs, proline-rich
regions, cysteine repeat motifs, and the like.
[0210] Orthologs and paralogs of presently disclosed transcription
factors may be cloned using compositions provided by the present
invention according to methods well known in the art. cDNAs can be
cloned using mRNA from a plant cell or tissue that expresses one of
the present transcription factors. Appropriate mRNA sources may be
identified by interrogating Northern blots with probes designed
from the present transcription factor sequences, after which a
library is prepared from the mRNA obtained from a positive cell or
tissue. Transcription factor-encoding cDNA is then isolated using,
for example, PCR, using primers designed from a presently disclosed
transcription factor gene sequence, or by probing with a partial or
complete cDNA or with one or more sets of degenerate probes based
on the disclosed sequences. The cDNA library may be used to
transform plant cells. Expression of the cDNAs of interest is
detected using, for example, methods disclosed herein such as
microarrays, Northern blots, quantitative PCR, or any other
technique for monitoring changes in expression. Genomic clones may
be isolated using similar techniques to those.
Identifying Polynucleotides or Nucleic Acids by Hybridization
[0211] Polynucleotides homologous to the sequences illustrated in
the Sequence Listing and tables can be identified, for example, by
hybridization to each other under stringent or under highly
stringent conditions. Single-stranded polynucleotides hybridize
when they associate based on a variety of well characterized
physical-chemical forces, such as hydrogen bonding, solvent
exclusion, base stacking and the like. The stringency of a
hybridization reflects the degree of sequence identity of the
nucleic acids involved, such that the higher the stringency, the
more similar are the two polynucleotide strands. Stringency is
influenced by a variety of factors, including temperature, salt
concentration and composition, organic and non-organic additives,
solvents, etc. present in both the hybridization and wash solutions
and incubations (and number thereof), as described in more detail
in the references cited above.
[0212] Stability of DNA duplexes is affected by such factors as
base composition, length, and degree of base pair mismatch.
Hybridization conditions may be adjusted to allow DNAs of different
sequence relatedness to hybridize. The melting temperature
(T.sub.m) is defined as the temperature when 50% of the duplex
molecules have dissociated into their constituent single strands.
The melting temperature of a perfectly matched duplex, where the
hybridization buffer contains formamide as a denaturing agent, may
be estimated by the following equations:
(I) DNA-DNA:
[0213] T.sub.m(.degree. C.)=81.5+16.6(log [Na+])+0.41(% G+C)-0.62(%
formamide)-500/L
(II) DNA-RNA:
[0214] T.sub.m(.degree. C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(%
G+C).sup.2-0.5(% formamide)-820/L
(III) RNA-RNA:
[0215] T.sub.m(.degree. C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(%
G+C).sup.2-0.35(% formamide)-820/L
[0216] where L is the length of the duplex formed, [Na+] is the
molar concentration of the sodium ion in the hybridization or
washing solution, and % G+C is the percentage of (guanine+cytosine)
bases in the hybrid. For imperfectly matched hybrids, approximately
1.degree. C. is required to reduce the melting temperature for each
1% mismatch.
[0217] Hybridization experiments are generally conducted in a
buffer of pH between 6.8 to 7.4, although the rate of hybridization
is nearly independent of pH at ionic strengths likely to be used in
the hybridization buffer (Anderson and Young (1985) "Quantitative
Filter Hybridisation." In: Hames and Higgins, ed., Nucleic Acid
Hybridisation, A Practical Approach. Oxford, IRL Press, 73-111). In
addition, one or more of the following may be used to reduce
non-specific hybridization: sonicated salmon sperm DNA or another
non-complementary DNA, bovine serum albumin, sodium pyrophosphate,
sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and
Denhardt's solution. Dextran sulfate and polyethylene glycol 6000
act to exclude DNA from solution, thus raising the effective probe
DNA concentration and the hybridization signal within a given unit
of time. In some instances, conditions of even greater stringency
may be desirable or required to reduce non-specific and/or
background hybridization. These conditions may be created with the
use of higher temperature, lower ionic strength and higher
concentration of a denaturing agent such as formamide.
[0218] Stringency conditions can be adjusted to screen for
moderately similar fragments such as homologous sequences from
distantly related organisms, or to highly similar fragments such as
genes that duplicate functional enzymes from closely related
organisms. The stringency can be adjusted either during the
hybridization step or in the post-hybridization washes. Salt
concentration, formamide concentration, hybridization temperature
and probe lengths are variables that can be used to alter
stringency (as described by the formula above). As a general
guidelines high stringency is typically performed at
T.sub.m-5.degree. C. to T.sub.m-20.degree. C., moderate stringency
at T.sub.m-20.degree. C. to T.sub.m-35.degree. C. and low
stringency at T.sub.m-35.degree. C. to T.sub.m-50.degree. C. for
duplex >150 base pairs. Hybridization may be performed at low to
moderate stringency (25-50.degree. C. below T.sub.m), followed by
post-hybridization washes at increasing stringencies. Maximum rates
of hybridization in solution are determined empirically to occur at
T.sub.m-25.degree. C. for DNA-DNA duplex and T.sub.m-15.degree. C.
for RNA-DNA duplex. Optionally, the degree of dissociation may be
assessed after each wash step to determine the need for subsequent,
higher stringency wash steps.
[0219] High stringency conditions may be used to select for nucleic
acid sequences with high degrees of identity to the disclosed
sequences. An example of stringent hybridization conditions
obtained in a filter-based method such as a Southern or northern
blot for hybridization of complementary nucleic acids that have
more than 100 complementary residues is about 5.degree. C. to
20.degree. C. lower than the thermal melting point (T.sub.m) for
the specific sequence at a defined ionic strength and pH.
Conditions used for hybridization may include about 0.02 M to about
0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02%
SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M
sodium citrate, at hybridization temperatures between about
50.degree. C. and about 70.degree. C. More preferably, high
stringency conditions are about 0.02 M sodium chloride, about 0.5%
casein, about 0.02% SDS, about 0.001 M sodium citrate, at a
temperature of about 50.degree. C. Nucleic acid molecules that
hybridize under stringent conditions will typically hybridize to a
probe based on either the entire DNA molecule or selected portions,
for example, to a unique subsequence, of the DNA.
[0220] Stringent salt concentration will ordinarily be less than
about 750 mM NaCl and 75 mM trisodium citrate. Increasingly
stringent conditions may be obtained with less than about 500 mM
NaCl and 50 mM trisodium citrate, to even greater stringency with
less than about 250 mM NaCl and 25 mM trisodium citrate. Low
stringency hybridization can be obtained in the absence of organic
solvent, for example, formamide, whereas high stringency
hybridization may be obtained in the presence of at least about 35%
formamide, and more preferably at least about 50% formamide.
Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. with formamide present. Varying additional
parameters, such as hybridization time, the concentration of
detergent, for example, sodium dodecyl sulfate (SDS) and ionic
strength, are well known to those skilled in the art. Various
levels of stringency are accomplished by combining these various
conditions as needed.
[0221] The washing steps that follow hybridization may also vary in
stringency; the post-hybridization wash steps primarily determine
hybridization specificity, with the most critical factors being
temperature and the ionic strength of the final wash solution. Wash
stringency can be increased by decreasing salt concentration or by
increasing temperature. Stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate.
[0222] Thus, hybridization and wash conditions that may be used to
bind and remove polynucleotides with less than the desired homology
to the nucleic acid sequences or their complements that encode the
present transcription factors include, for example:
[0223] 6.times.SSC at 65.degree. C.;
[0224] 50% formamide, 4.times.SSC at 42.degree. C.; or
[0225] 0.5.times.SSC, 0.1% SDS at 65.degree. C.;
[0226] with, for example, two wash steps of 10-30 minutes each.
Useful variations on these conditions will be readily apparent to
those skilled in the art.
[0227] A person of skill in the art would not expect substantial
variation among polynucleotide species encompassed within the scope
of the present invention because the highly stringent conditions
set forth in the above formulae yield structurally similar
polynucleotides.
[0228] If desired, one may employ wash steps of even greater
stringency, including about 0.2.times.SSC, 0.1% SDS at 65.degree.
C. and washing twice, each wash step being about 30 min, or about
0.1.times.SSC, 0.1% SDS at 65.degree. C. and washing twice for 30
min. The temperature for the wash solutions will ordinarily be at
least about 25.degree. C., and for greater stringency at least
about 42.degree. C. Hybridization stringency may be increased
further by using the same conditions as in the hybridization steps,
with the wash temperature raised about 3.degree. C. to about
5.degree. C., and stringency may be increased even further by using
the same conditions except the wash temperature is raised about
6.degree. C. to about 9.degree. C. For identification of less
closely related homologs, wash steps may be performed at a lower
temperature, for example, 50.degree. C.
[0229] An example of a low stringency wash step employs a solution
and conditions of at least 25.degree. C. in 30 mM NaCl, 3 mM
trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may
be obtained at 42.degree. C. in 15 mM NaCl, with 1.5 mM trisodium
citrate, and 0.1% SDS over 30 min. Even higher stringency wash
conditions are obtained at 65.degree. C.-68.degree. C. in a
solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Wash procedures will generally employ at least two final wash
steps. Additional variations on these conditions will be readily
apparent to those skilled in the art (for example, in US Patent
Application No. 20010010913).
[0230] Stringency conditions can be selected such that an
oligonucleotide that is perfectly complementary to the coding
oligonucleotide hybridizes to the coding oligonucleotide with at
least about a 5-10.times. higher signal to noise ratio than the
ratio for hybridization of the perfectly complementary
oligonucleotide to a nucleic acid encoding a transcription factor
known as of the filing date of the application. It may be desirable
to select conditions for a particular assay such that a higher
signal to noise ratio, that is, about 15.times. or more, is
obtained. Accordingly, a subject nucleic acid will hybridize to a
unique coding oligonucleotide with at least a 2.times. or greater
signal to noise ratio as compared to hybridization of the coding
oligonucleotide to a nucleic acid encoding known polypeptide. The
particular signal will depend on the label used in the relevant
assay, for example, a fluorescent label, a colorimetric label, a
radioactive label, or the like. Labeled hybridization or PCR probes
for detecting related polynucleotide sequences may be produced by
oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
[0231] Encompassed by the invention are polynucleotide sequences
that are capable of hybridizing to the polynucleotide sequences of
the Sequence Listing, and fragments thereof under various
conditions of stringency (for example, in Wahl and Berger (1987)
Methods Enzymol. 152: 399-407, and Kimmel (1987) Methods Enzymol.
152: 507-511). Estimates of homology are provided by either DNA-DNA
or DNA-RNA hybridization under conditions of stringency as is well
understood by those skilled in the art (Hames and Higgins, Eds.
(1985) Nucleic Acid Hybridisation: A Practical Approach, IRL Press,
Oxford, U.K.). Stringency conditions can be adjusted to screen for
moderately similar fragments, such as homologous sequences from
distantly related organisms, to highly similar fragments, such as
genes that duplicate functional enzymes from closely related
organisms. Post-hybridization washes determine stringency
conditions.
Identifying Polynucleotides or Nucleic Acids with Expression
Libraries
[0232] In addition to hybridization methods, transcription factor
homolog polypeptides can be obtained by screening an expression
library using antibodies specific for one or more transcription
factors. With the provision herein of the disclosed transcription
factor, and transcription factor homolog nucleic acid sequences,
the encoded polypeptide(s) can be expressed and purified in a
heterologous expression system (for example, E. coli) and used to
raise antibodies (monoclonal or polyclonal) specific for the
polypeptide(s) in question. Antibodies can also be raised against
synthetic peptides derived from the amino acid sequences or
subsequences of a transcription factor or transcription factor
homolog. Methods of raising antibodies are well known in the art
and are described in Harlow and Lane (1988), Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such
antibodies can then be used to screen an expression library
produced from the plant from which it is desired to clone
additional transcription factor homologs, using the methods
described above. The selected cDNAs can be confirmed by sequencing
and enzymatic activity.
Sequence Variations
[0233] It will readily be appreciated by those of skill in the art,
that any of a variety of polynucleotide sequences are capable of
encoding the transcription factors and transcription factor homolog
polypeptides of the invention. Due to the degeneracy of the genetic
code, many different polynucleotides can encode identical and/or
substantially similar polypeptides in addition to those sequences
illustrated in the Sequence Listing. Nucleic acids having a
sequence that differs from the sequences shown in the Sequence
Listing, or complementary sequences, that encode functionally
equivalent peptides (i.e., peptides having some degree of
equivalent or similar biological activity) but differ in sequence
from the sequence shown in the Sequence Listing due to degeneracy
in the genetic code, are also within the scope of the
invention.
[0234] Altered polynucleotide sequences encoding polypeptides
include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide encoding a polypeptide with at least one functional
characteristic of the instant polypeptides. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding the instant polypeptides, and improper or
unexpected hybridization to allelic variants, with a locus other
than the normal chromosomal locus for the polynucleotide sequence
encoding the instant polypeptides.
[0235] Allelic variant refers to any of two or more alternative
forms of a gene occupying the same chromosomal locus. Allelic
variation arises naturally through mutation, and may result in
phenotypic polymorphism within populations. Gene mutations can be
silent (i.e., no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequence. The term allelic
variant is also used herein to denote a protein encoded by an
allelic variant of a gene. Splice variant refers to alternative
forms of RNA transcribed from a gene. Splice variation arises
naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0236] Those skilled in the art would recognize that, for example,
G2133, SEQ ID NO: 12, represents a single transcription factor;
allelic variation and alternative splicing may be expected to
occur. Allelic variants of SEQ ID NO: 11 can be cloned by probing
cDNA or genomic libraries from different individual organisms
according to standard procedures. Allelic variants of the DNA
sequence shown in SEQ ID NO: 11, including those containing silent
mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of SEQ ID NO: 12. cDNAs
generated from alternatively spliced mRNAs, which retain the
properties of the transcription factor are included within the
scope of the present invention, as are polypeptides encoded by such
cDNAs and mRNAs. Allelic variants and splice variants of these
sequences can be cloned by probing cDNA or genomic libraries from
different individual organisms or tissues according to standard
procedures known in the art (U.S. Pat. No. 6,388,064).
[0237] Thus, in addition to the sequences set forth in the Sequence
Listing, the invention also encompasses related nucleic acid
molecules that include allelic or splice variants of the sequences
of the Sequence Listing, and include sequences that are
complementary to any of the above nucleotide sequences. Related
nucleic acid molecules also include nucleotide sequences encoding a
polypeptide comprising a substitution, modification, addition
and/or deletion of one or more amino acid residues compared to the
polypeptide sequences of the Sequence Listing and equivalogs. Such
related polypeptides may comprise, for example, additions and/or
deletions of one or more N-linked or O-linked glycosylation sites,
or an addition and/or a deletion of one or more cysteine
residues.
[0238] For example, Table 5 illustrates, for example, that the
codons AGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino
acid: serine. Accordingly, at each position in the sequence where
there is a codon encoding serine, any of the above trinucleotide
sequences can be used without altering the encoded polypeptide.
TABLE-US-00006 TABLE 5 Amino acid Possible Codons Alanine Ala A GCA
GCC GCG GCT Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT
Glutamic acid Glu E GAA GAG Phenylalanine Phe F TTC TTT Glycine Gly
G GGA GGC GGG GGT Histidine His H CAC CAT Isoleucine Ile I ATA ATC
ATT Lysine Lys K AAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTT
Methionine Met M ATG Asparagine Asn N AAC AAT Proline Pro P CCA CCC
CCG CCT Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGT Serine Ser S AGC AGT TCA TCC TCG TCT Threonine Thr T ACA ACC
ACG ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGG Tyrosine
Tyr Y TAC TAT
[0239] Sequence alterations that do not change the amino acid
sequence encoded by the polynucleotide are termed "silent"
variations. With the exception of the codons ATG and TGG, encoding
methionine and tryptophan, respectively, any of the possible codons
for the same amino acid can be substituted by a variety of
techniques, for example, site-directed mutagenesis, available in
the art. Accordingly, any and all such variations of a sequence
selected from the above table are a feature of the invention.
[0240] In addition to silent variations, other conservative
variations that alter one, or a few amino acid residues in the
encoded polypeptide, can be made without altering the function of
the polypeptide, these conservative variants are, likewise, a
feature of the invention.
[0241] For example, substitutions, deletions and insertions
introduced into the sequences provided in the Sequence Listing, are
also envisioned by the invention. Such sequence modifications can
be engineered into a sequence by site-directed mutagenesis (Wu
(ed.) Methods Enzymol. (1993) vol. 217, Academic Press) or the
other methods noted below Amino acid substitutions are typically of
single residues; insertions usually will be on the order of about
from 1 to 10 amino acid residues; and deletions will range about
from 1 to 30 residues. In one embodiment, deletions or insertions
are made in adjacent pairs, for example, a deletion of two residues
or insertion of two residues. Substitutions, deletions, insertions
or any combination thereof can be combined to arrive at a sequence.
The mutations that are made in the polynucleotide encoding the
transcription factor should not place the sequence out of reading
frame and should not create complementary regions that could
produce secondary mRNA structure. Preferably, the polypeptide
encoded by the DNA performs the desired function.
[0242] Conservative substitutions are those in which at least one
residue in the amino acid sequence has been removed and a different
residue inserted in its place. Such substitutions generally are
made in accordance with the Table 6 when it is desired to maintain
the activity of the protein. Table 6 shows amino acids which can be
substituted for an amino acid in a protein and which are typically
regarded as conservative substitutions.
TABLE-US-00007 TABLE 6 Residue Substitutions Conservative Ala Ser
Arg Lys Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His
Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe
Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0243] The polypeptides provided in the Sequence Listing have a
novel activity, such as, for example, regulatory activity. Although
all conservative amino acid substitutions (for example, one basic
amino acid substituted for another basic amino acid) in a
polypeptide will not necessarily result in the polypeptide
retaining its activity, it is expected that many of these
conservative mutations would result in the polypeptide retaining
its activity. Most mutations, conservative or non-conservative,
made to a protein but outside of a conserved domain required for
function and protein activity will not affect the activity of the
protein to any great extent.
[0244] Similar substitutions are those in which at least one
residue in the amino acid sequence has been removed and a different
residue inserted in its place. Such substitutions generally are
made in accordance with the Table 7 when it is desired to maintain
the activity of the protein. Table 7 shows amino acids which can be
substituted for an amino acid in a protein and which are typically
regarded as structural and functional substitutions. For example, a
residue in column 1 of Table 7 may be substituted with a residue in
column 2; in addition, a residue in column 2 of Table 7 may be
substituted with the residue of column 1.
TABLE-US-00008 TABLE 7 Residue Similar Substitutions Ala Ser; Thr;
Gly; Val; Leu; Ile Arg Lys; His; Gly Asn Gln; His; Gly; Ser; Thr
Asp Glu, Ser; Thr Gln Asn; Ala Cys Ser; Gly Glu Asp Gly Pro; Arg
His Asn; Gln; Tyr; Phe; Lys; Arg Ile Ala; Leu; Val; Gly; Met Leu
Ala; Ile; Val; Gly; Met Lys Arg; His; Gln; Gly; Pro Met Leu; Ile;
Phe Phe Met; Leu; Tyr; Trp; His; Val; Ala Ser Thr; Gly; Asp; Ala;
Val; Ile; His Thr Ser; Val; Ala; Gly Trp Tyr; Phe; His Tyr Trp;
Phe; His Val Ala; Ile; Leu; Gly; Thr; Ser; Glu
[0245] Substitutions that are less conservative than those in Table
7 can be selected by picking residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in protein properties will be those
in which (a) a hydrophilic residue, for example, seryl or threonyl,
is substituted for (or by) a hydrophobic residue, for example,
leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or
proline is substituted for (or by) any other residue; (c) a residue
having an electropositive side chain, for example, lysyl, arginyl,
or histidyl, is substituted for (or by) an electronegative residue,
for example, glutamyl or aspartyl; or (d) a residue having a bulky
side chain, for example, phenylalanine, is substituted for (or by)
one not having a side chain, for example, glycine.
Further Modifying Sequences of the Invention--Mutation/Forced
Evolution
[0246] In addition to generating silent or conservative
substitutions as noted, above, the present invention optionally
includes methods of modifying the sequences of the Sequence
Listing. In the methods, nucleic acid or protein modification
methods are used to alter the given sequences to produce new
sequences and/or to chemically or enzymatically modify given
sequences to change the properties of the nucleic acids or
proteins.
[0247] Thus, in one embodiment, given nucleic acid sequences are
modified, for example, according to standard mutagenesis or
artificial evolution methods to produce modified sequences. The
modified sequences may be created using purified natural
polynucleotides isolated from any organism or may be synthesized
from purified compositions and chemicals using chemical means well
known to those of skill in the art. For example, Ausubel, supra,
provides additional details on mutagenesis methods. Artificial
forced evolution methods are described, for example, by Stemmer
(1994) Nature 370: 389-391, Stemmer (1994) Proc. Natl. Acad. Sci.
91: 10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and
6,242,568. Methods for engineering synthetic transcription factors
and other polypeptides are described, for example, by Zhang et al.
(2000) J. Biol. Chem. 275: 33850-33860, Liu et al. (2001) J. Biol.
Chem. 276: 11323-11334, and Isalan et al. (2001) Nature Biotechnol.
19: 656-660. Many other mutation and evolution methods are also
available and expected to be within the skill of the
practitioner.
[0248] Similarly, chemical or enzymatic alteration of expressed
nucleic acids and polypeptides can be performed by standard
methods. For example, sequence can be modified by addition of
lipids, sugars, peptides, organic or inorganic compounds, by the
inclusion of modified nucleotides or amino acids, or the like. For
example, protein modification techniques are illustrated in
Ausubel, supra. Further details on chemical and enzymatic
modifications can be found herein. These modification methods can
be used to modify any given sequence, or to modify any sequence
produced by the various mutation and artificial evolution
modification methods noted herein.
[0249] Accordingly, the invention provides for modification of any
given nucleic acid by mutation, evolution, chemical or enzymatic
modification, or other available methods, as well as for the
products produced by practicing such methods, for example, using
the sequences herein as a starting substrate for the various
modification approaches.
[0250] For example, optimized coding sequence containing codons
preferred by a particular prokaryotic or eukaryotic host can be
used for example, to increase the rate of translation or to produce
recombinant RNA transcripts having desirable properties, such as a
longer half-life, as compared with transcripts produced using a
non-optimized sequence. Translation stop codons can also be
modified to reflect host preference. For example, preferred stop
codons for Saccharomyces cerevisiae and mammals are TAA and TGA,
respectively. The preferred stop codon for monocotyledonous plants
is TGA, whereas insects and E. coli prefer to use TAA as the stop
codon.
[0251] The polynucleotide sequences of the present invention can
also be engineered in order to alter a coding sequence for a
variety of reasons, including but not limited to, alterations which
modify the sequence to facilitate cloning, processing and/or
expression of the gene product. For example, alterations are
optionally introduced using techniques which are well known in the
art, for example, site-directed mutagenesis, to insert new
restriction sites, to alter glycosylation patterns, to change codon
preference, to introduce splice sites, etc.
[0252] Furthermore, a fragment or domain derived from any of the
polypeptides of the invention can be combined with domains derived
from other transcription factors or synthetic domains to modify the
biological activity of a transcription factor. For instance, a
DNA-binding domain derived from a transcription factor of the
invention can be combined with the activation domain of another
transcription factor or with a synthetic activation domain. A
transcription activation domain assists in initiating transcription
from a DNA-binding site. Examples include the transcription
activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl.
Acad. Sci. 95: 376-381; Aoyama et al. (1995) Plant Cell 7:
1773-1785), peptides derived from bacterial sequences (Ma and
Ptashne (1987) Cell 51: 113-119) and synthetic peptides (Giniger
and Ptashne (1987) Nature 330: 670-672).
Expression and Modification of Polypeptides
[0253] Typically, polynucleotide sequences of the invention are
incorporated into recombinant DNA (or RNA) molecules that direct
expression of polypeptides of the invention in appropriate host
cells, transgenic plants, in vitro translation systems, or the
like. Due to the inherent degeneracy of the genetic code, nucleic
acid sequences which encode substantially the same or a
functionally equivalent amino acid sequence can be substituted for
any listed sequence to provide for cloning and expressing the
relevant homolog.
[0254] The transgenic plants of the present invention comprising
recombinant polynucleotide sequences are generally derived from
parental plants, which may themselves be non-transformed (or
non-transgenic) plants. These transgenic plants may either have a
transcription factor gene "knocked out" (for example, with a
genomic insertion by homologous recombination, an antisense or
ribozyme construct) or expressed to a normal or wild-type extent.
However, overexpressing transgenic "progeny" plants will exhibit
greater mRNA levels, wherein the mRNA encodes a transcription
factor, that is, a DNA-binding protein that is capable of binding
to a DNA regulatory sequence and inducing transcription, and
preferably, expression of a plant trait gene. Preferably, the mRNA
expression level will be at least three-fold greater than that of
the parental plant, or more preferably at least ten-fold greater
mRNA levels compared to said parental plant, and most preferably at
least fifty-fold greater compared to said parental plant.
Vectors, Promoters, and Expression Systems
[0255] The present invention includes recombinant constructs
comprising one or more of the nucleic acid sequences herein. The
constructs typically comprise a vector, such as a plasmid, a
cosmid, a phage, a virus (for example, a plant virus), a bacterial
artificial chromosome (BAC), a yeast artificial chromosome (YAC),
or the like, into which a nucleic acid sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available.
[0256] General texts that describe molecular biological techniques
useful herein, including the use and production of vectors,
promoters and many other relevant topics, include Berger, Sambrook,
supra and Ausubel, supra. Any of the identified sequences can be
incorporated into a cassette or vector, for example, for expression
in plants. A number of expression vectors suitable for stable
transformation of plant cells or for the establishment of
transgenic plants have been described including those described in
Weissbach and Weissbach (1989) Methods for Plant Molecular Biology,
Academic Press, and Gelvin et al. (1990) Plant Molecular Biology
Manual, Kluwer Academic Publishers. Specific examples include those
derived from a Ti plasmid of Agrobacterium tumefaciens, as well as
those disclosed by Herrera-Estrella et al. (1983) Nature 303: 209,
Bevan (1984) Nucleic Acids Res. 12: 8711-8721, Klee (1985)
Bio/Technology 3: 637-642, for dicotyledonous plants.
[0257] Alternatively, non-Ti vectors can be used to transfer the
DNA into monocotyledonous plants and cells by using free DNA
delivery techniques. Such methods can involve, for example, the use
of liposomes, electroporation, microprojectile bombardment, silicon
carbide whiskers, and viruses. By using these methods transgenic
plants such as wheat, rice (Christou (1991) Bio/Technology 9:
957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be
produced. An immature embryo can also be a good target tissue for
monocots for direct DNA delivery techniques by using the particle
gun (Weeks et al. (1993) Plant Physiol. 102: 1077-1084; Vasil
(1993) Bio/Technology 10: 667-674; Wan and Lemeaux (1994) Plant
Physiol. 104: 37-48, and for Agrobacterium-mediated DNA transfer
(Ishida et al. (1996) Nature Biotechnol. 14: 745-750).
[0258] Typically, plant transformation vectors include one or more
cloned plant coding sequence (genomic or cDNA) under the
transcriptional control of 5' and 3' regulatory sequences and a
dominant selectable marker. Such plant transformation vectors
typically also contain a promoter (for example, a regulatory region
controlling inducible or constitutive, environmentally- or
developmentally-regulated, or cell- or tissue-specific expression),
a transcription initiation start site, an RNA processing signal
(such as intron splice sites), a transcription termination site,
and/or a polyadenylation signal.
[0259] A potential utility for the transcription factor
polynucleotides disclosed herein is the isolation of promoter
elements from these genes that can be used to program expression in
plants of any genes. Each transcription factor gene disclosed
herein is expressed in a unique fashion, as determined by promoter
elements located upstream of the start of translation, and
additionally within an intron of the transcription factor gene or
downstream of the termination codon of the gene. As is well known
in the art, for a significant portion of genes, the promoter
sequences are located entirely in the region directly upstream of
the start of translation. In such cases, typically the promoter
sequences are located within 2.0 kb of the start of translation, or
within 1.5 kb of the start of translation, frequently within 1.0 kb
of the start of translation, and sometimes within 0.5 kb of the
start of translation.
[0260] The promoter sequences can be isolated according to methods
known to one skilled in the art.
[0261] Examples of constitutive plant promoters which can be useful
for expressing the TF sequence include: the cauliflower mosaic
virus (CaMV) 35S promoter, which confers constitutive, high-level
expression in most plant tissues (for example, in Odell et al.
(1985) Nature 313: 810-812); the nopaline synthase promoter (An et
al. (1988) Plant Physiol. 88: 547-552); and the octopine synthase
promoter (Fromm et al. (1989) Plant Cell 1: 977-984).
[0262] The transcription factors of the invention may be operably
linked with a specific promoter that causes the transcription
factor to be expressed in response to environmental,
tissue-specific or temporal signals. A variety of plant gene
promoters that regulate gene expression in response to
environmental, hormonal, chemical, developmental signals, and in a
tissue-active manner can be used for expression of a TF sequence in
plants. Choice of a promoter is based largely on the phenotype of
interest and is determined by such factors as tissue (for example,
seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.),
inducibility (for example, in response to wounding, heat, cold,
drought, light, pathogens, etc.), timing, developmental stage, and
the like. Numerous known promoters have been characterized and can
favorably be employed to promote expression of a polynucleotide of
the invention in a transgenic plant or cell of interest. For
example, tissue-specific promoters include: seed-specific promoters
(such as the napin, phaseolin or DC3 promoter described in U.S.
Pat. No. 5,773,697), fruit-specific promoters that are active
during fruit ripening (such as the dru 1 promoter (U.S. Pat. No.
5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the
tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol.
Biol. 11: 651-662), root-specific promoters, such as those
disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186,
pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat.
No. 5,792,929), promoters active in vascular tissue (Ringli and
Keller (1998) Plant Mol. Biol. 37: 977-988), flower-specific
(Kaiser et al. (1995) Plant Mol. Biol. 28: 231-243), pollen
(Baerson et al. (1994) Plant Mol. Biol. 26: 1947-1959), carpels
(Ohl et al. (1990) Plant Cell 2: 837-848), pollen and ovules
(Baerson et al. (1993) Plant Mol. Biol. 22: 255-267),
auxin-inducible promoters (such as that described in van der Kop et
al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al., (1999)
Plant Cell 11: 323-334), cytokinin-inducible promoter
(Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promoters
responsive to gibberellin (Shi et al. (1998) Plant Mol. Biol. 38:
1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825)
and the like. Additional promoters are those that elicit expression
in response to heat (Ainley et al. (1993) Plant Mol. Biol. 22:
13-23), light (for example, the pea rbcS-3A promoter, Kuhlemeier et
al. (1989) Plant Cell 1: 471-478), and the maize rbcS promoter,
Schaffner and Sheen (1991) Plant Cell 3: 997-1012); wounding (for
example, wunI, Siebertz et al. (1989) Plant Cell 1: 961-968);
pathogens (such as the PR-1 promoter described in Buchel et al.
(1999) Plant Mol. Biol. 40: 387-396, and the PDF1.2 promoter
described in Manners et al. (1998) Plant Mol. Biol. 38: 1071-1080),
and chemicals such as methyl jasmonate or salicylic acid (Gatz
(1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 89-108). In
addition, the timing of the expression can be controlled by using
promoters such as those acting at senescence (Gan and Amasino
(1995) Science 270: 1986-1988); or late seed development (Odell et
al. (1994) Plant Physiol. 106: 447-458).
[0263] Plant expression vectors can also include RNA processing
signals that can be positioned within, upstream or downstream of
the coding sequence. In addition, the expression vectors can
include additional regulatory sequences from the 3'-untranslated
region of plant genes, for example, a 3' terminator region to
increase mRNA stability of the mRNA, such as the PI-II terminator
region of potato or the octopine or nopaline synthase 3' terminator
regions.
Additional Expression Elements
[0264] Specific initiation signals can aid in efficient translation
of coding sequences. These signals can include, for example, the
ATG initiation codon and adjacent sequences. No additional
translational control signals may be needed where a coding
sequence, its initiation codon and upstream sequences are inserted
into the appropriate expression vector. However, in cases where
only coding sequence (for example, a mature protein coding
sequence) or a portion thereof is inserted, exogenous
transcriptional control signals including the ATG initiation codon
can be separately provided. The initiation codon is provided in the
correct reading frame to facilitate transcription. Exogenous
transcriptional elements and initiation codons can be of various
origins, both natural and synthetic. The efficiency of expression
can be enhanced by the inclusion of enhancers appropriate to the
cell system in use.
Expression Hosts
[0265] The present invention also relates to host cells which are
transduced with vectors of the invention, and the production of
polypeptides of the invention (including fragments thereof) by
recombinant techniques. Host cells are genetically engineered
(i.e., nucleic acids are introduced, for example, transduced,
transformed or transfected) with the vectors of this invention,
which may be, for example, a cloning vector or an expression vector
comprising the relevant nucleic acids herein. The vector is
optionally a plasmid, a viral particle, a phage, a naked nucleic
acid, etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants, or amplifying the relevant
gene. The culture conditions, such as temperature, pH and the like,
are those previously used with the host cell selected for
expression, and will be apparent to those skilled in the art and in
the references cited herein, including, Sambrook, supra and
Ausubel, supra.
[0266] The host cell can be a eukaryotic cell, such as a yeast
cell, or a plant cell, or the host cell can be a prokaryotic cell,
such as a bacterial cell. Plant protoplasts are also suitable for
some applications. For example, the DNA fragments are introduced
into plant tissues, cultured plant cells or plant protoplasts by
standard methods including electroporation (Fromm et al. (1985)
Proc. Natl. Acad. Sci. 82: 5824-5828), infection by viral vectors
such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982)
Molecular Biology of Plant Tumors Academic Press, New York, N.Y.,
pp. 549-560; U.S. Pat. No. 4,407,956), high velocity ballistic
penetration by small particles with the nucleic acid either within
the matrix of small beads or particles, or on the surface (Klein et
al. (1987) Nature 327: 70-73), use of pollen as vector (WO
85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes
carrying a T-DNA plasmid in which DNA fragments are cloned. The
T-DNA plasmid is transmitted to plant cells upon infection by
Agrobacterium tumefaciens, and a portion is stably integrated into
the plant genome (Horsch et al. (1984) Science 233: 496-498; Fraley
et al. (1983) Proc. Natl. Acad. Sci. 80: 4803-4807).
[0267] The cell can include a nucleic acid of the invention that
encodes a polypeptide, wherein the cell expresses a polypeptide of
the invention. The cell can also include vector sequences, or the
like. Furthermore, cells and transgenic plants that include any
polypeptide or nucleic acid above or throughout this specification,
for example, produced by transduction of a vector of the invention,
are an additional feature of the invention.
[0268] For long-term, high-yield production of recombinant
proteins, stable expression can be used. Host cells transformed
with a nucleotide sequence encoding a polypeptide of the invention
are optionally cultured under conditions suitable for the
expression and recovery of the encoded protein from cell culture.
The protein or fragment thereof produced by a recombinant cell may
be secreted, membrane-bound, or contained intracellularly,
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides encoding mature proteins of the
invention can be designed with signal sequences which direct
secretion of the mature polypeptides through a prokaryotic or
eukaryotic cell membrane.
Modified Amino Acid Residues
[0269] Polypeptides of the invention may contain one or more
modified amino acid residues. The presence of modified amino acids
may be advantageous in, for example, increasing polypeptide
half-life, reducing polypeptide antigenicity or toxicity,
increasing polypeptide storage stability, or the like. Amino acid
residue(s) are modified, for example, co-translationally or
post-translationally during recombinant production or modified by
synthetic or chemical means.
[0270] Non-limiting examples of a modified amino acid residue
include incorporation or other use of acetylated amino acids,
glycosylated amino acids, sulfated amino acids, prenylated (for
example, farnesylated, geranylgeranylated) amino acids, PEG
modified (for example, "PEGylated") amino acids, biotinylated amino
acids, carboxylated amino acids, phosphorylated amino acids, etc.
References adequate to guide one of skill in the modification of
amino acid residues are replete throughout the literature.
[0271] The modified amino acid residues may prevent or increase
affinity of the polypeptide for another molecule, including, but
not limited to, polynucleotide, proteins, carbohydrates, lipids and
lipid derivatives, and other organic or synthetic compounds.
Identification of Additional Protein Factors
[0272] A transcription factor provided by the present invention can
also be used to identify additional endogenous or exogenous
molecules that can affect a phenotype or trait of interest. Such
molecules include endogenous molecules that are acted upon either
at a transcriptional level by a transcription factor of the
invention to modify a phenotype as desired. For example, the
transcription factors can be employed to identify one or more
downstream genes that are subject to a regulatory effect of the
transcription factor. In one approach, a transcription factor or
transcription factor homolog of the invention is expressed in a
host cell, for example, a transgenic plant cell, tissue or explant,
and expression products, either RNA or protein, of likely or random
targets are monitored, for example, by hybridization to a
microarray of nucleic acid probes corresponding to genes expressed
in a tissue or cell type of interest, by two-dimensional gel
electrophoresis of protein products, or by any other method known
in the art for assessing expression of gene products at the level
of RNA or protein. Alternatively, a transcription factor of the
invention can be used to identify promoter sequences (such as
binding sites on DNA sequences) involved in the regulation of a
downstream target. After identifying a promoter sequence,
interactions between the transcription factor and the promoter
sequence can be modified by changing specific nucleotides in the
promoter sequence or specific amino acids in the transcription
factor that interact with the promoter sequence to alter a plant
trait. Typically, transcription factor DNA-binding sites are
identified by gel shift assays. After identifying the promoter
regions, the promoter region sequences can be employed in
double-stranded DNA arrays to identify molecules that affect the
interactions of the transcription factors with their promoters
(Bulyk et al. (1999) Nature Biotechnol. 17: 573-577).
[0273] The identified transcription factors are also useful to
identify proteins that modify the activity of the transcription
factor. Such modification can occur by covalent modification, such
as by phosphorylation, or by protein-protein (homo or
-heteropolymer) interactions. Any method suitable for detecting
protein-protein interactions can be employed. Among the methods
that can be employed are co-immunoprecipitation, cross-linking and
co-purification through gradients or chromatographic columns, and
the two-hybrid yeast system.
[0274] The two-hybrid system detects protein interactions in vivo
and is described in Chien et al. ((1991) Proc. Natl. Acad. Sci. 88:
9578-9582) and is commercially available from Clontech (Palo Alto,
Calif.). In such a system, plasmids are constructed that encode two
hybrid proteins: one consists of the DNA-binding domain of a
transcription activator protein fused to the TF polypeptide and the
other consists of the transcription activator protein's activation
domain fused to an unknown protein that is encoded by a cDNA that
has been recombined into the plasmid as part of a cDNA library. The
DNA-binding domain fusion plasmid and the cDNA library are
transformed into a strain of the yeast Saccharomyces cerevisiae
that contains a reporter gene (for example, lacZ) whose regulatory
region contains the transcription activator's binding site. Either
hybrid protein alone cannot activate transcription of the reporter
gene. Interaction of the two hybrid proteins reconstitutes the
functional activator protein and results in expression of the
reporter gene, which is detected by an assay for the reporter gene
product. Then, the library plasmids responsible for reporter gene
expression are isolated and sequenced to identify the proteins
encoded by the library plasmids. After identifying proteins that
interact with the transcription factors, assays for compounds that
interfere with the TF protein-protein interactions can be
performed.
Subsequences
[0275] Also contemplated are uses of polynucleotides, also referred
to herein as oligonucleotides, typically having at least 12 or more
bases that h hybridize under stringent or highly stringent
conditions to a polynucleotide sequence described above. The
polynucleotides may be used as probes, primers, sense and antisense
agents, and the like, according to methods as noted above.
[0276] Subsequences of the polynucleotides of the invention,
including polynucleotide fragments and oligonucleotides are useful
as nucleic acid probes and primers. An oligonucleotide suitable for
use as a probe or primer is at least about 15 nucleotides in
length, more often at least about 18 nucleotides, often at least
about 21 nucleotides, frequently at least about 30 nucleotides, or
about 40 nucleotides, or more in length. A nucleic acid probe is
useful in hybridization protocols, for example, to identify
additional polypeptide homologs of the invention, including
protocols for microarray experiments. Primers can be annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, and
then extended along the target DNA strand by a DNA polymerase
enzyme. Primer pairs can be used for amplification of a nucleic
acid sequence, for example, by the polymerase chain reaction (PCR)
or other nucleic-acid amplification methods (Sambrook, supra, and
Ausubel, supra).
[0277] In addition, the invention includes an isolated or
recombinant polypeptide including a subsequence of at least about
15 contiguous amino acids encoded by the recombinant or isolated
polynucleotides of the invention. For example, such polypeptides,
or domains or fragments thereof, can be used as immunogens, for
example, to produce antibodies specific for the polypeptide
sequence, or as probes for detecting a sequence of interest. A
subsequence can range in size from about 15 amino acids in length
up to and including the full length of the polypeptide.
[0278] To be encompassed by the present invention, an expressed
polypeptide which comprises such a polypeptide subsequence performs
at least one biological function of the intact polypeptide in
substantially the same manner, or to a similar extent, as does the
intact polypeptide. For example, a polypeptide fragment can
comprise a recognizable structural motif or functional domain such
as a DNA binding domain that activates transcription, for example,
by binding to a specific DNA promoter region an activation domain,
or a domain for protein-protein interactions.
[0279] Production of Transgenic Plants and Modification of
Traits.
[0280] The polynucleotides of the invention are favorably employed
to produce transgenic plants with various traits or characteristics
that have been modified in a desirable manner, for example, to
improve the seed characteristics of a plant. For example,
alteration of expression levels or patterns (for example, spatial
or temporal expression patterns) of one or more of the
transcription factors (or transcription factor homologs) of the
invention, as compared with the levels of the same protein found in
a wild-type plant, can be used to modify a plant's traits. An
illustrative example of trait modification, improved
characteristics, by altering expression levels of a particular
transcription factor is described further in the Examples and the
Sequence Listing.
[0281] Arabidopsis as a Model System.
[0282] Arabidopsis thaliana is the object of rapidly growing
attention as a model for genetics and metabolism in plants.
Arabidopsis has a small genome, and well-documented studies are
available. It is easy to grow in large numbers and mutants defining
important genetically controlled mechanisms are either available,
or can readily be obtained. Various methods to introduce and
express isolated homologous genes are available (Koncz et al.,
eds., Methods in Arabidopsis Research (1992) World Scientific, New
Jersey, N.J., in "Preface"). Because of its small size, short life
cycle, obligate autogamy and high fertility, Arabidopsis is also a
choice organism for the isolation of mutants and studies in
morphogenetic and development pathways, and control of these
pathways by transcription factors (Koncz (1992) supra, p. 72). A
number of studies introducing transcription factors into A.
thaliana have demonstrated the utility of this plant for
understanding the mechanisms of gene regulation and trait
alteration in plants (for example, in Koncz (1992) supra, and in
U.S. Pat. No. 6,417,428).
[0283] Arabidopsis Genes in Transgenic Plants.
[0284] Expression of genes which encode transcription factors
modify expression of endogenous genes, polynucleotides, and
proteins are well known in the art. In addition, transgenic plants
comprising isolated polynucleotides encoding transcription factors
may also modify expression of endogenous genes, polynucleotides,
and proteins. Examples include Peng et al. (1997) Genes and
Development 11: 3194-3205 and Peng et al. (1999) Nature 400:
256-261. In addition, many others have demonstrated that an
Arabidopsis transcription factor expressed in an exogenous plant
species elicits the same or very similar phenotypic response (Fu et
al. (2001) Plant Cell 13: 1791-1802; Nandi et al. (2000) Curr.
Biol. 10: 215-218; Coupland (1995) Nature 377: 482-483; and Weigel
and Nilsson (1995) Nature 377: 482-500.
[0285] Homologous Genes Introduced into Transgenic Plants.
[0286] Homologous genes that may be derived from any plant, or from
any source whether natural, synthetic, semi-synthetic or
recombinant, and that share significant sequence identity or
similarity to those provided by the present invention, may be
introduced into plants, for example, crop plants, to confer
desirable or improved traits. Consequently, transgenic plants may
be produced that comprise a recombinant expression vector or
cassette with a promoter operably linked to one or more sequences
homologous to presently disclosed sequences. The promoter may be,
for example, a plant or viral promoter.
[0287] The invention thus provides for methods for preparing
transgenic plants, and for modifying plant traits. These methods
include introducing into a plant a recombinant expression vector or
cassette comprising a functional promoter operably linked to one or
more sequences homologous to presently disclosed sequences. Plants
and kits for producing these plants that result from the
application of these methods are also encompassed by the present
invention.
[0288] Transcription Factors of Interest for the Modification of
Plant Traits.
[0289] Currently, the existence of a series of maturity groups for
different latitudes represents a major barrier to the introduction
of new valuable traits. Any trait (for example disease resistance)
has to be bred into each of the different maturity groups
separately, a laborious and costly exercise. The availability of
single strain, which could be grown at any latitude, would
therefore greatly increase the potential for introducing new traits
to crop species such as soybean and cotton.
[0290] For the specific effects, traits and utilities conferred to
plants, one or more transcription factor genes of the present
invention may be used to increase or decrease, or improve or prove
deleterious to a given trait. For example, knocking out a
transcription factor gene that naturally occurs in a plant, or
suppressing the gene (with, for example, antisense suppression),
may cause decreased tolerance to shade or a drought stress relative
to non-transformed or wild-type plants. By overexpressing this
gene, the plant may experience increased tolerance to the same
stress. More than one transcription factor gene may be introduced
into a plant, either by transforming the plant with one or more
vectors comprising two or more transcription factors, or by
selective breeding of plants to yield hybrid crosses that comprise
more than one introduced transcription factor.
[0291] Genes, Traits and Utilities that Affect Plant
Characteristics.
[0292] Plant transcription factors can modulate gene expression,
and, in turn, be modulated by the environmental experience of a
plant. Significant alterations in a plant's environment invariably
result in a change in the plant's transcription factor gene
expression pattern. Altered transcription factor expression
patterns generally result in phenotypic changes in the plant.
Transcription factor gene product(s) in transgenic plants then
differ(s) in amounts or proportions from that found in wild-type or
non-transformed plants, and those transcription factors likely
represent polypeptides that are used to alter the response to the
environmental change. By way of example, it is well accepted in the
art that analytical methods based on altered expression patterns
may be used to screen for phenotypic changes in a plant far more
effectively than can be achieved using traditional methods.
[0293] Potential Applications of Presently Disclosed Sequences that
Regulate Abiotic Stress Tolerance Sugar Sensing.
[0294] In addition to their important role as an energy source and
structural component of the plant cell, sugars are central
regulatory molecules that control several aspects of plant
physiology, metabolism and development (Hsieh et al. (1998) Proc.
Natl. Acad. Sci. 95: 13965-13970). It is thought that this control
is achieved by regulating gene expression and, in higher plants,
sugars have been shown to repress or activate plant genes involved
in many essential processes such as photosynthesis, glyoxylate
metabolism, respiration, starch and sucrose synthesis and
degradation, pathogen response, wounding response, cell cycle
regulation, pigmentation, flowering and senescence. The mechanisms
by which sugars control gene expression are not understood.
[0295] Several sugar sensing mutants have turned out to be allelic
to ABA and ethylene mutants. ABA is found in all photosynthetic
organisms and acts as a key regulator of transpiration, stress
responses, embryogenesis, and seed germination. Most ABA effects
are related to the compound acting as a signal of decreased water
availability, whereby it triggers a reduction in water loss, slows
growth, and mediates adaptive responses. However, ABA also
influences plant growth and development via interactions with other
phytohormones. Physiological and molecular studies indicate that
maize and Arabidopsis have almost identical pathways with regard to
ABA biosynthesis and signal transduction (for example, in
Finkelstein and Rock (2002) "Abscisic acid biosynthesis and
response", in The Arabidopsis Book, Somerville and Meyerowitz,
editors (American Society of Plant Biologists, Rockville, Md.).
[0296] This potentially implicates the sequences of the invention
that, when overexpressed, confer a sugar sensing or hormone
signaling phenotype in plants. On the other hand, the sucrose
treatment used in these experiments (9.4% w/v) could also be an
osmotic stress. Therefore, one could interpret these data as an
indication that these transgenic lines are more tolerant to osmotic
stress. However, it is well known that plant responses to ABA,
osmotic and other stress may be linked, and these different
treatments may even act in a synergistic manner to increase the
degree of a response. For example, Xiong, Ishitani, and Zhu ((1999)
Plant Physiol. 119: 205-212) have shown that genetic and molecular
studies may be used to show extensive interaction between osmotic
stress, temperature stress, and ABA responses in plants. These
investigators analyzed the expression of RD29A-LUC in response to
various treatment regimes in Arabidopsis. The RD29A promoter
contains both the ABA-responsive and the dehydration-responsive
element--also termed the C-repeat--and can be activated by osmotic
stress, low temperature, or ABA treatment; transcription of the
RD29A gene in response to osmotic and cold stresses is mediated by
both ABA-dependent and ABA-independent pathways (Xiong, Ishitani,
and Zhu (1999) supra). LUC refers to the firefly luciferase coding
sequence, which, in this case, was driven by the stress responsive
RD29A promoter. The results revealed both positive and negative
interactions, depending on the nature and duration of the
treatments. Low temperature stress was found to impair osmotic
signaling but moderate heat stress strongly enhanced osmotic stress
induction, thus acting synergistically with osmotic signaling
pathways. In this study, the authors reported that osmotic stress
and ABA can act synergistically by showing that the treatments
simultaneously induced transgene and endogenous gene expression.
Similar results were reported by Bostock and Quatrano ((1992) Plant
Physiol. 98: 1356-1363), who found that osmotic stress and ABA act
synergistically and induce maize Em gene expression. Ishitani et al
(1997) Plant Cell 9: 1935-1949) isolated a group of Arabidopsis
single-gene mutations that confer enhanced responses to both
osmotic stress and ABA. The nature of the recovery of these mutants
from osmotic stress and ABA treatment suggested that although
separate signaling pathways exist for osmotic stress and ABA, the
pathways share a number of components; these common components may
mediate synergistic interactions between osmotic stress and ABA.
Thus, contrary to the previously-held belief that ABA-dependent and
ABA-independent stress signaling pathways act in a parallel manner,
our data reveal that these pathways cross-talk and converge to
activate stress gene expression.
[0297] Because sugars are important signaling molecules, the
ability to control either the concentration of a signaling sugar or
how the plant perceives or responds to a signaling sugar could be
used to control plant development, physiology or metabolism. For
example, the flux of sucrose (a disaccharide sugar used for
systemically transporting carbon and energy in most plants) has
been shown to affect gene expression and alter storage compound
accumulation in seeds. Manipulation of the sucrose signaling
pathway in seeds may therefore cause seeds to have more protein,
oil or carbohydrate, depending on the type of manipulation.
Similarly, in tubers, sucrose is converted to starch which is used
as an energy store. It is thought that sugar signaling pathways may
partially determine the levels of starch synthesized in the tubers.
The manipulation of sugar signaling in tubers could lead to tubers
with a higher starch content.
[0298] Thus, altering the expression of the presently disclosed
transcription factor genes that manipulate the sugar signal
transduction pathway, including, for example, G175, G303, G354,
G481, G916, G922, G1069, G1073, G1820, G2053, G2701, G2789, G2839,
G2854, along with their equivalogs, or that exhibit an osmotic
stress phenotype, including, for example, G47, G482, G489 or G1069,
G1073, as evidenced by their tolerance to, for example, high
mannitol, salt or PEG, may be used to produce plants with desirable
traits, including increased drought tolerance. In particular,
manipulation of sugar signal transduction pathways could be used to
alter source-sink relationships in seeds, tubers, roots and other
storage organs leading to increase in yield.
[0299] Abiotic Stress: Drought and Low Humidity Tolerance.
[0300] Exposure to dehydration invokes similar survival strategies
in plants as does freezing stress (for example, in Yelenosky (1989)
Plant Physiol 89: 444-451) and drought stress induces freezing
tolerance (for example, in Siminovitch et al. (1982) Plant Physiol
69: 250-255; and Guy et al. (1992) Planta 188: 265-270). In
addition to the induction of cold-acclimation proteins, strategies
that allow plants to survive in low water conditions may include,
for example, reduced surface area, or surface oil or wax
production. Modifying the expression of the presently disclosed
transcription factor genes, including G2133, G1274, G922, G2999,
G3086, G354, G1792, G2053, G975, G1069, G916, G1820, G2701, G47,
G2854, G2789, G634, G175, G2839, G1452, G3083, G489, G303, G2992,
and G682, and their equivalogs, may be used to increase a plant's
tolerance to low water conditions and provide the benefits of
improved survival, increased yield and an extended geographic and
temporal planting range.
[0301] Osmotic Stress.
[0302] Modification of the expression of a number of presently
disclosed transcription factor genes, for example, G47, G482, G489
or G1069, G2053 and their equivalogs, may be used to increase
germination rate or growth under adverse osmotic conditions, which
could impact survival and yield of seeds and plants. Osmotic
stresses may be regulated by specific molecular control mechanisms
that include genes controlling water and ion movements, functional
and structural stress-induced proteins, signal perception and
transduction, and free radical scavenging, and many others (Wang et
al. (2001) Acta Hort. (ISHS) 560: 285-292). Instigators of osmotic
stress include freezing, drought and high salinity, each of which
are discussed in more detail below.
[0303] In many ways, freezing, high salt and drought have similar
effects on plants, not the least of which is the induction of
common polypeptides that respond to these different stresses. For
example, freezing is similar to water deficit in that freezing
reduces the amount of water available to a plant. Exposure to
freezing temperatures may lead to cellular dehydration as water
leaves cells and forms ice crystals in intercellular spaces
(Buchanan et al. (2000) in Biochemistry and Molecular Biology of
Plants, American Society of Plant Physiologists, Rockville, Md.).
As with high salt concentration and freezing, the problems for
plants caused by low water availability include mechanical stresses
caused by the withdrawal of cellular water. Thus, the incorporation
of transcription factors that modify a plant's response to osmotic
stress into, for example, a crop or ornamental plant, may be useful
in reducing damage or loss. Specific effects caused by freezing,
high salt and drought are addressed below.
[0304] The Relationship Between Salt, Drought and Freezing
Tolerance.
[0305] Plants are subject to a range of environmental challenges.
Several of these, including drought stress, have the ability to
impact whole plant and cellular water availability. Not
surprisingly, then, plant responses to this collection of stresses
are related. In a recent review, Zhu notes that "most studies on
water stress signaling have focused on salt stress primarily
because plant responses to salt and drought are closely related and
the mechanisms overlap" (Zhu (2002) Ann. Rev. Plant Biol. 53:
247-273). Many examples of similar responses and pathways to this
set of stresses have been documented. For example, the CBF
transcription factors have been shown to condition resistance to
salt, freezing and drought (Kasuga et al. (1999) Nature Biotech.
17: 287-291). The Arabidopsis rd29B gene is induced in response to
both salt and dehydration stress, a process that is mediated
largely through an ABA signal transduction process (Uno et al.
(2000) Proc. Natl. Acad. Sci. USA 97: 11632-11637), resulting in
altered activity of transcription factors that bind to an upstream
element within the rd29B promoter. In Mesembryanthemum crystallinum
(ice plant), Patharker and Cushman have shown that a
calcium-dependent protein kinase (McCDPK1) is induced by exposure
to both drought and salt stresses (Patharker and Cushman (2000)
Plant J. 24: 679-691). The stress-induced kinase was also shown to
phosphorylate a transcription factor, presumably altering its
activity, although transcript levels of the target transcription
factor are not altered in response to salt or drought stress.
Similarly, Saijo et al. demonstrated that a rice
salt/drought-induced calmodulin-dependent protein kinase (OsCDPK7)
conferred increased salt and drought tolerance to rice when
overexpressed (Saijo et al. (2000) Plant J. 23: 319-327).
[0306] Exposure to dehydration invokes similar survival strategies
in plants as does freezing stress (for example, in Yelenosky (1989)
Plant Physiol 89: 444-451) and drought stress induces freezing
tolerance (for example, in Siminovitch et al. (1982) Plant Physiol
69: 250-255; and Guy et al. (1992) Planta 188: 265-270). In
addition to the induction of cold-acclimation proteins, strategies
that allow plants to survive in low water conditions may include,
for example, reduced surface area, or surface oil or wax
production.
[0307] Consequently, one skilled in the art would expect that some
pathways involved in resistance to one of these stresses, and hence
regulated by an individual transcription factor, will also be
involved in resistance to another of these stresses, regulated by
the same or homologous transcription factors. Of course, the
overall resistance pathways are related, not identical, and
therefore not all transcription factors controlling resistance to
one stress will control resistance to the other stresses.
Nonetheless, if a transcription factor conditions resistance to one
of these stresses, it would be apparent to one skilled in the art
to test for resistance to these related stresses.
[0308] Thus, the genes of the sequence listing, including, for
example, G175, G922, G1452, G1820, G2701, G2999, G3086 and their
equivalogs that provide tolerance to salt may be used to engineer
salt tolerant crops and trees that can flourish in soils with high
saline content or under drought conditions. In particular,
increased salt tolerance during the germination stage of a plant
enhances survival and yield. Presently disclosed transcription
factor genes that provide increased salt tolerance during
germination, the seedling stage, and throughout a plant's life
cycle, would find particular value for imparting survival and yield
in areas where a particular crop would not normally prosper.
[0309] Summary of Altered Drought-Related Plant
Characteristics.
[0310] The clades of structurally and functionally related
sequences that derive from a wide range of plants, including
polynucleotides of the Sequence Listing and their encoded
polypeptides, fragments thereof, paralogs, orthologs, equivalogs,
and fragments thereof, is provided. These sequences have been shown
in laboratory and field experiments to confer altered size and
abiotic stress tolerance phenotypes in plants. The invention also
provides the polypeptides of the Sequence Listing, and fragments
thereof, conserved domains thereof, paralogs, orthologs,
equivalogs, and fragments thereof. Plants that overexpress these
sequences have been observed to exhibit a sugar sensing phenotype
and/or be more tolerant to a wide variety of abiotic stresses,
including drought and high salt stress. Many of the orthologs of
these sequences are listed in the Sequence Listing, and due to the
high degree of structural similarity to the sequences of the
invention, it is expected that these sequences will also function
to increase drought stress tolerance. The invention also
encompasses the complements of the polynucleotides. The
polynucleotides are useful for screening libraries of molecules or
compounds for specific binding and for creating transgenic plants
having increased drought stress tolerance.
[0311] Potential Applications of Polynucleotides and Polypeptides
that Regulate C/N Sensing.
[0312] The genes identified by the experiments detailed in this
report represent potential regulators of plant responses to low
nutrient conditions. As such, these genes (or their putative
orthologs and paralogs) could be applied to commercial species in
order to improve yield, improve performance under conditions of
nutrient limitation, and substantially reduce the necessity for
fertilizer application.
[0313] The data of Lam et al. (Lam (2003) Plant Physiol. 132:
926-935) suggest that quantitative changes in seed nitrogen
reserves may require enhanced transportation of nitrogen resources.
These data further suggest that the C/N sensing screen detailed in
the below Examples can provide leads which, based on low
anthocyanin accumulation, could be used to create transgenic plants
with enhanced seed nitrogen reserves.
[0314] The experiments performed with specific sequences and
transgenic plants, described in Example IX (below), also identified
genes which produced elevated levels of anthocyanin, relative to
controls, when lines were tested in the C/N assay. In a number of
instances, such an effect was not alleviated by the provision of an
organic nitrogen source such as glutamine, suggesting that the
genes were producing a non-specific increase in anthocyanin levels.
Although such results might not be related to nutrient limitation
they likely reveal genes that have important roles in the
production of or accumulation of secondary metabolites related to
the phenylpropanoid pathway. A variety of applications can be
envisaged for such regulatory genes. Uses include altering pigment
production for horticultural purposes and increasing stress
resistance. For example, flavonoids have antimicrobial activity and
could be used to engineer pathogen resistance. In addition, several
flavonoid compounds have health promoting effects such as the
inhibition of tumor growth, prevention of bone loss and the
prevention of the oxidation of lipids. Since the phenylpropanoid
biosynthetic pathway feeds into the pathways for the production of
a number of other classes of secondary metabolites, such as lignins
and tannins, changing the activity of these genes or their
paralogs/orthologs might also influence the levels of those types
of compounds. For example, increased levels of condensed tannins in
forage legumes can prevent pasture bloat in cattle by collapsing
protein foams within the rumen. Additionally, lignins are of major
interest to the forestry and pulp and paper industries. Elevated
levels of lignin increase the quality of wood used for furniture
and building materials. However, paper manufacturers desire reduced
lignin levels, since these compounds are costly to remove during
the pulping process.
[0315] Both light and the C/N metabolic status of the plant tightly
regulate the uptake, assimilation, and transport of nitrogen from
sources (e.g. leaves) to sinks (e.g. developing seeds). We used an
assay that has been developed to detect alterations in the
mechanisms that plants use to sense internal levels of carbon and
nitrogen metabolites and presumably activate signal transduction
cascades which regulate the transcription of N-assimilatory genes
(Hsieh et al. (1998) Proc. Natl. Acad. Sci. 95: 13965-13970). To
determine whether the mechanisms used to sense nitrogen status are
altered in a particular mutant or transgenic line, we exploited the
observation that seedlings of wild-type plants accumulate high
levels of anthocyanins when the C/N balance is disturbed. This was
achieved by germinating these plants on media containing high
levels of sucrose (3%) without a nitrogen source. Sucrose-induced
anthocyanin accumulation may be relieved by the addition of either
inorganic or organic nitrogen. Thus, media containing glutamine as
a nitrogen source was also used in C/N sensing assays since
glutamine also serves as a compound used to transport N in
plants.
[0316] The clades of sequences shown in laboratory experiments to
confer altered C/N sensing in plants, and structurally and
functionally related sequences that derive from a wide range of
plants, including the polynucleotides and polypeptides of the
invention (for example, SEQ ID NO: 234, 286, 312, 324, 420, 422,
424, 294, 426, 428, 430, 432, 434, 436, 438, 240, 440, 442, 444,
446, 448, 450, 452, 454, 456, 458, 460, 462, 248, 464, 466, 468,
470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,
496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,
522, 238, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544,
546, 548, 550, 10, 552, 12, 554, 556, 558, 560, 562, 564, 566, and
568), polypeptides that are encoded by the polynucleotides of the
invention, functional fragments thereof, paralogs, orthologs,
equivalogs, and conserved domains thereof are provided. Many of the
orthologs of these sequences are listed in the Sequence Listing,
and due to the high degree of structural similarity to the
sequences of the invention, it is expected that these sequences may
also function to modify C/N sensing. The invention also encompasses
the complements of the polynucleotides. The polynucleotides are
useful for screening libraries of molecules or compounds for
specific binding and for creating transgenic plants having altered
C/N sensing.
[0317] Potential Applications of the Presently Disclosed Sequences
that Regulate Shade Tolerance.
[0318] The genes identified by the experiment presently disclosed
represent potential regulators of plant responses to shade
conditions. As such, these genes (or their orthologs and paralogs)
could be applied to commercial species in order to improve yield,
and potentially allow certain crops to be grown at higher
density.
[0319] While a shade avoidance phenotype has obvious advantages for
plants competing to survive in the wild, in a crop of identical
plants to be harvested at the end of the season, it can be a waste
of energy and detract resources from storage organs, the
accumulation of biomass, and the production of fruits and seeds.
Importantly, many plant species initiate a response to shade, due
to reflected far-red light from neighbors, well before light
availability becomes a growth-limiting factor. These effects have a
negative impact on yield, and result in increased volumes of waste
by-products, such as straw. In order to compensate for the
inefficiencies produced by shading responses, increased fertilizer
applications are required to maintain yield. Thus, genes that
suppress innate plant shading responses will offer the additional
advantages of permitting a reduction in fertilizer usage and a
reduction in undesirable waste products.
[0320] It should be noted that the transcription factor leads
revealed in this study likely represent key components of light
response pathways and as such might be used to manipulate
additional aspects of plant development and physiology. For
example, light response regulators might be applied to manipulate
the timing of major growth transitions like the onset of flowering.
It should also be recognized that a number of the genes identified,
result in generally compact plant morphologies, and as such could
be used to produce dwarf varieties that might be attractive for
both grain crops or for ornamental species.
Antisense and Co-Suppression
[0321] In addition to expression of the nucleic acids of the
invention as gene replacement or plant phenotype modification
nucleic acids, the nucleic acids are also useful for sense and
anti-sense suppression of expression, for example, to down-regulate
expression of a nucleic acid of the invention. That is, the nucleic
acids of the invention, or subsequences or anti-sense sequences
thereof, can be used to block expression of naturally occurring
homologous nucleic acids. A variety of sense and anti-sense
technologies are known in the art, for example, as set forth in
Lichtenstein and Nellen (1997) Antisense Technology: A Practical
Approach, IRL Press at Oxford University Press, Oxford, U.K.
Antisense regulation is also described in Crowley et al. (1985)
Cell 43: 633-641; Rosenberg et al. (1985) Nature 313: 703-706;
Preiss et al. (1985) Nature 313: 27-32; Melton (1985) Proc. Natl.
Acad. Sci. 82: 144-148; Izant and Weintraub (1985) Science 229:
345-352; and Kim and Wold (1985) Cell 42: 129-138. Additional
methods for antisense regulation are known in the art. Antisense
regulation has been used to reduce or inhibit expression of plant
genes in, for example in European Patent Publication No. 271988.
Antisense RNA may be used to reduce gene expression to produce a
visible or biochemical phenotypic change in a plant (Smith et al.
(1988) Nature, 334: 724-726; Smith et al. (1990) Plant Mol. Biol.
14: 369-379). In general, sense or anti-sense sequences are
introduced into a cell, where they are optionally amplified, for
example, by transcription. Such sequences include both simple
oligonucleotide sequences and catalytic sequences such as
ribozymes.
[0322] For example, a reduction or elimination of expression (i.e.,
a "knock-out") of a transcription factor or transcription factor
homolog polypeptide in a transgenic plant, for example, to modify a
plant trait, can be obtained by introducing an antisense construct
corresponding to the polypeptide of interest as a cDNA. For
antisense suppression, the transcription factor or homolog cDNA is
arranged in reverse orientation (with respect to the coding
sequence) relative to the promoter sequence in the expression
vector. The introduced sequence need not be the full length cDNA or
gene, and need not be identical to the cDNA or gene found in the
plant type to be transformed. Typically, the antisense sequence
need only be capable of hybridizing to the target gene or RNA of
interest. Thus, where the introduced sequence is of shorter length,
a higher degree of homology to the endogenous transcription factor
sequence will be needed for effective antisense suppression. While
antisense sequences of various lengths can be utilized, preferably,
the introduced antisense sequence in the vector will be at least 30
nucleotides in length, and improved antisense suppression will
typically be observed as the length of the antisense sequence
increases. Preferably, the length of the antisense sequence in the
vector will be greater than 100 nucleotides. Transcription of an
antisense construct as described results in the production of RNA
molecules that are the reverse complement of mRNA molecules
transcribed from the endogenous transcription factor gene in the
plant cell.
[0323] Suppression of endogenous transcription factor gene
expression can also be achieved using RNA interference, or RNAi.
RNAi is a post-transcriptional, targeted gene-silencing technique
that uses double-stranded RNA (dsRNA) to incite degradation of
messenger RNA (mRNA) containing the same sequence as the dsRNA
(Constans, (2002) The Scientist 16:36). Small interfering RNAs, or
siRNAs are produced in at least two steps: an endogenous
ribonuclease cleaves longer dsRNA into shorter, 21-23
nucleotide-long RNAs. The siRNA segments then mediate the
degradation of the target mRNA (Zamore, (2001) Nature Struct.
Biol., 8:746-50). RNAi has been used for gene function
determination in a manner similar to antisense oligonucleotides
(Constans, (2002) The Scientist 16:36). Expression vectors that
continually express siRNAs in transiently and stably transfected
cells have been engineered to express small hairpin RNAs (shRNAs),
which get processed in vivo into siRNAs-like molecules capable of
carrying out gene-specific silencing (Brummelkamp et al., (2002)
Science 296:550-553, and Paddison, et al. (2002) Genes & Dev.
16:948-958). Post-transcriptional gene silencing by double-stranded
RNA is discussed in further detail by Hammond et al. (2001) Nature
Rev Gen 2: 110-119, Fire et al. (1998) Nature 391: 806-811 and
Timmons and Fire (1998) Nature 395: 854. Vectors in which RNA
encoded by a transcription factor or transcription factor homolog
cDNA is over-expressed can also be used to obtain co-suppression of
a corresponding endogenous gene, for example, in the manner
described in U.S. Pat. No. 5,231,020 by Jorgensen. Such
co-suppression (also termed sense suppression) does not require
that the entire transcription factor cDNA be introduced into the
plant cells, nor does it require that the introduced sequence be
exactly identical to the endogenous transcription factor gene of
interest. However, as with antisense suppression, the suppressive
efficiency will be enhanced as specificity of hybridization is
increased, for example, as the introduced sequence is lengthened,
and/or as the sequence similarity between the introduced sequence
and the endogenous transcription factor gene is increased.
[0324] Vectors expressing an untranslatable form of the
transcription factor mRNA (for example, sequences comprising one or
more stop codon or nonsense mutation) can also be used to suppress
expression of an endogenous transcription factor, thereby reducing
or eliminating its activity and modifying one or more traits.
Methods for producing such constructs are described in U.S. Pat.
No. 5,583,021. Preferably, such constructs are made by introducing
a premature stop codon into the transcription factor gene.
Alternatively, a plant trait can be modified by gene silencing
using double-strand RNA (Sharp (1999) Genes and Development 13:
139-141). Another method for abolishing the expression of a gene is
by insertion mutagenesis using the T-DNA of Agrobacterium
tumefaciens. After generating the insertion mutants, the mutants
can be screened to identify those containing the insertion in a
transcription factor or transcription factor homolog gene. Plants
containing a single transgene insertion event at the desired gene
can be crossed to generate homozygous plants for the mutation. Such
methods are well known to those of skill in the art (for example,
in Koncz et al. (1992) Methods in Arabidopsis Research, World
Scientific Publishing Co. Pte. Ltd., River Edge, N.J.).
[0325] Alternatively, a plant phenotype can be altered by
eliminating an endogenous gene, such as a transcription factor or
transcription factor homolog, for example, by homologous
recombination (Kempin et al. (1997) Nature 389: 802-803).
[0326] A plant trait can also be modified by using the Cre-lox
system (for example, as described in U.S. Pat. No. 5,658,772). A
plant genome can be modified to include first and second lox sites
that are then contacted with a Cre recombinase. If the lox sites
are in the same orientation, the intervening DNA sequence between
the two sites is excised. If the lox sites are in the opposite
orientation, the intervening sequence is inverted.
[0327] The polynucleotides and polypeptides of this invention can
also be expressed in a plant in the absence of an expression
cassette by manipulating the activity or expression level of the
endogenous gene by other means, such as, for example, by
ectopically expressing a gene by T-DNA activation tagging (Ichikawa
et al. (1997) Nature 390 698-701; Kakimoto et al. (1996) Science
274: 982-985). This method entails transforming a plant with a gene
tag containing multiple transcriptional enhancers and once the tag
has inserted into the genome, expression of a flanking gene coding
sequence becomes deregulated. In another example, the
transcriptional machinery in a plant can be modified so as to
increase transcription levels of a polynucleotide of the invention
(for example, in PCT Publications WO 96/06166 and WO 98/53057,
which describe the modification of the DNA-binding specificity of
zinc finger proteins by changing particular amino acids in the
DNA-binding motif).
[0328] The transgenic plant can also include the machinery
necessary for expressing or altering the activity of a polypeptide
encoded by an endogenous gene, for example, by altering the
phosphorylation state of the polypeptide to maintain it in an
activated state.
[0329] Transgenic plants (or plant cells, or plant explants, or
plant tissues) incorporating the polynucleotides of the invention
and/or expressing the polypeptides of the invention can be produced
by a variety of well established techniques as described above.
Following construction of a vector, most typically an expression
cassette, including a polynucleotide, for example, encoding a
transcription factor or transcription factor homolog, of the
invention, standard techniques can be used to introduce the
polynucleotide into a plant, a plant cell, a plant explant or a
plant tissue of interest. Optionally, the plant cell, explant or
tissue can be regenerated to produce a transgenic plant.
[0330] The plant can be any higher plant, including gymnosperms,
monocotyledonous and dicotyledonous plants. Suitable protocols are
available for Leguminosae (alfalfa, soybean, clover, etc.),
Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage,
radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and
cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.),
Solanaceae (potato, tomato, tobacco, peppers, etc.), and various
other crops (for example, in protocols described in Ammirato et
al., eds., (1984) Handbook of Plant Cell Culture--Crop Species,
Macmillan Publ. Co., New York, N.Y.; Shimamoto et al. (1989) Nature
338: 274-276; Fromm et al. (1990) Bio/Technol. 8: 833-839; and
Vasil et al. (1990) Bio/Technol. 8: 429-434.
[0331] Transformation and regeneration of both monocotyledonous and
dicotyledonous plant cells is now routine, and the selection of the
most appropriate transformation technique will be determined by the
practitioner. The choice of method will vary with the type of plant
to be transformed; those skilled in the art will recognize the
suitability of particular methods for given plant types. Suitable
methods can include, but are not limited to: electroporation of
plant protoplasts; liposome-mediated transformation; polyethylene
glycol (PEG) mediated transformation; transformation using viruses;
micro-injection of plant cells; micro-projectile bombardment of
plant cells; vacuum infiltration; and Agrobacterium tumefaciens
mediated transformation. Transformation means introducing a
nucleotide sequence into a plant in a manner to cause stable or
transient expression of the sequence.
[0332] Successful examples of the modification of plant
characteristics by transformation with cloned sequences which serve
to illustrate the current knowledge in this field of technology,
and which are herein incorporated by reference, include: U.S. Pat.
Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945;
5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269;
5,736,369 and 5,610,042.
[0333] Following transformation, plants are preferably selected
using a dominant selectable marker incorporated into the
transformation vector. Typically, such a marker will confer
antibiotic or herbicide resistance on the transformed plants, and
selection of transformants can be accomplished by exposing the
plants to appropriate concentrations of the antibiotic or
herbicide.
[0334] After transformed plants are selected and grown to maturity,
those plants showing a modified trait are identified. The modified
trait can be any of those traits described above. Additionally, to
confirm that the modified trait is due to changes in expression
levels or activity of the polypeptide or polynucleotide of the
invention can be determined by analyzing mRNA expression using
Northern blots, RT-PCR or microarrays, or protein expression using
immunoblots or Western blots or gel shift assays.
Integrated Systems--Sequence Identity
[0335] Additionally, the present invention may be an integrated
system, computer or computer readable medium that comprises an
instruction set for determining the identity of one or more
sequences in a database. In addition, the instruction set can be
used to generate or identify sequences that meet any specified
criteria. Furthermore, the instruction set may be used to associate
or link certain functional benefits, such improved characteristics,
with one or more identified sequence.
[0336] For example, the instruction set can include, for example, a
sequence comparison or other alignment program, for example, an
available program such as, for example, the Wisconsin Package
Version 10.0, such as BLAST, FASTA, PILEUP, FINDPAT FERNS or the
like (GCG, Madison, Wis.). Public sequence databases such as
GenBank, EMBL, Swiss-Prot and PIR or private sequence databases
such as PHYTOSEQ sequence database (Incyte Genomics, Palo Alto,
Calif.) can be searched.
[0337] Alignment of sequences for comparison can be conducted by
the local homology algorithm of Smith and Waterman (1981) Adv.
Appl. Math. 2: 482-489, by the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, by the
search for similarity method of Pearson and Lipman (1988) Proc.
Natl. Acad. Sci. 85: 2444-2448, by computerized implementations of
these algorithms. After alignment, sequence comparisons between two
(or more) polynucleotides or polypeptides are typically performed
by comparing sequences of the two sequences over a comparison
window to identify and compare local regions of sequence
similarity. The comparison window can be a segment of at least
about 20 contiguous positions, usually about 50 to about 200, more
usually about 100 to about 150 contiguous positions. A description
of the method is provided in Ausubel et al. supra.
[0338] A variety of methods for determining sequence relationships
can be used, including manual alignment and computer assisted
sequence alignment and analysis. This later approach is a preferred
approach in the present invention, due to the increased throughput
afforded by computer assisted methods. As noted above, a variety of
computer programs for performing sequence alignment are available,
or can be produced by one of skill.
[0339] One example algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al. (1990) J. Mol.
Biol. 215: 403-410. Software for performing BLAST analyses is
publicly available, for example, through the National Library of
Medicine's National Center for Biotechnology Information
(ncbi.nlm.nih; world wide web (www) National Institutes of Health
US government (gov) website). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al. supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a wordlength (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (Henikoff and Henikoff (1992) Proc.
Natl. Acad. Sci. 89: 10915-10919). Unless otherwise indicated,
"sequence identity" here refers to the % sequence identity
generated from a tblastx using the NCBI version of the algorithm at
the default settings using gapped alignments with the filter "off"
(for example, in the NIH NLM NCBI website at ncbi.nlm.nih,
supra).
[0340] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (for example, in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5787). One measure
of similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence (and, therefore, in this context,
homologous) if the smallest sum probability in a comparison of the
test nucleic acid to the reference nucleic acid is less than about
0.1, or less than about 0.01, and or even less than about 0.001. An
additional example of a useful sequence alignment algorithm is
PILEUP. PILEUP creates a multiple sequence alignment from a group
of related sequences using progressive, pairwise alignments. The
program can align, for example, up to 300 sequences of a maximum
length of 5,000 letters.
[0341] The integrated system, or computer typically includes a user
input interface allowing a user to selectively view one or more
sequence records corresponding to the one or more character
strings, as well as an instruction set which aligns the one or more
character strings with each other or with an additional character
string to identify one or more region of sequence similarity. The
system may include a link of one or more character strings with a
particular phenotype or gene function. Typically, the system
includes a user readable output element that displays an alignment
produced by the alignment instruction set.
[0342] The methods of this invention can be implemented in a
localized or distributed computing environment. In a distributed
environment, the methods may be implemented on a single computer
comprising multiple processors or on a multiplicity of computers.
The computers can be linked, for example, through a common bus, but
more preferably the computer(s) are nodes on a network. The network
can be a generalized or a dedicated local or wide-area network and,
in certain preferred embodiments, the computers may be components
of an intra-net or an internet.
[0343] Thus, the invention provides methods for identifying a
sequence similar or homologous to one or more polynucleotides as
noted herein, or one or more target polypeptides encoded by the
polynucleotides, or otherwise noted herein and may include linking
or associating a given plant phenotype or gene function with a
sequence. In the methods, a sequence database is provided (locally
or across an inter or intra net) and a query is made against the
sequence database using the relevant sequences herein and
associated plant phenotypes or gene functions.
[0344] Any sequence herein can be entered into the database, before
or after querying the database. This provides for both expansion of
the database and, if done before the querying step, for insertion
of control sequences into the database. The control sequences can
be detected by the query to ensure the general integrity of both
the database and the query. As noted, the query can be performed
using a web browser based interface. For example, the database can
be a centralized public database such as those noted herein, and
the querying can be done from a remote terminal or computer across
an internet or intranet.
[0345] Any sequence herein can be used to identify a similar,
homologous, paralogous, or orthologous sequence in another plant.
This provides means for identifying endogenous sequences in other
plants that may be useful to alter a trait of progeny plants, which
results from crossing two plants of different strain. For example,
sequences that encode an ortholog of any of the sequences herein
that naturally occur in a plant with a desired trait can be
identified using the sequences disclosed herein. The plant is then
crossed with a second plant of the same species but which does not
have the desired trait to produce progeny which can then be used in
further crossing experiments to produce the desired trait in the
second plant. Therefore the resulting progeny plant contains no
transgenes; expression of the endogenous sequence may also be
regulated by treatment with a particular chemical or other means,
such as EMR. Some examples of such compounds well known in the art
include: ethylene; cytokinins; phenolic compounds, which stimulate
the transcription of the genes needed for infection; specific
monosaccharides and acidic environments which potentiate vir gene
induction; acidic polysaccharides which induce one or more
chromosomal genes; and opines; other mechanisms include light or
dark treatment (for example, in Winans (1992) Microbiol. Rev. 56:
12-31; Eyal et al. (1992) Plant Mol. Biol. 19: 589-599; Chrispeels
et al. (2000) Plant Mol. Biol. 42: 279-290; Piazza et al. (2002)
Plant Physiol. 128: 1077-1086).
[0346] Table 8 lists sequences within the UniGene database
determined to be orthologous to a number of transcription factor
sequences of the present invention. The column headings include the
transcription factors listed by (a) the Clade Identifier SEQ ID NO:
(the Reference Arabidopsis sequence used to identify each clade);
(b) the GID of each Clade Identifier; (c) the AGI Identifier for
each Clade Identifier; (d) the UniGene identifier for each
orthologous sequence identified in this study; (e) the species from
which the orthologs to the transcription factors are derived; and
(f) the smallest sum probability relationship of the homologous
sequence to Arabidopsis Clade Identifier sequence in a given row,
determined by BLAST analysis.
TABLE-US-00009 TABLE 8 Orthologs of Representative Arabidopsis
Transcription Factor Genes Identified Using BLAST Clade AGI
Identifier Clade Identifier for (SEQ ID Identifier Clade NO:) (GID)
Identifier UniGene Identifier Species p-Value 223 G175 AT4G26440
Les_S5295446 Lycopersicon esculentum 1.00E-174 223 G175 AT4G26440
Os_S121030 Oryza sativa 2.00E-77 223 G175 AT4G26440
SGN-UNIGENE-57877 Lycopersicon esculentum 1.00E-75 223 G175
AT4G26440 Zm_S11524014 Zea mays 9.00E-50 223 G175 AT4G26440
SGN-UNIGENE-52888 Lycopersicon esculentum 7.00E-40 223 G175
AT4G26440 SGN-UNIGENE-50193 Lycopersicon esculentum 6.00E-36 223
G175 AT4G26440 Os_S50781 Oryza sativa 3.00E-19 255 G184 AT4G22070
SGN-UNIGENE-47543 Lycopersicon esculentum 1.00E-104 255 G184
AT4G22070 SGN-UNIGENE-47034 Lycopersicon esculentum 1.00E-100 255
G184 AT4G22070 Gma_S6668474 Glycine max 2.00E-77 255 G184 AT4G22070
SGN-UNIGENE- Lycopersicon esculentum 2.00E-71 SINGLET-18500 255
G184 AT4G22070 SGN-UNIGENE- Lycopersicon esculentum 5.00E-50
SINGLET-1941 255 G184 AT4G22070 SGN-UNIGENE- Lycopersicon
esculentum 8.00E-37 SINGLET-20683 255 G184 AT4G22070
SGN-UNIGENE-52279 Lycopersicon esculentum 5.00E-24 255 G184
AT4G22070 Gma_S4878547 Glycine max 2.00E-12 255 G184 AT4G22070
SGN-UNIGENE- Lycopersicon esculentum 2.00E-11 SINGLET-2301 255 G184
AT4G22070 Hv_S119532 Hordeum vulgare 2.00E-10 255 G184 AT4G22070
Zm_S11388469 Zea mays 2.00E-06 257 G186 AT1G62300 SGN-UNIGENE-47543
Lycopersicon esculentum 1.00E-104 257 G186 AT1G62300
SGN-UNIGENE-47034 Lycopersicon esculentum 1.00E-100 257 G186
AT1G62300 Gma_S6668474 Glycine max 2.00E-77 257 G186 AT1G62300
SGN-UNIGENE- Lycopersicon esculentum 2.00E-71 SINGLET-18500 257
G186 AT1G62300 SGN-UNIGENE- Lycopersicon esculentum 5.00E-50
SINGLET-1941 257 G186 AT1G62300 SGN-UNIGENE- Lycopersicon
esculentum 8.00E-37 SINGLET-20683 257 G186 AT1G62300
SGN-UNIGENE-52279 Lycopersicon esculentum 5.00E-24 257 G186
AT1G62300 Gma_S4878547 Glycine max 2.00E-12 257 G186 AT1G62300
SGN-UNIGENE- Lycopersicon esculentum 2.00E-11 SINGLET-2301 257 G186
AT1G62300 Hv_S119532 Hordeum vulgare 2.00E-10 257 G186 AT1G62300
Zm_S11388469 Zea mays 2.00E-06 259 G353 AT5G59820 SGN-UNIGENE-56766
Lycopersicon esculentum 6.00E-32 259 G353 AT5G59820 Gma_S4898433
Glycine max 3.00E-26 259 G353 AT5G59820 Ta_S200273 Triticum
aestivum 1.00E-24 259 G353 AT5G59820 Os_S109163 Oryza sativa
2.00E-20 259 G353 AT5G59820 Gma_S4973977 Glycine max 9.00E-17 259
G353 AT5G59820 Ta_S111267 Triticum aestivum 3.00E-16 259 G353
AT5G59820 Mtr_S5397852 Medicago truncatula 2.00E-14 259 G353
AT5G59820 Hv_S207187 Hordeum vulgare 5.00E-10 259 G353 AT5G59820
Ta_S296415 Triticum aestivum 1.00E-05 227 G354 AT3G46090
SGN-UNIGENE-56766 Lycopersicon esculentum 6.00E-32 227 G354
AT3G46090 Gma_S4898433 Glycine max 3.00E-26 227 G354 AT3G46090
Ta_S200273 Triticum aestivum 1.00E-24 227 G354 AT3G46090 Os_S109163
Oryza sativa 2.00E-20 227 G354 AT3G46090 Gma_S4973977 Glycine max
9.00E-17 227 G354 AT3G46090 Ta_S111267 Triticum aestivum 3.00E-16
227 G354 AT3G46090 Mtr_S5397852 Medicago truncatula 2.00E-14 227
G354 AT3G46090 Hv_S207187 Hordeum vulgare 5.00E-10 227 G354
AT3G46090 Ta_S296415 Triticum aestivum 1.00E-05 229 G489 AT1G08970
Vvi_S16526885 Vitis vinifera 1.00E-77 229 G489 AT1G08970
SGN-UNIGENE-45265 Lycopersicon esculentum 4.00E-75 229 G489
AT1G08970 Mtr_S5463839 Medicago truncatula 6.00E-73 229 G489
AT1G08970 Les_S5293479 Lycopersicon esculentum 2.00E-69 229 G489
AT1G08970 Mtr_S7092400 Medicago truncatula 9.00E-66 229 G489
AT1G08970 Pta_S17047341 Pinus taeda 7.00E-48 229 G489 AT1G08970
SGN-UNIGENE-45266 Lycopersicon esculentum 2.00E-36 229 G489
AT1G08970 Os_S37232 Oryza sativa 5.00E-09 229 G489 AT1G08970
Vvi_S15374122 Vitis vinifera 2.00E-08 263 G596 AT2G45430
Pta_S16786360 Pinus taeda 2.00E-70 263 G596 AT2G45430 Gma_S4935598
Glycine max 2.00E-67 263 G596 AT2G45430 Pta_S16788492 Pinus taeda
7.00E-63 263 G596 AT2G45430 Pta_S16802054 Pinus taeda 1.00E-57 263
G596 AT2G45430 Pta_S15799222 Pinus taeda 6.00E-43 231 G634
AT1G33240 Pta_S17050439 Pinus taeda 3.00E-39 231 G634 AT1G33240
Zm_S11449298 Zea mays 3.00E-35 233 G682 AT4G01060 Vvi_S15356289
Vitis vinifera 2.00E-30 233 G682 AT4G01060 Ta_S45274 Triticum
aestivum 3.00E-14 233 G682 AT4G01060 Vvi_S16820566 Vitis vinifera
3.00E-12 233 G682 AT4G01060 Gma_S4901946 Glycine max 0.004 265 G714
AT1G54830 Vvi_S16526885 Vitis vinifera 1.00E-77 265 G714 AT1G54830
SGN-UNIGENE-45265 Lycopersicon esculentum 4.00E-75 265 G714
AT1G54830 Mtr_S5463839 Medicago truncatula 6.00E-73 265 G714
AT1G54830 Les_S5293479 Lycopersicon esculentum 2.00E-69 265 G714
AT1G54830 Mtr_S7092400 Medicago truncatula 9.00E-66 265 G714
AT1G54830 Pta_S17047341 Pinus taeda 7.00E-48 265 G714 AT1G54830
SGN-UNIGENE-45266 Lycopersicon esculentum 2.00E-36 265 G714
AT1G54830 Os_S37232 Oryza sativa 5.00E-09 267 G877 AT5G56270
Les_S5295446 Lycopersicon esculentum 1.00E-174 267 G877 AT5G56270
Os_S121030 Oryza sativa 2.00E-77 267 G877 AT5G56270
SGN-UNIGENE-57877 Lycopersicon esculentum 1.00E-75 267 G877
AT5G56270 Zm_S11524014 Zea mays 9.00E-50 267 G877 AT5G56270
SGN-UNIGENE-52888 Lycopersicon esculentum 7.00E-40 267 G877
AT5G56270 SGN-UNIGENE-50193 Lycopersicon esculentum 6.00E-36 267
G877 AT5G56270 Os_S50781 Oryza sativa 3.00E-19 267 G877 AT5G56270
SGN-UNIGENE-56707 Lycopersicon esculentum 7.00E-10 235 G916
AT4G04450 SGN-UNIGENE-47543 Lycopersicon esculentum 1.00E-104 235
G916 AT4G04450 SGN-UNIGENE-47034 Lycopersicon esculentum 1.00E-100
235 G916 AT4G04450 Gma_S6668474 Glycine max 2.00E-77 235 G916
AT4G04450 SGN-UNIGENE- Lycopersicon esculentum 2.00E-71
SINGLET-18500 235 G916 AT4G04450 SGN-UNIGENE- Lycopersicon
esculentum 5.00E-50 SINGLET-1941 235 G916 AT4G04450 SGN-UNIGENE-
Lycopersicon esculentum 8.00E-37 SINGLET-20683 235 G916 AT4G04450
SGN-UNIGENE-52279 Lycopersicon esculentum 5.00E-24 235 G916
AT4G04450 Gma_S4878547 Glycine max 2.00E-12 235 G916 AT4G04450
Hv_S119532 Hordeum vulgare 2.00E-10 235 G916 AT4G04450 Zm_S11388469
Zea mays 2.00E-06 237 G975 AT1G15360 SGN-UNIGENE- Lycopersicon
esculentum 9.00E-59 SINGLET-335836 237 G975 AT1G15360 SGN-UNIGENE-
Lycopersicon esculentum 2.00E-52 SINGLET-14957 239 G1069 AT4G14465
SGN-UNIGENE-59076 Lycopersicon esculentum 6.00E-55 239 G1069
AT4G14465 Vvi_S16805621 Vitis vinifera 1.00E-04 271 G1387 AT5G25390
SGN-UNIGENE- Lycopersicon esculentum 9.00E-59 SINGLET-335836 271
G1387 AT5G25390 SGN-UNIGENE- Lycopersicon esculentum 2.00E-52
SINGLET-14957 273 G1634 AT5G05790 Vvi_S16872328 Vitis vinifera
4.00E-63 273 G1634 AT5G05790 SGN-UNIGENE- Lycopersicon esculentum
5.00E-34 SINGLET-48341 273 G1634 AT5G05790 SGN-UNIGENE-
Lycopersicon esculentum 4.00E-12 SINGLET-41892 275 G1889 AT2G28710
SGN-UNIGENE-56766 Lycopersicon esculentum 6.00E-32 275 G1889
AT2G28710 Gma_S4898433 Glycine max 3.00E-26 275 G1889 AT2G28710
Ta_S200273 Triticum aestivum 1.00E-24 275 G1889 AT2G28710
Os_S109163 Oryza sativa 2.00E-20 275 G1889 AT2G28710 Gma_S4973977
Glycine max 9.00E-17 275 G1889 AT2G28710 Ta_S111267 Triticum
aestivum 3.00E-16 275 G1889 AT2G28710 Mtr_S5397852 Medicago
truncatula 2.00E-14 275 G1889 AT2G28710 Hv_S207187 Hordeum vulgare
5.00E-10 277 G1940 AT5G54900 SGN-UNIGENE-44207 Lycopersicon
esculentum 1.00E-144 277 G1940 AT5G54900 Zm_S11525357 Zea mays
1.00E-130 277 G1940 AT5G54900 Zm_S11522955 Zea mays 1.00E-100 277
G1940 AT5G54900 Vvi_S16865171 Vitis vinifera 1.00E-85 277 G1940
AT5G54900 Hv_S153237 Hordeum vulgare 9.00E-72 277 G1940 AT5G54900
Ta_S152820 Triticum aestivum 1.00E-66 277 G1940 AT5G54900
SGN-UNIGENE- Lycopersicon esculentum 3.00E-55 SINGLET-396174 277
G1940 AT5G54900 SGN-UNIGENE- Lycopersicon esculentum 4.00E-53
SINGLET-333119 277 G1940 AT5G54900 Gma_S4975207 Glycine max
6.00E-51 277 G1940 AT5G54900 SGN-UNIGENE- Lycopersicon esculentum
1.00E-51 SINGLET-17539 277 G1940 AT5G54900 Hv_S63965 Hordeum
vulgare 4.00E-43 277 G1940 AT5G54900 SGN-UNIGENE-56600 Lycopersicon
esculentum 2.00E-43 277 G1940 AT5G54900 Os_S32676 Oryza sativa
2.00E-31 277 G1940 AT5G54900 Ta_S125786 Triticum aestivum 6.00E-26
277 G1940 AT5G54900 Ta_S267457 Triticum aestivum 5.00E-24 277 G1940
AT5G54900 Vvi_S16866336 Vitis vinifera 7.00E-18 277 G1940 AT5G54900
Os_S75860 Oryza sativa 4.00E-11 277 G1940 AT5G54900 SGN-UNIGENE-
Lycopersicon esculentum 2.00E-04 SINGLET-49629 279 G1974 AT3G46070
SGN-UNIGENE-56766 Lycopersicon esculentum 6.00E-32 279 G1974
AT3G46070 Gma_S4898433 Glycine max 3.00E-26 279 G1974 AT3G46070
Ta_S200273 Triticum aestivum 1.00E-24 279 G1974 AT3G46070
Os_S109163 Oryza sativa 2.00E-20 279 G1974 AT3G46070 Gma_S4973977
Glycine max 9.00E-17 279 G1974 AT3G46070 Ta_S111267 Triticum
aestivum 3.00E-16 279 G1974 AT3G46070 Mtr_S5397852 Medicago
truncatula 2.00E-14 279 G1974 AT3G46070 Hv_S207187 Hordeum vulgare
5.00E-10 279 G1974 AT3G46070 Ta_S296415 Triticum aestivum 1.00E-05
281 G2153 AT3G04570 SGN-UNIGENE-59076 Lycopersicon esculentum
6.00E-55 281 G2153 AT3G04570 Mtr_S5308977 Medicago truncatula
2.00E-31 281 G2153 AT3G04570 Hv_S52928 Hordeum vulgare 5 283 G2583
AT5G11190 SGN-UNIGENE- Lycopersicon esculentum 9.00E-59
SINGLET-335836 283 G2583 AT5G11190 SGN-UNIGENE- Lycopersicon
esculentum 2.00E-52 SINGLET-14957 245 G2701 AT3G11280 Vvi_S16872328
Vitis vinifera 4.00E-63 245 G2701 AT3G11280 SGN-UNIGENE-
Lycopersicon esculentum 5.00E-34 SINGLET-48341 245 G2701 AT3G11280
SGN-UNIGENE- Lycopersicon esculentum 4.00E-12 SINGLET-41892 247
G2789 AT3G60870 Pta_S16786360 Pinus taeda 2.00E-70 247 G2789
AT3G60870 Gma_S4935598 Glycine max 2.00E-67 247 G2789 AT3G60870
Pta_S16788492 Pinus taeda 7.00E-63 247 G2789 AT3G60870
Pta_S16802054 Pinus taeda 1.00E-57 247 G2789 AT3G60870
Pta_S15799222 Pinus taeda 6.00E-43 249 G2839 AT3G46080
SGN-UNIGENE-56766 Lycopersicon esculentum 6.00E-32 249 G2839
AT3G46080 Gma_S4898433 Glycine max 3.00E-26 249 G2839 AT3G46080
Ta_S200273 Triticum aestivum 1.00E-24 249 G2839 AT3G46080
Os_S109163 Oryza sativa 2.00E-20 249 G2839 AT3G46080 Gma_S4973977
Glycine max 9.00E-17 249 G2839 AT3G46080 Ta_S111267 Triticum
aestivum 3.00E-16 249 G2839 AT3G46080 Mtr_S5397852 Medicago
truncatula 2.00E-14 249 G2839 AT3G46080 Hv_S207187 Hordeum vulgare
5.00E-10 249 G2839 AT3G46080 Ta_S296415 Triticum aestivum 1.00E-05
251 G2854 AT4G27000 SGN-UNIGENE-44207 Lycopersicon esculentum
1.00E-144 251 G2854 AT4G27000 Zm_S11525357 Zea mays 1.00E-130 251
G2854 AT4G27000 Zm_S11522955 Zea mays 1.00E-100 251 G2854 AT4G27000
Vvi_S16865171 Vitis vinifera 1.00E-85 251 G2854 AT4G27000
Hv_S153237 Hordeum vulgare 9.00E-72 251 G2854 AT4G27000 Ta_S152820
Triticum aestivum 1.00E-66 251 G2854 AT4G27000 SGN-UNIGENE-
Lycopersicon esculentum 3.00E-55 SINGLET-396174 251 G2854 AT4G27000
SGN-UNIGENE- Lycopersicon esculentum 4.00E-53 SINGLET-333119 251
G2854 AT4G27000 Gma_S4975207 Glycine max 6.00E-51 251 G2854
AT4G27000 SGN-UNIGENE- Lycopersicon esculentum 1.00E-51
SINGLET-17539 251 G2854 AT4G27000 Hv_S63965 Hordeum vulgare
4.00E-43 251 G2854 AT4G27000 SGN-UNIGENE-56600 Lycopersicon
esculentum 2.00E-43 251 G2854 AT4G27000 Os_S32676 Oryza sativa
2.00E-31 251 G2854 AT4G27000 Ta_S125786 Triticum aestivum 6.00E-26
251 G2854 AT4G27000 Ta_S267457 Triticum aestivum 5.00E-24 251 G2854
AT4G27000 Vvi_S16866336 Vitis vinifera 7.00E-18 251 G2854 AT4G27000
Os_S75860 Oryza sativa 4.00E-11 251 G2854 AT4G27000 SGN-UNIGENE-
Lycopersicon esculentum 2.00E-04 SINGLET-49629 253 G3083 AT3G14880
Gma_S4880456 Glycine max 1.00E-25 253 G3083 AT3G14880 Ta_S179586
Triticum aestivum 1.00E-13 253 G3083 AT3G14880 Os_S54214 Oryza
sativa 5.00E-08 253 G3083 AT3G14880 Hv_S60182 Hordeum vulgare
3.00E-06 463 G8 AT2G28550 SGN-UNIGENE- Lycopersicon esculentum
1.00E-64 SINGLET-395477 463 G8 AT2G28550 Ta_S177690 Triticum
aestivum 2.00E-21 463 G8 AT2G28550 Vvi_S15411435 Vitis vinifera
6.00E-07 419 G24 AT2G23340 Gma_S5071803 Glycine max 8.00E-40 419
G24 AT2G23340 SGN-UNIGENE-49683 Lycopersicon esculentum 1.00E-14
419 G24 AT2G23340 SGN-UNIGENE-54594 Lycopersicon esculentum
4.00E-41 419 G24 AT2G23340 SGN-UNIGENE- Lycopersicon esculentum
7.00E-19 SINGLET-47313 419 G24 AT2G23340 Mtr_S5349908 Medicago
truncatula 4.00E-32 419 G24 AT2G23340 Os_S32369 Oryza sativa
1.00E-13 419 G24 AT2G23340 Os_S80194 Oryza sativa 4.00E-08 419 G24
AT2G23340 Vvi_S15370190 Vitis vinifera 1.00E-38 419 G24 AT2G23340
Vvi_S16806812 Vitis vinifera 6.00E-25 421 G154 AT2G45660
Gma_S5094568 Glycine max 2.00E-13 421 G154 AT2G45660 Les_S5295933
Lycopersicon esculentum 2.00E-57 421 G154 AT2G45660
SGN-UNIGENE-50586 Lycopersicon esculentum 4.00E-56 421 G154
AT2G45660 SGN-UNIGENE-52410 Lycopersicon esculentum 2.00E-54 421
G154 AT2G45660 SGN-UNIGENE- Lycopersicon esculentum 2.00E-27
SINGLET-366830 421 G154 AT2G45660 SGN-UNIGENE- Lycopersicon
esculentum 3.00E-47 SINGLET-394847 421 G154 AT2G45660 Mtr_S5357829
Medicago truncatula 2.00E-53 421 G154 AT2G45660 Os_S60918 Oryza
sativa 1.00E-57 421 G154 AT2G45660 Pta_S15732813 Pinus taeda
5.00E-13 421 G154 AT2G45660 Pta_S15736271 Pinus taeda 2.00E-37 421
G154 AT2G45660 Pta_S15739572 Pinus taeda 4.00E-22 421 G154
AT2G45660 Pta_S15740527 Pinus taeda 8.00E-31 421 G154 AT2G45660
Pta_S15746398 Pinus taeda 6.00E-26 421 G154 AT2G45660 Pta_S15751737
Pinus taeda 2.00E-39 421 G154 AT2G45660 Pta_S15777399 Pinus taeda
3.00E-22
421 G154 AT2G45660 Pta_S15780122 Pinus taeda 1.00E-36 421 G154
AT2G45660 Pta_S15795745 Pinus taeda 1.00E-23 421 G154 AT2G45660
Pta_S16849782 Pinus taeda 3.00E-55 421 G154 AT2G45660 Ta_S203038
Triticum aestivum 3.00E-47 421 G154 AT2G45660 Ta_S424724 Triticum
aestivum 8.00E-19 421 G154 AT2G45660 Vvi_S15373999 Vitis vinifera
4.00E-72 421 G154 AT2G45660 Vvi_S16872184 Vitis vinifera 7.00E-35
421 G154 AT2G45660 Zm_S11418746 Zea mays 2.00E-58 421 G154
AT2G45660 Zm_S11527819 Zea mays 6.00E-55 467 G156 AT5G23260
SGN-UNIGENE-54690 Lycopersicon esculentum 5.00E-40 469 G161
AT5G60440 SGN-UNIGENE-57990 Lycopersicon esculentum 3.00E-20 475
G189 AT2G23320 Gma_S4901804 Glycine max 3.00E-15 475 G189 AT2G23320
Les_S6657758 Lycopersicon esculentum 2.00E-22 475 G189 AT2G23320
Pta_S16793418 Pinus taeda 1.00E-36 475 G189 AT2G23320 Vvi_S15353287
Vitis vinifera 1.00E-29 475 G189 AT2G23320 Vvi_S15374453 Vitis
vinifera 9.00E-32 477 G200 AT1G08810 SGN-UNIGENE-57276 Lycopersicon
esculentum 9.00E-10 477 G200 AT1G08810 SGN-UNIGENE- Lycopersicon
esculentum 1.00E-61 SINGLET-385670 477 G200 AT1G08810 Os_S60479
Oryza sativa 9.00E-71 477 G200 AT1G08810 Zm_S11529138 Zea mays
9.00E-18 477 G200 AT1G08810 Zm_S11529143 Zea mays 1.00E-19 477 G200
AT1G08810 Zm_S11529165 Zea mays 8.00E-19 479 G234 AT3G49690
SGN-UNIGENE- Lycopersicon esculentum 3.00E-57 SINGLET-21166 479
G234 AT3G49690 Zm_S11529159 Zea mays 3.00E-15 479 G234 AT3G49690
Zm_S11529194 Zea mays 3.00E-16 483 G275 AT5G64030 Gma_S4898629
Glycine max 1.00E-93 483 G275 AT5G64030 Gma_S4907362 Glycine max
1.00E-16 483 G275 AT5G64030 Hv_S8292 Hordeum vulgare 2.00E-71 483
G275 AT5G64030 SGN-UNIGENE-47489 Lycopersicon esculentum 1.0e-999
483 G275 AT5G64030 SGN-UNIGENE-47510 Lycopersicon esculentum
1.00E-121 483 G275 AT5G64030 SGN-UNIGENE-51256 Lycopersicon
esculentum 1.00E-142 483 G275 AT5G64030 SGN-UNIGENE-56050
Lycopersicon esculentum 2.00E-54 483 G275 AT5G64030 Mtr_S10821012
Medicago truncatula 1.00E-117 483 G275 AT5G64030 Pta_S15736214
Pinus taeda 1.00E-48 483 G275 AT5G64030 Pta_S15776645 Pinus taeda
1.00E-74 483 G275 AT5G64030 Vvi_S15426449 Vitis vinifera 1.00E-118
483 G275 AT5G64030 Vvi_S16870363 Vitis vinifera 6.00E-23 483 G275
AT5G64030 Zm_S11528144 Zea mays 1.0e-999 485 G326 AT2G33500
Hv_S67575 Hordeum vulgare 4.00E-12 485 G326 AT2G33500 SGN-UNIGENE-
Lycopersicon esculentum 1.00E-45 SINGLET-19083 485 G326 AT2G33500
Pta_S17049915 Pinus taeda 9.00E-17 485 G326 AT2G33500 Ta_S148486
Triticum aestivum 2.00E-12 485 G326 AT2G33500 Zm_S11450524 Zea mays
1.00E-18 485 G326 AT2G33500 Zm_S11510508 Zea mays 1.00E-11 487 G347
AT4G20380 Gma_S4934838 Glycine max 1.00E-12 487 G347 AT4G20380
Les_S5275585 Lycopersicon esculentum 3.00E-22 487 G347 AT4G20380
SGN-UNIGENE-51747 Lycopersicon esculentum 5.00E-29 487 G347
AT4G20380 Mtr_S5454462 Medicago truncatula 1.00E-72 487 G347
AT4G20380 Os_S100515 Oryza sativa 9.00E-09 487 G347 AT4G20380
Ta_S64707 Triticum aestivum 2.00E-54 487 G347 AT4G20380
Vvi_S16531517 Vitis vinifera 3.00E-66 487 G347 AT4G20380
Zm_S11437336 Zea mays 1.00E-19 487 G347 AT4G20380 Zm_S11520104 Zea
mays 3.00E-53 423 G384 AT4G21750 Gma_S4992142 Glycine max 3.00E-23
423 G384 AT4G21750 Hv_S30279 Hordeum vulgare 7.00E-22 423 G384
AT4G21750 SGN-UNIGENE- Lycopersicon esculentum 4.00E-60
SINGLET-17776 423 G384 AT4G21750 Mtr_S5447672 Medicago truncatula
1.00E-123 423 G384 AT4G21750 Os_S112966 Oryza sativa 1.0e-999 423
G384 AT4G21750 Os_S113503 Oryza sativa 2.00E-93 423 G384 AT4G21750
Ta_S133393 Triticum aestivum 3.00E-12 423 G384 AT4G21750
Zm_S11333633 Zea mays 1.00E-28 423 G384 AT4G21750 Zm_S11401894 Zea
mays 9.00E-16 423 G384 AT4G21750 Zm_S11418286 Zea mays 1.0e-999 423
G384 AT4G21750 Zm_S11418453 Zea mays 1.0e-999 423 G384 AT4G21750
Zm_S11418455 Zea mays 1.0e-999 423 G384 AT4G21750 Zm_S11523949 Zea
mays 4.00E-09 489 G427 AT5G11060 Gma_S4867945 Glycine max 7.00E-49
489 G427 AT5G11060 Hv_S23303 Hordeum vulgare 3.00E-82 489 G427
AT5G11060 Les_S5295728 Lycopersicon esculentum 1.00E-125 489 G427
AT5G11060 Les_S5295749 Lycopersicon esculentum 1.00E-137 489 G427
AT5G11060 SGN-UNIGENE-51523 Lycopersicon esculentum 2.00E-46 489
G427 AT5G11060 SGN-UNIGENE-54900 Lycopersicon esculentum 5.00E-12
489 G427 AT5G11060 SGN-UNIGENE-55550 Lycopersicon esculentum
1.00E-140 489 G427 AT5G11060 SGN-UNIGENE-55551 Lycopersicon
esculentum 4.00E-49 489 G427 AT5G11060 SGN-UNIGENE- Lycopersicon
esculentum 4.00E-16 SINGLET-397654 489 G427 AT5G11060 SGN-UNIGENE-
Lycopersicon esculentum 8.00E-09 SINGLET-446384 489 G427 AT5G11060
SGN-UNIGENE- Lycopersicon esculentum 2.00E-75 SINGLET-50339 489
G427 AT5G11060 SGN-UNIGENE- Lycopersicon esculentum 3.00E-49
SINGLET-9520 489 G427 AT5G11060 Mtr_S5306926 Medicago truncatula
7.00E-38 489 G427 AT5G11060 Mtr_S5449876 Medicago truncatula
2.00E-82 489 G427 AT5G11060 Mtr_S7092065 Medicago truncatula
5.00E-85 489 G427 AT5G11060 Os_S60901 Oryza sativa 5.00E-89 489
G427 AT5G11060 Os_S64872 Oryza sativa 2.00E-94 489 G427 AT5G11060
Os_S64899 Oryza sativa 1.00E-118 489 G427 AT5G11060 Os_S64900 Oryza
sativa 1.00E-114 489 G427 AT5G11060 Pta_S16847381 Pinus taeda
1.00E-110 489 G427 AT5G11060 Pta_S17051722 Pinus taeda 4.00E-66 489
G427 AT5G11060 Ta_S16327 Triticum aestivum 3.00E-93 489 G427
AT5G11060 Ta_S201090 Triticum aestivum 2.00E-47 489 G427 AT5G11060
Vvi_S15401282 Vitis vinifera 8.00E-19 489 G427 AT5G11060
Vvi_S15423741 Vitis vinifera 4.00E-58 489 G427 AT5G11060
Zm_S11442066 Zea mays 2.00E-08 489 G427 AT5G11060 Zm_S11452342 Zea
mays 3.00E-48 489 G427 AT5G11060 Zm_S11527509 Zea mays 4.00E-86 425
G545 AT1G27730 Gma_S4873409 Glycine max 1.00E-50 425 G545 AT1G27730
Gma_S5146663 Glycine max 2.00E-55 425 G545 AT1G27730
SGN-UNIGENE-44163 Lycopersicon esculentum 1.00E-56 425 G545
AT1G27730 SGN-UNIGENE-44287 Lycopersicon esculentum 2.00E-35 425
G545 AT1G27730 SGN-UNIGENE- Lycopersicon esculentum 4.00E-33
SINGLET-6983 425 G545 AT1G27730 Mtr_S5317695 Medicago truncatula
4.00E-55 425 G545 AT1G27730 Mtr_S5431156 Medicago truncatula
5.00E-40 425 G545 AT1G27730 Ta_S147812 Triticum aestivum 9.00E-16
425 G545 AT1G27730 Ta_S66284 Triticum aestivum 5.00E-35 425 G545
AT1G27730 Vvi_S15355617 Vitis vinifera 1.00E-52 425 G545 AT1G27730
Vvi_S15382170 Vitis vinifera 7.00E-47 425 G545 AT1G27730
Zm_S11441492 Zea mays 7.00E-30 425 G545 AT1G27730 Zm_S11443346 Zea
mays 1.00E-34 425 G545 AT1G27730 Zm_S11465527 Zea mays 2.00E-18 493
G590 AT4G36930 SGN-UNIGENE-47483 Lycopersicon esculentum 2.00E-34
493 G590 AT4G36930 SGN-UNIGENE-47925 Lycopersicon esculentum
2.00E-41 495 G602 AT2G45820 Gma_S4863794 Glycine max 5.00E-55 495
G602 AT2G45820 SGN-UNIGENE- Lycopersicon esculentum 5.00E-04
SINGLET-2565 495 G602 AT2G45820 Mtr_S5431439 Medicago truncatula
3.00E-37 495 G602 AT2G45820 Pta_S16797626 Pinus taeda 4.00E-46 495
G602 AT2G45820 Vvi_S15353882 Vitis vinifera 4.00E-63 495 G602
AT2G45820 Zm_S11527752 Zea mays 5.00E-57 497 G618 AT1G53230
Gma_S5029115 Glycine max 9.00E-30 497 G618 AT1G53230 Les_S5295478
Lycopersicon esculentum 1.00E-95 497 G618 AT1G53230
SGN-UNIGENE-50577 Lycopersicon esculentum 1.00E-52 497 G618
AT1G53230 SGN-UNIGENE-58580 Lycopersicon esculentum 1.00E-41 497
G618 AT1G53230 SGN-UNIGENE- Lycopersicon esculentum 1.00E-21
SINGLET-24189 497 G618 AT1G53230 SGN-UNIGENE- Lycopersicon
esculentum 8.00E-30 SINGLET-394109 497 G618 AT1G53230 SGN-UNIGENE-
Lycopersicon esculentum 2.00E-40 SINGLET-401522 497 G618 AT1G53230
Os_S113396 Oryza sativa 1.00E-48 497 G618 AT1G53230 Os_S113398
Oryza sativa 1.00E-78 499 G635 AT5G63430 Mtr_S5399163 Medicago
truncatula 1.00E-40 499 G635 AT5G63430 Ta_S2764 Triticum aestivum
6.00E-24 501 G643 AT4G31270 SGN-UNIGENE-56459 Lycopersicon
esculentum 1.00E-32 503 G653 AT2G39900 Hv_S136844 Hordeum vulgare
1.00E-72 503 G653 AT2G39900 SGN-UNIGENE-46400 Lycopersicon
esculentum 4.00E-93 503 G653 AT2G39900 SGN-UNIGENE- Lycopersicon
esculentum 2.00E-12 SINGLET-64524 503 G653 AT2G39900 Mtr_S7091176
Medicago truncatula 3.00E-51 503 G653 AT2G39900 Os_S76089 Oryza
sativa 1.00E-37 503 G653 AT2G39900 Pta_S16790444 Pinus taeda
1.00E-40 503 G653 AT2G39900 Pta_S17050802 Pinus taeda 2.00E-14 503
G653 AT2G39900 Ta_S166473 Triticum aestivum 5.00E-71 503 G653
AT2G39900 Vvi_S15426604 Vitis vinifera 2.00E-94 503 G653 AT2G39900
Zm_S11528938 Zea mays 7.00E-81 427 G760 AT5G04410 Gma_S4883349
Glycine max 3.00E-09 427 G760 AT5G04410 SGN-UNIGENE-47781
Lycopersicon esculentum 1.00E-106 427 G760 AT5G04410
SGN-UNIGENE-52634 Lycopersicon esculentum 6.00E-65 427 G760
AT5G04410 SGN-UNIGENE-53754 Lycopersicon esculentum 4.00E-72 427
G760 AT5G04410 SGN-UNIGENE- Lycopersicon esculentum 5.00E-29
SINGLET-23750 427 G760 AT5G04410 SGN-UNIGENE- Lycopersicon
esculentum 1.00E-07 SINGLET-310313 427 G760 AT5G04410 SGN-UNIGENE-
Lycopersicon esculentum 3.00E-12 SINGLET-447414 427 G760 AT5G04410
Mtr_S5340844 Medicago truncatula 6.00E-06 427 G760 AT5G04410
Mtr_S7090764 Medicago truncatula 2.00E-14 427 G760 AT5G04410
Pta_S16789085 Pinus taeda 7.00E-36 427 G760 AT5G04410 Ta_S202572
Triticum aestivum 5.00E-37 427 G760 AT5G04410 Vvi_S16873427 Vitis
vinifera 4.00E-21 427 G760 AT5G04410 Zm_S11526816 Zea mays 1.00E-16
427 G760 AT5G04410 Zm_S11529038 Zea mays 1.00E-45 429 G773
AT3G15500 Gma_S5050636 Glycine max 5.00E-84 429 G773 AT3G15500
Les_S5295623 Lycopersicon esculentum 1.00E-105 429 G773 AT3G15500
SGN-UNIGENE-45948 Lycopersicon esculentum 1.00E-105 429 G773
AT3G15500 SGN-UNIGENE-48215 Lycopersicon esculentum 1.00E-105 507
G837 AT1G29470 Gma_S4898629 Glycine max 1.00E-93 507 G837 AT1G29470
Gma_S4907362 Glycine max 1.00E-16 507 G837 AT1G29470 Hv_S8292
Hordeum vulgare 2.00E-71 507 G837 AT1G29470 SGN-UNIGENE-47489
Lycopersicon esculentum 1.0e-999 507 G837 AT1G29470
SGN-UNIGENE-47510 Lycopersicon esculentum 1.00E-121 507 G837
AT1G29470 SGN-UNIGENE-51256 Lycopersicon esculentum 1.00E-142 507
G837 AT1G29470 SGN-UNIGENE-56050 Lycopersicon esculentum 2.00E-54
507 G837 AT1G29470 Mtr_S10821012 Medicago truncatula 1.00E-117 507
G837 AT1G29470 Pta_S15736214 Pinus taeda 1.00E-48 507 G837
AT1G29470 Pta_S15776645 Pinus taeda 1.00E-74 507 G837 AT1G29470
Vvi_S15426449 Vitis vinifera 1.00E-118 507 G837 AT1G29470
Vvi_S16870363 Vitis vinifera 6.00E-23 507 G837 AT1G29470
Zm_S11528144 Zea mays 1.0e-999 509 G866 AT2G24570 Gma_S4874203
Glycine max 2.00E-47 509 G866 AT2G24570 Gma_S4886425 Glycine max
5.00E-19 509 G866 AT2G24570 Gma_S5106568 Glycine max 2.00E-53 509
G866 AT2G24570 Les_S6657761 Lycopersicon esculentum 2.00E-19 509
G866 AT2G24570 Les_S6657762 Lycopersicon esculentum 2.00E-16 509
G866 AT2G24570 SGN-UNIGENE-45903 Lycopersicon esculentum 2.00E-86
509 G866 AT2G24570 SGN-UNIGENE- Lycopersicon esculentum 1.00E-26
SINGLET-439904 509 G866 AT2G24570 Mtr_S5305224 Medicago truncatula
2.00E-44 509 G866 AT2G24570 Mtr_S7091692 Medicago truncatula
1.00E-66 509 G866 AT2G24570 Os_S44434 Oryza sativa 8.00E-42 509
G866 AT2G24570 Ta_S174179 Triticum aestivum 8.00E-46 509 G866
AT2G24570 Ta_S280279 Triticum aestivum 1.00E-27 509 G866 AT2G24570
Vvi_S15374416 Vitis vinifera 9.00E-39 509 G866 AT2G24570
Zm_S11523935 Zea mays 1.00E-75 511 G872 AT1G74930 SGN-UNIGENE-50296
Lycopersicon esculentum 7.00E-44 511 G872 AT1G74930 Pta_S15754706
Pinus taeda 7.00E-25 511 G872 AT1G74930 Pta_S15767728 Pinus taeda
2.00E-29 511 G872 AT1G74930 Pta_S15779272 Pinus taeda 2.00E-28 511
G872 AT1G74930 Vvi_S16870232 Vitis vinifera 1.00E-15 515 G912
AT5G51990 Hv_S152300 Hordeum vulgare 2.00E-46 515 G912 AT5G51990
Hv_S158942 Hordeum vulgare 3.00E-33 515 G912 AT5G51990 Hv_S74288
Hordeum vulgare 4.00E-36 515 G912 AT5G51990 Hv_S74289 Hordeum
vulgare 4.00E-35 515 G912 AT5G51990 Les_S5295301 Lycopersicon
esculentum 6.00E-61 515 G912 AT5G51990 SGN-UNIGENE-46974
Lycopersicon esculentum 4.00E-50 515 G912 AT5G51990
SGN-UNIGENE-46975 Lycopersicon esculentum 2.00E-56 515 G912
AT5G51990 SGN-UNIGENE-58571 Lycopersicon esculentum 8.00E-47 515
G912 AT5G51990 SGN-UNIGENE- Lycopersicon esculentum 3.00E-35
SINGLET-398604 515 G912 AT5G51990 Os_S116938 Oryza sativa 1.00E-36
515 G912 AT5G51990 Os_S116940 Oryza sativa 9.00E-33 515 G912
AT5G51990 Os_S117813 Oryza sativa 3.00E-44 515 G912 AT5G51990
Os_S65912 Oryza sativa 5.00E-25 515 G912 AT5G51990 Ta_S47586
Triticum aestivum 2.00E-20 515 G912 AT5G51990 Ta_S75229 Triticum
aestivum 2.00E-33 515 G912 AT5G51990 Vvi_S15357313 Vitis vinifera
7.00E-09 515 G912 AT5G51990 Vvi_S15391707 Vitis vinifera 1.00E-41
515 G912 AT5G51990 Zm_S11519368 Zea mays 3.00E-31 517 G932
AT3G47600 Les_S5295595 Lycopersicon esculentum 7.00E-82 517 G932
AT3G47600 SGN-UNIGENE-52504 Lycopersicon esculentum 7.00E-81 517
G932 AT3G47600 SGN-UNIGENE-52540 Lycopersicon esculentum 1.00E-46
517 G932 AT3G47600 SGN-UNIGENE-57232 Lycopersicon esculentum
6.00E-68 517 G932 AT3G47600 Vvi_S16532074 Vitis vinifera 2.00E-87
517 G932 AT3G47600 Zm_S11524655 Zea mays 4.00E-80 517 G932
AT3G47600 Zm_S11529150 Zea mays 7.00E-18 517 G932 AT3G47600
Zm_S11529161 Zea mays 8.00E-16 517 G932 AT3G47600 Zm_S11529174 Zea
mays 3.00E-15 517 G932 AT3G47600 Zm_S11529193 Zea mays 9.00E-18 431
G937 AT1G49560 Gma_S5129137 Glycine max 4.00E-20 431 G937 AT1G49560
Vvi_S15431951 Vitis vinifera 2.00E-39 431 G937 AT1G49560
Vvi_S16805106 Vitis vinifera 1.00E-16 519 G958 AT1G65910 Os_S61189
Oryza sativa 3.00E-55 519 G958 AT1G65910 Os_S69951 Oryza sativa
8.00E-10 519 G958 AT1G65910 Pta_S15738910 Pinus taeda 4.00E-10 519
G958 AT1G65910 Pta_S15774939 Pinus taeda 2.00E-33 519 G958
AT1G65910 Zm_S11437468 Zea mays 4.00E-19 521 G964 AT5G47370
Gma_S5001940 Glycine max 3.00E-04 521 G964 AT5G47370 Pta_S15797996
Pinus taeda 1.00E-38 237 G975 AT1G15360 SGN-UNIGENE- Lycopersicon
esculentum 2.00E-52 SINGLET-14957 237 G975 AT1G15360 SGN-UNIGENE-
Lycopersicon esculentum 9.00E-59 SINGLET-335836 523 G979 AT3G54320
SGN-UNIGENE- Lycopersicon esculentum 5.00E-74 SINGLET-517
523 G979 AT3G54320 Zm_S11528772 Zea mays 3.00E-77 435 G988
AT1G55580 Les_S5295726 Lycopersicon esculentum 1.00E-114 525 G1049
AT3G30530 Gma_S5131758 Glycine max 2.00E-30 525 G1049 AT3G30530
SGN-UNIGENE- Lycopersicon esculentum 5.00E-38 SINGLET-333614 525
G1049 AT3G30530 Zm_S11445843 Zea mays 1.00E-17 239 G1069 AT4G14465
SGN-UNIGENE-59076 Lycopersicon esculentum 6.00E-55 239 G1069
AT4G14465 Vvi_S16805621 Vitis vinifera 1.00E-04 439 G1090 AT1G33760
SGN-UNIGENE-54402 Lycopersicon esculentum 3.00E-40 529 G1255
AT1G25440 SGN-UNIGENE-48698 Lycopersicon esculentum 5.00E-55 529
G1255 AT1G25440 SGN-UNIGENE-53476 Lycopersicon esculentum 1.00E-41
529 G1255 AT1G25440 SGN-UNIGENE-54828 Lycopersicon esculentum
9.00E-37 529 G1255 AT1G25440 Mtr_S5409553 Medicago truncatula
1.00E-17 529 G1255 AT1G25440 Ta_S203158 Triticum aestivum 5.00E-19
529 G1255 AT1G25440 Ta_S363550 Triticum aestivum 2.00E-21 529 G1255
AT1G25440 Vvi_S15427527 Vitis vinifera 4.00E-24 529 G1255 AT1G25440
Vvi_S15431583 Vitis vinifera 1.00E-24 529 G1255 AT1G25440
Zm_S11485770 Zea mays 1.00E-26 531 G1266 AT3G23240 Les_S5269007
Lycopersicon esculentum 2.00E-18 531 G1266 AT3G23240 Les_S5295266
Lycopersicon esculentum 2.00E-37 531 G1266 AT3G23240 Les_S5295755
Lycopersicon esculentum 8.00E-30 531 G1266 AT3G23240 Les_S6682822
Lycopersicon esculentum 8.00E-56 531 G1266 AT3G23240
SGN-UNIGENE-48067 Lycopersicon esculentum 3.00E-38 531 G1266
AT3G23240 SGN-UNIGENE-49923 Lycopersicon esculentum 9.00E-30 531
G1266 AT3G23240 SGN-UNIGENE-52630 Lycopersicon esculentum 2.00E-37
531 G1266 AT3G23240 SGN-UNIGENE- Lycopersicon esculentum 6.00E-19
SINGLET-38956 441 G1322 AT3G01530 Gma_S4904682 Glycine max 1.00E-17
441 G1322 AT3G01530 SGN-UNIGENE-58620 Lycopersicon esculentum
7.00E-67 441 G1322 AT3G01530 SGN-UNIGENE- Lycopersicon esculentum
4.00E-42 SINGLET-16950 441 G1322 AT3G01530 Vvi_S15388842 Vitis
vinifera 4.00E-48 441 G1322 AT3G01530 Zm_S11529147 Zea mays
9.00E-13 533 G1331 AT4G13480 Zm_S11529198 Zea mays 7.00E-18 537
G1494 AT2G43010 Vvi_S16871195 Vitis vinifera 4.00E-46 539 G1535
AT5G46880 SGN-UNIGENE- Lycopersicon esculentum 4.00E-70
SINGLET-13754 539 G1535 AT5G46880 Os_S98061 Oryza sativa 9.00E-11
539 G1535 AT5G46880 Zm_S11418454 Zea mays 1.00E-180 539 G1535
AT5G46880 Zm_S11522858 Zea mays 1.00E-155 445 G1666 AT4G09820
Pta_S17046663 Pinus taeda 7.00E-21 543 G1750 AT4G27950 Les_S5295754
Lycopersicon esculentum 9.00E-38 543 G1750 AT4G27950
SGN-UNIGENE-49801 Lycopersicon esculentum 9.00E-19 543 G1750
AT4G27950 SGN-UNIGENE- Lycopersicon esculentum 1.00E-10
SINGLET-2078 543 G1750 AT4G27950 SGN-UNIGENE- Lycopersicon
esculentum 3.00E-28 SINGLET-446513 547 G1835 AT3G54810 Gma_S4889036
Glycine max 2.00E-33 547 G1835 AT3G54810 Gma_S4911179 Glycine max
2.00E-11 547 G1835 AT3G54810 SGN-UNIGENE-48476 Lycopersicon
esculentum 1.00E-54 547 G1835 AT3G54810 SGN-UNIGENE-51325
Lycopersicon esculentum 4.00E-25 547 G1835 AT3G54810 Ta_S142289
Triticum aestivum 2.00E-25 547 G1835 AT3G54810 Ta_S266353 Triticum
aestivum 1.00E-31 547 G1835 AT3G54810 Vvi_S16865934 Vitis vinifera
1.00E-36 451 G1868 AT4G37740 SGN-UNIGENE-48848 Lycopersicon
esculentum 4.00E-82 451 G1868 AT4G37740 SGN-UNIGENE- Lycopersicon
esculentum 5.00E-25 SINGLET-453383 451 G1868 AT4G37740 Os_S96499
Oryza sativa 7.00E-04 451 G1868 AT4G37740 Pta_S16800293 Pinus taeda
2.00E-08 451 G1868 AT4G37740 Ta_S178842 Triticum aestivum 3.00E-11
451 G1868 AT4G37740 Zm_S11522646 Zea mays 2.00E-14 451 G1868
AT4G37740 Zm_S11522707 Zea mays 9.00E-11 451 G1868 AT4G37740
Zm_S11525236 Zea mays 2.00E-21 453 G1888 AT4G39070
SGN-UNIGENE-47593 Lycopersicon esculentum 7.00E-60 453 G1888
AT4G39070 Mtr_S10820905 Medicago truncatula 2.00E-48 453 G1888
AT4G39070 Os_S60490 Oryza sativa 6.00E-45 453 G1888 AT4G39070
Zm_S11432778 Zea mays 3.00E-19 549 G1930 AT3G25730
SGN-UNIGENE-47598 Lycopersicon esculentum 3.00E-52 549 G1930
AT3G25730 SGN-UNIGENE- Lycopersicon esculentum 5.00E-57
SINGLET-393621 549 G1930 AT3G25730 SGN-UNIGENE- Lycopersicon
esculentum 6.00E-27 SINGLET-44327 549 G1930 AT3G25730 Mtr_S5430627
Medicago truncatula 1.00E-63 549 G1930 AT3G25730 Os_S75175 Oryza
sativa 3.00E-17 549 G1930 AT3G25730 Zm_S11506592 Zea mays 1.00E-37
551 G2057 AT3G15030 Gma_S5029115 Glycine max 9.00E-30 551 G2057
AT3G15030 Les_S5295478 Lycopersicon esculentum 1.00E-95 551 G2057
AT3G15030 SGN-UNIGENE-50577 Lycopersicon esculentum 1.00E-52 551
G2057 AT3G15030 SGN-UNIGENE-58580 Lycopersicon esculentum 1.00E-41
551 G2057 AT3G15030 SGN-UNIGENE- Lycopersicon esculentum 1.00E-21
SINGLET-24189 551 G2057 AT3G15030 SGN-UNIGENE- Lycopersicon
esculentum 8.00E-30 SINGLET-394109 551 G2057 AT3G15030 SGN-UNIGENE-
Lycopersicon esculentum 2.00E-40 SINGLET-401522 551 G2057 AT3G15030
Os_S113396 Oryza sativa 1.00E-48 551 G2057 AT3G15030 Os_S113398
Oryza sativa 1.00E-78 457 G2131 AT1G79700 SGN-UNIGENE- Lycopersicon
esculentum 5.00E-74 SINGLET-517 457 G2131 AT1G79700 Zm_S11528772
Zea mays 3.00E-77 553 G2144 AT3G57800 SGN-UNIGENE-51335
Lycopersicon esculentum 1.00E-21 553 G2144 AT3G57800 Vvi_S16529913
Vitis vinifera 3.00E-39 555 G2145 AT1G27740 Ta_S174040 Triticum
aestivum 3.00E-40 559 G2512 AT1G06160 Hv_S20601 Hordeum vulgare
9.00E-15 559 G2512 AT1G06160 SGN-UNIGENE- Lycopersicon esculentum
6.00E-24 SINGLET-2865 459 G2520 AT1G59640 Gma_S5045510 Glycine max
2.00E-46 459 G2520 AT1G59640 Les_S5183164 Lycopersicon esculentum
1.00E-55 459 G2520 AT1G59640 Les_S5203454 Lycopersicon esculentum
1.00E-44 459 G2520 AT1G59640 SGN-UNIGENE-44928 Lycopersicon
esculentum 4.00E-72 459 G2520 AT1G59640 Ta_S84222 Triticum aestivum
3.00E-38 459 G2520 AT1G59640 Vvi_S15421316 Vitis vinifera 7.00E-07
459 G2520 AT1G59640 Vvi_S16529182 Vitis vinifera 6.00E-61 459 G2520
AT1G59640 Zm_S11524369 Zea mays 1.00E-40 461 G2522 AT3G61310
Gma_S4864518 Glycine max 5.00E-24 461 G2522 AT3G61310 Hv_S36040
Hordeum vulgare 4.00E-35 461 G2522 AT3G61310 SGN-UNIGENE-50326
Lycopersicon esculentum 6.00E-40 461 G2522 AT3G61310 Pta_S15767209
Pinus taeda 3.00E-17 461 G2522 AT3G61310 Ta_S115031 Triticum
aestivum 9.00E-09 461 G2522 AT3G61310 Ta_S65435 Triticum aestivum
4.00E-48 461 G2522 AT3G61310 Vvi_S15370801 Vitis vinifera 1.00E-55
563 G2535 AT3G61910 Gma_S5137324 Glycine max 4.00E-12 563 G2535
AT3G61910 SGN-UNIGENE- Lycopersicon esculentum 1.00E-72
SINGLET-366637 567 G2719 AT3G55730 SGN-UNIGENE- Lycopersicon
esculentum 5.00E-52 SINGLET-357168 567 G2789 AT3G60870 Gma_S4935598
Glycine max 2.00E-67 247 G2789 AT3G60870 Pta_S15799222 Pinus taeda
6.00E-43 247 G2789 AT3G60870 Pta_S16786360 Pinus taeda 2.00E-70 247
G2789 AT3G60870 Pta_S16788492 Pinus taeda 7.00E-63 247 G2789
AT3G60870 Pta_S16802054 Pinus taeda 1.00E-57 423 G38 AT5G05410
Gma_S4861946 Glycine max 6.00E-42 423 G38 AT5G05410 Hv_S230730
Hordeum vulgare 4.00E-47 423 G38 AT5G05410 Hv_S230731 Hordeum
vulgare 3.00E-44 423 G38 AT5G05410 Les_S6682824 Lycopersicon
esculentum 2.00E-52 423 G38 AT5G05410 Os_S116939 Oryza sativa
9.00E-45 423 G38 AT5G05410 Ta_S266443 Triticum aestivum 9.00E-42
423 G38 AT5G05410 Zm_S11524426 Zea mays 4.00E-41 827 G44 AT5G61600
Vvi_S15378188 Vitis vinifera 1.00E-04 827 G44 AT5G61600
Vvi_S15402707 Vitis vinifera 4.00E-42 827 G44 AT5G61600
Vvi_S16082016 Vitis vinifera 1.00E-41 829 G230 AT2G23290
Gma_S4873244 Glycine max 8.00E-08 829 G230 AT2G23290 Gma_S4897857
Glycine max 2.00E-69 829 G230 AT2G23290 SGN-UNIGENE-46140
Lycopersicon esculentum 1.00E-79 829 G230 AT2G23290
SGN-UNIGENE-46445 Lycopersicon esculentum 3.00E-71 829 G230
AT2G23290 SGN-UNIGENE- Lycopersicon esculentum 3.00E-45
SINGLET-396033 829 G230 AT2G23290 Mtr_S5318648 Medicago truncatula
5.00E-67 829 G230 AT2G23290 Mtr_S5421663 Medicago truncatula
1.00E-48 829 G230 AT2G23290 Mtr_S5454442 Medicago truncatula
4.00E-09 829 G230 AT2G23290 Vvi_S15351083 Vitis vinifera 2.00E-15
829 G230 AT2G23290 Vvi_S15373434 Vitis vinifera 4.00E-10 479 G234
AT3G49690 SGN-UNIGENE- Lycopersicon esculentum 3.00E-57
SINGLET-21166 479 G234 AT3G49690 Zm_S11529159 Zea mays 3.00E-15 479
G234 AT3G49690 Zm_S11529194 Zea mays 3.00E-16 831 G261 AT4G18880
Gma_S5144289 Glycine max 4.00E-34 831 G261 AT4G18880
SGN-UNIGENE-51749 Lycopersicon esculentum 9.00E-76 831 G261
AT4G18880 SGN-UNIGENE-59194 Lycopersicon esculentum 3.00E-24 831
G261 AT4G18880 Mtr_S7091605 Medicago truncatula 2.00E-50 831 G261
AT4G18880 Os_S23803 Oryza sativa 8.00E-12 831 G261 AT4G18880
Os_S83230 Oryza sativa 1.00E-66 831 G261 AT4G18880 Pta_S15769714
Pinus taeda 2.00E-45 831 G261 AT4G18880 Vvi_S15370308 Vitis
vinifera 5.00E-21 831 G261 AT4G18880 Vvi_S15413763 Vitis vinifera
4.00E-08 831 G261 AT4G18880 Zm_S11521772 Zea mays 6.00E-26 839 G388
AT1G79840 SGN-UNIGENE- Lycopersicon esculentum 3.00E-36
SINGLET-2889 839 G388 AT1G79840 SGN-UNIGENE- Lycopersicon
esculentum 8.00E-24 SINGLET-393604 839 G388 AT1G79840 Vvi_S15431305
Vitis vinifera 3.00E-66 841 G435 AT5G53980 SGN-UNIGENE-
Lycopersicon esculentum 1.00E-24 SINGLET-385221 845 G468 AT2G46990
Os_S100653 Oryza sativa 6.00E-05 845 G468 AT2G46990 Vvi_S16820866
Vitis vinifera 5.00E-37 847 G571 AT5G06839 SGN-UNIGENE-
Lycopersicon esculentum 9.00E-49 SINGLET-312251 847 G571 AT5G06839
SGN-UNIGENE- Lycopersicon esculentum 3.00E-56 SINGLET-39818 231
G634 AT1G33240 Pta_S17050439 Pinus taeda 3.00E-39 231 G634
AT1G33240 Zm_S11449298 Zea mays 3.00E-35 849 G652 AT2G21060
Gma_S4871214 Glycine max 2.00E-20 849 G652 AT2G21060 Gma_S4965905
Glycine max 3.00E-26 849 G652 AT2G21060 Gma_S5135351 Glycine max
1.00E-18 849 G652 AT2G21060 Hv_S142991 Hordeum vulgare 6.00E-47 849
G652 AT2G21060 Hv_S147464 Hordeum vulgare 1.00E-22 849 G652
AT2G21060 Les_S5162139 Lycopersicon esculentum 3.00E-15 849 G652
AT2G21060 SGN-UNIGENE-56979 Lycopersicon esculentum 1.00E-40 849
G652 AT2G21060 Os_S46064 Oryza sativa 4.00E-36 849 G652 AT2G21060
Pta_S15741898 Pinus taeda 7.00E-49 849 G652 AT2G21060 Ta_S2509
Triticum aestivum 5.00E-05 849 G652 AT2G21060 Ta_S45732 Triticum
aestivum 1.00E-22 849 G652 AT2G21060 Ta_S60357 Triticum aestivum
6.00E-14 849 G652 AT2G21060 Ta_S75244 Triticum aestivum 3.00E-66
849 G652 AT2G21060 Vvi_S16864906 Vitis vinifera 5.00E-16 849 G652
AT2G21060 Vvi_S16965349 Vitis vinifera 1.00E-18 849 G652 AT2G21060
Zm_S11487070 Zea mays 1.00E-51 851 G664 AT4G38620 Gma_S4875209
Glycine max 6.00E-71 851 G664 AT4G38620 Gma_S5069370 Glycine max
3.00E-78 851 G664 AT4G38620 Hv_S73887 Hordeum vulgare 3.00E-77 851
G664 AT4G38620 Hv_S73888 Hordeum vulgare 3.00E-71 851 G664
AT4G38620 Les_S5295913 Lycopersicon esculentum 1.00E-89 851 G664
AT4G38620 SGN-UNIGENE-48139 Lycopersicon esculentum 1.00E-89 851
G664 AT4G38620 SGN-UNIGENE-52314 Lycopersicon esculentum 3.00E-76
851 G664 AT4G38620 SGN-UNIGENE-58669 Lycopersicon esculentum
5.00E-49 851 G664 AT4G38620 SGN-UNIGENE- Lycopersicon esculentum
3.00E-04 SINGLET-56292 851 G664 AT4G38620 Mtr_S5321074 Medicago
truncatula 2.00E-76 851 G664 AT4G38620 Mtr_S5436024 Medicago
truncatula 1.00E-38 851 G664 AT4G38620 Os_S60586 Oryza sativa
7.00E-67 851 G664 AT4G38620 Os_S96599 Oryza sativa 4.00E-06 851
G664 AT4G38620 Pta_S15736913 Pinus taeda 7.00E-65 851 G664
AT4G38620 Pta_S16787963 Pinus taeda 3.00E-74 851 G664 AT4G38620
Pta_S16796777 Pinus taeda 7.00E-16 851 G664 AT4G38620 Pta_S16796852
Pinus taeda 4.00E-52 851 G664 AT4G38620 Pta_S16800437 Pinus taeda
1.00E-21 851 G664 AT4G38620 Pta_S16802819 Pinus taeda 2.00E-66 851
G664 AT4G38620 Pta_S17046107 Pinus taeda 1.00E-64 851 G664
AT4G38620 Pta_S17052332 Pinus taeda 6.00E-29 851 G664 AT4G38620
Ta_S207746 Triticum aestivum 2.00E-73 851 G664 AT4G38620
Vvi_S15352484 Vitis vinifera 1.00E-10 851 G664 AT4G38620
Vvi_S15427762 Vitis vinifera 3.00E-88 851 G664 AT4G38620
Zm_S11519370 Zea mays 2.00E-46 851 G664 AT4G38620 Zm_S11521958 Zea
mays 1.00E-73 851 G664 AT4G38620 Zm_S11524344 Zea mays 1.00E-79 851
G664 AT4G38620 Zm_S11529167 Zea mays 2.00E-18 851 G664 AT4G38620
Zm_S11529181 Zea mays 5.00E-18 853 G772 AT3G10480 Gma_S5023840
Glycine max 1.00E-25 853 G772 AT3G10480 SGN-UNIGENE-49809
Lycopersicon esculentum 2.00E-87 853 G772 AT3G10480 Mtr_S5395615
Medicago truncatula 4.00E-10 855 G798 AT3G50410 Mtr_S5415694
Medicago truncatula 2.00E-15 859 G974 AT1G22190 Gma_S4897318
Glycine max 3.00E-18 859 G974 AT1G22190 Gma_S4897472 Glycine max
3.00E-30 859 G974 AT1G22190 Gma_S4898590 Glycine max 2.00E-57 859
G974 AT1G22190 Hv_S10412 Hordeum vulgare 2.00E-08 859 G974
AT1G22190 Hv_S70023 Hordeum vulgare 1.00E-14 859 G974 AT1G22190
Les_S5182292 Lycopersicon esculentum 4.00E-60 859 G974 AT1G22190
SGN-UNIGENE-44095 Lycopersicon esculentum 4.00E-76 859 G974
AT1G22190 SGN-UNIGENE-44231 Lycopersicon esculentum 1.00E-68 859
G974 AT1G22190 Mtr_S7093809 Medicago truncatula 4.00E-28 859 G974
AT1G22190 Os_S37084 Oryza sativa 2.00E-05 859 G974 AT1G22190
Pta_S16845578 Pinus taeda 3.00E-26 859 G974 AT1G22190 Ta_S120947
Triticum aestivum 5.00E-11 859 G974 AT1G22190 Ta_S184473 Triticum
aestivum 1.00E-17 859 G974 AT1G22190 Ta_S278378 Triticum aestivum
1.00E-09 859 G974 AT1G22190 Vvi_S15351270 Vitis vinifera 3.00E-39
859 G974 AT1G22190 Vvi_S15407610 Vitis vinifera 1.00E-60 859 G974
AT1G22190 Zm_S11323940 Zea mays 2.00E-29 859 G974 AT1G22190
Zm_S11490783 Zea mays 2.00E-45 859 G974 AT1G22190 Zm_S11528582 Zea
mays 1.00E-44 435 G988 AT1G55580 Les_S5295726 Lycopersicon
esculentum 1.00E-114 807 G1048 AT1G42990 Gma_S4871472 Glycine max
3.00E-07 807 G1048 AT1G42990 SGN-UNIGENE-45931 Lycopersicon
esculentum 2.00E-38 807 G1048 AT1G42990 Mtr_S5316975 Medicago
truncatula 7.00E-29 807 G1048 AT1G42990 Ta_S244122 Triticum
aestivum 3.00E-18 807 G1048 AT1G42990 Vvi_S15353884 Vitis vinifera
4.00E-30 807 G1048 AT1G42990 Zm_S11527760 Zea mays 1.00E-24 861
G1062 AT3G26744 Gma_S4932282 Glycine max 5.00E-43 861 G1062
AT3G26744 Les_S5250575 Lycopersicon esculentum 5.00E-70 861 G1062
AT3G26744 SGN-UNIGENE-45946 Lycopersicon esculentum 1.00E-102 861
G1062 AT3G26744 SGN-UNIGENE- Lycopersicon esculentum 5.00E-07
SINGLET-106 861 G1062 AT3G26744 SGN-UNIGENE- Lycopersicon
esculentum 2.00E-10 SINGLET-107 861 G1062 AT3G26744 SGN-UNIGENE-
Lycopersicon esculentum 2.00E-60 SINGLET-395584 861 G1062 AT3G26744
SGN-UNIGENE- Lycopersicon esculentum 1.00E-20 SINGLET-399204 861
G1062 AT3G26744 SGN-UNIGENE- Lycopersicon esculentum 2.00E-07
SINGLET-459012 861 G1062 AT3G26744 Pta_S15739278 Pinus taeda
4.00E-18 861 G1062 AT3G26744 Vvi_S15370409 Vitis vinifera 5.00E-47
239 G1069 AT4G14465 SGN-UNIGENE-59076 Lycopersicon esculentum
6.00E-55 239 G1069 AT4G14465 Mtr_S5308977 Medicago truncatula
2.00E-31 239 G1069 AT4G14465 Vvi_S16805621 Vitis vinifera 1.00E-04
863 G1129 AT4G34530 Vvi_S16532165 Vitis vinifera 4.00E-53 865 G1137
AT5G64340 SGN-UNIGENE-48745 Lycopersicon esculentum 3.00E-11 865
G1137 AT5G64340 SGN-UNIGENE-48746 Lycopersicon esculentum 6.00E-23
657 G1412 AT4G27410 Gma_S5050636 Glycine max 5.00E-84 657 G1412
AT4G27410 Les_S5295623 Lycopersicon esculentum 1.00E-105 657 G1412
AT4G27410 SGN-UNIGENE-45948 Lycopersicon esculentum 1.00E-105 657
G1412 AT4G27410 SGN-UNIGENE-48215 Lycopersicon esculentum 1.00E-105
657 G1412 AT4G27410 Vvi_S15352716 Vitis vinifera 0.41 867 G1425
AT1G52880 Les_S5247376 Lycopersicon esculentum 6.00E-83 867 G1425
AT1G52880 SGN-UNIGENE-44943 Lycopersicon esculentum 1.00E-95 867
G1425 AT1G52880 SGN-UNIGENE-46578 Lycopersicon esculentum 8.00E-93
867 G1425 AT1G52880 Pta_S16844825 Pinus taeda 1.00E-56 867 G1425
AT1G52880 Pta_S17050992 Pinus taeda 2.00E-54 871 G1655 AT1G09250
Gma_S4865861 Glycine max 4.00E-17 871 G1655 AT1G09250
SGN-UNIGENE-47983 Lycopersicon esculentum 1.00E-37 875 G1789
AT2G21650 Gma_S4886781 Glycine max 7.00E-25 875 G1789 AT2G21650
Les_S5295408 Lycopersicon esculentum 2.00E-28 875 G1789 AT2G21650
Ta_S102809 Triticum aestivum 3.00E-22 875 G1789 AT2G21650 Ta_S56880
Triticum aestivum 1.00E-18 875 G1789 AT2G21650 Vvi_S15406920 Vitis
vinifera 3.00E-13 875 G1789 AT2G21650 Vvi_S15424752 Vitis vinifera
7.00E-26 875 G1789 AT2G21650 Vvi_S16784697 Vitis vinifera 1.00E-27
875 G1789 AT2G21650 Zm_S11328185 Zea mays 8.00E-14 875 G1789
AT2G21650 Zm_S11474298 Zea mays 2.00E-16 877 G1806 AT1G68640
Gma_S4902665 Glycine max 3.00E-19 877 G1806 AT1G68640 Gma_S4911209
Glycine max 5.00E-65 877 G1806 AT1G68640 Gma_S5146796 Glycine max
1.00E-139 877 G1806 AT1G68640 Hv_S227616 Hordeum vulgare 2.00E-42
877 G1806 AT1G68640 Hv_S27170 Hordeum vulgare 4.00E-52 877 G1806
AT1G68640 Les_S5295407 Lycopersicon esculentum 1.00E-120 877 G1806
AT1G68640 Les_S5295673 Lycopersicon esculentum 9.00E-99 877 G1806
AT1G68640 SGN-UNIGENE-46372 Lycopersicon esculentum 3.00E-78 877
G1806 AT1G68640 SGN-UNIGENE-46373 Lycopersicon esculentum 1.00E-134
877 G1806 AT1G68640 SGN-UNIGENE-47327 Lycopersicon esculentum
1.00E-139 877 G1806 AT1G68640 SGN-UNIGENE-49500 Lycopersicon
esculentum 9.00E-51 877 G1806 AT1G68640 SGN-UNIGENE-50258
Lycopersicon esculentum 4.00E-89 877 G1806 AT1G68640
SGN-UNIGENE-57605 Lycopersicon esculentum 4.00E-06 877 G1806
AT1G68640 SGN-UNIGENE-57705 Lycopersicon esculentum 3.00E-84 877
G1806 AT1G68640 SGN-UNIGENE-58538 Lycopersicon esculentum 6.00E-97
877 G1806 AT1G68640 SGN-UNIGENE- Lycopersicon esculentum 6.00E-04
SINGLET-318510 877 G1806 AT1G68640 SGN-UNIGENE- Lycopersicon
esculentum 6.00E-26 SINGLET-340722 877 G1806 AT1G68640 SGN-UNIGENE-
Lycopersicon esculentum 8.00E-63 SINGLET-43282 877 G1806 AT1G68640
Mtr_S7091737 Medicago truncatula 8.00E-29 877 G1806 AT1G68640
Os_S107700 Oryza sativa 4.00E-04 877 G1806 AT1G68640 Os_S83289
Oryza sativa 1.00E-144 877 G1806 AT1G68640 Os_S83290 Oryza sativa
1.00E-139 877 G1806 AT1G68640 Os_S83291 Oryza sativa 1.00E-139 877
G1806 AT1G68640 Os_S83292 Oryza sativa 1.00E-138 877 G1806
AT1G68640 Pta_S17047774 Pinus taeda 1.00E-56 877 G1806 AT1G68640
Pta_S17049082 Pinus taeda 5.00E-17 877 G1806 AT1G68640 Ta_S115084
Triticum aestivum 1.00E-19 877 G1806 AT1G68640 Ta_S141705 Triticum
aestivum 5.00E-10 877 G1806 AT1G68640 Ta_S142610 Triticum aestivum
2.00E-15 877 G1806 AT1G68640 Ta_S66308 Triticum aestivum 1.00E-136
877 G1806 AT1G68640 Ta_S66461 Triticum aestivum 1.00E-142 877 G1806
AT1G68640 Vvi_S15429865 Vitis vinifera 2.00E-76 877 G1806 AT1G68640
Vvi_S16526894 Vitis vinifera 1.00E-80 877 G1806 AT1G68640
Zm_S11418176 Zea mays 1.00E-141 877 G1806 AT1G68640 Zm_S11418177
Zea mays 1.00E-138 877 G1806 AT1G68640 Zm_S11418513 Zea mays
1.00E-118 877 G1806 AT1G68640 Zm_S11425511 Zea mays 6.00E-58 877
G1806 AT1G68640 Zm_S11432162 Zea mays 4.00E-29 879 G1911 AT4G39250
Gma_S4886781 Glycine max 7.00E-25 879 G1911 AT4G39250 Les_S5295408
Lycopersicon esculentum 2.00E-28 879 G1911 AT4G39250 Ta_S102809
Triticum aestivum 3.00E-22 879 G1911 AT4G39250 Ta_S56880 Triticum
aestivum 1.00E-18 879 G1911 AT4G39250 Vvi_S15406920 Vitis vinifera
3.00E-13 879 G1911 AT4G39250 Vvi_S15424752 Vitis vinifera 7.00E-26
879 G1911 AT4G39250 Vvi_S16784697 Vitis vinifera 1.00E-27 879 G1911
AT4G39250 Zm_S11328185 Zea mays 8.00E-14 879 G1911 AT4G39250
Zm_S11474298 Zea mays 2.00E-16 813 G1995 AT3G58070
SGN-UNIGENE-54039 Lycopersicon esculentum 1.00E-22 813 G1995
AT3G58070 SGN-UNIGENE-54252 Lycopersicon esculentum 8.00E-32 813
G1995 AT3G58070 SGN-UNIGENE- Lycopersicon esculentum 7.00E-36
SINGLET-392715 813 G1995 AT3G58070 Pta_S15742384 Pinus taeda
1.00E-09 881 G2011 AT5G03720 Les_S5182191 Lycopersicon esculentum
8.00E-29 881 G2011 AT5G03720 SGN-UNIGENE-47254 Lycopersicon
esculentum 1.00E-28 881 G2011 AT5G03720 Os_S100853 Oryza sativa
1.00E-56 881 G2011 AT5G03720 Ta_S147235 Triticum aestivum 4.00E-14
315 G2155 AT1G14490 Gma_S5081748 Glycine max 2.00E-48 315 G2155
AT1G14490 SGN-UNIGENE-48878 Lycopersicon esculentum 5.00E-81 315
G2155 AT1G14490 SGN-UNIGENE- Lycopersicon esculentum 8.00E-48
SINGLET-471786 315 G2155 AT1G14490 Pta_S16802278 Pinus taeda
2.00E-34 885 G2452 AT5G01200 Zm_S11467963 Zea mays 4.00E-15 815
G2467 AT3G63350 SGN-UNIGENE-45592 Lycopersicon esculentum 7.00E-57
889 G2510 AT1G01250 Gma_S4877810 Glycine max 4.00E-41 889 G2510
AT1G01250 Gma_S5065417 Glycine max 2.00E-15 889 G2510 AT1G01250
Mtr_S5455425 Medicago truncatula 1.00E-49 889 G2510 AT1G01250
Pta_S15772552 Pinus taeda 6.00E-29 889 G2510 AT1G01250
Pta_S15778451 Pinus taeda 6.00E-21 889 G2510 AT1G01250
Vvi_S15351722 Vitis vinifera 2.00E-27 819 G2550 AT1G75410
Mtr_S7094331 Medicago truncatula 8.00E-41 819 G2550 AT1G75410
Zm_S11465618 Zea mays 1.00E-47 893 G2571 AT1G64380
SGN-UNIGENE-56732 Lycopersicon esculentum 0.25 821 G2640 AT3G51060
SGN-UNIGENE-58699 Lycopersicon esculentum 8.00E-42 821 G2640
AT3G51060 SGN-UNIGENE- Lycopersicon esculentum 9.00E-36
SINGLET-461966 895 G2702 AT3G08500 Gma_S5127272 Glycine max
2.00E-05 895 G2702 AT3G08500 Pta_S16807545 Pinus taeda 3.00E-56 897
G2763 AT3G17100 Pta_S16793632 Pinus taeda 2.00E-10 897 G2763
AT3G17100 Vvi_S16806536 Vitis vinifera 6.00E-26 899 G2774 AT4G05170
Mtr_S5310210 Medicago truncatula 9.00E-13 247 G2789 AT3G60870
Gma_S4935598 Glycine max 2.00E-67 247 G2789 AT3G60870 Pta_S15799222
Pinus taeda 6.00E-43 247 G2789 AT3G60870 Pta_S16786360 Pinus taeda
2.00E-70 247 G2789 AT3G60870 Pta_S16788492 Pinus taeda 7.00E-63 247
G2789 AT3G60870 Pta_S16802054 Pinus taeda 1.00E-57 901 G2888
AT1G25250 SGN-UNIGENE- Lycopersicon esculentum 3.00E-37
SINGLET-25079 901 G2888 AT1G25250 Os_S101092 Oryza sativa 4.00E-10
901 G2888 AT1G25250 Zm_S11429840 Zea mays 2.00E-41
[0347] Table 9 lists the gene identification number (GID) and
homologous relationships found using analyses according to the
Examples for the sequences of the Sequence Listing. Those sequences
listed as "reference sequences" were originally determined by
experimentation to confer drought tolerance when their expression
was altered. Generally, each reference sequence was used to
identify the clade in which functionally related homologous
sequences may be found.
TABLE-US-00010 TABLE 9 Homologs and Other Related Genes of
Representative Arabidopsis Transcription Factor Genes Identified
using BLAST Polynucleotide SEQ (DNA) or Species from Which ID
polypeptide Homologous Relationship of SEQ ID NO: to Other NO: GID
No: (PRT) Sequence is Derived Genes 1 G47 DNA Arabidopsis thaliana
Reference sequence; predicted polypeptide sequence is paralogous to
G2133 2 G47 PRT Arabidopsis thaliana Reference sequence; paralogous
to G2133 3 G922 DNA Arabidopsis thaliana Reference sequence 4 G922
PRT Arabidopsis thaliana Reference sequence 5 G1274 DNA Arabidopsis
thaliana Reference sequence 6 G1274 PRT Arabidopsis thaliana
Reference sequence 7 G1792 DNA Arabidopsis thaliana Reference
sequence 8 G1792 PRT Arabidopsis thaliana Reference sequence 9
G2053 DNA Arabidopsis thaliana Reference sequence 10 G2053 PRT
Arabidopsis thaliana Reference sequence 11 G2133 DNA Arabidopsis
thaliana Reference sequence; predicted polypeptide sequence is
paralogous to G47 12 G2133 PRT Arabidopsis thaliana Reference
sequence; paralogous to G47 13 G2999 DNA Arabidopsis thaliana
Reference sequence 14 G2999 PRT Arabidopsis thaliana Reference
sequence 15 G3086 DNA Arabidopsis thaliana Reference sequence 16
G3086 PRT Arabidopsis thaliana Reference sequence 17 G30 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1792 18 G30 PRT Arabidopsis thaliana Paralogous to G1792 19
G515 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2053 20 G515 PRT Arabidopsis thaliana Paralogous to
G2053 21 G516 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2053 22 G516 PRT Arabidopsis thaliana
Paralogous to G2053 23 G517 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2053 24 G517 PRT Arabidopsis
thaliana Paralogous to G2053 25 G592 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G3086 26 G592 PRT
Arabidopsis thaliana Paralogous to G3086 27 G1134 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G3086 28
G1134 PRT Arabidopsis thaliana Paralogous to G3086 29 G1275 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1274 30 G1275 PRT Arabidopsis thaliana Paralogous to G1274 31
G1758 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1274 32 G1758 PRT Arabidopsis thaliana Paralogous to
G1274 33 G1791 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1792 34 G1791 PRT Arabidopsis thaliana
Paralogous to G1792 35 G1795 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1792 36 G1795 PRT
Arabidopsis thaliana Paralogous to G1792 37 G2149 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G3086 38
G2149 PRT Arabidopsis thaliana Paralogous to G3086 39 G2555 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G3086 40 G2555 PRT Arabidopsis thaliana Paralogous to G3086 41
G2766 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G3086 42 G2766 PRT Arabidopsis thaliana Paralogous to
G3086 43 G2989 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2999 44 G2989 PRT Arabidopsis thaliana
Paralogous to G2999 45 G2990 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2999 46 G2990 PRT
Arabidopsis thaliana Paralogous to G2999 47 G2991 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G2999 48
G2991 PRT Arabidopsis thaliana Paralogous to G2999 49 G2992 DNA
Arabidopsis thaliana Reference sequence; predicted polypeptide
sequence is paralogous to G2999 50 G2992 PRT Arabidopsis thaliana
Reference sequence; paralogous to G2999 51 G2993 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G2999 52
G2993 PRT Arabidopsis thaliana Paralogous to G2999 53 G2994 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2999 54 G2994 PRT Arabidopsis thaliana Paralogous to G2999 55
G2995 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2999 56 G2995 PRT Arabidopsis thaliana Paralogous to
G2999 57 G2996 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2999 58 G2996 PRT Arabidopsis thaliana
Paralogous to G2999 59 G2997 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2999 60 G2997 PRT
Arabidopsis thaliana Paralogous to G2999 61 G2998 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G2999 62
G2998 PRT Arabidopsis thaliana Paralogous to G2999 63 G3000 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2999 64 G3000 PRT Arabidopsis thaliana Paralogous to G2999 65
G3001 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2999 66 G3001 PRT Arabidopsis thaliana Paralogous to
G2999 67 G3002 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2999 68 G3002 PRT Arabidopsis thaliana
Paralogous to G2999 69 G3380 DNA Oryza sativa Predicted polypeptide
sequence is orthologous to G1792 70 G3380 PRT Oryza sativa
Orthologous to G1792 71 G3381 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G1792 72 G3381 PRT Oryza
sativa Orthologous to G1792 73 G3383 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G1792 74 G3383 PRT Oryza
sativa Orthologous to G1792 75 G3515 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G1792 76 G3515 PRT Oryza
sativa Orthologous to G1792 77 G3516 DNA Zea mays Predicted
polypeptide sequence is orthologous to G1792 78 G3516 PRT Zea mays
Orthologous to G1792 79 G3517 DNA Zea mays Predicted polypeptide
sequence is orthologous to G1792 80 G3517 PRT Zea mays Orthologous
to G1792 81 G3518 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1792 82 G3518 PRT Glycine max Orthologous to G1792
83 G3519 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1792 84 G3519 PRT Glycine max Orthologous to G1792
85 G3520 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1792 86 G3520 PRT Glycine max Orthologous to G1792
87 G3643 DNA Glycine max Predicted polypeptide sequence is
orthologous to G47 88 G3643 PRT Glycine max Orthologous to G47 89
G3644 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G47 90 G3644 PRT Oryza sativa Orthologous to G47 91
G3645 DNA Brassica rapa subsp. Predicted polypeptide sequence is
Pekinensis orthologous to G47 92 G3645 PRT Brassica rapa subsp.
Orthologous to G47 Pekinensis 93 G3646 DNA Brassica oleracea
Predicted polypeptide sequence is orthologous to G47 94 G3646 PRT
Brassica oleracea Orthologous to G47 95 G3647 DNA Zinnia elegans
Predicted polypeptide sequence is orthologous to G47 96 G3647 PRT
Zinnia elegans Orthologous to G47 97 G3649 DNA Oryza sativa
Predicted polypeptide sequence is orthologous to G47 98 G3649 PRT
Oryza sativa Orthologous to G47 99 G3651 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G47 100 G3651 PRT Oryza
sativa Orthologous to G47 101 G3663 DNA Lotus corniculatus
Predicted polypeptide sequence is var. japonicus orthologous to
G2999 102 G3663 PRT Lotus corniculatus Orthologous to G2999 var.
japonicus 103 G3668 DNA Flaveria bidentis Predicted polypeptide
sequence is orthologous to G2999 104 G3668 PRT Flaveria bidentis
Orthologous to G2999 105 G3670 DNA Lotus corniculatus Predicted
polypeptide sequence is var. japonicus orthologous to G2999 106
G3670 PRT Lotus corniculatus Orthologous to G2999 var. japonicus
107 G3671 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 108 G3671 PRT Oryza sativa Orthologous to
G2999 109 G3674 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 110 G3674 PRT Oryza sativa Orthologous to
G2999 111 G3675 DNA Brassica napus Predicted polypeptide sequence
is orthologous to G2999 112 G3675 PRT Brassica napus Orthologous to
G2999 113 G3680 DNA Zea mays Predicted polypeptide sequence is
orthologous to G2999 114 G3680 PRT Zea mays Orthologous to G2999
115 G3683 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 116 G3683 PRT Oryza sativa Orthologous to
G2999 117 G3685 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 118 G3685 PRT Oryza sativa Orthologous to
G2999 119 G3686 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 120 G3686 PRT Oryza sativa Orthologous to
G2999 121 G3690 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 122 G3690 PRT Oryza sativa Orthologous to
G2999 123 G3692 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 124 G3692 PRT Oryza sativa Orthologous to
G2999 125 G3694 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 126 G3694 PRT Oryza sativa Orthologous to
G2999 127 G3695 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G2999 128 G3695 PRT Oryza sativa Orthologous to
G2999 129 G3719 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1274 130 G3719 PRT Zea mays Orthologous to G1274
131 G3720 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1274 132 G3720 PRT Zea mays Orthologous to G1274
133 G3721 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1274 134 G3721 PRT Oryza sativa Orthologous to
G1274 135 G3722 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1274 136 G3722 PRT Zea mays Orthologous to G1274
137 G3723 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1274 138 G3723 PRT Glycine max Orthologous to G1274
139 G3724 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1274 140 G3724 PRT Glycine max Orthologous to G1274
141 G3725 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1274 142 G3725 PRT Oryza sativa Orthologous to
G1274 143 G3726 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1274 144 G3726 PRT Oryza sativa Orthologous to
G1274 145 G3727 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1274 146 G3727 PRT Zea mays Orthologous to G1274
147 G3728 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1274 148 G3728 PRT Zea mays Orthologous to G1274
149 G3729 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1274 150 G3729 PRT Oryza sativa Orthologous to
G1274 151 G3730 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1274 152 G3730 PRT Oryza sativa Orthologous to
G1274 153 G3731 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G1274 154 G3731 PRT Lycopersicon
Orthologous to G1274 esculentum 155 G3732 DNA Solanum tuberosum
Predicted polypeptide sequence is orthologous to G1274 156 G3732
PRT Solanum tuberosum Orthologous to G1274 157 G3733 DNA Hordeum
vulgare Predicted polypeptide sequence is orthologous to G1274
158 G3733 PRT Hordeum vulgare Orthologous to G1274 159 G3735 DNA
Medicago truncatula Predicted polypeptide sequence is orthologous
to G1792 160 G3735 PRT Medicago truncatula Orthologous to G1792 161
G3736 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G1792 162 G3736 PRT Triticum aestivum Orthologous to
G1792 163 G3737 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1792 164 G3737 PRT Oryza sativa Orthologous to
G1792 165 G3739 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1792 166 G3739 PRT Zea mays Orthologous to G1792
167 G3740 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G3086 168 G3740 PRT Oryza sativa Orthologous to
G3086 169 G3741 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G3086 170 G3741 PRT Oryza sativa Orthologous to
G3086 171 G3742 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G3086 172 G3742 PRT Oryza sativa Orthologous to
G3086 173 G3744 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G3086 174 G3744 PRT Oryza sativa Orthologous to
G3086 175 G3746 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G3086 176 G3746 PRT Oryza sativa Orthologous to
G3086 177 G3755 DNA Zea mays Predicted polypeptide sequence is
orthologous to G3086 178 G3755 PRT Zea mays Orthologous to G3086
179 G3763 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 180 G3763 PRT Glycine max Orthologous to G3086
181 G3764 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 182 G3764 PRT Glycine max Orthologous to G3086
183 G3765 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 184 G3765 PRT Glycine max Orthologous to G3086
185 G3766 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 186 G3766 PRT Glycine max Orthologous to G3086
187 G3767 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 188 G3767 PRT Glycine max Orthologous to G3086
189 G3768 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 190 G3768 PRT Glycine max Orthologous to G3086
191 G3769 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 192 G3769 PRT Glycine max Orthologous to G3086
193 G3771 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 194 G3771 PRT Glycine max Orthologous to G3086
195 G3772 DNA Glycine max Predicted polypeptide sequence is
orthologous to G3086 196 G3772 PRT Glycine max Orthologous to G3086
197 G3782 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G3086 198 G3782 PRT Pinus taeda Orthologous to G3086
199 G3794 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1792 200 G3794 PRT Zea mays Orthologous to G1792
201 G3795 DNA Capsicum annuum Predicted polypeptide sequence is
orthologous to G1274 202 G3795 PRT Capsicum annuum Orthologous to
G1274 203 G3797 DNA Lactuca sativa Predicted polypeptide sequence
is orthologous to G1274 204 G3797 PRT Lactuca sativa Orthologous to
G1274 205 G3802 DNA Sorghum bicolor Predicted polypeptide sequence
is orthologous to G1274 206 G3802 PRT Sorghum bicolor Orthologous
to G1274 207 G3803 DNA Glycine max Predicted polypeptide sequence
is orthologous to G1274 208 G3803 PRT Glycine max Orthologous to
G1274 209 G3804 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1274 210 G3804 PRT Zea mays Orthologous to G1274
211 G3810 DNA Glycine max Predicted polypeptide sequence is
orthologous to G922 212 G3810 PRT Glycine max Orthologous to G922
213 G3811 DNA Glycine max Predicted polypeptide sequence is
orthologous to G922 214 G3811 PRT Glycine max Orthologous to G922
215 G3813 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G922 216 G3813 PRT Oryza sativa Orthologous to G922
217 G3814 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G922 218 G3814 PRT Oryza sativa Orthologous to G922
219 G3824 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G922 220 G3824 PRT Lycopersicon
Orthologous to G922 esculentum 221 G3827 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G922 222 G3827 PRT Oryza
sativa Orthologous to G922 223 G175 DNA Arabidopsis thaliana
Reference sequence 224 G175 PRT Arabidopsis thaliana Reference
sequence 225 G303 DNA Arabidopsis thaliana Reference sequence 226
G303 PRT Arabidopsis thaliana Reference sequence 227 G354 DNA
Arabidopsis thaliana Reference sequence 228 G354 PRT Arabidopsis
thaliana Reference sequence 229 G489 DNA Arabidopsis thaliana
Reference sequence 230 G489 PRT Arabidopsis thaliana Reference
sequence 231 G634 DNA Arabidopsis thaliana Reference sequence 232
G634 PRT Arabidopsis thaliana Reference sequence 233 G682 DNA
Arabidopsis thaliana Reference sequence 234 G682 PRT Arabidopsis
thaliana Reference sequence 235 G916 DNA Arabidopsis thaliana
Reference sequence 236 G916 PRT Arabidopsis thaliana Reference
sequence 237 G975 DNA Arabidopsis thaliana Reference sequence;
predicted polypeptide sequence is paralogous to G2583 238 G975 PRT
Arabidopsis thaliana Reference sequence; paralogous to G2583 239
G1069 DNA Arabidopsis thaliana Reference sequence; functionally
related, homologous to G1073 240 G1069 PRT Arabidopsis thaliana
Reference sequence; functionally related, homologous to G1073 241
G1452 DNA Arabidopsis thaliana Reference sequence; functionally
related, homologous to G512 242 G1452 PRT Arabidopsis thaliana
Reference sequence; functionally related, homologous to G512 243
G1820 DNA Arabidopsis thaliana Reference sequence 244 G1820 PRT
Arabidopsis thaliana Reference sequence 245 G2701 DNA Arabidopsis
thaliana Reference sequence; predicted polypeptide sequence is
paralogous to G1634 246 G2701 PRT Arabidopsis thaliana Reference
sequence; paralogous to G1634 247 G2789 DNA Arabidopsis thaliana
Reference sequence; predicted polypeptide sequence is paralogous to
G596 248 G2789 PRT Arabidopsis thaliana Reference sequence;
paralogous to G596 249 G2839 DNA Arabidopsis thaliana Reference
sequence; predicted polypeptide sequence is paralogous to G354 250
G2839 PRT Arabidopsis thaliana Reference sequence; paralogous to
G354 251 G2854 DNA Arabidopsis thaliana Reference sequence;
predicted polypeptide sequence is paralogous to G1940 252 G2854 PRT
Arabidopsis thaliana Reference sequence; paralogous to G1940 253
G3083 DNA Arabidopsis thaliana Reference sequence 254 G3083 PRT
Arabidopsis thaliana Reference sequence 255 G184 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G916 256
G184 PRT Arabidopsis thaliana Paralogous to G916 257 G186 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G916 258 G186 PRT Arabidopsis thaliana Paralogous to G916 259
G353 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G354 260 G353 PRT Arabidopsis thaliana Paralogous to
G354 261 G512 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1452 262 G512 PRT Arabidopsis thaliana
Paralogous to G1452 263 G596 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2789 264 G596 PRT
Arabidopsis thaliana Paralogous to G2789 265 G714 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G489 266
G714 PRT Arabidopsis thaliana Paralogous to G489 267 G877 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G175 268 G877 PRT Arabidopsis thaliana Paralogous to G175 269
G1357 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1452 270 G1357 PRT Arabidopsis thaliana Paralogous
to G1452 271 G1387 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G975 272 G1387 PRT Arabidopsis thaliana
Paralogous to G975 273 G1634 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2701 274 G1634 PRT
Arabidopsis thaliana Paralogous to G2701 275 G1889 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G354 276
G1889 PRT Arabidopsis thaliana Paralogous to G354 277 G1940 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2854 278 G1940 PRT Arabidopsis thaliana Paralogous to G2854 279
G1974 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G354 280 G1974 PRT Arabidopsis thaliana Paralogous to
G354 281 G2153 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1073 282 G2153 PRT Arabidopsis thaliana
Paralogous to G1073 283 G2583 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G975 284 G2583 PRT
Arabidopsis thaliana Paralogous to G975 285 G226 DNA Arabidopsis
thaliana Reference sequence; predicted polypeptide sequence is
paralogous to G682 286 G226 PRT Arabidopsis thaliana Reference
sequence; paralogous to G682 287 G481 DNA Arabidopsis thaliana
Reference sequence; predicted polypeptide sequence is paralogous to
G482 288 G481 PRT Arabidopsis thaliana Reference sequence;
paralogous to G482 289 G482 DNA Arabidopsis thaliana Reference
sequence; predicted polypeptide sequence is paralogous to G481 290
G482 PRT Arabidopsis thaliana Reference sequence; paralogous to
G481 291 G485 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G481 and G482 292 G485 PRT Arabidopsis
thaliana Paralogous to G481 and G482 293 G486 DNA Arabidopsis
thaliana Functionally related and homologous to G481 and G482 294
G486 PRT Arabidopsis thaliana Functionally related and homologous
to G481 and G482 295 G1067 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1073 296 G1067 PRT
Arabidopsis thaliana Paralogous to G1073 297 G1070 DNA Arabidopsis
thaliana Functionally related and homologous to G1073 298 G1070 PRT
Arabidopsis thaliana Functionally related and homologous to G1073
299 G1073 DNA Arabidopsis thaliana Reference sequence 300 G1073 PRT
Arabidopsis thaliana Reference sequence 301 G1075 DNA Arabidopsis
thaliana Functionally related and homologous to G1073 302 G1075 PRT
Arabidopsis thaliana Functionally related and homologous to G1073
303 G1076 DNA Arabidopsis thaliana Functionally related and
homologous to G1073 304 G1076 PRT Arabidopsis thaliana Functionally
related and homologous to G1073 305 G1248 DNA Arabidopsis thaliana
Functionally related and homologous to G481 and G482 306 G1248 PRT
Arabidopsis thaliana Functionally related and homologous to G481
and G482 307 G1364 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G481 and G482 308 G1364 PRT Arabidopsis
thaliana Paralogous to G481 and G482 309 G1781 DNA Arabidopsis
thaliana Functionally related and homologous to G481 and G482 310
G1781 PRT Arabidopsis thaliana Functionally related and homologous
to G481 and G482 311 G1816 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G226 and G682 312 G1816 PRT
Arabidopsis thaliana Paralogous to G226 and G682 313 G1945 DNA
Arabidopsis thaliana Functionally related and homologous to G1073
314 G1945 PRT Arabidopsis thaliana Functionally related and
homologous to G1073
315 G2155 DNA Arabidopsis thaliana Functionally related and
homologous to G1073 316 G2155 PRT Arabidopsis thaliana Functionally
related and homologous to G1073 317 G2156 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1073 318 G2156 PRT
Arabidopsis thaliana Paralogous to G1073 319 G2345 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G481 and
G482 320 G2345 PRT Arabidopsis thaliana Paralogous to G481 and G482
321 G2657 DNA Arabidopsis thaliana Functionally related and
homologous to G1073 322 G2657 PRT Arabidopsis thaliana Functionally
related and homologous to G1073 323 G2718 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G481 and G482 324
G2718 PRT Arabidopsis thaliana Paralogous to G481 and G482 325
G3392 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G682 326 G3392 PRT Oryza sativa Orthologous to G682
327 G3393 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G682 328 G3393 PRT Oryza sativa Orthologous to G682
329 G3394 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G481 and G482 330 G3394 PRT Oryza sativa Orthologous
to G481 and G482 331 G3395 DNA Oryza sativa Predicted polypeptide
sequence is orthologous to G481 and G482 332 G3395 PRT Oryza sativa
Orthologous to G481 and G482 333 G3396 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G481 and G482 334 G3396 PRT
Oryza sativa Orthologous to G481 and G482 335 G3397 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G481 and
G482 336 G3397 PRT Oryza sativa Orthologous to G481 and G482 337
G3398 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G481 and G482 338 G3398 PRT Oryza sativa Orthologous
to G481 and G482 339 G3399 DNA Oryza sativa Predicted polypeptide
sequence is orthologous to G1073 340 G3399 PRT Oryza sativa
Orthologous to G1073 341 G3400 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G1073 342 G3400 PRT Oryza
sativa Orthologous to G1073 343 G3401 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G1073 344 G3401 PRT Oryza
sativa Orthologous to G1073 345 G3403 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G1073 346 G3403 PRT Oryza
sativa Orthologous to G1073 347 G3404 DNA Oryza sativa Functionally
related and homologous to G1073 348 G3404 PRT Oryza sativa
Functionally related and homologous to G1073 349 G3405 DNA Oryza
sativa Functionally related and homologous to G1073 350 G3405 PRT
Oryza sativa Functionally related and homologous to G1073 351 G3406
DNA Oryza sativa Functionally related and homologous to G1073 352
G3406 PRT Oryza sativa Functionally related and homologous to G1073
353 G3407 DNA Oryza sativa Functionally related and homologous to
G1073 354 G3407 PRT Oryza sativa Functionally related and
homologous to G1073 355 G3408 DNA Oryza sativa Functionally related
and homologous to G1073 356 G3408 PRT Oryza sativa Functionally
related and homologous to G1073 357 G3429 DNA Oryza sativa
Predicted polypeptide sequence is orthologous to G481 and G482 358
G3429 PRT Oryza sativa Orthologous to G481 and G482 359 G3431 DNA
Zea mays Predicted polypeptide sequence is orthologous to G682 360
G3431 PRT Zea mays Orthologous to G682 361 G3434 DNA Zea mays
Predicted polypeptide sequence is orthologous to G481 and G482 362
G3434 PRT Zea mays Orthologous to G481 and G482 363 G3435 DNA Zea
mays Predicted polypeptide sequence is orthologous to G481 and G482
364 G3435 PRT Zea mays Orthologous to G481 and G482 365 G3436 DNA
Zea mays Predicted polypeptide sequence is orthologous to G481 and
G482 366 G3436 PRT Zea mays Orthologous to G481 and G482 367 G3437
DNA Zea mays Predicted polypeptide sequence is orthologous to G481
and G482 368 G3437 PRT Zea mays Orthologous to G481 and G482 369
G3444 DNA Zea mays Predicted polypeptide sequence is orthologous to
G682 370 G3444 PRT Zea mays Orthologous to G682 371 G3445 DNA
Glycine max Predicted polypeptide sequence is orthologous to G682
372 G3445 PRT Glycine max Orthologous to G682 373 G3446 DNA Glycine
max Predicted polypeptide sequence is orthologous to G682 374 G3446
PRT Glycine max Orthologous to G682 375 G3447 DNA Glycine max
Predicted polypeptide sequence is orthologous to G682 376 G3447 PRT
Glycine max Orthologous to G682 377 G3448 DNA Glycine max Predicted
polypeptide sequence is orthologous to G682 378 G3448 PRT Glycine
max Orthologous to G682 379 G3449 DNA Glycine max Predicted
polypeptide sequence is orthologous to G682 380 G3449 PRT Glycine
max Orthologous to G682 381 G3450 DNA Glycine max Predicted
polypeptide sequence is orthologous to G682 382 G3450 PRT Glycine
max Orthologous to G682 383 G3456 DNA Glycine max Predicted
polypeptide sequence is orthologous to G1073 384 G3456 PRT Glycine
max Orthologous to G1073 385 G3458 DNA Glycine max Functionally
related and homologous to G1073 386 G3458 PRT Glycine max
Functionally related and homologous to G1073 387 G3459 DNA Glycine
max Predicted polypeptide sequence is functionally related and
homologous to G1073 388 G3459 PRT Glycine max Functionally related
and homologous to G1073 389 G3460 DNA Glycine max Predicted
polypeptide sequence is functionally related and homologous to
G1073 390 G3460 PRT Glycine max Functionally related and homologous
to G1073 391 G3462 DNA Glycine max Predicted polypeptide sequence
is orthologous to G1073 392 G3462 PRT Glycine max Orthologous to
G1073 393 G3470 DNA Glycine max Predicted polypeptide sequence is
orthologous to G481 and G482 394 G3470 PRT Glycine max Orthologous
to G481 and G482 395 G3471 DNA Glycine max Predicted polypeptide
sequence is orthologous to G481 and G482 396 G3471 PRT Glycine max
Orthologous to G481 and G482 397 G3472 DNA Glycine max Predicted
polypeptide sequence is orthologous to G481 and G482 398 G3472 PRT
Glycine max Orthologous to G481 and G482 399 G3473 DNA Glycine max
Predicted polypeptide sequence is orthologous to G481 and G482 400
G3473 PRT Glycine max Orthologous to G481 and G482 401 G3474 DNA
Glycine max Predicted polypeptide sequence is orthologous to G481
and G482 402 G3474 PRT Glycine max Orthologous to G481 and G482 403
G3475 DNA Glycine max Predicted polypeptide sequence is orthologous
to G481 and G482 404 G3475 PRT Glycine max Orthologous to G481 and
G482 405 G3476 DNA Glycine max Predicted polypeptide sequence is
orthologous to G481 and G482 406 G3476 PRT Glycine max Orthologous
to G481 and G482 407 G3477 DNA Glycine max Predicted polypeptide
sequence is orthologous to G481 and G482 408 G3477 PRT Glycine max
Orthologous to G481 and G482 409 G3478 DNA Glycine max Predicted
polypeptide sequence is orthologous to G481 and G482 410 G3478 PRT
Glycine max Orthologous to G481 and G482 411 G3556 DNA Oryza sativa
Predicted polypeptide sequence is orthologous to G1073 412 G3556
PRT Oryza sativa Orthologous to G1073 413 G3835 DNA Oryza sativa
Predicted polypeptide sequence is orthologous to G481 and G482 414
G3835 PRT Oryza sativa Orthologous to G481 and G482 415 G3836 DNA
Oryza sativa Predicted polypeptide sequence is orthologous to G481
and G482 416 G3836 PRT Oryza sativa Orthologous to G481 and G482
417 G3837 DNA Glycine max Predicted polypeptide sequence is
orthologous to G481 and G482 418 G3837 PRT Glycine max Orthologous
to G481 and G482 419 G24 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G12, G1277, G1379;
orthologous to G3656 420 G24 PRT Arabidopsis thaliana Paralogous to
G12, G1277, G1379; orthologous to G3656 421 G154 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1011 422
G154 PRT Arabidopsis thaliana Paralogous to G1011 423 G384 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1588, G385 424 G384 PRT Arabidopsis thaliana Paralogous to
G1588, G385 425 G545 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G350, G351 426 G545 PRT Arabidopsis
thaliana Paralogous to G350, G351 427 G760 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G3041 428 G760 PRT
Arabidopsis thaliana Paralogous to G3041 429 G773 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1412,
G759 430 G773 PRT Arabidopsis thaliana Paralogous to G1412, G759
433 G971 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G914 434 G971 PRT Arabidopsis thaliana Paralogous to
G914 435 G988 DNA Arabidopsis thaliana 436 G988 PRT Arabidopsis
thaliana 441 G1322 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G221, G249 442 G1322 PRT Arabidopsis
thaliana Paralogous to G221, G249 449 G1818 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1836 450
G1818 PRT Arabidopsis thaliana Paralogous to G1836 451 G1868 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1439 452 G1868 PRT Arabidopsis thaliana Paralogous to G1439 453
G1888 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1482 454 G1888 PRT Arabidopsis thaliana Paralogous
to G1482 457 G2131 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2106, G979 458 G2131 PRT Arabidopsis
thaliana Paralogous to G2106, G979 461 G2522 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1071 462
G2522 PRT Arabidopsis thaliana Paralogous to G1071 465 G27 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1386, G441 466 G27 PRT Arabidopsis thaliana Paralogous to
G1386, G441 471 G168 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G170, G2065 472 G168 PRT Arabidopsis
thaliana Paralogous to G170, G2065 479 G234 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G232 480
G234 PRT Arabidopsis thaliana Paralogous to G232 481 G237 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1309 482 G237 PRT Arabidopsis thaliana Paralogous to G1309 483
G275 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G829, G837 484 G275 PRT Arabidopsis thaliana
Paralogous to G829, G837 485 G326 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1337 486 G326 PRT
Arabidopsis thaliana Paralogous to G1337 489 G427 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G2545,
G425, G426 490 G427 PRT Arabidopsis thaliana Paralogous to G2545,
G425, G426 495 G602 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1065 496 G602 PRT Arabidopsis thaliana
Paralogous to G1065 497 G618 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2057 498 G618 PRT
Arabidopsis thaliana Paralogous to G2057 503 G653 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G654 504
G653 PRT Arabidopsis thaliana Paralogous to G654 507 G837 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G275, G829 508 G837 PRT Arabidopsis thaliana Paralogous to G275,
G829 509 G866 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G883 510 G866 PRT Arabidopsis thaliana
Paralogous to G883 511 G872 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2576; orthologous to G3652,
G3653, G3654, G3655 512 G872 PRT Arabidopsis thaliana Paralogous to
G2576; orthologous to
G3652, G3653, G3654, G3655 515 G912 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G40, G2107, G2513,
G41, G42; orthologous to G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 516 G912 PRT
Arabidopsis thaliana Paralogous to G40, G2107, G2513, G41, G42;
orthologous to G3362, G3364, G3365, G3366, G3367, G3368, G3370,
G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379,
G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 517 G932 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G256, G666, G668; orthologous to G3384, G3385, G3386, G3500,
G3501, G3502, G3537, G3538, G3539, G3540, G3541 518 G932 PRT
Arabidopsis thaliana Paralogous to G256, G666, G668; orthologous to
G3384, G3385, G3386, G3500, G3501, G3502, G3537, G3538, G3539,
G3540, G3541 519 G958 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2180, G518 520 G958 PRT
Arabidopsis thaliana Paralogous to G2180, G518 521 G964 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G398, G399 522 G964 PRT Arabidopsis thaliana Paralogous to G398,
G399 523 G979 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2106, G2131 524 G979 PRT Arabidopsis
thaliana Paralogous to G2106, G2131 525 G1049 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G572 526
G1049 PRT Arabidopsis thaliana Paralogous to G572 529 G1255 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1484 530 G1255 PRT Arabidopsis thaliana Paralogous to G1484 537
G1494 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G789 538 G1494 PRT Arabidopsis thaliana Paralogous to
G789 539 G1535 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G389 540 G1535 PRT Arabidopsis thaliana
Paralogous to G389 543 G1750 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G864, G440 544 G1750 PRT
Arabidopsis thaliana Paralogous to G864, G440 549 G1930 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G867, G9, G993; orthologous to G3388, G3389, G3390, G3391,
G3432, G3433, G3451, G3452, G3453, G3454 550 G1930 PRT Arabidopsis
thaliana Paralogous to G867, G9, G993; orthologous to G3388, G3389,
G3390, G3391, G3432, G3433, G3451, G3452, G3453, G3454 551 G2057
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G618 552 G2057 PRT Arabidopsis thaliana Paralogous to
G618 553 G2144 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1942 554 G2144 PRT Arabidopsis thaliana
Paralogous to G1942 555 G2145 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2148 556 G2145 PRT
Arabidopsis thaliana Paralogous to G2148 559 G2512 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1752 560
G2512 PRT Arabidopsis thaliana Paralogous to G1752 563 G2535 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G957, G961 564 G2535 PRT Arabidopsis thaliana Paralogous to
G957, G961 567 G2719 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G216 568 G2719 PRT Arabidopsis thaliana
Paralogous to G216 569 G9 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1930, G867, G993;
orthologous to G3388, G3389, G3390, G3391, G3432, G3433, G3451,
G3452, G3453, G3454 570 G9 PRT Arabidopsis thaliana Paralogous to
G1930, G867, G993; orthologous to G3388, G3389, G3390, G3391,
G3432, G3433, G3451, G3452, G3453, G3454 571 G12 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1277,
G1379, G24; orthologous to G3656 572 G12 PRT Arabidopsis thaliana
Paralogous to G1277, G1379, G24; orthologous to G3656 573 G40 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2107, G2513, G41, G42, G912; orthologous to G3362, G3364,
G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373, G3374,
G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441,
G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465, G3466,
G3467, G3468, G3469 574 G40 PRT Arabidopsis thaliana Paralogous to
G2107, G2513, G41, G42, G912; orthologous to G3362, G3364, G3365,
G3366, G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375,
G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442,
G3369, G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467,
G3468, G3469 575 G41 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G40, G2107, G2513, G42, G912; orthologous
to G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372,
G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3438, G3439,
G3440, G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464,
G3465, G3466, G3467, G3468, G3469 576 G41 PRT Arabidopsis thaliana
Paralogous to G40, G2107, G2513, G42, G912; orthologous to G3362,
G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 577 G42 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G40, G2107, G2513,
G41, G912; orthologous to G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 578 G42 PRT
Arabidopsis thaliana Paralogous to G40, G2107, G2513, G41, G912;
orthologous to G3362, G3364, G3365, G3366, G3367, G3368, G3370,
G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379,
G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 579 G170 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G168, G2065 580 G170 PRT Arabidopsis thaliana Paralogous to
G168, G2065 581 G216 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2719 582 G216 PRT Arabidopsis thaliana
Paralogous to G2719 583 G221 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1322, G249 584 G221 PRT
Arabidopsis thaliana Paralogous to G1322, G249 585 G232 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G234 586 G232 PRT Arabidopsis thaliana Paralogous to G234 587
G249 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1322, G221 588 G249 PRT Arabidopsis thaliana
Paralogous to G1322, G221 589 G256 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G666, G668, G932;
orthologous to G3384, G3385, G3386, G3500, G3501, G3502, G3537,
G3538, G3539, G3540, G3541 590 G256 PRT Arabidopsis thaliana
Paralogous to G666, G668, G932; orthologous to G3384, G3385, G3386,
G3500, G3501, G3502, G3537, G3538, G3539, G3540, G3541 591 G350 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G351, G545 592 G350 PRT Arabidopsis thaliana Paralogous to G351,
G545 593 G351 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G350, G545 594 G351 PRT Arabidopsis
thaliana Paralogous to G350, G545 595 G385 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1588, G384 596
G385 PRT Arabidopsis thaliana Paralogous to G1588, G384 597 G389
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1535 598 G389 PRT Arabidopsis thaliana Paralogous to
G1535 599 G398 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G399, G964 600 G398 PRT Arabidopsis
thaliana Paralogous to G399, G964 601 G399 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G398, G964 602 G399
PRT Arabidopsis thaliana Paralogous to G398, G964 603 G425 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2545, G426, G427 604 G425 PRT Arabidopsis thaliana Paralogous
to G2545, G426, G427 605 G426 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2545, G425, G427 606 G426
PRT Arabidopsis thaliana Paralogous to G2545, G425, G427 607 G440
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G864, G1750 608 G440 PRT Arabidopsis thaliana
Paralogous to G864, G1750 609 G441 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1386, G27 610 G441
PRT Arabidopsis thaliana Paralogous to G1386, G27 611 G518 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2180, G958 612 G518 PRT Arabidopsis thaliana Paralogous to
G2180, G958 613 G572 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1049 614 G572 PRT Arabidopsis thaliana
Paralogous to G1049 615 G654 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G653 616 G654 PRT Arabidopsis
thaliana Paralogous to G653 617 G666 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G256, G668, G932;
orthologous to G3384, G3385, G3386, G3500, G3501, G3502, G3537,
G3538, G3539, G3540, G3541 618 G666 PRT Arabidopsis thaliana
Paralogous to G256, G668, G932; orthologous to G3384, G3385, G3386,
G3500, G3501, G3502, G3537, G3538, G3539, G3540, G3541 619 G668 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G256, G666, G932; orthologous to G3384, G3385, G3386, G3500,
G3501, G3502, G3537, G3538, G3539, G3540, G3541 620 G668 PRT
Arabidopsis thaliana Paralogous to G256, G666, G932; orthologous to
G3384, G3385, G3386, G3500, G3501, G3502, G3537, G3538, G3539,
G3540, G3541 621 G759 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1412, G773 622 G759 PRT
Arabidopsis thaliana Paralogous to G1412, G773 623 G789 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1494 624 G789 PRT Arabidopsis thaliana Paralogous to G1494 625
G829 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G275, G837 626 G829 PRT Arabidopsis thaliana
Paralogous to G275, G837 627 G864 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1750, G440
628 G864 PRT Arabidopsis thaliana Paralogous to G1750, G440 629
G867 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1930, G9, G993; orthologous to G3388, G3389, G3390,
G3391, G3432, G3433, G3451, G3452, G3453, G3454 630 G867 PRT
Arabidopsis thaliana Paralogous to G1930, G9, G993; orthologous to
G3388, G3389, G3390, G3391, G3432, G3433, G3451, G3452, G3453,
G3454 631 G883 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G866 632 G883 PRT Arabidopsis thaliana
Paralogous to G866 633 G914 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G971 634 G914 PRT Arabidopsis
thaliana Paralogous to G971 635 G957 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G2535, G961 636
G957 PRT Arabidopsis thaliana Paralogous to G2535, G961 637 G961
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2535, G957 638 G961 PRT Arabidopsis thaliana
Paralogous to G2535, G957 639 G993 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1930, G867, G9;
orthologous to G3388, G3389, G3390, G3391, G3432, G3433, G3451,
G3452, G3453, G3454 640 G993 PRT Arabidopsis thaliana Paralogous to
G1930, G867, G9; orthologous to G3388, G3389, G3390, G3391, G3432,
G3433, G3451, G3452, G3453, G3454 641 G1011 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G154 642
G1011 PRT Arabidopsis thaliana Paralogous to G154 643 G1065 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G602 644 G1065 PRT Arabidopsis thaliana Paralogous to G602 645
G1071 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2522 646 G1071 PRT Arabidopsis thaliana Paralogous
to G2522 647 G1277 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G12, G1379, G24; orthologous to G3656 648
G1277 PRT Arabidopsis thaliana Paralogous to G12, G1379, G24;
orthologous to G3656 649 G1309 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G237 650 G1309 PRT
Arabidopsis thaliana Paralogous to G237 651 G1337 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G326 652
G1337 PRT Arabidopsis thaliana Paralogous to G326 653 G1379 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G12, G1277, G24; orthologous to G3656 654 G1379 PRT Arabidopsis
thaliana Paralogous to G12, G1277, G24; orthologous to G3656 655
G1386 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G27, G441 656 G1386 PRT Arabidopsis thaliana
Paralogous to G27, G441 657 G1412 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G759, G773 658
G1412 PRT Arabidopsis thaliana Paralogous to G759, G773 659 G1439
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1868 660 G1439 PRT Arabidopsis thaliana Paralogous
to G1868 661 G1482 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1888 662 G1482 PRT Arabidopsis thaliana
Paralogous to G1888 663 G1484 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1255 664 G1484 PRT
Arabidopsis thaliana Paralogous to G1255 665 G1588 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G384, G385
666 G1588 PRT Arabidopsis thaliana Paralogous to G384, G385 667
G1752 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2512 668 G1752 PRT Arabidopsis thaliana Paralogous
to G2512 669 G1836 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1818 670 G1836 PRT Arabidopsis thaliana
Paralogous to G1818 671 G1942 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2144 672 G1942 PRT
Arabidopsis thaliana Paralogous to G2144 673 G2065 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G168, G170
674 G2065 PRT Arabidopsis thaliana Paralogous to G168, G170 675
G2106 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2131, G979 676 G2106 PRT Arabidopsis thaliana
Paralogous to G2131, G979 677 G2107 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G40, G2513, G41,
G42, G912; orthologous to G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 678 G2107
PRT Arabidopsis thaliana Paralogous to G40, G2513, G41, G42, G912;
orthologous to G3362, G3364, G3365, G3366, G3367, G3368, G3370,
G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379,
G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 679 G2148 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2145 680 G2148 PRT Arabidopsis thaliana Paralogous to G2145 681
G2180 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G518, G958 682 G2180 PRT Arabidopsis thaliana
Paralogous to G518, G958 683 G2513 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G40, G2107, G41,
G42, G912; orthologous to G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 684 G2513
PRT Arabidopsis thaliana Paralogous to G40, G2107, G41, G42, G912;
orthologous to G3362, G3364, G3365, G3366, G3367, G3368, G3370,
G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379,
G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 685 G2545 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G425, G426, G427 686 G2545 PRT Arabidopsis thaliana Paralogous
to G425, G426, G427 687 G2576 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G872; orthologous to G3652,
G3653, G3654, G3655 688 G2576 PRT Arabidopsis thaliana Paralogous
to G872; orthologous to G3652, G3653, G3654, G3655 689 G3041 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G760 690 G3041 PRT Arabidopsis thaliana Paralogous to G760 691
G3362 DNA Medicago truncatula Predicted polypeptide sequence is
paralogous to G3364, G3365, G3366, G3367, G3368, G3369; orthologous
to G40, G2107, G2513, G41, G42, G912, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440,
G3441, G3442, G3497, G3498, G3499, G3463, G3464, G3465, G3466,
G3467, G3468, G3469 692 G3362 PRT Medicago truncatula Paralogous to
G3364, G3365, G3366, G3367, G3368, G3369; orthologous to G40,
G2107, G2513, G41, G42, G912, G3370, G3371, G3372, G3373, G3374,
G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441,
G3442, G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467,
G3468, G3469 693 G3364 DNA Medicago truncatula Predicted
polypeptide sequence is paralogous to G3362, G3365, G3366, G3367,
G3368, G3369; orthologous to G40, G2107, G2513, G41, G42, G912,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 694 G3364 PRT
Medicago truncatula Paralogous to G3362, G3365, G3366, G3367,
G3368, G3369; orthologous to G40, G2107, G2513, G41, G42, G912,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 695 G3365 DNA
Medicago truncatula Predicted polypeptide sequence is paralogous to
G3362, G3364, G3366, G3367, G3368, G3369; orthologous to G40,
G2107, G2513, G41, G42, G912, G3370, G3371, G3372, G3373, G3374,
G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441,
G3442, G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467,
G3468, G3469 696 G3365 PRT Medicago truncatula Paralogous to G3362,
G3364, G3366, G3367, G3368, G3369; orthologous to G40, G2107,
G2513, G41, G42, G912, G3370, G3371, G3372, G3373, G3374, G3375,
G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 697 G3366 DNA Medicago truncatula Predicted polypeptide
sequence is paralogous to G3362, G3364, G3365, G3367, G3368, G3369;
orthologous to G40, G2107, G2513, G41, G42, G912, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3438,
G3439, G3440, G3441, G3442, G3497, G3498, G3499, G3463, G3464,
G3465, G3466, G3467, G3468, G3469 698 G3366 PRT Medicago truncatula
Paralogous to G3362, G3364, G3365, G3367, G3368, G3369; orthologous
to G40, G2107, G2513, G41, G42, G912, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440,
G3441, G3442, G3497, G3498, G3499, G3463, G3464, G3465, G3466,
G3467, G3468, G3469 699 G3367 DNA Medicago truncatula Predicted
polypeptide sequence is paralogous to G3362, G3364, G3365, G3366,
G3368, G3369; orthologous to G40, G2107, G2513, G41, G42, G912,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 700 G3367 PRT
Medicago truncatula Paralogous to G3362, G3364, G3365, G3366,
G3368, G3369; orthologous to G40, G2107, G2513, G41, G42, G912,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3497, G3498, G3499,
G3463, G3464,
G3465, G3466, G3467, G3468, G3469 701 G3368 DNA Medicago truncatula
Predicted polypeptide sequence is paralogous to G3362, G3364,
G3365, G3366, G3367, G3369; orthologous to G40, G2107, G2513, G41,
G42, G912, G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377,
G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 702 G3368
PRT Medicago truncatula Paralogous to G3362, G3364, G3365, G3366,
G3367, G3369; orthologous to G40, G2107, G2513, G41, G42, G912,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3497, G3498, G3499,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 703 G3369 DNA
Medicago truncatula Predicted polypeptide sequence is paralogous to
G3362, G3364, G3365, G3366, G3367, G3368; orthologous to G40,
G2107, G2513, G41, G42, G912, G3370, G3371, G3372, G3373, G3374,
G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441,
G3442, G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467,
G3468, G3469 704 G3369 PRT Medicago truncatula Paralogous to G3362,
G3364, G3365, G3366, G3367, G3368; orthologous to G40, G2107,
G2513, G41, G42, G912, G3370, G3371, G3372, G3373, G3374, G3375,
G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 705 G3370 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3371, G3374, G3376, G3378; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3372, G3373, G3375, G3377, G3379, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 706 G3370 PRT Oryza sativa Paralogous to
G3371, G3374, G3376, G3378; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3372, G3373,
G3375, G3377, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 707 G3371 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3370, G3374, G3376, G3378; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3372, G3373, G3375, G3377, G3379, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 708 G3371 PRT Oryza sativa Paralogous to
G3370, G3374, G3376, G3378; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3372, G3373,
G3375, G3377, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 709 G3372 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3373, G3375, G3377, G3379; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3370, G3371, G3374, G3376, G3378, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 710 G3372 PRT Oryza sativa Paralogous to
G3373, G3375, G3377, G3379; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3374, G3376, G3378, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 711 G3373 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3372, G3375, G3377, G3379; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3370, G3371, G3374, G3376, G3378, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 712 G3373 PRT Oryza sativa Paralogous to
G3372, G3375, G3377, G3379; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3374, G3376, G3378, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 713 G3374 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3370, G3371, G3376, G3378; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3372, G3373, G3375, G3377, G3379, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 714 G3374 PRT Oryza sativa Paralogous to
G3370, G3371, G3376, G3378; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3372, G3373,
G3375, G3377, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 715 G3375 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3372, G3373, G3377, G3379; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3370, G3371, G3374, G3376, G3378, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 716 G3375 PRT Oryza sativa Paralogous to
G3372, G3373, G3377, G3379; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3374, G3376, G3378, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 717 G3376 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3370, G3371, G3374, G3378; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3372, G3373, G3375, G3377, G3379, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 718 G3376 PRT Oryza sativa Paralogous to
G3370, G3371, G3374, G3378; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3372, G3373,
G3375, G3377, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 719 G3377 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3372, G3373, G3375, G3379; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3370, G3371, G3374, G3376, G3378, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 720 G3377 PRT Oryza sativa Paralogous to
G3372, G3373, G3375, G3379; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3374, G3376, G3378, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 721 G3378 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3370, G3371, G3374, G3376; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3372, G3373, G3375, G3377, G3379, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 722 G3378 PRT Oryza sativa Paralogous to
G3370, G3371, G3374, G3376; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3372, G3373,
G3375, G3377, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 723 G3379 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3372, G3373, G3375, G3377; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3370, G3371, G3374, G3376, G3378, G3438, G3439, G3440,
G3441, G3442, G3369, G3497, G3498, G3499, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 724 G3379 PRT Oryza sativa Paralogous to
G3372, G3373, G3375, G3377; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3374, G3376, G3378, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 725 G3384 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3385, G3386, G3502; orthologous to G256, G666, G668,
G932, G3500, G3501, G3537, G3538, G3539, G3540, G3541 726 G3384 PRT
Oryza sativa Paralogous to G3385, G3386, G3502; orthologous to
G256, G666, G668, G932, G3500, G3501, G3537, G3538, G3539, G3540,
G3541 727 G3385 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3384, G3386, G3502; orthologous to G256, G666, G668,
G932, G3500, G3501, G3537, G3538, G3539, G3540, G3541 728 G3385 PRT
Oryza sativa Paralogous to G3384, G3386, G3502; orthologous to
G256, G666, G668, G932, G3500, G3501, G3537, G3538, G3539, G3540,
G3541 729 G3386 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3384, G3385, G3502; orthologous to G256, G666, G668,
G932, G3500, G3501, G3537, G3538, G3539, G3540, G3541 730 G3386 PRT
Oryza sativa Paralogous to G3384, G3385, G3502; orthologous to
G256, G666, G668, G932, G3500, G3501, G3537, G3538, G3539, G3540,
G3541 731 G3388 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3389, G3390, G3391; orthologous to G1930, G867, G9,
G993, G3432, G3433, G3451, G3452, G3453, G3454 732 G3388 PRT Oryza
sativa Paralogous to G3389, G3390, G3391; orthologous to G1930,
G867, G9, G993, G3432, G3433, G3451, G3452, G3453, G3454 733 G3389
DNA Oryza sativa Predicted polypeptide sequence is paralogous to
G3388, G3390, G3391; orthologous to G1930, G867, G9, G993, G3432,
G3433, G3451, G3452, G3453, G3454 734 G3389 PRT Oryza sativa
Paralogous to G3388, G3390, G3391; orthologous to G1930, G867, G9,
G993, G3432, G3433, G3451, G3452, G3453, G3454 735 G3390 DNA Oryza
sativa Predicted polypeptide sequence is paralogous to G3388,
G3389, G3391; orthologous to G1930, G867, G9, G993, G3432, G3433,
G3451, G3452, G3453, G3454 736 G3390 PRT Oryza sativa Paralogous to
G3388, G3389, G3391; orthologous to G1930, G867, G9, G993, G3432,
G3433, G3451, G3452, G3453, G3454 737 G3391 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3388, G3389,
G3390; orthologous to G1930, G867, G9, G993, G3432, G3433, G3451,
G3452, G3453, G3454 738 G3391 PRT Oryza sativa Paralogous to G3388,
G3389, G3390; orthologous to G1930, G867, G9, G993, G3432, G3433,
G3451, G3452, G3453, G3454 739 G3432 DNA Zea mays Predicted
polypeptide sequence is paralogous to G3433; orthologous to G1930,
G867, G9, G993, G3388, G3389, G3390, G3391, G3451, G3452, G3453,
G3454 740 G3432 PRT Zea mays Paralogous to G3433; orthologous to
G1930, G867, G9, G993, G3388, G3389, G3390, G3391, G3451, G3452,
G3453, G3454 741 G3433 DNA Zea mays Predicted polypeptide sequence
is paralogous to G3432; orthologous to G1930, G867, G9, G993,
G3388, G3389, G3390, G3391, G3451, G3452, G3453, G3454 742 G3433
PRT Zea mays Paralogous to G3432; orthologous to G1930, G867, G9,
G993, G3388, G3389, G3390, G3391, G3451, G3452, G3453, G3454 743
G3438 DNA Zea mays Predicted polypeptide sequence is paralogous to
G3439, G3440, G3441, G3442; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 744 G3438 PRT Zea mays Paralogous to G3439, G3440, G3441,
G3442; orthologous to G40, G2107, G2513, G41, G42, G912, G3362,
G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 745 G3439
DNA Zea mays Predicted polypeptide sequence is paralogous to G3438,
G3440, G3441, G3442; orthologous to G40, G2107, G2513, G41, G42,
G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 746 G3439 PRT Zea mays Paralogous to G3438, G3440, G3441,
G3442; orthologous to G40, G2107, G2513, G41, G42, G912, G3362,
G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 747 G3440
DNA Zea mays Predicted polypeptide sequence is paralogous to G3438,
G3439, G3441, G3442; orthologous to G40, G2107, G2513, G41, G42,
G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 748 G3440 PRT Zea mays Paralogous to G3438, G3439, G3441,
G3442; orthologous to G40, G2107, G2513, G41, G42, G912, G3362,
G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 749 G3441
DNA Zea mays Predicted polypeptide sequence is paralogous to G3438,
G3439, G3440, G3442; orthologous to G40, G2107, G2513, G41, G42,
G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 750 G3441 PRT Zea mays Paralogous to G3438, G3439, G3440,
G3442; orthologous to G40, G2107, G2513, G41, G42, G912, G3362,
G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 751 G3442
DNA Zea mays Predicted polypeptide sequence is paralogous to G3438,
G3439, G3440, G3441; orthologous to G40, G2107, G2513, G41, G42,
G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3369,
G3497, G3498, G3499, G3463, G3464, G3465, G3466, G3467, G3468,
G3469 752 G3442 PRT Zea mays Paralogous to G3438, G3439, G3440,
G3441; orthologous to G40, G2107, G2513, G41, G42, G912, G3362,
G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373,
G3374, G3375, G3376, G3377, G3378, G3379, G3369, G3497, G3498,
G3499, G3463, G3464, G3465, G3466, G3467, G3468, G3469 753 G3451
DNA Glycine max Predicted polypeptide sequence is paralogous to
G3452, G3453, G3454; orthologous to G1930, G867, G9, G993, G3388,
G3389, G3390, G3391, G3432, G3433 754 G3451 PRT Glycine max
Paralogous to G3452, G3453, G3454; orthologous to G1930, G867, G9,
G993, G3388, G3389, G3390, G3391, G3432, G3433 755 G3452 DNA
Glycine max Predicted polypeptide sequence is paralogous to G3451,
G3453, G3454; orthologous to G1930, G867, G9, G993, G3388, G3389,
G3390, G3391, G3432, G3433 756 G3452 PRT Glycine max Paralogous to
G3451, G3453, G3454; orthologous to G1930, G867, G9, G993, G3388,
G3389, G3390, G3391, G3432, G3433 757 G3453 DNA Glycine max
Predicted polypeptide sequence is paralogous to G3451, G3452,
G3454; orthologous to G1930, G867, G9, G993, G3388, G3389, G3390,
G3391, G3432, G3433 758 G3453 PRT Glycine max Paralogous to G3451,
G3452, G3454; orthologous to G1930, G867, G9, G993, G3388, G3389,
G3390, G3391, G3432, G3433 759 G3454 DNA Glycine max Predicted
polypeptide sequence is paralogous to G3451, G3452, G3453;
orthologous to G1930, G867, G9, G993, G3388, G3389, G3390, G3391,
G3432, G3433 760 G3454 PRT Glycine max Paralogous to G3451, G3452,
G3453; orthologous to G1930, G867, G9, G993, G3388, G3389, G3390,
G3391, G3432, G3433 761 G3463 DNA Glycine max Predicted polypeptide
sequence is paralogous to G3464, G3465, G3466, G3467, G3468, G3469;
orthologous to G40, G2107, G2513, G41, G42, G912, G3362, G3364,
G3365, G3366, G3367, G3368, G3370, G3371, G3372, G3373, G3374,
G3375, G3376, G3377, G3378, G3379, G3438, G3439, G3440, G3441,
G3442, G3369, G3497, G3498, G3499 762 G3463 PRT Glycine max
Paralogous to G3464, G3465, G3466, G3467, G3468, G3469; orthologous
to G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 763 G3464 DNA Glycine max Predicted polypeptide
sequence is paralogous to G3463, G3465, G3466, G3467, G3468, G3469;
orthologous to G40, G2107, G2513, G41, G42, G912, G3362, G3364,
G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 764 G3464 PRT Glycine max Paralogous to G3463,
G3465, G3466, G3467, G3468, G3469; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499 765 G3465 DNA Glycine max Predicted polypeptide sequence is
paralogous to G3463, G3464, G3466, G3467, G3468, G3469; orthologous
to G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 766 G3465 PRT Glycine max Paralogous to G3463,
G3464, G3466, G3467, G3468, G3469; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499 767 G3466 DNA Glycine max Predicted polypeptide sequence is
paralogous to G3463, G3464, G3465, G3467, G3468, G3469; orthologous
to G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 768 G3466 PRT Glycine max Paralogous to G3463,
G3464, G3465, G3467, G3468, G3469; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499 769 G3467 DNA Glycine max Predicted polypeptide sequence is
paralogous to G3463, G3464, G3465, G3466, G3468, G3469; orthologous
to G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 770 G3467 PRT Glycine max Paralogous to G3463,
G3464, G3465, G3466, G3468, G3469; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499 771 G3468 DNA Glycine max Predicted polypeptide sequence is
paralogous to G3463, G3464, G3465, G3466, G3467, G3469; orthologous
to G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 772 G3468 PRT Glycine max Paralogous to G3463,
G3464, G3465, G3466, G3467, G3469; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499 773 G3469 DNA Glycine max Predicted polypeptide sequence is
paralogous to G3463, G3464, G3465, G3466, G3467, G3468; orthologous
to G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3497, G3498, G3499 774 G3469 PRT Glycine max Paralogous to G3463,
G3464, G3465, G3466, G3467, G3468; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3497, G3498,
G3499 775 G3497 DNA Medicago sativa Predicted polypeptide sequence
is paralogous to G3498, G3499; orthologous to G40, G2107, G2513,
G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370,
G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379,
G3438, G3439, G3440, G3441, G3442, G3369, G3463, G3464, G3465,
G3466, G3467, G3468, G3469 776 G3497 PRT Medicago sativa Paralogous
to G3498, G3499; orthologous to G40, G2107, G2513, G41, G42, G912,
G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371, G3372,
G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3438, G3439,
G3440, G3441, G3442, G3369, G3463, G3464, G3465, G3466, G3467,
G3468, G3469 777 G3498 DNA Medicago sativa Predicted polypeptide
sequence is paralogous to G3497, G3499; orthologous to G40, G2107,
G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367, G3368,
G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377, G3378,
G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3463, G3464,
G3465, G3466, G3467, G3468, G3469 778 G3498 PRT Medicago sativa
Paralogous to G3497, G3499; orthologous to G40, G2107, G2513, G41,
G42, G912, G3362, G3364, G3365, G3366, G3367, G3368, G3370, G3371,
G3372, G3373, G3374, G3375, G3376, G3377, G3378, G3379, G3438,
G3439, G3440, G3441, G3442, G3369, G3463, G3464, G3465, G3466,
G3467, G3468, G3469 779 G3499 DNA Medicago sativa Predicted
polypeptide sequence is paralogous to G3497, G3498; orthologous to
G40, G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366,
G3367, G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376,
G3377, G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369,
G3463, G3464, G3465, G3466, G3467, G3468, G3469 780 G3499 PRT
Medicago sativa Paralogous to G3497, G3498; orthologous to G40,
G2107, G2513, G41, G42, G912, G3362, G3364, G3365, G3366, G3367,
G3368, G3370, G3371, G3372, G3373, G3374, G3375, G3376, G3377,
G3378, G3379, G3438, G3439, G3440, G3441, G3442, G3369, G3463,
G3464, G3465, G3466, G3467, G3468, G3469 781 G3500 DNA Lycopersicon
Predicted polypeptide sequence is esculentum paralogous to G3501;
orthologous to G256, G666, G668, G932, G3384, G3385, G3386, G3502,
G3537, G3538, G3539, G3540, G3541 782 G3500 PRT Lycopersicon
Paralogous to G3501; orthologous to esculentum G256, G666, G668,
G932, G3384, G3385, G3386, G3502, G3537, G3538, G3539, G3540, G3541
783 G3501 DNA Lycopersicon Predicted polypeptide sequence is
esculentum paralogous to G3500; orthologous to G256, G666, G668,
G932, G3384, G3385, G3386, G3502, G3537, G3538, G3539, G3540, G3541
784 G3501 PRT Lycopersicon Paralogous to G3500; orthologous to
esculentum G256, G666, G668, G932, G3384, G3385, G3386, G3502,
G3537, G3538, G3539, G3540, G3541 785 G3502 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3384, G3385,
G3386; orthologous to G256, G666, G668, G932, G3500, G3501, G3537,
G3538, G3539, G3540, G3541 786 G3502 PRT Oryza sativa Paralogous to
G3384, G3385, G3386; orthologous to G256, G666, G668, G932, G3500,
G3501, G3537, G3538, G3539, G3540, G3541 787 G3537 DNA Glycine max
Predicted polypeptide sequence is paralogous to G3538, G3539;
orthologous to G256, G666, G668, G932, G3384, G3385, G3386, G3500,
G3501, G3502, G3540, G3541 788 G3537 PRT Glycine max Paralogous to
G3538, G3539; orthologous to G256, G666, G668, G932, G3384, G3385,
G3386, G3500, G3501, G3502, G3540, G3541 789 G3538 DNA Glycine max
Predicted polypeptide sequence is paralogous to G3537, G3539;
orthologous to G256, G666, G668, G932, G3384, G3385, G3386, G3500,
G3501, G3502, G3540, G3541 790 G3538 PRT Glycine max Paralogous to
G3537, G3539; orthologous to G256, G666, G668, G932, G3384, G3385,
G3386, G3500, G3501, G3502, G3540, G3541 791 G3539 DNA Glycine max
Predicted polypeptide sequence is paralogous to G3537, G3538;
orthologous to G256, G666, G668, G932, G3384, G3385, G3386, G3500,
G3501, G3502, G3540, G3541 792 G3539 PRT Glycine max Paralogous to
G3537, G3538; orthologous to G256, G666, G668, G932, G3384, G3385,
G3386, G3500, G3501, G3502, G3540, G3541 793 G3540 DNA Zea mays
Predicted polypeptide sequence is paralogous to G3541; orthologous
to G256, G666, G668, G932, G3384, G3385, G3386, G3500, G3501,
G3502, G3537, G3538, G3539 794 G3540 PRT Zea mays Paralogous to
G3541; orthologous to G256, G666, G668, G932, G3384, G3385, G3386,
G3500, G3501, G3502, G3537, G3538, G3539 795 G3541 DNA Zea mays
Predicted polypeptide sequence is paralogous to G3540; orthologous
to G256, G666, G668, G932, G3384, G3385, G3386, G3500, G3501,
G3502, G3537, G3538, G3539 796 G3541 PRT Zea mays Paralogous to
G3540; orthologous to G256, G666, G668, G932, G3384, G3385, G3386,
G3500, G3501, G3502, G3537, G3538, G3539 797 G3652 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3653, G3654,
G3655; orthologous to G2576, G872 798 G3652 PRT Oryza sativa
Paralogous to G3653, G3654, G3655; orthologous to G2576, G872 799
G3653 DNA Oryza sativa Predicted polypeptide sequence is paralogous
to G3652, G3654, G3655; orthologous to G2576, G872 800 G3653 PRT
Oryza sativa Paralogous to G3652, G3654, G3655; orthologous to
G2576, G872 801 G3654 DNA Oryza sativa Predicted polypeptide
sequence is paralogous to G3652, G3653, G3655; orthologous to
G2576, G872 802 G3654 PRT Oryza sativa Paralogous to G3652, G3653,
G3655; orthologous to G2576, G872 803 G3655 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3652, G3653,
G3654; orthologous to G2576, G872 804 G3655 PRT Oryza sativa
Paralogous to G3652, G3653, G3654; orthologous to G2576, G872 805
G3656 DNA Zea mays Predicted polypeptide sequence is orthologous to
G12, G1277, G1379, G24 806 G3656 PRT Zea mays Orthologous to G12,
G1277, G1379, G24 Os_S32369 DNA Oryza sativa Predicted polypeptide
sequence is orthologous to G24 Os_S80194 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G24 Os_S60918 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G154
Os_S112966 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G384 Os_S113503 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G384 Os_S96499 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G1868
Os_S60490 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1888 Os_S60479 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G200 Os_S100515 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G347
Os_S60901 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G427 Os_S64872 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G427 Os_S64899 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G427
Os_S64900 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G427 Os_S113396 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G618, G2057 Os_S113398 DNA
Oryza sativa Predicted polypeptide sequence is orthologous to G618,
G2057 Os_S76089 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G653 Os_S44434 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G866 Os_S116938 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G912
Os_S116940 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G912 Os_S117813 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G912 Os_S65912 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G912
Os_S61189 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G958 Os_S69951 DNA Oryza sativa Predicted
polypeptide sequence is orthologous to G958 Os_S98061 DNA Oryza
sativa Predicted polypeptide sequence is orthologous to G1535
Os_S75175 DNA Oryza sativa Predicted polypeptide sequence is
orthologous to G1930 Gma_S5071803 DNA Glycine max Predicted
polypeptide sequence is orthologous to G24 Gma_S5094568 DNA Glycine
max Predicted polypeptide sequence is orthologous to G154
Gma_S4992142 DNA Glycine max Predicted polypeptide sequence is
orthologous to G384 Gma_S4873409 DNA Glycine max Predicted
polypeptide sequence is orthologous to G545 Gma_S5146663 DNA
Glycine max Predicted polypeptide sequence is orthologous to G545
Gma_S4883349 DNA Glycine max Predicted polypeptide sequence is
orthologous to G760 Gma_S5050636 DNA Glycine max Predicted
polypeptide sequence is orthologous to G773 Gma_S5129137 DNA
Glycine max Predicted polypeptide sequence is orthologous to G937
Gma_S4904682 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1322 Gma_S5045510 DNA Glycine max Predicted
polypeptide sequence is orthologous to G2520 Gma_S4864518 DNA
Glycine max Predicted polypeptide sequence is orthologous to G2522
Gma_S4935598 DNA Glycine max Predicted polypeptide sequence is
orthologous to G2789 Gma_S4901804 DNA Glycine max Predicted
polypeptide sequence is orthologous to G189 Gma_S4898629 DNA
Glycine max Predicted polypeptide sequence is orthologous to G275,
G837 Gma_S4907362 DNA Glycine max Predicted polypeptide sequence is
orthologous to G275, G837 Gma_S4934838 DNA Glycine max Predicted
polypeptide sequence is orthologous to G347 Gma_S4867945 DNA
Glycine max Predicted polypeptide sequence is orthologous to G427
Gma_S4863794 DNA Glycine max Predicted polypeptide sequence is
orthologous to G602 Gma_S5029115 DNA Glycine max Predicted
polypeptide sequence is orthologous to G618, G2057 Gma_S4874203 DNA
Glycine max Predicted polypeptide sequence is orthologous to G866
Gma_S4886425 DNA Glycine max Predicted polypeptide sequence is
orthologous to G866 Gma_S5106568 DNA Glycine max Predicted
polypeptide sequence is orthologous to G866 Gma_S5001940 DNA
Glycine max Predicted polypeptide sequence is orthologous to G964
Gma_S5131758 DNA Glycine max Predicted polypeptide sequence is
orthologous to G1049 Gma_S4889036 DNA Glycine max Predicted
polypeptide sequence is orthologous to G1835 Gma_S4911179 DNA
Glycine max Predicted polypeptide sequence is orthologous to G1835
Gma_S5137324 DNA Glycine max Predicted polypeptide sequence is
orthologous to G2535 Mtr_S5349908 DNA Medicago truncatula Predicted
polypeptide sequence is orthologous to G24 Mtr_S5357829 DNA
Medicago truncatula Predicted polypeptide sequence is orthologous
to G154 Mtr_S5447672 DNA Medicago truncatula Predicted polypeptide
sequence is orthologous to G384 Mtr_S5317695 DNA Medicago
truncatula Predicted polypeptide sequence is orthologous to G545
Mtr_S5431156 DNA Medicago truncatula Predicted polypeptide sequence
is orthologous to G545 Mtr_S5340844 DNA Medicago truncatula
Predicted polypeptide sequence is orthologous to G760 Mtr_S7090764
DNA Medicago truncatula Predicted polypeptide sequence is
orthologous to G760 Mtr_S10820905 DNA Medicago truncatula Predicted
polypeptide sequence is orthologous to G1888 Mtr_S10821012 DNA
Medicago truncatula Predicted polypeptide sequence is orthologous
to G275, G837 Mtr_S5454462 DNA Medicago truncatula Predicted
polypeptide sequence is orthologous to G347 Mtr_S5306926 DNA
Medicago truncatula Predicted polypeptide sequence is orthologous
to G427 Mtr_S5449876 DNA Medicago truncatula Predicted polypeptide
sequence is orthologous to G427 Mtr_S7092065 DNA Medicago
truncatula Predicted polypeptide sequence is orthologous to G427
Mtr_S5431439 DNA Medicago truncatula Predicted polypeptide sequence
is orthologous to G602 Mtr_S5399163 DNA Medicago truncatula
Predicted polypeptide sequence is orthologous to G635 Mtr_S7091176
DNA Medicago truncatula Predicted polypeptide sequence is
orthologous to G653 Mtr_S5305224 DNA Medicago truncatula Predicted
polypeptide sequence is orthologous to G866 Mtr_S7091692 DNA
Medicago truncatula Predicted polypeptide sequence is orthologous
to G866 Mtr_S5409553 DNA Medicago truncatula Predicted polypeptide
sequence is orthologous to G1255 Mtr_S5430627 DNA Medicago
truncatula Predicted polypeptide sequence is orthologous to G1930
Hv_S30279 DNA Hordeum vulgare Predicted polypeptide sequence is
orthologous to G384 Hv_S36040 DNA Hordeum vulgare Predicted
polypeptide sequence is orthologous to G2522 Hv_S8292 DNA Hordeum
vulgare Predicted polypeptide sequence is orthologous to G275, G837
Hv_S67575 DNA Hordeum vulgare Predicted polypeptide sequence is
orthologous to G326 Hv_S23303 DNA Hordeum vulgare Predicted
polypeptide sequence is orthologous to G427 Hv_S136844 DNA Hordeum
vulgare Predicted polypeptide sequence is orthologous to G653
Hv_S152300 DNA Hordeum vulgare Predicted polypeptide sequence is
orthologous to G912 Hv_S158942 DNA Hordeum vulgare Predicted
polypeptide sequence is orthologous to G912 Hv_S74288 DNA Hordeum
vulgare Predicted polypeptide sequence is orthologous to G912
Hv_S74289 DNA Hordeum vulgare Predicted polypeptide sequence is
orthologous to G912 Hv_S20601 DNA Hordeum vulgare Predicted
polypeptide sequence is orthologous to G2512 Zm_S11418746 DNA Zea
mays Predicted polypeptide sequence is orthologous to G154
Zm_S11527819 DNA Zea mays Predicted polypeptide sequence is
orthologous to G154 Zm_S11333633 DNA Zea mays Predicted polypeptide
sequence is orthologous to G384 Zm_S11401894 DNA Zea mays Predicted
polypeptide sequence is orthologous to G384 Zm_S11418286 DNA Zea
mays Predicted polypeptide sequence is orthologous to G384
Zm_S11418453 DNA Zea mays Predicted polypeptide sequence is
orthologous to G384 Zm_S11418455 DNA Zea mays Predicted polypeptide
sequence is orthologous to G384 Zm_S11523949 DNA Zea mays Predicted
polypeptide sequence is orthologous to G384 Zm_S11441492 DNA Zea
mays Predicted polypeptide sequence is orthologous to G545
Zm_S11443346 DNA Zea mays Predicted polypeptide sequence is
orthologous to G545 Zm_S11465527 DNA Zea mays Predicted polypeptide
sequence is orthologous to G545 Zm_S11526816 DNA Zea mays Predicted
polypeptide sequence is orthologous to G760 Zm_S11529038 DNA Zea
mays Predicted polypeptide sequence is orthologous to G760
Zm_S11529147 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1322 Zm_S11522646 DNA Zea mays Predicted
polypeptide sequence is orthologous to G1868 Zm_S11522707 DNA Zea
mays Predicted polypeptide sequence is orthologous to G1868
Zm_S11525236 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1868 Zm_S11432778 DNA Zea mays Predicted
polypeptide sequence is orthologous to G1888 Zm_S11528772 DNA Zea
mays Predicted polypeptide sequence is orthologous to G2131, G979
Zm_S11524369 DNA Zea mays Predicted polypeptide sequence is
orthologous to G2520 Zm_S11529138 DNA Zea mays Predicted
polypeptide sequence is orthologous to G200 Zm_S11529143 DNA Zea
mays Predicted polypeptide sequence is orthologous to G200
Zm_S11529165 DNA Zea mays Predicted polypeptide sequence is
orthologous to G200 Zm_S11529159 DNA Zea mays Predicted polypeptide
sequence is orthologous to G234 Zm_S11529194 DNA Zea mays Predicted
polypeptide sequence is orthologous to G234 Zm_S11528144 DNA Zea
mays Predicted polypeptide sequence is orthologous to G275, G837
Zm_S11450524 DNA Zea mays Predicted polypeptide sequence is
orthologous to G326 Zm_S11510508 DNA Zea mays Predicted polypeptide
sequence is
orthologous to G326 Zm_S11437336 DNA Zea mays Predicted polypeptide
sequence is orthologous to G347 Zm_S11520104 DNA Zea mays Predicted
polypeptide sequence is orthologous to G347 Zm_S11442066 DNA Zea
mays Predicted polypeptide sequence is orthologous to G427
Zm_S11452342 DNA Zea mays Predicted polypeptide sequence is
orthologous to G427 Zm_S11527509 DNA Zea mays Predicted polypeptide
sequence is orthologous to G427 Zm_S11527752 DNA Zea mays Predicted
polypeptide sequence is orthologous to G602 Zm_S11528938 DNA Zea
mays Predicted polypeptide sequence is orthologous to G653
Zm_S11523935 DNA Zea mays Predicted polypeptide sequence is
orthologous to G866 Zm_S11519368 DNA Zea mays Predicted polypeptide
sequence is orthologous to G912 Zm_S11524655 DNA Zea mays Predicted
polypeptide sequence is orthologous to G932 Zm_S11529150 DNA Zea
mays Predicted polypeptide sequence is orthologous to G932
Zm_S11529161 DNA Zea mays Predicted polypeptide sequence is
orthologous to G932 Zm_S11529174 DNA Zea mays Predicted polypeptide
sequence is orthologous to G932 Zm_S11529193 DNA Zea mays Predicted
polypeptide sequence is orthologous to G932 Zm_S11437468 DNA Zea
mays Predicted polypeptide sequence is orthologous to G958
Zm_S11445843 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1049 Zm_S11485770 DNA Zea mays Predicted
polypeptide sequence is orthologous to G1255 Zm_S11529198 DNA Zea
mays Predicted polypeptide sequence is orthologous to G1331
Zm_S11418454 DNA Zea mays Predicted polypeptide sequence is
orthologous to G1535 Zm_S11522858 DNA Zea mays Predicted
polypeptide sequence is orthologous to G1535 Zm_S11506592 DNA Zea
mays Predicted polypeptide sequence is orthologous to G1930
Ta_S203038 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G154 Ta_S424724 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G154 Ta_S133393 DNA Triticum
aestivum Predicted polypeptide sequence is orthologous to G384
Ta_S147812 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G545 Ta_S66284 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G545 Ta_S202572 DNA Triticum
aestivum Predicted polypeptide sequence is orthologous to G760
Ta_S178842 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G1868 Ta_S84222 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G2520 Ta_S115031 DNA
Triticum aestivum Predicted polypeptide sequence is orthologous to
G2522 Ta_S65435 DNA Triticum aestivum Predicted polypeptide
sequence is orthologous to G2522 Ta_S177690 DNA Triticum aestivum
Predicted polypeptide sequence is orthologous to G8 Ta_S148486 DNA
Triticum aestivum Predicted polypeptide sequence is orthologous to
G326 Ta_S64707 DNA Triticum aestivum Predicted polypeptide sequence
is orthologous to G347 Ta_S16327 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G427 Ta_S201090 DNA Triticum
aestivum Predicted polypeptide sequence is orthologous to G427
Ta_S2764 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G635 Ta_S166473 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G653 Ta_S174179 DNA Triticum
aestivum Predicted polypeptide sequence is orthologous to G866
Ta_S280279 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G866 Ta_S47586 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G912 Ta_S75229 DNA Triticum
aestivum Predicted polypeptide sequence is orthologous to G912
Ta_S203158 DNA Triticum aestivum Predicted polypeptide sequence is
orthologous to G1255 Ta_S363550 DNA Triticum aestivum Predicted
polypeptide sequence is orthologous to G1255 Ta_S142289 DNA
Triticum aestivum Predicted polypeptide sequence is orthologous to
G1835 Ta_S266353 DNA Triticum aestivum Predicted polypeptide
sequence is orthologous to G1835 Ta_S174040 DNA Triticum aestivum
Predicted polypeptide sequence is orthologous to G2145 Les_S5295933
DNA Lycopersicon Predicted polypeptide sequence is esculentum
orthologous to G154 Les_S5295623 DNA Lycopersicon Predicted
polypeptide sequence is esculentum orthologous to G773 Les_S5295726
DNA Lycopersicon Predicted polypeptide sequence is esculentum
orthologous to G988 Les_S5183164 DNA Lycopersicon Predicted
polypeptide sequence is esculentum orthologous to G2520
Les_S5203454 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G2520 Les_S6657758 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G189
Les_S5275585 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G347 Les_S5295728 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G427
Les_S5295749 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G427 Les_S5295478 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G618,
G2057 Les_S6657761 DNA Lycopersicon Predicted polypeptide sequence
is esculentum orthologous to G866 Les_S6657762 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G866
Les_S5295301 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G912 Les_S5295595 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G932
Les_S5269007 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G1266 Les_S5295266 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G1266
Les_S5295755 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G1266 Les_S6682822 DNA Lycopersicon
Predicted polypeptide sequence is esculentum orthologous to G1266
Les_S5295754 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G1750 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G24
49683 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G24 54594 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G24 SINGLET-47313 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G154 50586 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G154 52410 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G154
SINGLET- 366830 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G154 SINGLET- 394847
SGN- DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G384 SINGLET-17776 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G545 44163 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G545 44287 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G545 SINGLET-6983 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G760
47781 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G760 52634 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G760 53754 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G760 SINGLET-23750 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G760 SINGLET- 310313 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G760
SINGLET- 447414 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G773 45948 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G773 48215 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1069
59076 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G1090 54402 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1322 58620 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1322
SINGLET-16950 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G1868 48848 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1868 SINGLET- 453383 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G1888 47593 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G2131, G979 SINGLET-517 SGN-
DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G2520 44928 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G2522 50326 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G8 SINGLET- 395477 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G156 54690 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G161
57990 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G200 57276 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G200 SINGLET- 385670 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G234 SINGLET-21166
SGN- DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G275, G837 47489 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G275, G837 47510
SGN- DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G275, G837 51256 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G275, G837 56050 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G326 SINGLET-19083
SGN- DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G347 51747 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G427 51523 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G427 54900 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G427 55550 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G427
55551 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G427 SINGLET- 397654 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G427 SINGLET- 446384 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G427
SINGLET-50339 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G427 SINGLET-9520 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G590 47483 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G590
47925 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G602 SINGLET-2565 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G618, G2057 50577 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G618,
G2057 58580 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G618, G2057 SINGLET-24189 SGN-
DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G618, G2057 SINGLET- 394109 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G618, G2057 SINGLET- 401522 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G643 56459 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G653 46400 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G653 SINGLET-64524 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G866
45903 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G866 SINGLET- 439904 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G872 50296 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G912
46974 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G912 46975 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G912 58571 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G912 SINGLET- 398604 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G932 52504 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G932
52540 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G932 57232 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G975 SINGLET-14957 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G975 SINGLET- 335836
SGN- DNA Lycopersicon Predicted polypeptide sequence is UNIGENE-
esculentum orthologous to G1049 SINGLET- 333614 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1255 48698 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1255
53476 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G1255 54828 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1266 48067 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1266
49923 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G1266 52630 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1266 SINGLET-38956 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1535
SINGLET-13754 SGN- DNA Lycopersicon Predicted polypeptide sequence
is UNIGENE- esculentum orthologous to G1750 49801 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1750 SINGLET-2078 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1750
SINGLET- 446513 SGN- DNA Lycopersicon Predicted polypeptide
sequence is UNIGENE- esculentum orthologous to G1835 48476 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1835 51325 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G1930
47598 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G1930 SINGLET- 393621 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G1930 SINGLET-44327 SGN- DNA Lycopersicon Predicted
polypeptide sequence is UNIGENE- esculentum orthologous to G2144
51335 SGN- DNA Lycopersicon Predicted polypeptide sequence is
UNIGENE- esculentum orthologous to G2512 SINGLET-2865 SGN- DNA
Lycopersicon Predicted polypeptide sequence is UNIGENE- esculentum
orthologous to G2535 SINGLET- 366637 SGN- DNA Lycopersicon
Predicted polypeptide sequence is UNIGENE- esculentum orthologous
to G2719 SINGLET- 357168 Vvi_S15370190 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G24 Vvi_S16806812 DNA Vitis
vinifera Predicted polypeptide sequence is orthologous to G24
Vvi_S15373999 DNA Vitis vinifera Predicted polypeptide sequence is
orthologous to G154 Vvi_S16872184 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G154 Vvi_S15355617 DNA Vitis
vinifera Predicted polypeptide sequence is orthologous to G545
Vvi_S15382170 DNA Vitis vinifera Predicted polypeptide sequence is
orthologous to G545 Vvi_S16873427 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G760 Vvi_S15431951 DNA Vitis
vinifera Predicted polypeptide sequence is orthologous to G937
Vvi_S16805106 DNA Vitis vinifera Predicted polypeptide sequence is
orthologous to G937 Vvi_S16805621 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G1069 Vvi_S15388842 DNA
Vitis vinifera Predicted polypeptide sequence is orthologous to
G1322 Vvi_S15421316 DNA Vitis vinifera Predicted polypeptide
sequence is orthologous to G2520 Vvi_S16529182 DNA Vitis vinifera
Predicted polypeptide sequence is orthologous to G2520
Vvi_S15370801 DNA Vitis vinifera Predicted polypeptide sequence is
orthologous to G2522 Vvi_S15411435 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G8 Vvi_S15353287 DNA Vitis
vinifera Predicted polypeptide sequence is orthologous to G189
Vvi_S15374453 DNA Vitis vinifera Predicted polypeptide sequence is
orthologous to G189 Vvi_S15426449 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G275, G837 Vvi_S16870363 DNA
Vitis vinifera Predicted polypeptide sequence is orthologous to
G275, G837 Vvi_S16531517 DNA Vitis vinifera Predicted polypeptide
sequence is orthologous to G347 Vvi_S15401282 DNA Vitis vinifera
Predicted polypeptide sequence is orthologous to G427 Vvi_S15423741
DNA Vitis vinifera Predicted polypeptide sequence is orthologous to
G427 Vvi_S15353882 DNA Vitis vinifera Predicted polypeptide
sequence is orthologous to G602 Vvi_S15426604 DNA Vitis vinifera
Predicted polypeptide sequence is orthologous to G653 Vvi_S15374416
DNA Vitis vinifera Predicted polypeptide sequence is orthologous to
G866 Vvi_S16870232 DNA Vitis vinifera Predicted polypeptide
sequence is orthologous to G872 Vvi_S15357313 DNA Vitis vinifera
Predicted polypeptide sequence is orthologous to G912 Vvi_S15391707
DNA Vitis vinifera Predicted polypeptide sequence is orthologous to
G912 Vvi_S16532074 DNA Vitis vinifera Predicted polypeptide
sequence is orthologous to G932 Vvi_S15427527 DNA Vitis vinifera
Predicted polypeptide sequence is orthologous to G1255
Vvi_S15431583 DNA Vitis vinifera Predicted polypeptide sequence is
orthologous to G1255 Vvi_S16871195 DNA Vitis vinifera Predicted
polypeptide sequence is orthologous to G1494 Vvi_S16865934 DNA
Vitis vinifera Predicted polypeptide sequence is orthologous to
G1835 Vvi_S16529913 DNA Vitis vinifera Predicted polypeptide
sequence is orthologous to G2144 Pta_S15732813 DNA Pinus taeda
Predicted polypeptide sequence is orthologous to G154 Pta_S15736271
DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G154 Pta_S15739572 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G154 Pta_S15740527 DNA Pinus
taeda Predicted polypeptide sequence is orthologous to G154
Pta_S15746398 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G154 Pta_S15751737 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G154 Pta_S15777399 DNA Pinus
taeda Predicted polypeptide sequence is orthologous to G154
Pta_S15780122 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G154 Pta_S15795745 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G154 Pta_S16849782 DNA Pinus
taeda Predicted polypeptide sequence is orthologous to G154
Pta_S16789085 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G760 Pta_S17046663 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G1666 Pta_S16800293 DNA
Pinus taeda Predicted polypeptide sequence is orthologous to G1868
Pta_S15767209 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G2522 Pta_S15799222 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G2789 Pta_S16786360 DNA
Pinus taeda Predicted polypeptide sequence is orthologous to G2789
Pta_S16788492 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G2789 Pta_S16802054 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G2789 Pta_S16793418 DNA
Pinus taeda Predicted polypeptide sequence is orthologous to G189
Pta_S15736214 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G275, G837 Pta_S15776645 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G275, G837 Pta_S17049915 DNA
Pinus taeda Predicted polypeptide sequence is orthologous to G326
Pta_S16847381 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G427 Pta_S17051722 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G427 Pta_S16797626 DNA Pinus
taeda Predicted polypeptide sequence is orthologous to G602
Pta_S16790444 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G653 Pta_S17050802 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G653 Pta_S15754706 DNA Pinus
taeda Predicted polypeptide sequence is orthologous to G872
Pta_S15767728 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G872 Pta_S15779272 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G872 Pta_S15738910 DNA Pinus
taeda Predicted polypeptide sequence is orthologous to G958
Pta_S15774939 DNA Pinus taeda Predicted polypeptide sequence is
orthologous to G958 Pta_S15797996 DNA Pinus taeda Predicted
polypeptide sequence is orthologous to G964 807 G1048 DNA
Arabidopsis thaliana 808 G1048 PRT Arabidopsis thaliana 809 G1100
DNA Arabidopsis thaliana 810 G1100 PRT Arabidopsis thaliana 811
G1796 DNA Arabidopsis thaliana 812 G1796 PRT Arabidopsis thaliana
813 G1995 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G2826, G2838, G361, G362, G370 814 G1995 PRT
Arabidopsis thaliana Paralogous to G2826, G2838, G361, G362, G370
815 G2467 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G812 816 G2467 PRT Arabidopsis thaliana Paralogous
to G812 817 G2505 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2635 818 G2505 PRT Arabidopsis thaliana
Paralogous to G2635 819 G2550 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2546 820 G2550 PRT
Arabidopsis thaliana Paralogous to G2546 821 G2640 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G2639,
G2642 822 G2640 PRT Arabidopsis thaliana Paralogous to G2639, G2642
823 G2686 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G2586, G2587 824 G2686 PRT Arabidopsis thaliana
Paralogous to G2586, G2587 825 G38 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1141 826 G38 PRT
Arabidopsis thaliana Paralogous to G1141 827 G44 DNA Arabidopsis
thaliana 828 G44 PRT Arabidopsis thaliana 829 G230 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G207,
G227, G242 830 G230 PRT Arabidopsis thaliana Paralogous to G207,
G227, G242 831 G261 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G265 832 G261 PRT Arabidopsis thaliana
Paralogous to G265 833 G271 DNA Arabidopsis thaliana 834 G271 PRT
Arabidopsis thaliana 835 G359 DNA Arabidopsis thaliana 836 G359 PRT
Arabidopsis thaliana 837 G377 DNA Arabidopsis thaliana 838 G377 PRT
Arabidopsis thaliana 839 G388 DNA Arabidopsis thaliana 840 G388 PRT
Arabidopsis thaliana 841 G435 DNA Arabidopsis thaliana 842 G435 PRT
Arabidopsis thaliana 843 G442 DNA Arabidopsis thaliana 844 G442 PRT
Arabidopsis thaliana 845 G468 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2866 846 G468 PRT
Arabidopsis thaliana Paralogous to G2866 847 G571 DNA Arabidopsis
thaliana 848 G571 PRT Arabidopsis thaliana 849 G652 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1335 850
G652 PRT Arabidopsis thaliana Paralogous to G1335 851 G664 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G197, G255; orthologous to G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3529, G3531, G3532, G3533, G3534, G3527, G3528 852
G664 PRT Arabidopsis thaliana Paralogous to G197, G255; Orthologous
to G3503, G3504, G3505, G3506, G3507, G3508, G3509, G3529, G3531,
G3532, G3533, G3534, G3527, G3528 853 G772 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G776 854 G772 PRT
Arabidopsis thaliana Paralogous to G776 855 G798 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1897 856
G798 PRT Arabidopsis thaliana Paralogous to G1897 857 G818 DNA
Arabidopsis thaliana 858 G818 PRT Arabidopsis thaliana 859 G974 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G5 860 G974 PRT Arabidopsis thaliana Paralogous to G5 861 G1062
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1664 862 G1062 PRT Arabidopsis thaliana Paralogous
to G1664 863 G1129 DNA Arabidopsis thaliana 864 G1129 PRT
Arabidopsis thaliana 865 G1137 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1133 866 G1137 PRT
Arabidopsis thaliana Paralogous to G1133 867 G1425 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1454,
G504; orthologous to G3809 868 G1425 PRT Arabidopsis thaliana
Paralogous to G1454, G504; Orthologous to G3809 869 G1517 DNA
Arabidopsis thaliana 870 G1517 PRT Arabidopsis thaliana 871 G1655
DNA Arabidopsis thaliana 872 G1655 PRT Arabidopsis thaliana 873
G1743 DNA Arabidopsis thaliana 874 G1743 PRT Arabidopsis thaliana
875 G1789 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G1911, G2721, G997 876 G1789 PRT Arabidopsis
thaliana Paralogous to G1911, G2721, G997 877 G1806 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1198,
G554, G555, G556, G558, G578, G629 878 G1806 PRT Arabidopsis
thaliana Paralogous to G1198, G554, G555, G556, G558, G578, G629
879 G1911 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G1789, G2721, G997 880 G1911 PRT Arabidopsis
thaliana Paralogous to G1789, G2721, G997 881 G2011 DNA Arabidopsis
thaliana 882 G2011 PRT Arabidopsis thaliana 883 G2215 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2216 884 G2215 PRT Arabidopsis thaliana Paralogous to G2216 885
G2452 DNA Arabidopsis thaliana 886 G2452 PRT Arabidopsis thaliana
887 G2455 DNA Arabidopsis thaliana 888 G2455 PRT Arabidopsis
thaliana 889 G2510 DNA Arabidopsis thaliana 890 G2510 PRT
Arabidopsis thaliana 891 G2515 DNA Arabidopsis thaliana 892 G2515
PRT Arabidopsis thaliana 893 G2571 DNA Arabidopsis thaliana 894
G2571 PRT Arabidopsis thaliana 895 G2702 DNA Arabidopsis thaliana
896 G2702 PRT Arabidopsis thaliana 897 G2763 DNA Arabidopsis
thaliana 898 G2763 PRT Arabidopsis thaliana 899 G2774 DNA
Arabidopsis thaliana 900 G2774 PRT Arabidopsis thaliana 901 G2888
DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1991 902 G2888 PRT Arabidopsis thaliana Paralogous
to G1991 903 G2958 DNA Arabidopsis thaliana 904 G2958 PRT
Arabidopsis thaliana 905 G5 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G974 906 G5 PRT Arabidopsis
thaliana Paralogous to G974 907 G197 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G255, G664;
orthologous to G3503, G3504, G3505, G3506, G3507, G3508, G3509,
G3529, G3531, G3532, G3533, G3534, G3527, G3528 908 G197 PRT
Arabidopsis thaliana Paralogous to G255, G664; Orthologous to
G3503, G3504, G3505, G3506, G3507, G3508, G3509, G3529, G3531,
G3532, G3533, G3534, G3527, G3528 909 G207 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G227, G230, G242
910 G207 PRT Arabidopsis thaliana Paralogous to G227, G230, G242
911 G227 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G207, G230, G242 912 G227 PRT Arabidopsis thaliana
Paralogous to G207, G230, G242 913 G242 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G207, G227, G230
914 G242 PRT Arabidopsis thaliana Paralogous to G207, G227, G230
915 G255 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G197, G664; orthologous to G3503, G3504, G3505,
G3506, G3507, G3508, G3509, G3529, G3531, G3532, G3533, G3534,
G3527, G3528 916 G255 PRT Arabidopsis thaliana Paralogous to G197,
G664; Orthologous to G3503, G3504, G3505, G3506, G3507, G3508,
G3509, G3529, G3531, G3532, G3533, G3534, G3527, G3528 917 G265 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G261 918 G265 PRT Arabidopsis thaliana Paralogous to G261 919
G361 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1995, G2826, G2838, G362, G370 920 G361 PRT
Arabidopsis thaliana Paralogous to G1995, G2826, G2838, G362, G370
921 G362 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1995, G2826, G2838, G361, G370 922 G362 PRT
Arabidopsis thaliana Paralogous to G1995, G2826, G2838, G361, G370
923 G370 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G1995, G2826, G2838, G361, G362 924 G370 PRT
Arabidopsis thaliana Paralogous to G1995, G2826, G2838, G361, G362
925 G504 DNA Arabidopsis thaliana Predicted polypeptide sequence
is
paralogous to G1425, G1454; orthologous to G3809 926 G504 PRT
Arabidopsis thaliana Paralogous to G1425, G1454; Orthologous to
G3809 927 G554 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1198, G1806, G555, G556, G558, G578,
G629 928 G554 PRT Arabidopsis thaliana Paralogous to G1198, G1806,
G555, G556, G558, G578, G629 929 G555 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1198, G1806, G554,
G556, G558, G578, G629 930 G555 PRT Arabidopsis thaliana Paralogous
to G1198, G1806, G554, G556, G558, G578, G629 931 G556 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1198, G1806, G554, G555, G558, G578, G629 932 G556 PRT
Arabidopsis thaliana Paralogous to G1198, G1806, G554, G555, G558,
G578, G629 933 G558 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G1198, G1806, G554, G555, G556, G578,
G629 934 G558 PRT Arabidopsis thaliana Paralogous to G1198, G1806,
G554, G555, G556, G578, G629 935 G578 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1198, G1806, G554,
G555, G556, G558, G629 936 G578 PRT Arabidopsis thaliana Paralogous
to G1198, G1806, G554, G555, G556, G558, G629 937 G629 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1198, G1806, G554, G555, G556, G558, G578 938 G629 PRT
Arabidopsis thaliana Paralogous to G1198, G1806, G554, G555, G556,
G558, G578 939 G776 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G772 940 G776 PRT Arabidopsis thaliana
Paralogous to G772 941 G812 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2467 942 G812 PRT
Arabidopsis thaliana Paralogous to G2467 943 G997 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G1789,
G1911, G2721 944 G997 PRT Arabidopsis thaliana Paralogous to G1789,
G1911, G2721 945 G1133 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1137 946 G1133 PRT
Arabidopsis thaliana Paralogous to G1137 947 G1141 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G38 948
G1141 PRT Arabidopsis thaliana Paralogous to G38 949 G1198 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G1806, G554, G555, G556, G558, G578, G629 950 G1198 PRT
Arabidopsis thaliana Paralogous to G1806, G554, G555, G556, G558,
G578, G629 951 G1335 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G652 952 G1335 PRT Arabidopsis thaliana
Paralogous to G652 953 G1454 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1425, G504; orthologous to
G3809 954 G1454 PRT Arabidopsis thaliana Paralogous to G1425, G504;
Orthologous to G3809 955 G1664 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G1062 956 G1664 PRT
Arabidopsis thaliana Paralogous to G1062 957 G1897 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G798 958
G1897 PRT Arabidopsis thaliana Paralogous to G798 959 G1991 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2888 960 G1991 PRT Arabidopsis thaliana Paralogous to G2888 961
G2216 DNA Arabidopsis thaliana Predicted polypeptide sequence is
paralogous to G2215 962 G2216 PRT Arabidopsis thaliana Paralogous
to G2215 963 G2546 DNA Arabidopsis thaliana Predicted polypeptide
sequence is paralogous to G2550 964 G2546 PRT Arabidopsis thaliana
Paralogous to G2550 965 G2586 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2587, G2686 966 G2586 PRT
Arabidopsis thaliana Paralogous to G2587, G2686 967 G2587 DNA
Arabidopsis thaliana Predicted polypeptide sequence is paralogous
to G2586, G2686 968 G2587 PRT Arabidopsis thaliana Paralogous to
G2586, G2686 969 G2635 DNA Arabidopsis thaliana Predicted
polypeptide sequence is paralogous to G2505 970 G2635 PRT
Arabidopsis thaliana Paralogous to G2505 971 G2639 DNA Arabidopsis
thaliana Predicted polypeptide sequence is paralogous to G2640,
G2642 972 G2639 PRT Arabidopsis thaliana Paralogous to G2640, G2642
973 G2642 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G2639, G2640 974 G2642 PRT Arabidopsis thaliana
Paralogous to G2639, G2640 975 G2721 DNA Arabidopsis thaliana
Predicted polypeptide sequence is paralogous to G1789, G1911, G997
976 G2721 PRT Arabidopsis thaliana Paralogous to G1789, G1911, G997
977 G2826 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G1995, G2838, G361, G362, G370 978 G2826 PRT
Arabidopsis thaliana Paralogous to G1995, G2838, G361, G362, G370
979 G2838 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G1995, G2826, G361, G362, G370 980 G2838 PRT
Arabidopsis thaliana Paralogous to G1995, G2826, G361, G362, G370
981 G2866 DNA Arabidopsis thaliana Predicted polypeptide sequence
is paralogous to G468 982 G2866 PRT Arabidopsis thaliana Paralogous
to G468 983 G3503 DNA Oryza sativa Predicted polypeptide sequence
is paralogous to G3504, G3505, G3506, G3507, G3508; orthologous to
G197, G255, G664, G3509, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 984 G3503 PRT Oryza sativa Paralogous to G3504, G3505, G3506,
G3507, G3508; Orthologous to G197, G255, G664, G3509, G3529, G3531,
G3532, G3533, G3534, G3527, G3528 985 G3504 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3503, G3505,
G3506, G3507, G3508; orthologous to G197, G255, G664, G3509, G3529,
G3531, G3532, G3533, G3534, G3527, G3528 986 G3504 PRT Oryza sativa
Paralogous to G3503, G3505, G3506, G3507, G3508; Orthologous to
G197, G255, G664, G3509, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 987 G3505 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3503, G3504, G3506, G3507, G3508; orthologous to
G197, G255, G664, G3509, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 988 G3505 PRT Oryza sativa Paralogous to G3503, G3504, G3506,
G3507, G3508; Orthologous to G197, G255, G664, G3509, G3529, G3531,
G3532, G3533, G3534, G3527, G3528 989 G3506 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3503, G3504,
G3505, G3507, G3508; orthologous to G197, G255, G664, G3509, G3529,
G3531, G3532, G3533, G3534, G3527, G3528 990 G3506 PRT Oryza sativa
Paralogous to G3503, G3504, G3505, G3507, G3508; Orthologous to
G197, G255, G664, G3509, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 991 G3507 DNA Oryza sativa Predicted polypeptide sequence is
paralogous to G3503, G3504, G3505, G3506, G3508; orthologous to
G197, G255, G664, G3509, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 992 G3507 PRT Oryza sativa Paralogous to G3503, G3504, G3505,
G3506, G3508; Orthologous to G197, G255, G664, G3509, G3529, G3531,
G3532, G3533, G3534, G3527, G3528 993 G3508 DNA Oryza sativa
Predicted polypeptide sequence is paralogous to G3503, G3504,
G3505, G3506, G3507; orthologous to G197, G255, G664, G3509, G3529,
G3531, G3532, G3533, G3534, G3527, G3528 994 G3508 PRT Oryza sativa
Paralogous to G3503, G3504, G3505, G3506, G3507; Orthologous to
G197, G255, G664, G3509, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 995 G3509 DNA Lycopersicon Predicted polypeptide sequence is
esculentum orthologous to G197, G255, G664, G3503, G3504, G3505,
G3506, G3507, G3508, G3529, G3531, G3532, G3533, G3534, G3527,
G3528 996 G3509 PRT Lycopersicon Orthologous to G197, G255, G664,
esculentum G3503, G3504, G3505, G3506, G3507, G3508, G3529, G3531,
G3532, G3533, G3534, G3527, G3528 997 G3527 DNA Glycine max
Predicted polypeptide sequence is paralogous to G3529, G3528;
orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3531, G3532, G3533, G3534 998 G3527 PRT Glycine max
Paralogous to G3529, G3528; Orthologous to G197, G255, G664, G3503,
G3504, G3505, G3506, G3507, G3508, G3509, G3531, G3532, G3533,
G3534 999 G3528 DNA Glycine max Predicted polypeptide sequence is
paralogous to G3529, G3527; orthologous to G197, G255, G664, G3503,
G3504, G3505, G3506, G3507, G3508, G3509, G3531, G3532, G3533,
G3534 1000 G3528 PRT Glycine max Paralogous to G3529, G3527;
Orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3531, G3532, G3533, G3534 1001 G3529 DNA Glycine max
Predicted polypeptide sequence is paralogous to G3527, G3528;
orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3531, G3532, G3533, G3534 1002 G3529 PRT Glycine max
Paralogous to G3527, G3528; Orthologous to G197, G255, G664, G3503,
G3504, G3505, G3506, G3507, G3508, G3509, G3531, G3532, G3533,
G3534 1003 G3531 DNA Zea mays Predicted polypeptide sequence is
paralogous to G3532, G3533, G3534; orthologous to G197, G255, G664,
G3503, G3504, G3505, G3506, G3507, G3508, G3509, G3529, G3527,
G3528 1004 G3531 PRT Zea mays Paralogous to G3532, G3533, G3534;
Orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3529, G3527, G3528 1005 G3532 DNA Zea mays Predicted
polypeptide sequence is paralogous to G3531, G3533, G3534;
orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3529, G3527, G3528 1006 G3532 PRT Zea mays
Paralogous to G3531, G3533, G3534; Orthologous to G197, G255, G664,
G3503, G3504, G3505, G3506, G3507, G3508, G3509, G3529, G3527,
G3528 1007 G3533 DNA Zea mays Predicted polypeptide sequence is
paralogous to G3531, G3532, G3534; orthologous to G197, G255, G664,
G3503, G3504, G3505, G3506, G3507, G3508, G3509, G3529, G3527,
G3528 1008 G3533 PRT Zea mays Paralogous to G3531, G3532, G3534;
Orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3529, G3527, G3528
1009 G3534 DNA Zea mays Predicted polypeptide sequence is
paralogous to G3531, G3532, G3533; orthologous to G197, G255, G664,
G3503, G3504, G3505, G3506, G3507, G3508, G3509, G3529, G3527,
G3528 1010 G3534 PRT Zea mays Paralogous to G3531, G3532, G3533;
Orthologous to G197, G255, G664, G3503, G3504, G3505, G3506, G3507,
G3508, G3509, G3529, G3527, G3528 1011 G3809 DNA Oryza sativa
Predicted polypeptide sequence is orthologous to G1425, G1454, G504
1012 G3809 PRT Oryza sativa Orthologous to G1425, G1454, G504
Molecular Modeling
[0348] Another means that may be used to confirm the utility and
function of transcription factor sequences that are orthologous or
paralogous to presently disclosed transcription factors is through
the use of molecular modeling software. Molecular modeling is
routinely used to predict polypeptide structure, and a variety of
protein structure modeling programs, such as "Insight II"
(Accelrys, Inc.) are commercially available for this purpose.
Modeling can thus be used to predict which residues of a
polypeptide can be changed without altering function (Crameri et
al. (2003) U.S. Pat. No. 6,521,453). Thus, polypeptides that are
sequentially similar can be shown to have a high likelihood of
similar function by their structural similarity, which may, for
example, be established by comparison of regions of superstructure.
The relative tendencies of amino acids to form regions of
superstructure (for example, helixes and (3-sheets) are well
established. For example, O'Neil et al. ((1990) Science 250:
646-651) have discussed in detail the helix forming tendencies of
amino acids. Tables of relative structure forming activity for
amino acids can be used as substitution tables to predict which
residues can be functionally substituted in a given region, for
example, in DNA-binding domains of known transcription factors and
equivalogs. Homologs that are likely to be functionally similar can
then be identified.
[0349] Of particular interest is the structure of a transcription
factor in the region of its conserved domains, such as those
identified in Table 1 and Table 3. Structural analyses may be
performed by comparing the structure of the known transcription
factor around its conserved domain with those of orthologs and
paralogs. Analysis of a number of polypeptides within a
transcription factor group or clade, including the functionally or
sequentially similar polypeptides provided in the Sequence Listing,
may also provide an understanding of structural elements required
to regulate transcription within a given family.
EXAMPLES
[0350] The invention, now being generally described, will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention and are not intended to
limit the invention. It will be recognized by one of skill in the
art that a transcription factor that is associated with a
particular first trait may also be associated with at least one
other, unrelated and inherent second trait which was not predicted
by the first trait.
[0351] The complete descriptions of the traits associated with each
polynucleotide of the invention are fully disclosed in Examples
VIII, IX and X.
Example I: Full Length Gene Identification and Cloning
[0352] Putative transcription factor sequences (genomic or ESTs)
related to known transcription factors were identified in the
Arabidopsis thaliana GenBank database using the tblastn sequence
analysis program using default parameters and a P-value cutoff
threshold of -4 or -5 or lower, depending on the length of the
query sequence. Putative transcription factor sequence hits were
then screened to identify those containing particular sequence
strings. If the sequence hits contained such sequence strings, the
sequences were confirmed as transcription factors.
[0353] Alternatively, Arabidopsis thaliana cDNA libraries derived
from different tissues or treatments, or genomic libraries were
screened to identify novel members of a transcription family using
a low stringency hybridization approach. Probes were synthesized
using gene specific primers in a standard PCR reaction (annealing
temperature 60.degree. C.) and labeled with .sup.32P dCTP using the
High Prime DNA Labeling Kit (Roche Diagnostics Corp., Indianapolis,
Ind.). Purified radiolabelled probes were added to filters immersed
in Church hybridization medium (0.5 M NaPO.sub.4 pH 7.0, 7% SDS, 1%
w/v bovine serum albumin) and hybridized overnight at 60.degree. C.
with shaking. Filters were washed two times for 45 to 60 minutes
with 1.times.SCC, 1% SDS at 60.degree. C.
[0354] To identify additional sequence 5' or 3' of a partial cDNA
sequence in a cDNA library, 5' and 3' rapid amplification of cDNA
ends (RACE) was performed using the MARATHON cDNA amplification kit
(Clontech, Palo Alto, Calif.). Generally, the method entailed first
isolating poly(A) mRNA, performing first and second strand cDNA
synthesis to generate double-stranded cDNA, blunting cDNA ends,
followed by ligation of the MARATHON Adaptor to the cDNA to form a
library of adaptor-ligated ds cDNA.
[0355] Gene-specific primers were designed to be used along with
adaptor specific primers for both 5' and 3' RACE reactions. Nested
primers, rather than single primers, were used to increase PCR
specificity. Using 5' and 3' RACE reactions, 5' and 3' RACE
fragments were obtained, sequenced and cloned. The process can be
repeated until 5' and 3' ends of the full-length gene were
identified. Then the full-length cDNA was generated by PCR using
primers specific to 5' and 3' ends of the gene by end-to-end
PCR.
Example II: Construction of Expression Vectors
[0356] The sequence was amplified from a genomic or cDNA library
using primers specific to sequences upstream and downstream of the
coding region. The expression vector was pMEN20 or pMEN65, which
are both derived from pMON316 (Sanders et al. (1987) Nucleic Acids
Res. 15:1543-1558) and contain the CaMV 35S promoter to express
transgenes. To clone the sequence into the vector, both pMEN20 and
the amplified DNA fragment were digested separately with SalI and
NotI restriction enzymes at 37.degree. C. for 2 hours. The
digestion products were subject to electrophoresis in a 0.8%
agarose gel and visualized by ethidium bromide staining. The DNA
fragments containing the sequence and the linearized plasmid were
excised and purified by using a QIAQUICK gel extraction kit
(Qiagen, Valencia Calif.). The fragments of interest were ligated
at a ratio of 3:1 (vector to insert). Ligation reactions using T4
DNA ligase (New England Biolabs, Beverly Mass.) were carried out at
16.degree. C. for 16 hours. The ligated DNAs were transformed into
competent cells of the E. coli strain DH5a by using the heat shock
method. The transformations were plated on LB plates containing 50
mg/l kanamycin (Sigma Chemical Co. St. Louis Mo.). Individual
colonies were grown overnight in five milliliters of LB broth
containing 50 mg/l kanamycin at 37.degree. C. Plasmid DNA was
purified by using Qiaquick Mini Prep kits (Qiagen).
Example III: Transformation of Agrobacterium with the Expression
Vector
[0357] After the plasmid vector containing the gene was
constructed, the vector was used to transform Agrobacterium
tumefaciens cells expressing the gene products. The stock of
Agrobacterium tumefaciens cells for transformation was made as
described by Nagel et al. (1990) FEMS Microbiol Letts. 67: 325-328.
Agrobacterium strain ABI was grown in 250 ml LB medium (Sigma)
overnight at 28.degree. C. with shaking until an absorbance over 1
cm at 600 nm (A.sub.600) of 0.5-1.0 was reached. Cells were
harvested by centrifugation at 4,000.times.g for 15 min at
4.degree. C. Cells were then resuspended in 250 .mu.l chilled
buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were
centrifuged again as described above and resuspended in 125 .mu.l
chilled buffer. Cells were then centrifuged and resuspended two
more times in the same HEPES buffer as described above at a volume
of 100 .mu.l and 750 respectively. Resuspended cells were then
distributed into 40 .mu.l aliquots, quickly frozen in liquid
nitrogen, and stored at -80.degree. C.
[0358] Agrobacterium cells were transformed with plasmids prepared
as described above following the protocol described by Nagel et al.
(1990) supra. For each DNA construct to be transformed, 50-100 ng
DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
was mixed with 40 .mu.l of Agrobacterium cells. The DNA/cell
mixture was then transferred to a chilled cuvette with a 2 mm
electrode gap and subject to a 2.5 kV charge dissipated at 25 .mu.F
and 200 .mu.F using a Gene Pulser II apparatus (Bio-Rad, Hercules,
Calif.). After electroporation, cells were immediately resuspended
in 1.0 ml LB and allowed to recover without antibiotic selection
for 2-4 hours at 28.degree. C. in a shaking incubator. After
recovery, cells were plated onto selective medium of LB broth
containing 100 .mu.g/ml spectinomycin (Sigma) and incubated for
24-48 hours at 28.degree. C. Single colonies were then picked and
inoculated in fresh medium. The presence of the plasmid construct
was verified by PCR amplification and sequence analysis.
Example IV: Transformation of Arabidopsis Plants with Agrobacterium
tumefaciens with Expression Vector
[0359] After transformation of Agrobacterium tumefaciens with
plasmid vectors containing the gene, single Agrobacterium colonies
were identified, propagated, and used to transform Arabidopsis
plants. Briefly, 500 ml cultures of LB medium containing 50 mg/l
kanamycin were inoculated with the colonies and grown at 28.degree.
C. with shaking for 2 days until an optical absorbance at 600 nm
wavelength over 1 cm (A.sub.600) of >2.0 is reached. Cells were
then harvested by centrifugation at 4,000.times.g for 10 min, and
resuspended in infiltration medium (1/2.times.Murashige and Skoog
salts (Sigma), 1.times. Gamborg's B-5 vitamins (Sigma), 5.0% (w/v)
sucrose (Sigma), 0.044 .mu.M benzylamino purine (Sigma), 200
.mu.l/l Silwet L-77 (Lehle Seeds)) until an A.sub.600 of 0.8 was
reached.
[0360] Prior to transformation, Arabidopsis thaliana seeds (ecotype
Columbia) were sown at a density of -10 plants per 4'' pot onto
Pro-Mix BX potting medium (Hummert International) covered with
fiberglass mesh (18 mm.times.16 mm). Plants were grown under
continuous illumination (50-75 .mu.E/m.sup.2/sec) at 22-23.degree.
C. with 65-70% relative humidity. After about 4 weeks, primary
inflorescence stems (bolts) are cut off to encourage growth of
multiple secondary bolts. After flowering of the mature secondary
bolts, plants were prepared for transformation by removal of all
siliques and opened flowers.
[0361] The pots were then immersed upside down in the mixture of
Agrobacterium infiltration medium as described above for 30 sec,
and placed on their sides to allow draining into a 1'.times.2' flat
surface covered with plastic wrap. After 24 h, the plastic wrap was
removed and pots are turned upright. The immersion procedure was
repeated one week later, for a total of two immersions per pot.
Seeds were then collected from each transformation pot and analyzed
following the protocol described below.
Example V: Identification of Arabidopsis Primary Transformants
[0362] Seeds collected from the transformation pots were sterilized
essentially as follows. Seeds were dispersed into in a solution
containing 0.1% (v/v) Triton X-100 (Sigma) and sterile water and
washed by shaking the suspension for 20 min. The wash solution was
then drained and replaced with fresh wash solution to wash the
seeds for 20 min with shaking. After removal of the
ethanol/detergent solution, a solution containing 0.1% (v/v) Triton
X-100 and 30% (v/v) bleach (CLOROX; Clorox Corp. Oakland Calif.)
was added to the seeds, and the suspension was shaken for 10 min.
After removal of the bleach/detergent solution, seeds were then
washed five times in sterile distilled water. The seeds were stored
in the last wash water at 4.degree. C. for 2 days in the dark
before being plated onto antibiotic selection medium (1.times.
Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH),
1.times. Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies),
and 50 mg/l kanamycin). Seeds were germinated under continuous
illumination (50-75 .mu.E/m.sup.2/sec) at 22-23.degree. C. After
7-10 days of growth under these conditions, kanamycin resistant
primary transformants (T1 generation) were visible and obtained.
These seedlings were transferred first to fresh selection plates
where the seedlings continued to grow for 3-5 more days, and then
to soil (Pro-Mix BX potting medium).
[0363] Primary transformants were crossed and progeny seeds (T2)
collected; kanamycin resistant seedlings were selected and
analyzed. The expression levels of the recombinant polynucleotides
in the transformants varies from about a 5% expression level
increase to a least a 100% expression level increase. Similar
observations are made with respect to polypeptide level
expression.
Example VI: Identification of Arabidopsis Plants with Transcription
Factor Gene Knockouts
[0364] The screening of insertion mutagenized Arabidopsis
collections for null mutants in a known target gene was essentially
as described in Krysan et al. (1999) Plant Cell 11: 2283-2290.
Briefly, gene-specific primers, nested by 5-250 base pairs to each
other, were designed from the 5' and 3' regions of a known target
gene. Similarly, nested sets of primers were also created specific
to each of the T-DNA or transposon ends (the "right" and "left"
borders). All possible combinations of gene specific and
T-DNA/transposon primers were used to detect by PCR an insertion
event within or close to the target gene. The amplified DNA
fragments were then sequenced which allows the precise
determination of the T-DNA/transposon insertion point relative to
the target gene. Insertion events within the coding or intervening
sequence of the genes were deconvoluted from a pool comprising a
plurality of insertion events to a single unique mutant plant for
functional characterization. The method is described in more detail
in Yu and Adam, U.S. application Ser. No. 09/177,733 filed Oct. 23,
1998.
Example VII: Identification of Modified Phenotypes in
Overexpression or Gene Knockout Plants
[0365] Arabidopsis thaliana ecotype Columbia (Col-0) was used to
create all overexpressing lines. The control plants for the assay
were Col-0 plants transformed with an empty transformation vector
(pMEN65).
Microarray Experiments
[0366] In some instances, expression patterns of the stress-induced
genes may be monitored by microarray experiments. In these
experiments, cDNAs are generated by PCR and resuspended at a final
concentration of 100 ng/.mu.l in 3.times.SSC or 150 mM Na-phosphate
(Eisen and Brown (1999) Methods Enzymol. 303: 179-205). The cDNAs
are spotted on microscope glass slides coated with polylysine. The
prepared cDNAs are aliquoted into 384 well plates and spotted on
the slides using, for example, an x-y-z gantry (OmniGrid) which may
be purchased from GeneMachines (Menlo Park, Calif.) outfitted with
quill type pins which may be purchased from Telechem International
(Sunnyvale, Calif.). After spotting, the arrays are cured for a
minimum of one week at room temperature, rehydrated and blocked
following the protocol recommended by Eisen and Brown (1999)
supra.
[0367] Sample total RNA (10 .mu.g) samples are labeled using
fluorescent Cy3 and Cy5 dyes. Labeled samples are resuspended in
4.times.SSC/0.03% SDS/4 .mu.g salmon sperm DNA/2 .mu.g tRNA/50 mM
Na-pyrophosphate, heated for 95.degree. C. for 2.5 minutes, spun
down and placed on the array. The array is then covered with a
glass coverslip and placed in a sealed chamber. The chamber is then
kept in a water bath at 62.degree. C. overnight. The arrays are
washed as described in Eisen and Brown (1999) supra) and scanned on
a General Scanning 3000 laser scanner. The resulting files are
subsequently quantified using IMAGENE, software (BioDiscovery, Los
Angeles Calif.).
[0368] RT-PCR experiments may be performed to identify those genes
induced after exposure to abiotic stresses. Generally, the gene
expression patterns from ground plant leaf tissue is examined.
[0369] Reverse transcriptase PCR was conducted using gene specific
primers within the coding region for each sequence identified. The
primers were designed near the 3' region of each DNA binding
sequence initially identified.
[0370] Total RNA from these ground leaf tissues was isolated using
the CTAB extraction protocol. Once extracted total RNA was
normalized in concentration across all the tissue types to ensure
that the PCR reaction for each tissue received the same amount of
cDNA template using the 28S band as reference. Poly(A+) RNA was
purified using a modified protocol from the Qiagen OLIGOTEX
purification kit batch protocol. cDNA was synthesized using
standard protocols. After the first strand cDNA synthesis, primers
for Actin 2 were used to normalize the concentration of cDNA across
the tissue types. Actin 2 is found to be constitutively expressed
in fairly equal levels across the tissue types being
investigated.
[0371] For RT PCR, cDNA template was mixed with corresponding
primers and Taq DNA polymerase. Each reaction consisted of 0.2
.mu.l cDNA template, 2 .mu.l 10.times. Tricine buffer, 2 .mu.l
10.times. Tricine buffer and 16.8 .mu.l water, 0.05 .mu.l Primer 1,
0.05 Primer 2, 0.3 .mu.l Taq DNA polymerase and 8.6 .mu.l
water.
[0372] The 96 well plate is covered with microfilm and set in the
thermocycler to start the reaction cycle. By way of illustration,
the reaction cycle may comprise the following steps:
[0373] STEP 1: 93.degree. C. for 3 minutes;
[0374] STEP 2: 93.degree. C. for 30 seconds;
[0375] STEP 3: 65.degree. C. for 1 minute;
[0376] STEP 4: 72.degree. C. for 2 minutes;
[0377] STEPS 2, 3 and 4 are repeated for 28 cycles;
[0378] STEP 5: 72.degree. C. for 5 minutes; and
[0379] STEP 6 4.degree. C.
[0380] To amplify more products, for example, to identify genes
that have very low expression, additional steps may be performed:
the following method illustrates a method that may be used in this
regard. the PCR plate is placed back in the thermocycler for 8 more
cycles of Steps 2-4.
[0381] STEP 2 93.degree. C. for 30 seconds;
[0382] STEP 3 65.degree. C. for 1 minute;
[0383] STEP 4 72.degree. C. for 2 minutes, repeated for 8 cycles;
and
[0384] STEP 5 4.degree. C.
[0385] Eight microliters of PCR product and 1.5 .mu.l of loading
dye are loaded on a 1.2% agarose gel for analysis after 28 cycles
and 36 cycles. Expression levels of specific transcripts are
considered low if they were only detectable after 36 cycles of PCR.
Expression levels are considered medium or high depending on the
levels of transcript compared with observed transcript levels for
an internal control such as actin2. Transcript levels are
determined in repeat experiments and compared to transcript levels
in control (e.g., non-transformed) plants.
Abiotic Stress Assays
[0386] Modified phenotypes observed for particular overexpressor
plants may include increased biomass, and/or increased or decreased
abiotic stress tolerance or resistance. For a particular
overexpressor that shows a less beneficial characteristic, such as
reduced abiotic stress tolerance or resistance, it may be more
useful to select a plant with a decreased expression of the
particular transcription factor. For a particular knockout that
shows a less beneficial characteristic, such as decreased abiotic
stress tolerance, it may be more useful to select a plant with an
increased expression of the particular transcription factor.
[0387] The germination assays in this example followed
modifications of the same basic protocol. Sterile seeds were sown
on the conditional media listed below. Plates were incubated at
22.degree. C. under 24-hour light (120-130 .mu.Ein/m.sup.2/s) in a
growth chamber. Evaluation of germination and seedling vigor was
conducted 3 to 15 days after planting. The basal media was 80%
Murashige-Skoog medium (MS)+vitamins.
[0388] For stress experiments conducted with more mature plants,
seeds were germinated and grown for seven days on MS+vitamins+1%
sucrose at 22.degree. C. and then transferred to cold and heat
stress conditions. The plants were either exposed to cold stress (6
hour exposure to 4-8.degree. C.), or heat stress (32.degree. C. was
applied for five days, after which the plants were transferred back
22.degree. C. for recovery and evaluated after 5 days relative to
controls not exposed to the depressed or elevated temperature).
[0389] The salt stress assays were intended to find genes that
confer better germination, seedling vigor or growth in high salt.
Evaporation from the soil surface causes upward water movement and
salt accumulation in the upper soil layer where the seeds are
placed. Thus, germination normally takes place at a salt
concentration much higher than the mean salt concentration of in
the whole soil profile. Plants differ in their tolerance to NaCl
depending on their stage of development, therefore seed
germination, seedling vigor, and plant growth responses were
evaluated.
[0390] Osmotic stress assays (including NaCl and mannitol assays)
were conducted to determine if an osmotic stress phenotype was
NaCl-specific or if it was a general osmotic stress related
phenotype. Plants tolerant to osmotic stress could also have more
tolerance to drought and/or freezing.
[0391] For salt and osmotic stress germination experiments, the
medium was supplemented with 150 mM NaCl or 300 mM mannitol. Growth
regulator sensitivity assays were performed in MS media, vitamins,
and either 0.3 .mu.M ABA, 9.4% sucrose, or 5% glucose.
[0392] Experiments were performed to identify those transformants
that exhibited modified sugar-sensing. For such studies, seeds from
transformants were germinated on high sugar-containing media (5%
glucose, 9.4% sucrose) that normally partially restrict hypocotyl
elongation. Plants with altered sugar sensing may have either
longer or shorter hypocotyls than normal plants when grown on this
media. Additionally, other plant traits may be varied such as root
mass. Sugar sensing assays were intended to find genes involved in
sugar sensing by germinating seeds on high concentrations of
sucrose and glucose and looking for degrees of hypocotyl
elongation. The germination assay on mannitol controlled for
responses related to osmotic stress. Sugars are key regulatory
molecules that affect diverse processes in higher plants including
germination, growth, flowering, senescence, sugar metabolism and
photosynthesis. Sucrose is the major transport form of
photosynthate and its flux through cells has been shown to affect
gene expression and alter storage compound accumulation in seeds
(source-sink relationships). Glucose-specific hexose-sensing has
also been described in plants and is implicated in cell division
and repression of "famine" genes (photosynthetic or glyoxylate
cycles).
[0393] Temperature stress assays were carried out to find genes
that confer better germination, seedling vigor or plant growth
under temperature stress (cold, freezing and heat). Temperature
stress cold germination experiments were carried out at 8.degree.
C. Heat stress germination experiments were conducted at 32.degree.
C. to 37.degree. C. for 6 hours of exposure.
[0394] Soil-based drought screens were performed with Arabidopsis
plants overexpressing the transcription factors listed in the
Sequence Listing. Seeds from wild-type Arabidopsis plants, or
plants overexpressing a polypeptide of the invention, were
stratified for three days at 4.degree. C. in 0.1% agarose. Fourteen
seeds of each overexpressor or wild-type were then sown in three
inch clay pots containing a 50:50 mix of vermiculite:perlite topped
with a small layer of MetroMix 200 and grown for fifteen days under
24 hr light. Pots containing wild-type and overexpressing seedlings
were placed in flats in random order. Drought stress was initiated
by placing pots on absorbent paper for seven to eight days. The
seedlings were considered to be sufficiently stressed when the
majority of the pots containing wild-type seedlings within a flat
had become severely wilted. Pots were then re-watered and survival
was scored four to seven days later. Plants were ranked against
wild-type controls for each of two criteria: tolerance to the
drought conditions and recovery (survival) following
re-watering
[0395] At the end of the initial drought period, each pot was
assigned a numeric value score depending on the above criteria.
Scores of 0-6 were assigned (Table 11), with a low value of "0"
assigned to plants with an extremely poor appearance (i.e., the
plants were uniformly brown) and a value of "6" given to plants
that were rated very healthy in appearance (i.e., the plants were
all green). After the plants were rewatered and incubated an
additional four to seven days, the plants were reevaluated to
indicate the degree of recovery from the water deprivation
treatment.
[0396] An analysis was then conducted to determine which plants
best survived water deprivation, identifying the transgenes that
consistently conferred drought-tolerant phenotypes and their
ability to recover from this treatment. The analysis was performed
by comparing overall and within-flat tabulations with a set of
statistical models to account for variations between batches.
Several measures of survival were tabulated, including: (a) the
average proportion of plants surviving relative to wild-type
survival within the same flat; (b) the median proportion surviving
relative to wild-type survival within the same flat; (c) the
overall average survival (taken over all batches, flats, and pots);
(d) the overall average survival relative to the overall wild-type
survival; and (e) the average visual score of plant health before
rewatering.
Analysis of Flowering Time
[0397] Flowering time was measured by the number of rosette leaves
present when a visible inflorescence of approximately 3 cm is
apparent. Rosette and total leaf number on the progeny stem are
tightly correlated with the timing of flowering (Koornneef et al.
(1991) Mol. Gen. Genet. 229: 57-66). The vernalization response was
also measured. For vernalization treatments, seeds were sown to MS
agar plates, sealed with micropore tape, and placed in a 4.degree.
C. cold room with low light levels for 6-8 weeks. The plates were
then transferred to the growth rooms alongside plates containing
freshly sown non-vernalized controls. Rosette leaves were counted
when a visible inflorescence of approximately 3 cm was
apparent.
C/N Sensing Assays
[0398] Germination assays were conducted to monitor the effects of
C on N signaling through anthocyanin production on high sucrose
plus and minus glutamine (Hsieh et al, (1998) Proc. Natl. Acad.
Sci. USA. 95: 13965-13970).
[0399] For overexpression lines examined in the assay, the screen
was primarily performed on a seed lot comprised of seed mixed
together from each of three independent primary transformants.
These seed batches were segregating, but selection was not
performed to avoid the extra stress that might be associated with
kanamycin selection. In the case of knockout (KO) lines, the screen
was performed on seed from plant(s) homozygous for a T-DNA
insertion within the gene of interest. Lines that gave positive
results in our previous studies were included here as positive
controls.
[0400] All assays were designed to detect plants that were more
tolerant or less tolerant of an alteration in C/N balance brought
about by an increase in sucrose levels in the absence of a nitrogen
source. Lines were scored as tolerant if they accumulated lower
levels of anthocyanins than controls and sensitive if they
accumulated higher levels of anthocyanins than controls. The
general vigor and size of the seedlings compared to controls was
also assessed.
[0401] Prior to plating, seed for all experiments were surface
sterilized and prepared for germination by:
[0402] 1. a 5 minute incubation with mixing in 70% ethanol;
[0403] 2. a 20 minute incubation with mixing in 30% bleach, 0.01%
triton-X 100;
[0404] 3. five rinses with sterile water; and
[0405] 4. seeds are re-suspended in 0.1% sterile agarose and
stratified at 4.degree. C. for 3 days.
[0406] The sterile seeds were then sown onto plates containing
media based on 80% MS without a nitrogen source. For C/N assays,
the media contained 3% sucrose. The -N/+Gln media was identical but
was supplemented with 1 mM glutamine. Plates were incubated in a
24-hour light C (120-130 .mu.Eins.sup.-2 m.sup.-1) growth chamber
at 22.degree. C. Evaluation of germination and seedling vigor was
done five days after planting for C/N assays. The production of
less anthocyanin on these media is generally associated with
increased tolerance to nitrogen limitation, and a transgene
responsible for the altered response is likely involved in the
plant's ability to perceive their carbon and nitrogen status.
[0407] Data was recorded for all phenotypes observed, regardless of
their strength, based on the assumption that any lead could
potentially result in a product either after a period of
development or improvement, or when used in combination with
another gene involved in the particular stress response
pathway.
[0408] All scores presented in the result lists (other than
wild-type) were based on data from two independent experiments on
the seed batches, assuming sufficient seed was available to repeat
the experiment twice.
Shade Tolerance Assays
[0409] The shade avoidance response was determined by the
perception of light quality. We used an assay which detects
alterations in the mechanisms that plants use to sense light
quality and presumably activate the signal transduction cascades
that regulate a shade avoidance response. Seeds were germinated
under white light versus light deficient in the red portion of the
visible spectrum. In a natural setting, reflected or transmitted
light would be deficient in both the red and blue portions of the
visible spectrum. However, because shading is detected using
phytochrome to sense the R:FR ratio in light, we mimicked the
effect of shading by using a filter designed to prevent only the
transmission of red wavelengths (to mimic loss of red light caused
by shading). To determine whether the mechanisms used to sense
shading were altered, we exploited the observation that seedlings
of wild-type plants grown under light deficient in red wavelengths
have extended hypocotyls. Plants overexpressing genes that produce
short hypocotyls under these conditions and exhibit a shade
tolerance phenotype are candidates for further examination in more
rigorous studies looking at components such as yield under high
densities in greenhouse studies.
[0410] The assay was intended to associate a transcription factor
with shade avoidance control mechanisms. All data were recorded,
regardless of phenotype strength, based on the assumption that any
lead (or its related paralogs/orthologs) could potentially result
in a product either after a period of development or improvement,
or when used in combination with another gene involved in the
particular stress response pathway.
[0411] Arabidopsis thaliana ecotype Columbia (Col-0) was used to
create all overexpressing lines. The control plants for the assay
were Col-0 plants transformed with an empty transformation vector
(pMEN65).
[0412] For overexpression lines examined in the assay, the screen
was primarily performed on a seed lot comprised of seed mixed
together from each of three independent primary transformants.
These seed batches were segregating, but selection was not
performed to avoid the extra stress that might be associated with
kanamycin selection. In the case of knockout (KO) lines, the screen
was performed on seed from plant(s) homozygous for a T-DNA
insertion within the gene of interest.
[0413] Prior to plating, seed for all experiments were surface
sterilized in the following manner:
[0414] 1. 5 minute incubation with mixing in 70% ethanol
[0415] 2. 20 minute incubation with mixing in 30% bleach, 0.01%
triton-X 100
[0416] 3. 5.times. rinses with sterile water
[0417] 4. Seeds are re-suspended in 0.1% sterile agarose and
stratified at 4.degree. C. for 3 days.
[0418] The basal media onto which Arabidopsis seeds were plated
comprised 80% MS+Vitamins. For shade avoidance assays, plates were
incubated at 22.degree. C. under 24-hour light (about 50
.mu.Einsteins.sup.-2 m.sup.-1) under both white light (control) and
under light depleted in red wavelengths. Seedlings were grown in a
chamber deficient in red light versus a standard white light
chamber. The assay was designed to detect plants that were more
tolerant of the low R:FR conditions. The growth chamber used in the
shade avoidance screen contained a filter that effectively removed
wavelengths in the red region of the visible light spectrum.
Seedlings were assessed for shade tolerance at 7 days.
[0419] Shade tolerance was scored by visually observing differences
in hypocotyl length compared with control seedlings grown under
white light and grown under light lacking the red wavelengths.
[0420] Examples of genes and homologs that confer significant
improvements to knockout or overexpressing plants are noted below.
Experimental observations made by us with regard to specific genes
whose expression has been modified in overexpressing or knock-out
plants, and potential applications based on these observations, are
also presented. In most cases, the conserved domains can be
determined and located in each of the sequences provided below with
the protein BLAST (BLASTp) page of the NCBI Conserved Domain
Database, presently found at: blast.ncbi.nlm.nih.gov/Blast.cgi.
(Marchler-Bauer A et al. (2009) Nucleic Acids Res. 37(D): 205-210;
Marchler-Bauer and Bryant (2004) Nucleic Acids Res. 32(W):
327-331).
Example VIII: Results of Drought Stress Analyses
[0421] This example provides experimental evidence for increased
abiotic stress tolerance controlled by transcription factor
polypeptides and polypeptides of the invention.
Results:
[0422] As noted below, overexpression of G2133, G1274, G922, G2999,
G3086, G354, G1792, G2053, G975, G1069, G916, G1820, G2701, G47,
G2854, G2789, G634, G175, G2839, G1452, G3083, G489, G303, G2992,
and G682 was shown to increase drought stress tolerance in plants.
A number of orthologs of some of these sequences were also able to
increase abiotic stress tolerance, as noted below.
The G47 Clade of Transcription Factor Polypeptides
G47 (SEQ ID NO: 1 and 2)
[0423] G47 corresponds to gene T22J18.2 (AAC25505). No information
is available about the function(s) of G47. G47 and closely-related
clade member sequences each comprise a conserved AP2 DNA binding
domain that is expected to function in a similar manner in each of
these related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0424] Experimental Observations.
[0425] The function of G47 was studied using transgenic Arabidopsis
plants in which the gene was expressed under the control of the 35S
promoter. Overexpression of G47 resulted in a variety of
morphological and physiological phenotypic alterations.
[0426] 35S::G47 plants showed enhanced tolerance to osmotic stress;
osmotic stress assays were conducted using growth medium containing
polyethylene glycol (PEG). After germination, the seedlings of
35S::G47 overexpressing lines generally appeared larger and had
more root growth than wild-type control seedlings.
[0427] As would be predicted by these osmotic stress assays, G47
plants also showed enhanced survival and drought tolerance in a
soil-based drought assay.
[0428] Overexpression of G47 also produced a substantial delay in
flowering time and caused a marked change in shoot architecture.
35S::G47 transformants were small at early stages and switched to
flowering more than a week later than wild-type controls
(continuous light conditions). Interestingly, the inflorescences
from these plants appeared thick and fleshy, had reduced apical
dominance, and exhibited reduced internode elongation leading to a
short compact stature. The branching pattern of the stems also
appeared abnormal, with the primary shoot becoming `kinked` at each
coflorescence node. Additionally, the plants showed slightly
reduced fertility and formed rather small siliques that were borne
on short pedicels and held vertically, close against the stem.
[0429] Additional alterations were detected in the inflorescence
stems of 35S::G47 plants. Stem sections from T2-21 and T2-24 plants
were of wider diameter, and had large irregular vascular bundles
containing a much greater number of xylem vessels than wild type.
Furthermore some of the xylem vessels within the bundles appeared
narrow and were possibly more lignified than were those of
controls.
[0430] G47 was expressed at higher levels in rosette leaves, and
transcripts can be detected in other tissues (flower, embryo,
silique, and germinating seedling), but apparently not in
roots.
[0431] Utilities.
[0432] G47 or its equivalogs can be used to increase the tolerance
of plants to drought and to other osmotic stresses. G47 or its
equivalogs could also be used to manipulate flowering time, to
modify plant architecture and stem structure, including development
of vascular tissues and lignin content. The use of G47 or its
equivalogs from tree species could offer the potential for
modulating lignin content. This might allow the quality of wood
used for furniture or construction to be improved. G47 equivalogs
include, for example, Arabidopsis thaliana SEQ ID NO: 12 (G2133);
Oryza sativa (japonica cultivar-group) SEQ ID NOs: 98 (G3649), SEQ
ID NO: 100 (G3651), and SEQ ID NO: 90 (G3644); Glycine max SEQ ID
NO: 88 (G3643); Zinnia elegans SEQ ID NO: 96 (G3647); Brassica rapa
subsp. Pekinensis SEQ ID NO: 92 (G3645); and Brassica oleracea SEQ
ID NO: 94 (G3646).
G2133 (SEQ ID NO: 11 and 12)
[0433] G2133 is a paralog of G47. G2133 corresponds to gene
F26A9.11 (AAF23336). No information is available about the
function(s) of G2133. G2133 and closely-related clade member
sequences each comprise a conserved AP2 DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0434] Experimental Observations.
[0435] The function of G2133 was studied using transgenic
Arabidopsis plants in which the gene was expressed under the
control of the 35S promoter.
[0436] G2133 expression was detected in a variety of tissues:
flower, leaf, embryo, and silique samples. Its expression might be
altered by several conditions, including auxin treatment, osmotic
stress, and Fusarium infection. Overexpression of G2133 caused a
variety of alterations in plant growth and development: delayed
flowering, altered inflorescence architecture, and a decrease in
overall size and fertility.
[0437] At early stages, 35S::G2133 transformants were markedly
smaller than controls and displayed curled, dark-green leaves. Most
of these plants remained in a vegetative phase of development
substantially longer than controls, and produced an increased
number of leaves before bolting. In the most severely affected
plants, bolting occurred more than a month later than in wild type
(24-hour light). In addition, the plants displayed a reduction in
apical dominance and formed large numbers of shoots simultaneously,
from the axils of rosette leaves. These inflorescence stems had
short internodes, and carried increased numbers of cauline leaf
nodes, giving them a very leafy appearance. The fertility of
35S::G2133 plants was generally very low. In addition, G2133
overexpressing lines were found to be more resistant to the
herbicide glyphosate in initial and repeat experiments.
[0438] No alterations were detected in 35S::G2133 plants in the
biochemical analyses that were performed.
[0439] G2133 is a paralog of G47, the latter having been known from
earlier studies to confer a drought tolerance phenotype when
overexpressed. It was thus not surprising when G2133 was also shown
to induce drought tolerance in a number of 35S::G2133 lines
challenged in soil-based drought assays (Tables 11 and 12).
Experiments comparing the recovery of wild-type controls and two
lines of Arabidopsis plants overexpressing G2133 (a paralog of G47)
from a drought treatment were conducted under constant light. The
35S::G2133 and control lines were grown in pots with each pot
containing several plants. All were deprived of water for eight
days, and then re-watered. After re-watering, all of the plants of
both G2133 overexpressor lines became reinvigorated, and all of the
control plants died or were severely affected by the drought
treatment (Table 12).
[0440] Utilities.
[0441] G2133 and its equivalogs can be used to increase the
tolerance of plants to drought and to other osmotic stresses. G2133
could also be used for the generation of glyphosate resistant
plants, and to increase plant resistance to oxidative stress. G2133
equivalogs include, for example, Arabidopsis thaliana SEQ ID NO: 2
(G47); Oryza sativa (japonica cultivar-group) SEQ ID NO: 98
(G3649), SEQ ID NO: 100 (G3651), and SEQ ID NO: 90 (G3644); Glycine
max SEQ ID NO: 88 (G3643); Zinnia elegans SEQ ID NO: 96 (G3647);
Brassica rapa subsp. Pekinensis SEQ ID NO: 92 (G3645); and Brassica
oleracea SEQ ID NO: 94 (G3646).
G3643 (SEQ ID NO: 87 and 88)
[0442] G3643 is a soy ortholog of G47 and G2133. G3643 and
closely-related clade member sequences each comprise a conserved
AP2 DNA-binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0443] Experimental Observations.
[0444] The function of G3643 was studied using transgenic
Arabidopsis plants in which the gene was expressed under the
control of the 35S promoter.
[0445] G3643-overexpressing Arabidopsis plants were more tolerant
to cold than wild-type control plants grown under similar
conditions in plate-based germination assays. One of these lines
was also more tolerant to desiccation and growth in cold conditions
in plate-based assays.
[0446] Utilities.
[0447] G3643 or its equivalogs can be used to increase the
tolerance of plants to cold conditions and low water conditions,
including drought.
G3644 (SEQ ID NO: 89 and 90)
[0448] G3644 is a rice ortholog of G47 and G2133. G3644 and
closely-related clade member sequences each comprise a conserved
AP2 DNA-binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0449] Experimental Observations.
[0450] The function of G3644 was studied using transgenic
Arabidopsis plants in which the gene was expressed under the
control of the 35S promoter.
[0451] Several G3644-overexpressing Arabidopsis plants were found
to be more tolerant to desiccation than wild-type control plants
grown under similar conditions in plate based-assays. Two lines
were shown to be more salt tolerant than wild type.
[0452] Utilities.
[0453] G3644 or its equivalogs can be used to increase the
tolerance of plants to high salt and low water conditions,
including drought.
G3649 (SEQ ID NO: 97 and 98)
[0454] G3649 is a rice ortholog of G47 and G2133. G3649 and
closely-related clade member sequences each comprise a conserved
AP2 DNA-binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0455] Experimental Observations.
[0456] The function of G3649 was studied using transgenic
Arabidopsis plants in which the gene was expressed under the
control of the 35S promoter.
[0457] Several G3649-overexpressing Arabidopsis plants were more
tolerant to cold than wild-type control plants grown under similar
conditions in plate-based germination assays. Two overexpressing
lines were more heat tolerant than wild-type plants, and one
35S::G3649 line was found to be more desiccation tolerant than wild
type.
[0458] Utilities.
[0459] G3649 or its equivalogs can be used to increase the
tolerance of plants to cold conditions and low water conditions,
including drought.
The G1274 Clade of Transcription Factor Polypeptides
G1274 (SEQ ID NO: 5 and 6)
[0460] G1274 is a member of the WRKY family of transcription
factors. The gene corresponds to WRKY51 (At5g64810). G1274 and
closely-related clade member sequences each comprise a conserved
WRKY DNA-binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0461] Experimental Observations.
[0462] RT-PCR analysis was used to determine the endogenous
expression pattern of G1274. Expression of G1274 was detected in
leaf, root and flower tissues. The biotic stress related
conditions, Erysiphe and SA treatment, induced expression of G1274
in leaf tissue. The gene also appeared to be slightly induced by
osmotic and cold stress treatments and perhaps by auxin.
[0463] The function of G1274 was studied using transgenic plants in
which the gene was expressed under the control of the 35S promoter.
G1274 overexpressing lines were more tolerant to growth on low
nitrogen containing media. In an assay intended to determine
whether the transgene expression could alter C/N sensing,
35S::G1274 seedlings contained less anthocyanins than wild-type
controls (grown on high sucrose/N- and high sucrose/N/Gln plates.
These data together indicated that overexpression of G1274 may
alter a plant's ability to modulate carbon and/or nitrogen uptake
and utilization.
[0464] G1274 overexpression and wild-type germination were also
compared in a cold germination assay, the overexpressors appearing
larger and greener than the controls.
[0465] 35S::G1274-overexpressing plants were significantly greener
and larger than wild-type control plants in a soil-based drought
assay (Tables 11 and 12). These assays confirmed the results
predicted after the performance of the plate-based osmotic stress
assays; 35S::G1274 lines fared much better after a period of water
deprivation than control plants. This distinction was particularly
evident in the overexpressor plants after once again being watered;
the overexpressor plants almost all fully recovered to a healthy
and vigorous state. Conversely, none of the wild-type plants
recovered after rewatering, as it was apparently too late for
rehydration to rescue these plants (Table 12).
[0466] In addition, 35S::G1274 transgenic plants were more tolerant
to chilling compared to the wild-type controls, in both germination
as well as seedling growth assays.
[0467] Overexpression of G1274 produced alterations in leaf
morphology and inflorescence architecture. Four out of eighteen
35S::G1274 primary transformants were slightly small and developed
inflorescences that were short, and showed reduced internode
elongation, leading to a bushier, more compact stature than in
wild-type.
[0468] In an experiment using T2 populations, it was observed that
the rosette leaves from many of the plants were distinctly broad
and appeared to have a greater rosette biomass than in wild
type.
[0469] A similar inflorescence phenotype was obtained from
overexpression of a potentially related WRKY gene, G1275. However,
G1275 also caused extreme dwarfing, which was not apparent when
G1274 was overexpressed.
[0470] Utilities.
[0471] The phenotypic effects of G1274 or equivalog overexpression
could have several potential applications:
[0472] The enhanced performance of 35S::G1274 plants in a
soil-based drought assay indicated that the gene or its equivalogs
may be used to enhance drought tolerance in plants.
[0473] The enhanced performance of 35S::G1274 seedlings under
chilling conditions indicates that the gene or its equivalogs might
be applied to engineer crops that show better growth under cold
conditions.
[0474] The morphological phenotype shown by 35S::G1274 lines
indicate that the gene or its equivalogs might be used to alter
inflorescence architecture, to produce more compact dwarf forms
that might afford yield benefits.
[0475] The effects on leaf size that were observed as a result of
G1274 or equivalog overexpression might also have commercial
applications. Increased leaf size, or an extended period of leaf
growth, could increase photosynthetic capacity, and biomass, and
have a positive effect on yield. G1274 equivalogs include, for
example, Arabidopsis thaliana SEQ ID NO: 30 (G1275) and SEQ ID NO:
32 (G1758); Oryza sativa (japonica cultivar-group) SEQ ID NO: 134
(G3721), SEQ ID NO: 142 (G3725), SEQ ID NO: 144 (G3726), SEQ ID NO:
150 (G3729), and SEQ ID NO: 152 (G3730); Glycine max SEQ ID NO: 138
(G3723), SEQ ID NO: 140 (G3724), and SEQ ID NO: 208 (G3803);
Solanum tuberosum SEQ ID NO: 156 (G3732); Capsicum annuum SEQ ID
NO: 202 (G3795); Lactuca sativa SEQ ID NO: 204 (G3797); Hordeum
vulgare SEQ ID NO: 158 (G3733); Zea mays SEQ ID NO: 130 (G3719),
SEQ ID NO: 132 (G3720), SEQ ID NO: 136 (G3722), SEQ ID NO: 146
(G3727), SEQ ID NO: 148 (G3728), and SEQ ID NO: 210 (G3804);
Sorghum bicolor SEQ ID NO: 206 (G3802); and Lycopersicon esculentum
SEQ ID NO: 154 (G3731).
The G922 Clade of Transcription Factor Polypeptides
G922 (SEQ ID NO: 3 and 4)
[0476] G922 corresponds to Scarecrow-like 3 (SCL3) first described
by Pysh et al. (GenBank accession number AF036301; (1999) Plant J.
18: 111-119). Northern blot analysis results show that G922 is
expressed in siliques, roots, and to a lesser extent in shoot
tissue from 14 day old seedlings. Pysh et al did not test any other
tissues for G922 expression. In situ hybridization results showed
that G922 was expressed predominantly in the endodermis in the root
tissue. This pattern of expression was very similar to that of
SCARECROW (SCR), G306. Experimental evidence indicated that the
co-localization of the expression is not due to cross-hybridization
of the G922 probe with G306. Pysh et al proposed that G922 may play
a role in epidermal cell specification and that G922 may either
regulate or be regulated by G306. G922 and closely-related clade
member sequences each comprise at least one conserved SCR domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0477] The sequence for G922 can also be found in the annotated BAC
clone F11F12 from chromosome 1 (GenBank accession number AC012561).
The sequence for F11F12 was submitted to GenBank by the DNA
Sequencing and Technology Center at Stanford University.
[0478] Experimental Observations.
[0479] The function of this gene was analyzed using transgenic
plants in which G922 was expressed under the control of the 35S
promoter.
[0480] Morphologically, plants overexpressing G922 had altered leaf
morphology, coloration, fertility, and overall plant size. In
wild-type plants, expression of G922 was induced by auxin, ABA,
heat, and drought treatments. In non-induced wild-type plants, G922
was expressed constitutively at low levels.
[0481] Transgenic plants overexpressing G922 were more salt
tolerant than wild-type plants as determined by a root growth assay
on MS media supplemented with 150 mM NaCl; 35S::G922 overexpressors
exhibited greener seedlings with longer roots than wild-type
seedlings.
[0482] G922 overexpressors were more cold tolerant than wild-type
controls, with overexpressor lines accumulating less anthocyanin
than wild-type plants.
[0483] G922 overexpressors were also more desiccation tolerant in
plate-based assays than wild-type control plants, as the seedlings
of the former were larger and greener in these experiments.
[0484] Almost all of the G922 overexpressors were exhibited a
degree of insensitivity to ABA; on ABA-containing plates,
overexpressor seedlings were larger and greener than wild-type
controls. For some lines, the difference between overexpressors and
wild-type plants was dramatic.
[0485] Arabidopsis plants overexpressing G922 also were more
tolerant to osmotic stress as determined by germination assays in
sucrose (9.4%)-containing media than controls; overexpressors had
greener cotyledons and longer roots than wild-type seedlings on the
same media.
[0486] The high salt, ABA, osmotic stress and plate-based
desiccation assays suggested that this gene would confer drought
tolerance, a supposition confirmed by soil-based assays, in which
G922-overexpressing plants were significantly healthier after water
deprivation treatment than wild-type control plants (Tables 11 and
12).
[0487] Utilities.
[0488] Based upon results observed in plants overexpressing G922 or
its equivalogs could be used to alter salt tolerance, tolerance to
osmotic stress, and leaf morphology in other plant species.
Evaporation from the soil surface causes upward water movement and
salt accumulation in the upper soil layer where the seeds are
placed. Thus, germination normally takes place at a salt
concentration much higher than the mean salt concentration in the
whole soil profile. Increased salt tolerance during the germination
stage of a crop plant would impact survivability and yield.
[0489] Altered leaf morphology conferred by overexpression of G922
or its equivalogs could be desirable in ornamental horticulture.
G922 equivalogs include, for example, Oryza sativa (japonica
cultivar-group) SEQ ID NO: 218 (G3814), SEQ ID NO: 216 (G3813), and
SEQ ID NO: 222 (G3827); Lycopersicon esculentum SEQ ID NO: 220
(G3824); and Glycine max SEQ ID NO: 212 (G3810) and SEQ ID NO: 214
(G3811).
The G2999 Clade of Transcription Factor Polypeptides
G2999 (SEQ ID NO: 13 and 14)
[0490] G2999 was identified within a sequence released by the
Arabidopsis Genome Initiative (Chromosome 2, GenBank accession
AC006439). G2999 and closely-related clade member sequences each
comprise a conserved ZF-HD protein dimerization domain and a
homeo_ZF_HD homeobox domain that are expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0491] Experimental Observations.
[0492] The boundaries of G2999 were determined by RACE experiments
and a full-length clone was PCR-amplified out of cDNA derived from
mixed tissues. The function of G2999 was then assessed by analysis
of transgenic Arabidopsis lines in which the cDNA was
constitutively expressed from a 35S CaMV promoter. 35S::G2999
transformants displayed wild-type morphology, but two of three T2
lines showed increased tolerance to salt stress. Root growth assays
with G2999 overexpressing seedlings and controls in a high sodium
chloride medium showed that a majority of 35S::G2999 Arabidopsis
seedlings appeared larger, greener, and had more root growth than
the control seedlings. G2998, a paralogous Arabidopsis sequence,
also showed a salt tolerance phenotype in a plate-based salt stress
assay, where these overexpressors were greener and had more
cotyledon expansion than wild-type seedlings. Thus, G2998 and G2999
could act in the same pathways, and have a role in the response to
abiotic stress.
[0493] G2999 overexpressing lines were also more osmotic stress
tolerant, as evidenced by comparing their growth with wild-type
plants on 9.4% sucrose, and more cold tolerant than wild-type
plants.
[0494] These assays suggested that this gene would confer drought
tolerance, a supposition confirmed in a soil-based assay in which
G2999 overexpressing-plants were significantly more drought
tolerant than wild-type control plants (Tables 11 and 12).
[0495] Utilities.
[0496] Given the pattern of abiotic stress tolerance exhibited by
35S::G2999 transformants, the gene and its equivalogs can be used
to engineer drought and salt tolerant crops and trees that can
flourish in conditions of osmotic stress. G2999 equivalogs include,
for example, Arabidopsis thaliana SEQ ID NO: 50 (G2992), SEQ ID NO:
48 (G2991), SEQ ID NO: 68 (G3002), SEQ ID NO: 66 (G3001), SEQ ID
NO: 46 (G2990), SEQ ID NO: 44 (G2989), SEQ ID NO: 62 (G2998), SEQ
ID NO: 64 (G3000), SEQ ID NO: 54 (G2994), SEQ ID NO: 52 (G2993),
SEQ ID NO: 60 (G2997), SEQ ID NO: 58 (G2996), SEQ ID NO: 56
(G2995); Zea mays SEQ ID NO: 114 (G3680); Oryza sativa (japonica
cultivar group) SEQ ID NO: 128 (G3695), SEQ ID NO: 126 (G3694), SEQ
ID NO: 122 (G3690), SEQ ID NO: 118 (G3685), SEQ ID NO: 108 (G3671),
SEQ ID NO: 116 (G3683), and SEQ ID NO: 124 (G3692); Oryza sativa
(indica cultivar group) SEQ ID NO: 120 (G3686) and SEQ ID NO: 110
(G3674); Lotus corniculatus var. japonicus SEQ ID NO: 102 (G3663)
and SEQ ID NO: 106 (G3670); Brassica napus SEQ ID NO: 112 (G3675);
and Flaveria bidentis SEQ ID NO: 104 (G3668).
G2989 (SEQ ID NO: 43 and 44)
[0497] G2989 is a paralog of G2999 from Arabidopsis. G2989 and
closely-related clade member sequences each comprise a conserved
ZF-HD protein dimerization domain and a homeo_ZF_HD homeobox domain
that are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
Experimental Observations.
[0498] G2989 overexpressors were more desiccation and cold tolerant
than wild-type controls in plate-based assays.
[0499] Utilities.
[0500] Given the pattern of abiotic stress tolerance exhibited by
35S::G2989 transformants, the gene and its equivalogs can be used
to engineer drought and cold tolerant crops and trees.
G2990 (SEQ ID NO: 45 and 46)
[0501] G2990 is a paralog of G2999 from Arabidopsis. G2990 and
closely-related clade member sequences each comprise a conserved
ZF-HD protein dimerization domain and a homeo_ZF_HD homeobox domain
that are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
Experimental Observations.
[0502] G2990 overexpressors were more ABA insensitive and
desiccation and cold tolerant than wild-type controls in
plate-based assays.
[0503] Utilities.
[0504] Given the pattern of abiotic stress tolerance exhibited by
35S::G2990 transformants, the gene and its equivalogs can be used
to engineer drought and cold tolerant crops and trees.
G2992 (SEQ ID NO: 49 and 50)
[0505] G2992 corresponds to gene F24J1.29 within BAC clone F24J1
(GenBank accession AC021046) derived from chromosome 1. We
identified this locus as a novel member of the ZF-HB family and no
data regarding its function are currently in the public domain (as
of 8/5/02). G2992 and closely-related clade member sequences each
comprise a conserved ZF-HD protein dimerization domain and a
homeo_ZF_HD homeobox domain that are expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0506] Experimental Observations.
[0507] The boundaries of G2992 were determined by RACE, and a clone
was PCR-amplified from cDNA derived from mixed tissue samples. The
function of G2992 was then assessed by analysis of transgenic
Arabidopsis lines in which the cDNA was constitutively expressed
from a 35S CaMV promoter.
[0508] Morphological studies revealed that overexpression of G2992
can accelerate the onset of reproductive development, reduce plant
size, and produce changes in leaf shape.
[0509] 35S::G2992 T2 populations displayed an enhanced ability to
germinate on plates containing high levels of sodium chloride. The
role of G2992 in a response pathway to abiotic stress was affirmed
by a soil-based drought assay, in which it was shown that G2992
overexpressors were, on average, more tolerant to water deprivation
conditions in soil-based drought assays than wild-type plants
(Table 12), and one of the lines tested was significantly more
drought tolerant than the wild-type controls.
[0510] Utilities.
[0511] Based on the phenotypes observed in morphological and
physiological assays, G2992 might have a number of
applications.
[0512] Given the drought and salt tolerance exhibited by 35S::G2992
transformants, the gene and its equivalogs might be used to
engineer drought and salt tolerant crops and trees that can
flourish in drought conditions and salinified soils.
[0513] The early flowering exhibited by 35S::G2992 lines, indicates
that the gene might be used to manipulate flowering time in
commercial species. In particular, G2992 could be applied to
accelerate flowering or eliminate any requirements for
vernalization. In some instances, a faster cycling time might allow
additional harvests of a crop to be made within a given growing
season. Shortening generation times could also help speed-up
breeding programs, particularly in species such as trees, which
typically grow for many years before flowering. Conversely, it
might be possible to modify the activity of G2992 (or its
equivalogs) to delay flowering in order to achieve an increase in
biomass and yield.
[0514] Finally, the effects of G2992 overexpression on leaf shape
suggest that the gene might be used to modify plant
architecture.
G2994 (SEQ ID NO: 53 and 54)
[0515] G2994 is a paralog of G2999 from Arabidopsis. G2994 and
closely-related clade member sequences each comprise a conserved
ZF-HD protein dimerization domain and a homeo_ZF_HD homeobox domain
that are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
Experimental Observations.
[0516] Almost all of the G2994 overexpressors tested were more ABA
insensitive than wild-type controls in plate-based assays.
[0517] Utilities.
[0518] Given the ABA insensitivity exhibited by 35S::G2994
transformants, the gene and its equivalogs can be used to engineer
osmotic stress and drought tolerant crops and trees.
G2996 (SEQ ID NO: 57 and 58)
[0519] G2996 is a paralog of G2999 from Arabidopsis. G2996 and
closely-related clade member sequences each comprise a conserved
ZF-HD protein dimerization domain and a homeo_ZF_HD homeobox domain
that are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
Experimental Observations.
[0520] Many of the G2996 overexpressors tested were larger on 9.4%
sucrose than wild-type controls in plate-based assays.
[0521] Utilities.
[0522] Given the sugar sensing phenotype exhibited by 35S::G2996
transformants, the gene and its equivalogs can be used to engineer
osmotic stress and drought tolerant crops and trees.
G2997 (SEQ ID NO: 59 and 60)
[0523] G2997 is a paralog of G2999 from Arabidopsis. G2997 and
closely-related clade member sequences each comprise a conserved
ZF-HD protein dimerization domain and a homeo_ZF_HD homeobox domain
that are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
Experimental Observations.
[0524] Almost all of the G2997 overexpressors tested were more ABA
insensitive than wild-type controls in plate-based assays.
[0525] Utilities.
[0526] Given the ABA insensitivity exhibited by 35S::G2997
transformants, the gene and its equivalogs can be used to engineer
osmotic stress and drought tolerant crops and trees.
G3002 (SEQ ID NO: 67 and 68)
[0527] G3002 is a paralog of G2999 from Arabidopsis. Seedlings of
G3002 overexpressors were generally slightly larger than wild-type
controls. G3002 and closely-related clade member sequences each
comprise a conserved ZF-HD protein dimerization domain and a
homeo_ZF_HD homeobox domain that are expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
Experimental Observations.
[0528] G3002 overexpressors were more heat and cold tolerant than
wild-type controls in plate-based germination and growth
assays.
[0529] Utilities.
[0530] Given the pattern of abiotic stress tolerance exhibited by
35S::G3002 transformants, the gene and its equivalogs can be used
to engineer heat, drought and cold tolerant crops and trees.
The G3086 Clade of Transcription Factor Polypeptides
G3086 (SEQ ID NO: 15 and 16)
[0531] G3086 corresponds to gene AT1G51140, annotated by the
Arabidopsis Genome Initiative. No information is available about
the function(s) of G3086. G3086 and closely-related clade member
sequences each comprise a conserved bHLH DNA-binding and
dimerization domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0532] Experimental Observations.
[0533] The function of G3086 was studied using transgenic plants in
which the gene was expressed under the control of the 35S promoter.
Overexpression of G3086 in Arabidopsis produced a pronounced
acceleration in the onset of flowering. 35S::G3086 transformants
produced visible flower buds 5-7 days early (in inductive 24-hour
light conditions). Some lines were markedly smaller than wild-type
controls, although a number of lines at the seedling stage were
slightly larger than wild-type plants at the same stage.
[0534] G3086 overexpressing lines were larger and more tolerant of
cold stress; the overexpressors were generally larger than the wild
type plants when grown in cold conditions.
[0535] 35S::G3086 transformants were also larger and displayed more
root growth when grown under high salt conditions. G3086
overexpressors were larger, greener, and had more root growth than
control plants.
[0536] Several G3086 overexpressing lines were more tolerant to
desiccation in plate-based assays than wild-type control
plants.
[0537] These abiotic stress assays suggested that this gene may
confer drought tolerance, a supposition confirmed in a soil-based
assay in which G3086 overexpressing-plants were significantly more
tolerant of drought stress than control plants in soil-based
drought assays (Tables 11 and 12).
[0538] Utilities.
[0539] Based on the phenotypes observed in morphological and
physiological assays, G3086 and its equivalogs might have a number
of utilities.
[0540] Given the salt resistance exhibited by 35S::G3086
transformants, the gene or its equivalogs might be used to engineer
salt tolerant crops and trees that can flourish in saline soils, or
under drought conditions.
[0541] Based on the response of 35S::G3086 lines to cold stress,
the gene or its equivalogs might be used to engineer crop plants
with increased tolerance to abiotic stresses such as low
temperatures, and may thus improve the range available for planting
of many crop species.
[0542] The early flowering displayed by 35S::G3086 transformants
indicated that the gene or its equivalogs might be used to
accelerate the flowering of commercial species, or to eliminate any
requirements for vernalization.
[0543] G3086 equivalogs include, for example, Arabidopsis thaliana
SEQ ID NO: 26 (G592), SEQ ID NO: 28 (G1134), SEQ ID NO: 38 (G2149),
SEQ ID NO: 40 (G2555); and SEQ ID NO: 42 (G2766); Oryza sativa
(japonica cultivar-group) SEQ ID NO: 168 (G3740), SEQ ID NO: 170
(G3741), SEQ ID NO: 172 (G3742), SEQ ID NO: 174 (G3744), and SEQ ID
NO: 176 (G3746); Glycine max SEQ ID NO: 180 (G3763), SEQ ID NO: 182
(G3764), SEQ ID NO: 184 (G3765), SEQ ID NO: 186 (G3766), SEQ ID NO:
188 (G3767), SEQ ID NO: 190 (G3768), SEQ ID NO: 192 (G3769), SEQ ID
NO: 194 (G3771), and SEQ ID NO: 196 (G3772); Zea mays SEQ ID NO:
178 (G3755); and Pinus taeda SEQ ID NO: 197 (G3782).
The G354 Clade of Transcription Factor Polypeptides
G354 (SEQ ID NO: 227 and 228)
[0544] G354 was identified in the sequence of BAC clone F12M12,
GenBank accession number AL355775, released by the Arabidopsis
Genome Initiative. G354 corresponds to ZAT7 (Meissner and Michael
(1997) Plant Mol. Biol. 33: 615-624). G354 and closely-related
clade member sequences each comprise a conserved C2H2 zinc finger
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0545] Experimental Observations.
[0546] The highest level of expression of G354 was observed in
rosette leaves, embryos, and siliques. Some expression of G354 was
also observed in flowers.
[0547] The function of this gene was analyzed using transgenic
plants in which G353 was expressed under the control of the 35S
promoter. 35S::G354 plants had a reduction in flower pedicel
length, and downward pointing siliques. This phenotype was very
similar to that described for the brevipedicellus (bp) mutant
(Koornneef et al. (1983) J. Hered. 74: 265-272) and in
overexpression of a related gene G353. Other morphological changes
in shoots were also observed in 35S::G354 plants. Many 35S::G354
seedlings had abnormal cotyledons, elongated, thickened hypocotyls,
and short roots. The majority of T1 plants had a very extreme
phenotype, were tiny, and arrested development without forming
inflorescences. T1 plants showing more moderate effects had poor
seed yield.
[0548] Overexpression of G354 in Arabidopsis resulted in seedlings
with an altered response to light. In a germination assay conducted
in darkness, G354 seedlings failed to show an etiolation response.
In some cases the phenotype was severe; overexpression of the
transgene resulted in reduced open and greenish cotyledons.
[0549] G354 overexpressors were also shown to be tolerant to water
deprivation in soil-based drought assays (Tables 11 and 12).
Closely related paralogs of this gene, G353 and G2839, also showed
an osmotic stress tolerance phenotype in a germination assay on
media containing high sucrose; one line of 35S::G353 seedlings and
several lines of 35S::G2839 were greener and had higher germination
rates than controls. Thus, G354 and its paralogs G353 and G2839
appear to influence osmotic stress responses.
[0550] Utilities.
[0551] G354 and its equivalogs can be could be used to increase a
plant's tolerance to drought and other osmotic stress, and can be
used alter inflorescence structure, which may have value in
production of novel ornamental plants.
G353 (SEQ ID NO: 259 and 260)
[0552] G353 is a paralog of G354 from Arabidopsis. G353 and
closely-related clade member sequences each comprise a conserved
C2H2 DNA-binding zinc finger domain that is expected to function in
a similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0553] Experimental Observations.
[0554] Overexpressors of G353 have shown an osmotic stress
tolerance phenotype in a germination assay on media containing high
sucrose. These results suggested that the gene may also confer
drought tolerance, an indication confirmed in soil-based drought
assays. In the latter assays, G353 overexpressing Arabidopsis
plants were more tolerant to initial water deprivation, and after
rewatering, exhibited superior recovery than wild-type
controls.
[0555] Utilities.
[0556] G353 and its equivalogs can be could be used to increase a
plant's tolerance to drought and other osmotic stress.
G2839 (SEQ ID NO: 249 and 250)
[0557] G2839 is a paralog of G354 from Arabidopsis. G2839 and
closely-related clade member sequences each comprise a conserved
C2H2 DNA-binding zinc finger domain that is expected to function in
a similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0558] G2839 (At3g46080) was identified in the sequence of BAC
F12M12 (GenBank accession number AL355775) based on its sequence
similarity within the conserved domain to other C2H2 related
proteins in Arabidopsis. There is no published or public
information about the function of G2839.
[0559] Experimental Observations.
[0560] The function of G2839 was studied using transgenic plants in
which the gene was expressed under the control of the 35S promoter.
Few primary transformants were generated, suggesting that G2839
overexpression can be lethal. T1 lines displayed stunted growth and
development, and yielded very few or zero seeds. Inflorescences
were poorly developed. In one line, flower pedicels were very short
and flowers and siliques were oriented downwards. G2839
overexpressors showed a phenotype in a germination assay on media
containing high sucrose: seedlings were green and had high
germination rates. Thus, the gene appeared to influence sugar
sensing and/or osmotic stress responses.
[0561] G2839 is similar to two other Arabidopsis sequences, G354
and G353. Flower phenotypes in which pedicels were very short and
flowers and siliques were oriented downwards have been described
for G353 and G354 and are also similar to the brevipedicellus
mutant (Koornneef et al. (1983) J. Hered. 74: 265-272; Venglat et
al. (2002) Proc. Natl. Acad. Sci. USA. 99:4730-4735; Douglas et al.
(2002) Plant Cell. 14:547-558. Interestingly 35S::G353 lines also
showed increased resistance to osmotic stress.
[0562] Supplementing the results of the high sucrose germination
assay, G2839 was shown to be more tolerant to water deprivation
than wild-type control plants in soil-based drought assays (Tables
11 and 12).
[0563] Utilities.
[0564] The phenotypes observed in physiology assays indicate that
G2839 might be used to generate crop plants with altered sugar
sensing. Since the gene appears to be associated with the response
to osmotic stress, the gene could be used to engineer cold and
dehydration tolerance. The latter was confirmed by the soil-based
drought assay.
[0565] The morphological phenotype shown by 35S::G2839 lines
indicate that the gene might be used to alter inflorescence
architecture. In particular, a reduction in pedicel length and a
change in the position at which flowers and fruits are held, might
influence harvesting or pollination efficiency. Additionally, such
changes might produce attractive novel forms for the ornamental
markets.
The G1792 Clade of Transcription Factor Polypeptides
G1792 (SEQ ID NO: 7 and 8)
[0566] G1792 was identified in the sequence of BAC clone K14B15
(AB025608, gene K14B15.14). G1792 and closely-related clade member
sequences each comprise a conserved AP2 DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0567] Experimental Observations.
[0568] G1792 was studied using transgenic plants in which the gene
was expressed under the control of the 35S promoter.
[0569] In soil-based assays, G1792 overexpressing plants were
significantly more drought tolerant than wild-type control plants;
35S::G1792 lines fared much better after a period of water
deprivation than control plants. This distinction was particularly
evident in the overexpressor plants when the drought period was
followed by rewatering; the overexpressor plants recovered to a
healthy and vigorous state. Conversely, none of the wild-type
plants in these experiments recovered after rewatering
[0570] 35S::G1792 plants were more tolerant to the fungal pathogens
Fusarium oxysporum and Botrytis cinerea and showed fewer symptoms
after inoculation with a low dose of each pathogen. This result was
confirmed using individual T2 lines. The effect of G1792
overexpression in increasing tolerance to pathogens received
further, incidental confirmation. T2 plants of two 35S::G1792 lines
had been growing in a room that suffered a serious powdery mildew
infection. For each line, a pot of six plants was present in a flat
containing nine other pots of lines from unrelated genes. In either
of the two different flats, the only plants that were free from
infection were those from the 35S::G1792 line. This observation
suggested that G1792 overexpression might be used to increase
resistance to powdery mildew. Additional experiments confirmed that
35S::G1792 plants showed increased tolerance to Erysiphe. G1792 was
ubiquitously expressed, but appeared to be induced by salicylic
acid.
[0571] 35S::G1792 overexpressing plants also showed more tolerance
to growth under nitrogen-limiting conditions. In a root growth
assay under conditions of limiting N, 35S::G1792 lines were
slightly less stunted. The lack of anthocyanin production by
35S::G1274 seedlings grown on low nitrogen media supplemented with
sucrose plus glutamine, as compared to wild-type seedlings which
accumulated significant anthocyanin, indicated that these lines
were less stressed than control seedlings under the same
conditions. These results indicate that G1792 can be involved in
monitoring carbon and nitrogen status in plants.
[0572] G1792 overexpressors and wild-type plants were also compared
in a cold germination assay, in which the overexpressors were found
to be generally larger and greener than the controls.
[0573] G1792 overexpressing plants showed several mild
morphological alterations: leaves were dark green and shiny, and
plants bolted, subsequently senesced, slightly later than wild-type
controls. Among the T1 plants, additional morphological variation
(not reproduced later in the T2 plants) was observed: many showed
reductions in size as well as aberrations in leaf shape,
phyllotaxy, and flower development.
[0574] Utilities.
[0575] G1792 or its equivalogs can be used to improve drought and
other osmotic stress tolerances, and engineer pathogen-resistant
plants. In addition, it can also be used to improve seedling
germination and performance under conditions of limited
nitrogen.
[0576] Potential utilities of this gene or its equivalogs also
include increasing chlorophyll content allowing more growth and
productivity in conditions of low light. With a potentially higher
photosynthetic rate, fruits could have higher sugar content.
Increased carotenoid content could be used as a nutraceutical to
produce foods with greater antioxidant capability.
[0577] G1792 or its equivalogs could be used to manipulate wax
composition, amount, or distribution, which in turn could modify
plant tolerance to drought and/or low humidity or resistance to
insects, as well as plant appearance (shiny leaves). Increased wax
deposition on leaves of a plant like cotton may improve drought
resistance or water use efficiency. A possible application for this
gene might be in reducing the wax coating on sunflower seeds (the
wax fouls the oil extraction system during sunflower seed
processing for oil). For this purpose, antisense or co-suppression
of the gene in a tissue-specific manner might be useful
[0578] G1792 equivalogs include, for example, Arabidopsis thaliana
SEQ ID NO: 18 (G30), SEQ ID NO: 34 (G1791), and SEQ ID NO: 36
(G1795); Medicago truncatula SEQ ID NO: 160 (G3735); Glycine max
SEQ ID NO: 82 (G3518), SEQ ID NO: 84 (G3519), SEQ ID NO: 86
(G3520); Oryza sativa (japonica cultivar-group) SEQ ID NO: 70
(G3380), SEQ ID NO: 72 (G3381), SEQ ID NO: 74 (G3383), SEQ ID NO:
76 (G3515), and SEQ ID NO: 164 (G3737); Zea mays), SEQ ID NO: 78
(G3516), SEQ ID NO: 80 (G3517), SEQ ID NO: 200 (G3794), SEQ ID NO:
166 (G3739) and Triticum aestivum SEQ ID NO: 162 (G3736).
G3381 (SEQ ID NO: 71 and 72)
[0579] G3381 is a rice ortholog of G1792. G3381 and closely-related
clade member sequences each comprise a conserved AP2 DNA-binding
domain that is expected to function in a similar manner in each of
these related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0580] Experimental Observations.
[0581] In plate-based assays, G3381 overexpressors were more
tolerant to mannitol and cold conditions than wild-type
controls.
[0582] Utilities.
[0583] G3381 and its equivalogs may be used to confer osmotic
stress, drought and cold tolerance in plants.
G3383 (SEQ ID NO: 73 and 74) G3383 is a rice ortholog of G1792.
G3383 and closely-related clade member sequences each comprise a
conserved AP2 DNA-binding domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0584] Experimental Observations.
[0585] In plate-based assays, G3383 overexpressors were more
tolerant to mannitol, cold and desiccation conditions than
wild-type controls.
[0586] Utilities.
[0587] G3383 and its equivalogs may be used to confer osmotic
stress, drought and cold tolerance in plants.
G3517 (SEQ ID NO: 73 and 74)
[0588] G3517 is a corn ortholog of G1792. G3517 and closely-related
clade member sequences each comprise a conserved AP2 DNA-binding
domain that is expected to function in a similar manner in each of
these related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0589] Experimental Observations.
[0590] In plate-based assays, G3517 overexpressors were more
tolerant to heat, cold and desiccation conditions than wild-type
controls.
[0591] Utilities.
[0592] G3517 and its equivalogs may be used to confer heat stress,
osmotic stress, drought and cold tolerance in plants.
The G2053 Clade of Transcription Factor Polypeptides
G2053 (SEQ ID NO: 9 and 10)
[0593] G2053 was identified in the sequence of BAC T27C4, GenBank
accession number AC022287, released by the Arabidopsis Genome
Initiative. G2053 and closely-related clade member sequences each
comprise a conserved NAC DNA-binding and dimerization domain that
is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0594] Experimental Observations.
[0595] The function of G2053 was analyzed using transgenic plants
in which the gene was expressed under the control of the 35S
promoter. Overexpression of G2053 in Arabidopsis resulted in plants
with altered osmotic stress tolerance. In a root growth assay on
media containing high concentrations of PEG, G2053 overexpressors
showed more root growth and were generally larger than wild-type
controls.
[0596] The osmotic stress tolerance assays suggested that this gene
may confer drought tolerance, a supposition confirmed in soil-based
assays in which G2053 overexpressors were significantly more
drought tolerant than wild-type control plants (Tables 11 and
12).
[0597] Utilities.
[0598] Based on the altered stress tolerance induced by G2053
overexpression, this transcription factor or its equivalogs could
be used to alter a plant's response water deficit conditions and,
therefore, could be used to engineer plants with enhanced tolerance
to drought, salt stress, and freezing.
[0599] G2053 equivalogs include, for example, Arabidopsis thaliana
SEQ ID NO: 20 (G515), SEQ ID NO: 22 (G516), and SEQ ID NO: 24
(G517)
G516 (SEQ ID NO: 21 and 22)
[0600] G516 is a paralog of G2053 from Arabidopsis. G516 and
closely-related clade member sequences each comprise a conserved
NAC DNA-binding and dimerization domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[0601] Experimental Observations.
[0602] 35S::G516 overexpressors were more tolerant to mannitol and
cold than wild-type control plants.
[0603] Utilities.
[0604] Based on the abiotic assay stress results, G516 could be
used to engineer plants with enhanced tolerance to osmotic stress,
drought and cold.
The G975 Clade of Transcription Factor Polypeptides
G975 (SEQ ID NO: 237 and 238)
[0605] After its discovery by us, G975 has appeared in the
sequences released by the Arabidopsis Genome Initiative (BAC F9L1,
GenBank accession number AC007591). G975 and closely-related clade
member sequences each comprise a conserved AP2 DNA binding domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared traits.
G975 and closely-related clade member sequences each comprise a
conserved AP2 DNA-binding domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0606] Experimental Observations.
[0607] G975 was discovered by us and is a new member of the
AP2/EREBP family (EREBP subfamily) of transcription factors. G975
is expressed in flowers and, at lower levels, in shoots, leaves,
and siliques. GC-FID and GC-MS analyses of leaves from G975
overexpressing plants have shown that the levels of C29, C31, and
C33 alkanes were substantially increased (up to 10-fold) compared
to control plants. A number of additional compounds of similar
molecular weight, presumably also wax components, also accumulated
to significantly higher levels in G975 overexpressing plants.
Although total amounts of wax in G975 overexpressing plants have
not yet been measured, C29 alkanes constitute close to 50% of the
wax content in wild-type plants (Millar et al. (1998) Plant Cell
11: 1889-1902), indicating that a major increase in total wax
content occurs in these transgenic plants. However, the transgenic
plants had an almost normal phenotype (small morphological
differences are detected in leaf appearance), indicating that
overexpression of G975 is not deleterious to the plant. It is
noteworthy that overexpression of G975 did not cause the dramatic
alterations in plant morphology that have been reported for
Arabidopsis plants in which the FATTY ACID ELONGATION1 gene was
overexpressed (Millar et al. (1998) supra). G975 could specifically
regulate the expression of some of the genes involved in wax
metabolism. One Arabidopsis AP2 gene was found that is
significantly more closely related to G975 than the rest of the
members of the AP2/EREBP family. This other gene, G1387, may have a
function, and therefore a utility, related to that of G975.
[0608] Plants overexpressing G975 were significantly larger and
greener than wild-type control plants in a soil-based drought assay
(Tables 11 and 12).
[0609] Utilities.
[0610] G975 or its equivalogs could be used to improve a plant's
tolerance to drought or low water conditions.
[0611] G975 or its equivalogs could be used to manipulate wax
composition, amount, or distribution, which in turn could modify
plant tolerance to drought and/or low humidity or resistance to
insects, as well as plant appearance (shiny leaves). A possible
application for this gene or its equivalogs might be in reducing
the wax coating on sunflower seeds (the wax fouls the oil
extraction system during sunflower seed processing for oil). For
this purpose, antisense or co-suppression of the gene in a
tissue-specific manner might be useful.
[0612] G975 could also be used to specifically alter wax
composition, amount, or distribution in those plants and crops from
which wax is a valuable product.
The G1073 Clade of Transcription Factor Polypeptides
G1073 (SEQ ID NO: 239 and 240), AtHRC1
[0613] G1073 has been identified in the sequence of a BAC clone
from chromosome 4 (BAC clone F23E12, gene F23E12.50, GenBank
accession number AL022604), released by EU Arabidopsis Sequencing
Project. G1073 and closely-related clade member sequences each
comprise a conserved At-hook domain and a second conserved domain
(amino acids 43-187) or the DUF296 domain (amino acids 61-180) that
are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0614] Experimental Observations.
[0615] The function of G1073 was analyzed using transgenic plants
in which G1073 was expressed under the control of the cauliflower
mosaic virus 35S promoter (these transgenic plants are referred to
as "35S::G1073"). Transgenic plants overexpressing G1073 were
substantially larger than wild-type controls, with at least a 60%
increase in biomass (Table 10). The increased mass of 35S::G1073
transgenic plants was attributed to enlargement of multiple organ
types including stems, roots and floral organs; other than the size
differences, these organs were not affected in their overall
morphology. 35S::G1073 plants exhibited an increase of the width
(but not length) of mature leaf organs, produced 2-3 more rosette
leaves, and had enlarged cauline leaves in comparison to
corresponding wild-type leaves. Overexpression of G1073 resulted in
an increase in both leaf mass and leaf area per plant, and leaf
morphology (G1073 overexpressors tended to produce more serrated
leaves). We also found that root mass was increased in the
transgenic plants, and that floral organs were also enlarged. An
increase of approximately 40% in stem diameter was observed in the
transgenic plants. Images from the stem cross-sections of
35S::G1073 plants revealed that cortical cells are large and that
vascular bundles contained more cells in the phloem and xylem
relative to wild type. Petal size in the 35S::G1073 lines was
increased by 40-50% compared to wild type controls. Petal epidermal
cells in those same lines were approximately 25-30% larger than
those of the control plants. Furthermore, 15-20% more epidermal
cells per petal were produced compared to wild type. Thus, in
petals and stems, the increase in size was associated with an
increase in cell size as well as in cell number.
[0616] Seed yield was also increased compared to control plants.
35S::G1073 lines showed an increase of at least 70% in seed yield
(Table 10). This increased seed production was associated with an
increased number of siliques per plant, rather than seeds per
silique.
TABLE-US-00011 TABLE 10 Comparison of biomass and seed yield
production in Arabidopsis wild-type and two 35S::G1073
overexpressing lines Line Fresh Weight (g) Dry Weight (g) Seed (g)
Wild-type 3.43 .+-. 0.70 0.73 .+-. 0.20 0.17 .+-. 0.07 35S::G1073-3
5.74 .+-. 1.74 1.17 .+-. 0.30 0.31 .+-. 0.08 35S::G1073-4 6.54 .+-.
2.19 1.38 .+-. 0.44 0.35 .+-. 0.12
[0617] All 35S::G1073 lines tested (10/10) exhibited significantly
improved salt tolerance. Most of these lines also showed a sugar
sensing phenotype, exhibiting improved germination on high sucrose
media. One line showed increased heat germination tolerance.
Flowering of G1073 overexpressing plants was delayed. Leaves of
G1073 overexpressing plants were generally more serrated than those
of wild-type plants. Improved drought tolerance was observed in
35S::G1073 transgenic lines.
[0618] A number of the CUT1::G1073 lines tested exhibited
significantly improved salt tolerance and sugar sensing on high
sucrose. One line showed improved germination on high mannitol.
[0619] Half of the ARSK::G1073 lines tested ( 5/10) showed improved
germination on high salt, and two lines showed improved germination
in cold relative to controls.
[0620] Utilities.
[0621] Large size and late flowering produced as a result of G1073
or equivalog overexpression would be extremely useful in crops
where the vegetative portion of the plant is the marketable portion
(often vegetative growth stops when plants make the transition to
flowering). In this case, it would be advantageous to prevent or
delay flowering with the use of this gene or its equivalogs in
order to increase yield (biomass). Prevention of flowering by this
gene or its equivalogs would be useful in these same crops in order
to prevent the spread of transgenic pollen and/or to prevent seed
set. This gene or its equivalogs could also be used to manipulate
leaf shape, abiotic stress tolerance, including drought and salt
tolerance, and seed yield.
G1069 (SEQ ID NO: 239 and 240)
[0622] G1069 is a sequence functionally and structurally related to
G1073 from Arabidopsis. G1069 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 76-218) or the DUF296 domain (amino
acids 93-211) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0623] The sequence of G1069 was obtained from EU Arabidopsis
sequencing project, GenBank accession number Z97336, based on its
sequence similarity within the conserved domain to other AT-Hook
related proteins in Arabidopsis.
[0624] Experimental Observations.
[0625] The sequence of G1069 was experimentally determined and the
function of G1069 was analyzed using transgenic plants in which
G1069 was expressed under the control of the 35S promoter.
[0626] Plants overexpressing G1069 showed changes in leaf
architecture, reduced overall plant size, and retarded progression
through the life cycle. This is a common phenomenon for most
transgenic plants in which AT-HOOK proteins are overexpressed if
the gene is predominantly expressed in root in the wild-type
background. G1069 was predominantly expressed in roots, based on
analysis of RT-PCR results. To minimize these detrimental effects,
G1069 may be overexpressed under a tissue-specific promoter such as
root- or leaf-specific promoter or under inducible promoter.
[0627] One of G1069 overexpressing lines showed more tolerance to
osmotic stress when they were germinated in high sucrose plates.
This line also showed insensitivity to ABA in a germination
assay.
[0628] The high sucrose and ABA assay results suggested that this
gene may confer increased tolerance to other abiotic stresses when
G1069 is overexpressed. This was subsequently confirmed in
soil-based drought assays in which 35S::G1069 plants were more
drought tolerant than wild-type control plants (Tables 11 and
12).
[0629] Utilities.
[0630] The drought and osmotic stress results indicate that G1069
could be used to alter a plant's response to water deficit
conditions and, therefore, the gene or its equivalogs could be used
to engineer plants with enhanced tolerance to drought, salt stress,
and freezing.
[0631] G1069 affects ABA sensitivity, and thus when transformed
into a plant the gene or its equivalogs may diminish cold, drought,
oxidative and other stress sensitivities, and also be used to alter
plant architecture, and yield.
G2789 (SEQ ID NO: 247 and 248)
[0632] G2789 is a sequence functionally and structurally related to
G1073 from Arabidopsis. G2789 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 68-208) or the DUF296 domain (amino
acids 86-201) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0633] The sequence of G2789 was obtained from Arabidopsis genomic
sequencing project, GenBank accession number AL162295, based on its
sequence similarity within the conserved domain to other AT-hook
related proteins in Arabidopsis. G2789 corresponds to gene
T4C21_280 (CAB82691). To date, there is no published information
regarding the functions of this gene.
[0634] Experimental Observations.
[0635] The complete sequence of G2789 was determined. G2789 is
expressed at moderate levels in roots, flowers, embryos, siliques,
and germinating seeds. It was not detectable in rosette leaves or
shoots. No significant induction of G2789 was observed in rosette
leaves by any condition tested.
[0636] The function of this gene was analyzed using transgenic
plants in which G2789 was expressed under the control of the 35S
promoter. Overexpression of G2789 in Arabidopsis resulted in
seedlings that are ABA insensitive and osmotic stress tolerant. In
a germination assay on ABA containing media, G2789 transgenic
seedlings showed enhanced seedling vigor. In a similar germination
assay on media containing high concentrations of sucrose, the G2789
overexpressors also showed enhanced seedling vigor. In a repeat
experiment on individual lines, all three lines show the phenotype.
The combination of ABA insensitivity and better germination under
osmotic stress was also observed for G1820. It is possible that ABA
insensitivity at the germination stage promotes germination despite
unfavorable conditions.
[0637] The osmotic stress tolerance and enhanced seedling vigor on
ABA phenotypes suggested that G2789 overexpressors would be more
tolerant to drought conditions This supposition was confirmed by
soil-based drought assays, in which plants overexpressing G2789
performed significantly better in conditions of water deprivation
than wild-type plants (Tables 11 and 12).
[0638] Utilities.
[0639] G2789 could be used to alter a plant's response to water
deficit conditions and therefore, could be used to engineer plants
with enhanced tolerance to drought, salt stress, and freezing.
Rice G3399 (SEQ ID NO: 339 and 340)
[0640] G3399 is a rice ortholog of G1073. Phylogenetic analysis
identifies G3399 along with G3400 as being the most closely related
rice orthologs of G1073. G3399 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 108-253) or the DUF296 domain (amino
acids 126-246) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0641] The morphologically similar effects caused by overexpression
of this rice gene versus G1073 and other Arabidopsis paralogs
suggest that they likely have related functions. A number of
Arabidopsis lines overexpressing G3399 and G3407 under the control
of the 35S promoter were found be larger, with broader leaves and
larger rosettes than wild-type control plants. Two of the lines
overexpressing G3399 were found to have greater tolerance to
desiccation and heat than wild-type controls in plate-based assays,
and drought in soil-based assays.
[0642] Utilities.
[0643] G3399 could be used to increase a plant's biomass and alter
a plant's response to cold and water deficit conditions and,
therefore, could be used to engineer plants with enhanced tolerance
to drought.
Rice G3407 (SEQ ID NO: 353 and 354)
[0644] G3407 is a rice ortholog of G1073. G3407 and closely-related
clade member sequences each comprise a conserved At-hook domain and
a second conserved domain (amino acids 72-220) or the DUF296 domain
(amino acids 90-213) that are expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0645] The morphologically similar effects caused by overexpression
of this rice gene versus G1073 and other Arabidopsis paralogs
suggest that they likely have related functions.
[0646] Experimental Observations.
[0647] At the seedling stage, about half of the 35S::G3407 lines
appeared larger than controls. At later stages of growth, lines
overexpressing G3407 showed no consistent morphological differences
from control plants, with the exception of one line which was 50%
larger than controls at the rosette stage.
[0648] Two lines of overexpressors were less sensitive germination
in cold conditions than wild type controls.
[0649] Utilities.
[0650] G3407 could be used to increase a plant's biomass and
engineer plants with enhanced tolerance to cold.
Soybean G3456 (SEQ ID NO: 383 and 384)
[0651] G3456 is a sequence functionally and structurally related to
G1073 from Arabidopsis. G3456 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 53-195) or the DUF296 domain (amino
acids 71-188) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0652] Experimental Observations.
[0653] A significant number of Arabidopsis lines overexpressing
G3456 under the control of the 35S promoter were found be larger,
with broader leaves and larger rosettes than wild-type control
plants.
[0654] Most of the lines overexpressing G3456 were significantly
more cold tolerant than wild-type controls. Several 35S::G3456
lines were found to have greater salt tolerance than wild type
controls. Several lines of overexpressors were much more tolerant
to drought than wild-type controls in soil-based assays.
[0655] Utilities.
[0656] G3456 can be used to increase a plant's biomass. G3456 may
be also used to alter a plant's response to water deficit
conditions and, therefore, could be used to engineer plants with
enhanced tolerance to drought and salt stress.
Soybean G3459 (SEQ ID NO: 387 and 388)
[0657] G3459 is a sequence functionally and structurally related to
G1073 from Arabidopsis. G3459 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 86-228) or the DUF296 domain (amino
acids 104-221) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0658] Experimental Observations.
[0659] A significant number of Arabidopsis lines overexpressing
G3459 under the control of the 35S promoter were found be larger,
with broader leaves and larger rosettes than wild-type control
plants.
[0660] Most of the lines overexpressing G3459 conferred tolerance
to one abiotic stress, and were significantly more salt, heat or
cold tolerant than wild-type controls.
[0661] Utilities.
[0662] G3459 can be used to increase a plant's biomass. G3459 may
be also used to alter a plant's response to water deficit
conditions and, therefore, could be used to engineer plants with
enhanced tolerance to salt, heat, cold and drought.
Soybean G3460 (SEQ ID NO: 389 and 390)
[0663] G3460 is a sequence functionally and structurally related to
G1073 from Arabidopsis. G3460 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 83-225) or the DUF296 domain (amino
acids 101-218) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0664] Experimental Observations.
[0665] A significant number of Arabidopsis lines overexpressing
G3460 under the control of the 35S promoter were found be larger,
with broader leaves and larger rosettes than wild-type control
plants.
[0666] Most of the lines overexpressing G3459 conferred tolerance
to one abiotic stress, and were significantly more heat,
desiccation or cold tolerant than wild-type controls. Several lines
of overexpressors were much more tolerant to drought than wild-type
controls in soil-based assays.
[0667] Utilities.
[0668] G3460 can be used to increase a plant's biomass.
[0669] G3460 may be also used to alter a plant's response to water
deficit conditions and, therefore, could be used to engineer plants
with enhanced tolerance to heat, drought, and cold.
The G482 Clade of Transcription Factor Polypeptides
G481 (Polynucleotide SEQ ID NO: 287 and 288)
[0670] G481 is equivalent to AtHAP3a which was identified by
Edwards et al., ((1998) Plant Physiol. 117: 1015-1022) as an EST
with extensive sequence homology to the yeast HAP3. G481 is a
member of the HAP3 subgroup of the CCAAT box-binding transcription
factor family. G481 and closely-related clade member sequences each
a conserved CCAAT box-binding domain that is expected to function
in a similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0671] Experimental Observations.
[0672] Northern blot data from five different tissue samples
indicates that G481 is primarily expressed in flower and/or
silique, and root tissue. The function of G481 was analyzed through
its ectopic overexpression in plants. Except for darker color in
one line (noted below), plants overexpressing G481 had a wild-type
morphology. G481 overexpressors were found to be more tolerant to
high sucrose and high salt, having better germination, longer
radicles, and more cotyledon expansion. There was a consistent
difference in the hypocotyl and root elongation in the
overexpressor compared to wild-type controls. These results
indicated that G481 is involved in sucrose-specific sugar sensing.
Sucrose-sensing has been implicated in the regulation of
source-sink relationships in plants.
[0673] In the T2 generation, one overexpressing line was darker
green than wild-type plants, which may indicate a higher
photosynthetic rate that would be consistent with the role of G481
in sugar sensing.
[0674] 35S::G481 plants were also significantly larger and greener
in a soil-based drought assay than wild-type controls plants After
eight days of drought treatment overexpressing lines had a darker
green and less withered appearance than those in the control group.
The differences in appearance between the control and
G481-overexpressing plants after they were rewatered was even more
striking. Eleven of twelve plants of this set of control plants
died after rewatering, indicating the inability to recover
following severe water deprivation, whereas all nine of the
overexpressor plants of the line shown recovered from this drought
treatment. These results were typical of a number of control and
35S::G481-overexpressing lines.
[0675] One line of plants in which G481 was overexpressed under the
control of the ARSK1 root-specific promoter was found to germinate
better under cold conditions than wild-type plants.
[0676] Interestingly, in one Arabidopsis line in which G481 was
knocked out, the plants were found to be more sensitive to high
salt in a plate-based assay than wild-type plants, which indicates
the importance of the role played by G481 in regulating osmotic
stress tolerance, and demonstrates that the gene is both necessary
and sufficient to fulfill that function.
[0677] A number of the 35S::G481 plants evaluated had a late
flowering phenotype.
[0678] Utilities.
[0679] The potential utility of G481 includes altering
photosynthetic rate, which could also impact yield in vegetative
tissues as well as seed. Sugars are key regulatory molecules that
affect diverse processes in higher plants including germination,
growth, flowering, senescence, sugar metabolism and photosynthesis.
Sucrose is the major transport form of photosynthate and its flux
through cells has been shown to affect gene expression and alter
storage compound accumulation in seeds (source-sink
relationships).
[0680] Since G481 overexpressing plants performed better than
controls in drought experiments, this gene or its equivalogs may be
used to improve seedling vigor, plant survival, as well as yield,
quality, and range.
G482 (Polynucleotide SEQ ID NO: 289 and 290)
[0681] G482, a paralog of G481, is equivalent to AtHAP3b which was
identified by Edwards et al. (1998) Plant Physiol. 117: 1015-1022)
as an EST with homology to the yeast gene HAP3b. Their northern
blot data suggests that AtHAP3b is expressed primarily in roots.
G482 is a member of the HAP3 subgroup of the CCAAT box-binding
transcription factor family. G482 and closely-related clade member
sequences each a conserved CCAAT box-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0682] Experimental Observations.
[0683] RT-PCR analysis of endogenous levels of G482 transcripts
indicated that this gene is expressed constitutively in all tissues
tested. A cDNA array experiment supports the RT-PCR derived tissue
distribution data. G482 is not induced above basal levels in
response to any environmental stress treatments tested.
[0684] A T-DNA insertion mutant for G482 was analyzed and was found
to flower slightly later than control plants.
[0685] The function of G482 was also analyzed through its ectopic
overexpression in plants. Plants overexpressing G482 had a
wild-type morphology. Germination assays to measure salt tolerance
demonstrated increased seedling growth when germinated on the high
salt medium.
[0686] 35S::G482 transgenic plants also displayed an osmotic stress
response phenotype similar to 35S::G481 transgenic lines. Five of
ten overexpressing lines had increased seedling growth on medium
containing 80% MS plus vitamins with 300 mM mannitol.
[0687] Three of ten 35S::G482 lines also demonstrated enhanced
germination relative to controls after a 6 hour exposure to
32.degree. C.
[0688] The majority of these 35S::G482 lines also demonstrated a
slightly early flowering phenotype.
[0689] Utilities.
[0690] The potential utilities of this gene include the ability to
confer osmotic stress tolerance, as measured by salt, heat
tolerance and improved germination in mannitol-containing media,
during the germination stage of a crop plant. This would most
likely impact survivability and yield. Evaporation of water from
the soil surface causes upward water movement and salt accumulation
in the upper soil layer, where the seeds are placed. Thus,
germination normally takes place at a salt concentration much
higher than the mean salt concentration in the whole soil
profile.
[0691] Improved osmotic stress tolerance is also likely to result
in enhanced seedling vigor, plant survival, improved yield,
quality, and range. Osmotic stress assays, including subjecting
plants to aqueous dissolved sugars, are often used as surrogate
assays for improved water-stress (for example, drought) response.
Thus, G482 may also be used to improve plant performance under
conditions of water deprivation, including increased seedling
vigor, plant survival, yield, quality, and range.
Rice G3395 (SEQ ID NO: 331 and 332)
[0692] G3395 (rice) is an ortholog of G481 and G482, and is a
member of the HAP3-like subfamily of CCAAT-box binding
transcription factors. G3395 corresponds to polypeptide BAC76331
("NF-YB subunit of rice"). G3395 and closely-related clade member
sequences each a conserved CCAAT box-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0693] Experimental Observations.
[0694] The function of G3395 was analyzed through its ectopic
overexpression in plants. One of the lines of 35S::G3395
overexpressors tested was found to be more tolerant to high salt
levels, producing larger and greener seedlings in a high salt
germination assay. Several lines were also significantly more
drought tolerant than wild type controls in soil-based assays.
[0695] Utilities.
[0696] The potential utilities of G3395 include the ability to
confer salt and drought stress tolerance
Soy G3470 (SEQ ID NO: 393 and 394)
[0697] G3470 (soybean) is an ortholog of G481 and G482, and is a
member of the HAP3-like subfamily of CCAAT-box binding
transcription factors. G3470 and closely-related clade member
sequences each a conserved CCAAT box-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0698] Experimental Observations.
[0699] The function of G3470 was analyzed through its ectopic
overexpression in plants. Seven of ten lines of 35S::G3470
overexpressors were found to be significantly more tolerant to high
salt in a plate-based germination assay.
[0700] Utilities.
[0701] The potential utilities of these two genes, G3395 and G3470,
and their equivalogs, include the ability to confer tolerance to
drought and other osmotic stresses, including during the
germination stage of a crop plant. Equivalogs of G3395 and G3470
include, for example, Arabidopsis sequences G481 (SEQ ID NO: 288),
G482 (SEQ ID NO: 290), G485 (SEQ ID NO: 292), G486 (SEQ ID NO:
294), G1248 (SEQ ID NO: 306), G1364 (SEQ ID NO: 308), G1781 (SEQ ID
NO: 310), G2345 (SEQ ID NO: 320), G2718 (SEQ ID NO: 324), rice
sequences G3394 (SEQ ID NO: 330), G3396 (SEQ ID NO: 334), G3397
(SEQ ID NO: 336), G3398 (SEQ ID NO: 338), G3429 (SEQ ID NO: 358),
G3835 (SEQ ID NO: 414), G3836 (SEQ ID NO: 416), corn sequences
G3434 (SEQ ID NO: 362), G3435 (SEQ ID NO: 364), G3436 (SEQ ID NO:
366), G3437 (SEQ ID NO: 368), and soy sequences G3470 (SEQ ID NO:
394), G3471 (SEQ ID NO: 396), G3472 (SEQ ID NO: 398), G3473 (SEQ ID
NO: 400), G3474 (SEQ ID NO: 402), G3475 (SEQ ID NO: 404), G3476
(SEQ ID NO: 406), G3477 (SEQ ID NO: 408), G3478 (SEQ ID NO: 410),
and G3837 (SEQ ID NO: 418).
HAP5 Transcription Factor Polypeptides
G1820 (SEQ ID NO: 243 and 244)
[0702] G1820 is a member of the Hap5 subfamily of CCAAT-box-binding
transcription factors. G1820 was identified as part of the BAC
clone MBA10, accession number AB025619 released by the Arabidopsis
Genome sequencing project. G1820 and closely-related clade member
sequences each comprise a conserved CCAAT binding factor domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0703] Experimental Observations.
[0704] The complete sequence of G1820 was determined. The function
of this gene was analyzed using transgenic plants in which G1820
was expressed under the control of the 35S promoter. G1820
overexpressing lines showed more tolerance to salt stress in a
germination assay. They also showed insensitivity to ABA, with the
three lines analyzed showing the phenotype. The salt and ABA
phenotypes could be related to the plants' increased tolerance to
osmotic stress, which was subsequently confirmed in soil-based
drought assays in which 35S::G1820 plants were significantly more
drought-tolerant than wild-type control plants (Tables 11 and
12).
[0705] Interestingly, overexpression of G1820 also consistently
reduced the time to flowering. Under continuous light conditions at
20-25 C, the 35S::G1820 transformants displayed visible flower buds
several days earlier than control plants. The primary shoots of
these plants typically started flower initiation 1-4 leaf
plastochrons sooner than those of wild type. Such effects were
observed in all three T2 populations and in a substantial number of
primary transformants.
[0706] When biochemical assays were performed, some changes in leaf
fames were detected. In one line, an increase in the percentage of
18:3 and a decrease in 16:1 were observed. Otherwise, G1820
overexpressors behaved similarly to wild-type controls in all
biochemical assays performed. As determined by RT-PCR, G1820 was
highly expressed in embryos and siliques. No expression of G1820
was detected in the other tissues tested. G1820 expression appeared
to be induced in rosette leaves by cold and drought stress
treatments, and overexpressing lines showed tolerance to water
deficit and high salt conditions.
[0707] One possible explanation for the complexity of the G1820
overexpression phenotype is that the gene is somehow involved in
the cross talk between ABA and GA signal transduction pathways. It
is well known that seed dormancy and germination are regulated by
the plant hormones ABA and gibberellin (GA). These two hormones act
antagonistically with each other. ABA induces seed dormancy in
maturing embryos and inhibits germination of seeds. GA breaks seed
dormancy and promotes germination. It is conceivable that the
flowering time and ABA insensitive phenotypes observed in the G1820
overexpressors are related to an enhanced sensitivity to GA, or an
increase in the level of GA, and that the phenotype of the
overexpressors is unrelated to ABA. In Arabidopsis, GA is thought
to be required to promote flowering in non-inductive photoperiods.
However, the drought and salt tolerant phenotypes would indicate
that ABA signal transduction is also perturbed in these plants. It
seems counterintuitive for a plant with salt and drought tolerance
to be ABA insensitive since ABA seems to activate signal
transduction pathways involved in tolerance to salt and dehydration
stresses. One explanation is that ABA levels in the G1820
overexpressors are also high but that the plant is unable to
perceive or transduce the signal.
[0708] G1820 overexpressors also had decreased seed oil content and
increased seed protein content compared to wild-type plants
[0709] Utilities.
[0710] G1820 and its equivalogs may be used to enhance a plant's
tolerance to drought conditions. The osmotic stress results
indicated that G1820 or its equivalogs could be used to alter a
plant's response to additional water deficit conditions and can be
used to engineer plants with enhanced tolerance to salt stress, and
freezing. Evaporation from the soil surface causes upward water
movement and salt accumulation in the upper soil layer where the
seeds are placed. Thus, germination normally takes place at a salt
concentration much higher than the mean salt concentration of in
the whole soil profile. Increased salt tolerance during the
germination stage of a crop plant would impact survivability and
yield.
[0711] G1820 affects ABA sensitivity, and thus when transformed
into a plant this transcription factor or its equivalogs may
diminish cold, drought, oxidative and other stress sensitivities,
and also be used to alter plant architecture, and yield.
[0712] G1820 or its equivalogs could also be used to accelerate
flowering time.
[0713] G1820 or its equivalogs may be used to modify levels of
saturation in oils.
[0714] G1820 or its equivalogs may be used to seed protein
content.
[0715] The promoter of G1820 could be used to drive seed-specific
gene expression.
[0716] G1820 or equivalog overexpression may be used to alter seed
protein content, which may be very important for the nutritional
value and production of various food products
G489 (SEQ ID NO: 229 and 230)
[0717] G489 was identified from a BAC sequence that showed high
sequence homology to AtHAP5-like transcription factors in
Arabidopsis. G489 and closely-related clade member sequences each
comprise a conserved CCAAT binding factor domain that is expected
to function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[0718] Experimental Observations.
[0719] The function of G489 was analyzed through its ectopic
overexpression in plants.
[0720] RT-PCR analysis of endogenous levels of G489 transcripts
indicates that this gene is expressed constitutively in all tissues
tested. A cDNA array experiment confirms the RT-PCR derived tissue
distribution data. G489 was not induced above basal levels in
response to the stress treatments tested.
[0721] G489 overexpressors were more tolerant to high NaCl stress,
showing more root growth and leaf expansion compared to the
controls in culture. Two well characterized ways in which NaCl
toxicity is manifested in the plant is through general osmotic
stress and potassium deficiency due to the inhibition of its
transport. These lines were more tolerant to osmotic stress,
showing more root growth on mannitol containing media; however,
they were not more tolerant to potassium deficiency.
[0722] The involvement of G489 in a response pathway to abiotic
stress was further confirmed in soil-based drought assays, where
the overexpressors were observed to be more tolerant to water
deprivation conditions than wild-type control plants (Table
12).
[0723] Utilities.
[0724] The potential utilities of this gene include the ability to
confer drought and salt tolerance during the growth and
developmental stages of a crop plant. This would most likely impact
yield and or biomass.
The G916 Clade of Transcription Factor Polypeptides
G916 (SEQ ID NO: 235 and 236)
[0725] G916 corresponds to gene At4g04450, and it has also been
described as WRKY42. No information is available about the
function(s) of G916. G916 and closely-related clade member
sequences each comprise a conserved WRKY DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0726] Experimental Observations.
[0727] The complete cDNA sequence of G916 was experimentally
determined. G916 appears to be expressed at low levels in a range
of tissues, and was not significantly induced by any of the
conditions tested.
[0728] A T-DNA insertion mutant for G916, displayed wild-type
morphology. Overexpression of G916 produced a wide spectrum of
developmental abnormalities in Arabidopsis. Many of the 35S::G916
seedlings were extremely tiny and showed an apparent lack of shoot
organization. Such plants arrested growth and died at very early
stages. Other individuals were small and displayed
disproportionately long hypocotyls and narrow cotyledons. At later
stages, the majority of surviving lines were markedly smaller than
wild type, and formed rather weedy inflorescence stems that yielded
very few flowers. Additionally, flowers often had poorly developed
organs.
[0729] In addition, G916 overexpressing lines were larger than
control wild-type seedlings in several germination assays. Larger
seedlings were observed under conditions of high sucrose. In
addition, 35S::G916 seedlings were larger and appeared to have less
anthocyanin on high sucrose plates that were nitrogen deficient,
with or without glutamine supplementation. The assays monitor the
effect of C on N signaling through anthocyanin production. That
35S::G916 seedlings performed better under conditions of high
sucrose alone makes it more difficult to interpret the better
seedling performance under conditions of low nitrogen.
Tissue-specific or inducible expression of this gene could aid in
sorting out the complex phenotypes caused by the constitutive
overexpression of this gene.
[0730] The results of the high sucrose assays indicated that
G916-overexpressing plants might be significantly more drought
tolerant than control plants, which was subsequently confirmed in
soil-based drought assays (Tables 11 and 12).
[0731] Utilities.
[0732] The results of physiological assays indicate that G916 could
be used to alter the sugar signaling in plants. The soil-based
drought and sugar sensing assays indicate that G916 and its
equivalogs may also be used to enhance a plant's drought or other
osmotic stress tolerance.
[0733] The enhanced performance of G916 overexpression lines under
low nitrogen conditions indicate that the gene could be used to
engineer crops that could thrive under conditions of reduced
nitrogen availability.
[0734] That 35S::G916 lines make less anthocyanin on high sucrose
plus glutamine, indicates G916 might be used to modify carbon and
nitrogen status, and hence assimilate partitioning.
[0735] Additionally, the morphological phenotypes shown by
35S::G916 seedlings indicate that the gene might be used to
manipulate light responses such as shade avoidance.
The G2701 Clade of Transcription Factor Polypeptides
G2701 (SEQ ID NO: 245 and 246)
[0736] G2701 was identified in the sequence of BAC F11B9, GenBank
accession number AC073395, released by the Arabidopsis Genome
Initiative. G2701 and closely-related clade member sequences each
comprise at least one conserved Myb-related DNA-binding domain that
is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0737] Experimental Observations.
[0738] The function of G2701 was analyzed using transgenic plants
in which the gene was expressed under the control of the 35S
promoter. Overexpression of G2701 is Arabidopsis resulted in plants
that were wild-type in morphology and in the biochemical analyses
performed. However, 35S::G2701 transgenic plants were more tolerant
to osmotic stress in a germination assay, the seedlings were
greener with expanded cotyledons and longer roots than wild-type
controls when germinated on plates containing either high salt or
high sucrose. The phenotype was repeated in all three lines.
[0739] The results of the high sucrose and salt assays suggested
that this gene would confer increased tolerance to other abiotic
stresses when G2701 is overexpressed, which was subsequently
confirmed in soil-based drought assays, in which 35S::G2701 plants
were significantly more drought tolerant than wild-type control
plants (Tables 11 and 12).
[0740] G2701 was expressed ubiquitously in Arabidopsis according to
RT-PCR, and the level of G2701 expression in leaf tissue was
essentially unchanged in response to environmental stress related
conditions.
[0741] Utilities.
[0742] G2701 or its equivalogs could be used to alter a plant's
response to water deficit conditions and therefore, could be used
to engineer plants with enhanced tolerance to drought, salt stress,
and freezing.
The G2854 Clade of Transcription Factor Polypeptides
G2854 (SEQ ID NO: 251 and 256)
[0743] The sequence of G2854 was obtained from the Arabidopsis
genome sequencing project, GenBank accession number AL161566,
nid=7269538, based on its sequence similarity within the conserved
domain to other ACBF-like related proteins in Arabidopsis. G2854
and closely-related clade member sequences each comprise at least
one conserved RNA Recognition Motif (RRM; also known as an RBD or
RNP domain) that is expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[0744] Experimental Observations.
[0745] The 5' and 3' ends of G2854 were determined by RACE. The
function of G2854 was analyzed using transgenic plants in which
G2854 was expressed under the control of the 35S promoter.
35S::G2854 transformants showed increased germination efficiency on
sucrose plates compared to wild-type controls. These results
suggested a possible role for G2854 in conferring drought tolerance
in plants. This supposition was confirmed in soil-based drought
assays, in which plants overexpressing G2854 performed
significantly better than wild-type plants (Tables 11 and 12).
[0746] Utilities.
[0747] G2854 and its equivalogs may be used to confer improved
drought tolerance in plants.
[0748] G2854 and its equivalogs might also be used to generate crop
plants with altered sugar sensing. Sugars are key regulatory
molecules that affect diverse processes in higher plants including
germination, growth, flowering, senescence, sugar metabolism and
photosynthesis. Sucrose is the major transport form of
photosynthate and its flux through cells has been shown to affect
gene expression and alter storage compound accumulation in seeds
(source-sink relationships). Glucose-specific hexose-sensing has
been described in plants and implicated in cell division and
repression of `famine` genes (photosynthetic or glyoxylate cycles).
The potential utilities of a gene involved in glucose-specific
sugar sensing are to alter energy balance, photosynthetic rate,
carbohydrate accumulation, biomass production, source-sink
relationships, and senescence.
The G634 Clade of Transcription Factor Polypeptides
G634 (SEQ ID NO: 231 and 232)
[0749] G634 was initially identified as public partial cDNAs
sequences for GTL1 and GTL2 which are splice variants of the same
gene (Small et al (1998) Proc. Natl. Acad. Sci. USA. 95:3318-3322).
The published expression pattern of GTL1 shows that G634 is highly
expressed in siliques and not expressed in leaves, stems, flowers
or roots. G634 and closely-related clade member sequences each
comprise at least one conserved TH domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[0750] Experimental Observations.
[0751] The boundaries of G634 in were experimentally determined and
the function of G634 was investigated by constitutively expressing
G634 using the CaMV 35S promoter.
[0752] Three constructs were made for G634: P1374, P324, and P1717
(SEQ ID NOs: 1013, 1015 and 1017, respectively). P324 was found to
encode a truncated protein. P1374 and P1717 represent full length
splice variants of G634; P1374, the shorter of the two splice
variants was used for the experiments described here. The longest
available cDNA (P1717), confirmed by RACE, has the same ATG and
stop codons as the genomic sequence.
[0753] Plants overexpressing G634 from construct P1374 showed a
dramatic increase the density of trichomes, which additionally
appear larger in size. The increase in trichome density was most
noticeable on later arising rosette leaves, cauline leaves,
inflorescence stems and sepals with the stem trichomes being more
highly branched than controls. Approximately half of the primary
transformants and two of three T2 lines showed the phenotype. Apart
from slight smallness, there did not appear to be any other clear
phenotype associated with the overexpression of G634. However, a
reduction in germination was observed in T2 seeds grown in culture.
It is not clear whether this defect was due to the quality of the
seed lot tested or whether this characteristic is related to the
transgene overexpression.
[0754] RT PCR data showed that G634 is potentially preferentially
expressed in flowers and germinating seedlings, and induced by
auxin. The role of auxin in trichome initiation and development has
not been established in the published literature.
[0755] The increase in trichome density observed in G634
overexpressors suggested a possible role for this gene in
drought-stress tolerance, a presumption subsequently confirmed in
soil-based drought assays (Tables 11 and 12).
[0756] Utilities.
[0757] Trichome glands on the surface of many higher plants produce
and secrete exudates that give protection from the elements and
pests such as insects, microbes and herbivores. These exudates may
physically immobilize insects and spores, may be insecticidal or
ant-microbial or they may allergens or irritants to protect against
herbivores. Trichomes have also been suggested to decrease
transpiration by decreasing leaf surface air flow, and by exuding
chemicals that protect the leaf from the sun.
[0758] Depending on the plant species, varying amounts of diverse
secondary biochemicals (often lipophilic terpenes) are produced and
exuded or volatilized by trichomes. These exotic secondary
biochemicals, which are relatively easy to extract because they are
on the surface of the leaf, have been widely used in such products
as flavors and aromas, drugs, pesticides and cosmetics. One class
of secondary metabolites, the diterpenes, can effect several
biological systems such as tumor progression, prostaglandin
synthesis and tissue inflammation. In addition, diterpenes can act
as insect pheromones, termite allomones, and can exhibit
neurotoxic, cytotoxic and antimitotic activities. As a result of
this functional diversity, diterpenes have been the target of
research several pharmaceutical ventures. In most cases where the
metabolic pathways are impossible to engineer, increasing trichome
density or size on leaves may be the only way to increase plant
productivity.
[0759] Thus, the use of G634 and its equivalogs to increase
trichome density, size or type may therefore have profound
utilities in so called molecular farming practices (i.e. the use of
trichomes as a manufacturing system for complex secondary
metabolites), and in producing resistant insect and herbivore
resistant plants.
[0760] G634 and its equivalogs may also be used to increase the
drought tolerance of plants.
The G175 Clade of Transcription Factor Polypeptides
G175 (SEQ ID NO: 223 and 224)
[0761] G175 was identified in the sequence of P1 clone M3E9 (Gene
AT4g26440/M3E9.130; GenBank accession number CAB79499). G175 and
closely-related clade member sequences each comprise a conserved
WRKY DNA-binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0762] Experimental Observations.
[0763] The complete cDNA sequence of G175 was determined by us. The
function of this gene was studied using transgenic plants in which
G175 was expressed under the control of the 35S promoter. 35S::G175
plants are more tolerant to osmotic stress conditions (better
germination in NaCl and sucrose containing media). The plants were
otherwise wild-type in morphology and development.
[0764] G175 appears to be specifically expressed in floral tissues,
and also appears to be induced elsewhere by heat and salt
stress.
[0765] The results of the osmotic stress assays and heat and salt
stress expression analyses suggested that G175 could be used to
confer drought tolerance in plants, a supposition that was
confirmed in soil-based assays in which G175-overexpressing plants
were shown to be more tolerant to water deprivation than wild-type
control plants (Tables 11 and 12).
[0766] Utilities.
[0767] G175 and its equivalogs can be used to improve drought
tolerance and increase germination under adverse osmotic stress
conditions, which could impact survivability and yield. The
promoter of G175 could also be used to drive flower specific
expression.
The G1452 Clade of Transcription Factor Polypeptides
G1452 (SEQ ID NO: 241 and 242)
[0768] G1452 was identified in the sequence of clones T22O13, F12K2
with accession number AC006233 released by the Arabidopsis Genome
Initiative. G1452 and closely-related clade member sequences each
comprise a conserved NAC domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0769] Experimental Observations.
[0770] The function of G1452 was analyzed using transgenic plants
in which the gene was expressed under the control of the 35S
promoter. G1452 and closely-related clade member sequences each
comprise a conserved NAC domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0771] Overexpression of G1452 produced changes in leaf development
and markedly delayed the onset of flowering. 35S::G1452 plants
produced dark green, flat, rounded leaves, and typically formed
flower buds between 2 and 14 days later than controls.
Additionally, some of the transformants were noted to have rather
low trichome density on leaves and stems. At later stages of life
cycle, 35S::G1452 appeared to develop slowly and senesced
considerably later than wild-type controls.
[0772] G1452 overexpressors were more tolerant to high
sucrose-induced osmotic stress than wild-type control plants, were
more tolerant to high salt than controls, and were insensitive to
ABA in separate germination assays. These results indicated that
G1452 may be used to confer improved survival in drought, which was
confirmed in soil-based drought assays where G1452-overexpressors
fared significantly better than wild-type control plants (Tables 11
and 12).
[0773] Utilities.
[0774] G1452 could be used to alter a plant's response to water
deficit conditions and therefore, could be used to engineer plants
with enhanced tolerance to drought and salt stress.
[0775] On the basis of the analyses performed to date, G1452 could
be use to alter plant growth and development.
The G3083 Clade of Transcription Factor Polypeptides
G3083 (SEQ ID NO: 253 and 254)
[0776] G3083 (At3g14880) was identified as part of the BAC clone
K15M2, GenBank accession number AP000370 (nid=5541653). G3083 and
closely-related clade member sequences each comprise a conserved
domain that is expected to function in a similar manner in each of
these related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0777] Experimental Observations.
[0778] The 5'- and 3'-ends of G3083 were determined by RACE and the
function of the gene was assessed by analysis of transgenic
Arabidopsis lines in which a genomic clone was constitutively
expressed from a 35S promoter. 35S::G3083 plants were
indistinguishable from wild-type controls in the morphological
analysis.
[0779] In the physiological analysis, two out of the three
35S::G3083 lines tested, displayed an enhanced ability to germinate
on plates containing high levels of sodium chloride. This suggested
that G3083 might function as part of a response pathway to abiotic
stress, which was further indicated in soil-based drought assays in
which one line of a G3083 overexpressor was shown to be
significantly more tolerant to water deprivation than wild-type
control plants.
Utilities
[0780] Based on the increased salt tolerance exhibited by the
35S::G3083 lines in physiology assays, this gene might be used to
engineer salt tolerant crops and trees that can flourish in drought
or in salinified soils. The latter condition is of particular
importance early in the lifecycle, since evaporation from the soil
surface causes upward water movement, and salt accumulates in the
upper soil layer where the seeds are placed. Thus, germination
normally takes place at a salt concentration much higher than the
mean salt level in the whole soil profile. Increased salt tolerance
during the germination stage of a crop plant would therefore
enhance survivability and yield.
The G303 Clade of Transcription Factor Polypeptides
G303 (SEQ ID NO: 225 and 226)
[0781] G303 corresponds to gene MNA5.5 (BAB11554.1). G303 and
closely-related clade member sequences each comprise a conserved
HLH DNA-binding and dimerization domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[0782] Experimental Observations.
[0783] The complete sequence of G303 was determined. G303 was
detected at very low levels in roots and rosette leaves. It did not
appear to be induced by any condition tested. No altered
morphological or biochemical phenotypes were detected in G303
overexpressing plants.
[0784] The function of this gene was analyzed using transgenic
plants in which G303 was expressed under the control of the 35S
promoter. G303 overexpressing plants showed more tolerance to
osmotic stress vigor than wild-type controls in a germination assay
in three separate experiments on high salt and high sucrose.
[0785] The involvement of G303 in a response pathway to abiotic
stress was further confirmed in soil-based drought assays, in which
the plants overexpressing G303 were found to be more tolerant to
drought than the wild-type controls in the experiment (Table
12).
[0786] Utilities.
[0787] G303 may be useful for enhancing drought tolerance and seed
germination under high salt conditions or other conditions of
osmotic stress (for example, freezing).
The G682 Subclade of Transcription Factor Polypeptides
G682 (SEQ ID NO: 233 and 234)
[0788] G682 was identified from the Arabidopsis BAC, AF007269,
based on sequence similarity to other members of the Myb family
within the conserved domain. G682 and closely-related clade member
sequences each comprise a conserved Myb-related DNA-binding domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0789] Experimental Observations.
[0790] The function of G682 was analyzed through its ectopic
overexpression in plants.
[0791] RT-PCR analysis of the endogenous levels of G682 transcripts
indicated that this gene is expressed in all tissues tested,
however, a very low level of transcript is detected in roots and
shoots. Array tissue print data suggests that G682 is expressed
primarily, but not exclusively, in flower tissue.
[0792] G682 overexpressors were glabrous and had tufts of more root
hairs.
[0793] An array experiment was performed on G682 overexpressing
line 5. The data from this one experiment indicates that this gene
could be a negative regulator of chloroplast development and/or
light dependent development because the gene Albino3 and many
chloroplast genes are repressed. Albino3 functions to regulate
chloroplast development (Plant Cell (1997) 9: 717-730). The gene
G682 is itself is induced 20-fold. Other than a few additional
transcription factors, very few genes are induced as a result of
the ectopic expression of G682. These plants are not pale in color,
making it uncertain how to relate the morphological and
physiological data with the gene profiling data. The array
experiment needs to be repeated with additional lines.
[0794] The effects of a high salt environment (MS medium
supplemented with 150 mM NaCl) on the germination of G682
overexpressors and control seedlings was studied. The results
demonstrated that the overexpressors were more tolerant to the high
salt concentration, being much larger and greener than controls.
High sodium chloride growth assays often are used to indicate
abiotic stress tolerance such as osmotic stress tolerance,
including drought tolerance, which was subsequently confirmed with
soil-based drought assays conducted with plants overexpressing
G682.
[0795] G682-overexpressing line were found to be larger and greener
than wild-type controls that were similarly treated in a cold
germination assay (8.degree. C.), indicating enhanced tolerance of
the former to germination in these cold conditions.
[0796] G682 overexpressors were larger and greener in sucrose
germination assays than wild-type controls, indicating that G682
overexpression can confer a sugar-sensing or abiotic stress
phenotype. This assay is used to determine whether a plant has an
altered sugar sensing response or altered abiotic stress tolerance,
and, in this case, indicates that overexpression of G682 can confer
this phenotype in plants.
[0797] In a heat germination assay (32.degree. C. to 37.degree. C.
for 6 hours of exposure), G682 overexpressing seedlings were
significantly larger, greener and had greater cotyledon expansion
than wild type seedlings. In subsequent experiments, it was found
that older plants were also more tolerant to heat stress compared
to wild-type controls. At the time these experiments were
performed, it was suggested that further experiments were needed to
address whether or not the heat germination phenotype of the G682
overexpressors was related to water deficit stress tolerance in the
germinating seedling, and correlated with a possible drought
tolerance phenotype. More recent experiments have shown that G682
overexpressors were, on average, more tolerant to water deprivation
conditions in soil-based drought assays than wild-type plants
(Table 12), and two of three lines were significantly more drought
tolerant than the wild-type controls.
[0798] Utilities.
[0799] The utility of this gene and its equivalogs would be to
confer salt, heat and cold tolerance to germinating seeds and
plants, and drought tolerance in plants.
G1816 (SEQ ID NO: 311 and 312)
[0800] G1816 is a paralog of G682 from Arabidopsis. G1816 is a
member of the MYB-related class of transcription factors. The gene
corresponds to TRIPTYCHON (TRY), and has recently been shown to be
involved in the lateral inhibition during epidermal cell
specification in the leaf and root (Schellmann et al. (2002) EMBO
J. 21: 5036-5046). The model proposes that TRY (G1816) and CPC
(G225) function as repressors of trichome and atrichoblast cell
fate. TRY loss-of-function mutants form ectopic trichomes on the
leaf surface. TRY gain-of-function mutants are glabrous and form
ectopic root hairs. G1816 and closely-related Glade member
sequences each comprise a conserved Myb-related DNA-binding domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0801] Experimental Observations.
[0802] The complete sequence of G1816 was determined. The function
of the gene was studied using transgenic plants in which G1816 was
expressed under the control of the 35S promoter. Consistent with
the morphological phenotypes published for the 35S::TRY
overexpressors, the transgenic plants were glabrous and form
ectopic root hairs.
[0803] The 35S::G1816 plants were also insensitive to growth
retardation effects of germination on conditions of high glucose
and sucrose (MS medium supplemented with 5% glucose and 9.4%
sucrose, respectively); the overexpressor seedlings were large and
green, as contrasted with the wild-type control seedlings which
were significantly smaller and more pigmented. This indicates that
G1816 plays a role in sugar sensing responses in the plant or
osmotic stress tolerance.
[0804] A number of G1816 overexpressing lines were more tolerant to
drought conditions than wild-type controls in soil-based
assays.
[0805] Utilities.
[0806] The phenotypic effects of G1816 overexpression, such as the
increase in root hair formation and the increase in seedling vigor
observed in a germination assay on high glucose media, indicated
that the gene or its orthologs can be used to engineer plants with
increased tolerance to abiotic stresses such as drought, salt, heat
or cold.
[0807] In addition, the enhanced performance of G1816
overexpression lines under low nitrogen conditions indicated that
the gene or its orthologs could be used to engineer crops that
could thrive under conditions of reduced nitrogen availability.
These assays also indicate that G1816 and its equivalogs are
potential regulators of a plant's C/N sensing, nitrogen uptake and
utilization, and its response to low nutrient conditions. For
further analysis, see the discussion above: "Potential Applications
of Polynucleotides and Polypeptides that Regulate C/N sensing".
[0808] The effect of G1816 overexpression on insensitivity to
glucose in a germination assay, indicated that the gene or its
orthologs could be involved in sugar sensing responses in the
plant.
[0809] G1816 or its orthologs could also be used to alter
anthocyanin production and trichome formation in leaves.
[0810] The potential utilities of genes involved in anthocyanin
production include alterations in pigment production for
horticultural purposes and increase stress resistance perhaps in
combination with other transcription factors. Flavonoids have
antimicrobial activity and could be used to engineer pathogen
resistance. In addition, several flavonoid compounds have health
promoting effects such as the inhibition of tumor growth and
cancer, prevention of bone loss and the prevention of the oxidation
of lipids.
G3450 (SEQ ID NO: 319 and 320)
[0811] G3450 is a soy ortholog or G682. Almost all of the
35S::G3450 lines examined were glabrous and had more root hair than
controls, thus exhibiting a morphology similar to G682. G3450 and
closely-related clade member sequences each comprise a conserved
Myb-related DNA-binding domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0812] Experimental Observations.
[0813] In plate-based assays, G3450 overexpressors were more
tolerant to germination and growth in cold conditions, and growth
in heat.
[0814] At least four lines of G3450 overexpressors were more
tolerant to drought treatment than wild-type controls in soil-based
assays. After rewatering, these same lines also exhibited much
superior recovery from the effects of the drought treatment than
the controls, as evidenced by their return to vigor (many of the
control plants were dead at this point).
[0815] Utilities.
[0816] Similar to other members of the G682 subclade, G3450 or its
equivalogs can be used to engineer plants with increased tolerance
to abiotic stresses such as drought, heat or cold.
Summary of Drought Assay Results
[0817] Table 11 presents the results obtained in an assay in which
Arabidopsis plants were subjected to water deprivation for seven to
eight days. At the end of this dry-down period, each pot was
assigned a numeric score depending on the health of its plants. A
score of 0 to 6 was assigned based on a plant's color and general
appearance, with plants that were all brown receiving a "0" and, at
the other end of the spectrum, plants that had an excellent
appearance (all green) receiving a "6". The mean of the recorded
numeric score of all pots of a given genotype per line of all flats
tested is presented in order of decreasing health.
TABLE-US-00012 TABLE 11 Comparison of recorded numeric score plants
subjected to drought treatment. GID Mean score G2133 5.875 G634
4.778 G922 4.667 G916 4.6 G1274 4.273 G864 3.733 G2999 3.7 G2992
3.7 G353 3.6 G47 3.459 G2053 3.404 G975 3.393 G489 3.364 G1792
3.281 G1820 3.2 G2453 3.2 G2140 3.139 G2701 3.108 G3086 3.056 G611
3.048 G1452 3.042 G481 3.041 G624 3.000 G2854 2.829 G303 2.812
G2839 2.783 G2789 2.708 G188 2.692 G325 2.556 G2776 2.513 G175
2.467 G2110 2.432 G1206 2.412 G682 2.381 G1730 2.341 G2969 2.333
G2998 2.333 G1069 2.316 Wild-type 2.284
[0818] Table 12 compares the survival ratings of Arabidopsis plants
overexpressing various polypeptides, evaluated after seven to eight
days of drought treatment, rewatering, and two to three days of a
recovery period Values indicate the median odds of survival within
a given flat (the 50th percentile of survival within each pot of a
given genotype per line divided by the average wild-type survival
in the flat).
TABLE-US-00013 TABLE 12 Survival ratings of Arabidopsis plants
after drought and rewatering treatment GID Median per flat G2133
3.365 G1274 2.059 G922 1.406 G2999 1.255 G3086 1.179 G354 1.167
G1792 1.161 G2053 1.091 G975 1.090 G1069 1.037 G916 1.023 G2701
1.000 G1820 1.000 G47 0.921 G2854 0.889 G2789 0.845 G481 0.843 G634
0.834 G175 0.814 G2839 0.805 G1452 0.803 Wild-type 0.800
Example IX: Results of C/N Sensing Assays
[0819] This example provides experimental evidence for altered
carbon-nitrogen balance controlled by transcription factor
polypeptides and polypeptides of the invention.
The G682 Subclade of Transcription Factor Polypeptides
G682 (SEQ ID NO: 233 and 234)
[0820] G682 was identified from the Arabidopsis BAC, AF007269,
based on sequence similarity to other members of the Myb family
within the conserved domain. G682 and closely-related clade member
sequences each comprise a conserved Myb-related DNA-binding domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0821] Experimental Observations.
[0822] The function of G682 was analyzed through its ectopic
overexpression in plants.
[0823] RT-PCR analysis of the endogenous levels of G682 transcripts
indicated that the gene is expressed in all tissues. However, only
a very low level of transcript was detected in roots and shoots.
The function of G682 was analyzed through its ectopic
overexpression. 35S::G682 lines were glabrous, had tufts of
increased root hair density and showed better germination under
drought related stress (heat). In one of the genomics experiments,
it was also noted that 35S::G682 lines showed a slightly enhanced
performance on potassium limited media.
[0824] We have now analyzed 35S::G682 seedlings in a C/N sensing
assay by comparing the effects of G682-overexpressing lines
germinating on N-/S medium (MS media minus nitrogen plus 3%
sucrose) with control wild-type seedlings on the same medium. The
overexpressors of these lines were found to produce much less
anthocyanin, indicating that G682 likely has a role in nitrogen
utilization and in the response to low nutrient conditions.
[0825] The phenotypic effects described for 35S::G682
overexpressing plants in the C/N sensing assay is similar to that
observed for 35S lines from other members of the G682 subclade
(G226, G682, G1816, and 2718). Similarly, plants that overexpress
any of the G682 Arabidopsis 35S CaMV clade members have been
observed to have increased root hair formation and reduced
anthocyanin levels in C/N sensing assays. Additionally, Arabidopsis
G682 subclade member (Table 13) and non-Arabidopsis G682 subclade
members, including soy and corn sequences, have also been shown in
laboratory experiments to confer tolerance to various abiotic
stresses (Table 13).
[0826] Thus, the entire clade of G682-related genes appear to have
very related functions and those that have been so tested have been
shown to be involved in the response to nitrogen limitation. As
such, these sequences are likely to be good candidates for
improving the efficiency of nutrient utilization and tolerance to
other stresses in commercial crops. Thus, the G682-related genes
could afford yield savings via multiple different traits.
[0827] Table 13 lists the results obtained in several abiotic
stress assays in which a number of members of the G682 subclade
were overexpressed in Arabidopsis plants. For all genes, assays
were performed in which expression was under the control of the
cauliflower mosaic virus 35S transcription initiation region. For
G682, assays were also performed with transgenic plants in which
expression was controlled as indicated in the second column.
Control of expression of G682 was performed using ARSK1, a
root-specific protein kinase gene promoter, the CUT1 promoter,
which controls production of epicuticular wax in bolting stems and
is used for epidermis-specific expression, and by superactivation,
in which an expression vector having a GAL4 activation domain is
fused to the G682 sequence to create an N-terminal GAL4 activation
domain protein fusion. The first and second columns identify the
sequence test by SEQ ID NO: and Gene Identification Number. The
third column identifies the species in which the gene originated.
The fourth through eleventh columns list the ratio of transgenic
Arabidopsis lines with an altered phenotype relative to controls,
over the number of lines tested. These results show increased
germination in high salt, increased germination in high mannitol,
increased germination in high sucrose, decreased sensitivity to
ABA, increased germination in heat, increased tolerance to heat in
a growth assay, increased germination in cold conditions, and
increased tolerance to cold in a growth assay (chilling),
respectively. The column labeled "C/N" identifies the sequences
that were tested and conferred altered C/N sensing of the plants
(in each of these case, less anthocyanin was produced by the
seedlings in the C/N sensing assays). "Low N tol." refers to
decreased sensitivity, relative to controls, to low nitrogen
conditions in plate-based assays. The column labeled "Morph"
identifies the sequences that exhibited a glabrous phenotype with
increased root hairs, the latter being of particular interest in
that this trait may help confer abiotic stress tolerance. The last
column indicates the lines that were positive in a soil-based
drought assay.
TABLE-US-00014 TABLE 13 Results of abiotic stress experiments with
G682-related sequences Germ Germ Grth Germ Grth SEQ ID in in in in
in C/N Low N Drought NO: GID Species NaCl Mann Sucr ABA Heat Heat
Cold cold sens tol. Morph tol. 234 35S::G682 A. thaliana 9/10 3/10
10/10 6/10 3/10 0/10 0/10 0/10 + nc + + 234 ARSK1::G682 A. thaliana
0/10 0/10 0/10 0/10 0/10 0/10 2/10 0/10 nc nc wt + 234 CUT1::G682
A. thaliana 6/10 0/10 0/10 0/10 0/10 0/10 1/10 0/10 nc nc wt wt 234
SA G682 A. thaliana 2/10 0/10 0/10 0/10 0/10 1/10 0/10 0/10 nc nc +
wt 285 G226 A. thaliana 0/9 0/9 5/9 8/9 0/9 0/9 2/9 2/9 + + + nc
286 G1816 A. thaliana 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 + +
+ nc 323 G2718 A. thaliana nc nc nc nc nc nc nc nc + + + nc 324
G3393 Oryza sativa 0/10 0/10 1/10 0/10 0/10 0/10 0/10 0/10 nc nc +
nc 360 G3431 Z. mays 0/10 0/10 4/10 0/10 2/10 0/10 0/10 0/10 nc nc
+ nc 360 G3444 Z. mays 0/10 0/10 0/10 0/10 2/10 2/10 0/10 1/10 nc
nc + nc 378 G3448 G. max 0/10 0/10 0/10 0/10 0/10 0/10 0/10 3/10 nc
nc + + 380 G3449 G. max 1/10 0/10 0/10 0/10 1/10 0/10 3/10 1/10 nc
nc + nc 382 G3450 G. max 2/10 0/10 0/10 0/10 1/10 3/10 6/10 5/10 nc
nc + + Symbols and abbreviations: + phenotype observed wt result
not significantly different from wild-type Grth Growth Germ
germination Tol tolerance Morph morphology C/N sens carbon/nitrogen
balance sensing Mann growth in high mannitol Sucr growth in high
sucrose ABA reduced sensitivity to abscisic acid SA superactivation
nc assay results not completed or performed to date
Utilities
[0828] The utility of this gene and its equivalogs would be to
confer heat tolerance to germinating seeds and drought tolerance in
plants.
[0829] These assays also indicate that G682 and its equivalogs are
potential regulators of a plant's C/N sensing, nitrogen uptake and
utilization, and its response to low nutrient conditions. For
further analysis, see the discussion above: "Potential Applications
of Polynucleotides and Polypeptides that Regulate C/N sensing".
[0830] G682 equivalogs include, for example, Arabidopsis thaliana
SEQ ID NO: 286, 312 and 324 (G226, G1816 and G2718); Oryza sativa
(japonica cultivar-group) SEQ ID NO: 326 and 328 (G3392 and G3393);
Glycine max SEQ ID NO: 372, 374, 376, 378, 380, and 382 (G3445,
G3446, G3447, G3448, G3449, and G3450); and Zea mays SEQ ID NO: 360
and 370 (G3431 and G3444).
G226 (SEQ ID NO: 285 and 286)
[0831] G226 is a paralog of G682 from Arabidopsis. G226 (AT2G30420)
was identified from the Arabidopsis BAC sequence (GenBank accession
AC002338), based on sequence similarity within the conserved domain
to other Myb family members in Arabidopsis. G226 and
closely-related clade member sequences each comprise a conserved
Myb-related DNA-binding domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0832] Experimental Observations.
[0833] RT-PCR expression analysis of the endogenous levels of G226
indicated that the gene is primarily expressed in leaf tissue. The
function of G226 was analyzed through its ectopic overexpression.
G226 overexpressors were more tolerant to conditions of high salt
(Table 13) and low nitrogen (Table 14). The overexpressors were
larger and greener and had more root growth and root hairs under
conditions of nitrogen limitation than wild-type controls. Many
plants were glabrous and lacked anthocyanin production when under
stress such as growth conditions of low nitrogen (the medium
contained 20 mg/L of NH.sub.4(NO.sub.3) as the nitrogen
source).
[0834] G226 also showed a salt tolerance phenotype in plate-based
salt stress assays (MS medium supplemented with 150 mM NaCl).
35S::G226 seedlings generally appeared larger and greener than
wild-type seedlings, the latter being generally smaller with less
root mass, and were more chlorotic.
[0835] We have now analyzed 35S::G226 seedlings in a C/N sensing
assay. Anthocyanin accumulation was significantly less than that
observed in control wild-type seedlings, confirming that this gene
has a role in the response to nutrient limited conditions. It
should be noted that other members of the clade (G226, G682, G1816,
G2718 and non-Arabidopsis orthologs) produce similar effects when
overexpressed (Tables 13 and 14).
[0836] Utilities.
[0837] These assays indicate that G226 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
[0838] In addition, this gene and its equivalogs could be used to
alter seed protein amounts and/or composition, which could impact
yield as well as the nutritional value and production of various
food products.
G1816 (SEQ ID NO: 311 and 312)
[0839] G1816 is a paralog of G682 from Arabidopsis. G1816 is a
member of the MYB-related class of transcription factors. The gene
corresponds to TRIPTYCHON (TRY), and has recently been shown to be
involved in the lateral inhibition during epidermal cell
specification in the leaf and root (Schellmann et al. (2002) EMBO
J. 21: 5036-5046). The model proposes that TRY (G1816) and CPC
(G225) function as repressors of trichome and atrichoblast cell
fate. TRY loss-of-function mutants form ectopic trichomes on the
leaf surface. TRY gain-of-function mutants are glabrous and form
ectopic root hairs. G1816 and closely-related Glade member
sequences each comprise a conserved Myb-related DNA-binding domain
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0840] Experimental Observations.
[0841] The complete sequence of G1816 was determined. The function
of the gene was studied using transgenic plants in which G1816 was
expressed under the control of the 35S promoter. Consistent with
the morphological phenotypes published for the 35S::TRY
overexpressors, the transgenic plants were glabrous and form
ectopic root hairs.
[0842] These transgenic lines were also more tolerant to growth
under nitrogen-limiting conditions, both in a germination assay as
well as a root growth assay on older seedlings.
[0843] In addition to the nitrogen-limiting tolerance phenotypes
observed in these transgenic lines, the 35S::G1816 plants were also
insensitive to growth retardation effects of germination on
conditions of high glucose (MS medium supplemented with 5%
glucose); the overexpressor seedlings were large and green, as
contrasted with the wild-type control seedlings which were
significantly smaller and more pigmented. This indicates that G1816
plays a role in sugar sensing responses in the plant or osmotic
stress tolerance. Genes for many sugar-sensing mutants are allelic
to genes involved in abscisic acid and ethylene signaling (Rolland
et al. (2002) Plant Cell 14: Suppl. S185-S205). Therefore, G1816
could also be involved in hormone signaling pathways.
[0844] We have now analyzed 35S::G1816 seedlings in a C/N sensing
assay. The seedlings in these experiments were germinated on
N-/S/Gln medium (MS media minus nitrogen plus 3% sucrose and 1 mM
glutamine). The G1816 overexpressing seedlings were found to have
less anthocyanin than the control seedlings, indicating that G1816
likely has a role in nitrogen utilization and in the response to
low nutrient conditions.
[0845] Germination assays were also used to compare G1816
overexpressors and wild-type control seedlings on a low nitrogen
medium. The overexpressors were much larger, had no anthocyanin and
produced more root growth and root hair density than the wild-type
controls.
[0846] Utilities.
[0847] The phenotypic effects of G1816 overexpression, such as the
increase in root hair formation and the increase in seedling vigor
observed in a germination assay on high glucose media, indicated
that the gene or its orthologs can be used to engineer plants with
increased tolerance to abiotic stresses such as drought, salt, heat
or cold.
[0848] In addition, the enhanced performance of G1816
overexpression lines under low nitrogen conditions indicated that
the gene or its orthologs could be used to engineer crops that
could thrive under conditions of reduced nitrogen availability.
These assays also indicate that G1816 and its equivalogs are
potential regulators of a plant's C/N sensing, nitrogen uptake and
utilization, and its response to low nutrient conditions. For
further analysis, see the discussion above: "Potential Applications
of Polynucleotides and Polypeptides that Regulate C/N sensing".
[0849] The effect of G1816 overexpression on insensitivity to
glucose in a germination assay, indicated that the gene or its
orthologs could be involved in sugar sensing responses in the
plant.
[0850] G1816 or its orthologs could also be used to alter
anthocyanin production and trichome formation in leaves.
[0851] The potential utilities of genes involved in anthocyanin
production include alterations in pigment production for
horticultural purposes and increase stress resistance perhaps in
combination with other transcription factors. Flavonoids have
antimicrobial activity and could be used to engineer pathogen
resistance. In addition, several flavonoid compounds have health
promoting effects such as the inhibition of tumor growth and
cancer, prevention of bone loss and the prevention of the oxidation
of lipids.
G2718 (SEQ ID NO: 323 and 324)
[0852] G2718 is a paralog of G682 from Arabidopsis. G2718
(AT1G01380) was identified in the BAC clone, F6F3 (GenBank
accession AC023628). Two highly related genes, TRY and CPC have
been implicated in epidermal cell specification. A lateral
inhibition model proposes that TRY (G1816) and CPC (G225) function
as repressors of trichome and atrichoblast cell fate (Shellmann et
al. (2002) EMBO J. 21: 5036-5046). A comprehensive review on
epidermal cell-fate specification has been published recently
(Schiefelbein (2003) Curr. Opin. Plant Biol. 6: 74-78). G2718 and
closely-related clade member sequences each comprise a conserved
Myb-related DNA-binding domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0853] Experimental Observations.
[0854] Results obtained by the overexpression of G1816 in plants,
including abiotic stress tolerance and low nitrogen tolerance
phenotypes have been previously reported in U.S. patent application
Ser. No. 10/714,887, filed Nov. 13, 2003.
[0855] The function of G2718 was studied using plants in which the
gene was expressed under the control of the 35S promoter.
Overexpression of G2718 resulted in a glabrous phenotype. The
effect was highly penetrant, being observed in all primary
transformants and each of three independent T2 lines. All of the T1
lines showed a very strong phenotype and completely lacked
trichomes on leaves and stems. A comparably severe effect was
observed in one of the three T2 populations, whereas the other two
T2 populations each exhibited a weaker phenotype, indicating that
the effect might have become partially silenced between the
generations. Trichomes were present in these weaker lines, but at a
much lower density than in wild type.
[0856] In addition to the effects on trichome density, 35S::G2718
transformants were also generally slightly smaller than wild type
controls.
[0857] The phenotypic effects above were observed in the 35S::G2718
as well as in all 35S lines from members of the G2718 clade (G225,
G226, G1816, and G682). Similarly, 35S::TF lines from the G2718
clade all had increased root hair formation, reduced anthocyanin
levels, and showed improved growth under nitrogen limiting
conditions.
[0858] Overexpressors were generally larger, had more root mass,
and were often greener than wild-type control seedlings on low
nitrogen media, indicating that overexpression of G2718 confers
enhanced tolerance of plants to this low nutrient condition,
possibly by improving nutrient uptake.
[0859] We have now analyzed 35S::G2718 seedlings in a C/N sensing
assay. Anthocyanin accumulation was significantly less than that
observed in control plants (Table 14), indicating that G2718 likely
has a role in nitrogen utilization and in the response to low
nutrient conditions.
[0860] Utilities.
[0861] The phenotypic effects of G2718 overexpression, such as the
increase in root hair formation and the increase in seedling vigor
observed in a root growth assay on N-limiting media, indicates that
the gene or its equivalogs could be used to engineer plants with
increased tolerance to abiotic stresses such as nutrient
limitation, drought, salt, heat or cold.
[0862] The enhanced performance of G2718 overexpression lines under
low nitrogen conditions indicates that the gene or its equivalogs
could be used to engineer crops that could thrive under conditions
of reduced nitrogen availability. These assays also indicate that
G2718 and its equivalogs are potential regulators of a plant's C/N
sensing, nitrogen uptake and utilization, and its response to low
nutrient conditions. For further analysis, see the discussion
above: "Potential Applications of Polynucleotides and Polypeptides
that Regulate C/N sensing".
[0863] G2718 or its equivalogs could also be used to alter
anthocyanin production or trichome formation. and production of
secondary biochemicals (e.g., lipophilic terpenes) by
trichomes.
G3392 (SEQ ID NO: 325 and 326)
[0864] G3392 is a rice ortholog of G682. G3392 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0865] Experimental Observations.
[0866] Similar to G682 and other homologs of G3392, a number of
G3392-overexpressing lines displayed reduced leaf trichomes and
more root hairs.
[0867] On low nitrogen media, Arabidopsis seedlings overexpressing
G3392 accumulated less anthocyanin than wild-type control
seedlings. G3392 overexpressors also accumulated less anthocyanin
on low nitrogen MS media minus nitrogen and supplemented with
either 3% sucrose, or 3% sucrose and 1 mM glutamine, indicating an
altered C/N sensing phenotype.
[0868] In heat germination assays and in assays conducted with more
mature plants conducted at 32.degree. C., G3392-overexpressing
Arabidopsis seedlings were greener than wild-type controls. The
results of this assay indicate that, similar to other members of
the clade, the monocot-derived G3392 has the ability to confer
tolerance to heat stress in plants.
[0869] G3392-overexpressing Arabidopsis plants were also more
tolerant to 300 mM mannitol and 9.4% sucrose than wild-type control
plants grown in plate based-assays under similar conditions,
indicating a sugar-sensing and osmotic stress tolerant
phenotype.
[0870] In heat germination assays and in assays conducted with more
mature plants conducted in media containing 150 mM NaCl,
G3392-overexpressing Arabidopsis seedlings were larger greener than
wild-type controls.
[0871] In cold germination assays for 6 hours at 8.degree. C. and
in assays conducted with more mature plants conducted with a 6 hour
exposure to 4-8.degree. C., G3392-overexpressing Arabidopsis
seedlings accumulated much less anthocyanin than wild-type
controls. The results of this assay indicate that, similar to other
members of the Glade, the monocot-derived G3392 has the ability to
confer tolerance to cold stress in plants.
[0872] Utilities.
[0873] The phenotypic effects of G3392 overexpression indicates
that the gene or its equivalogs could be used to engineer plants
with increased tolerance to several abiotic stresses and low
nitrogen conditions.
G3393 (SEQ ID NO: 327 and 328)
[0874] G3393 is a rice ortholog of G682. G3393 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0875] Experimental Observations.
[0876] Similar to G682 and other homologs of G3393, a number of
G3393-overexpressing lines displayed reduced leaf trichomes and
more root hairs.
[0877] On low nitrogen media, Arabidopsis seedlings overexpressing
G3393 accumulated significantly less anthocyanin than wild-type
control seedlings. G3393 overexpressors also accumulated less
anthocyanin on low nitrogen MS media minus nitrogen and
supplemented with either 3% sucrose, or 3% sucrose and 1 mM
glutamine, indicating an altered C/N sensing phenotype.
[0878] In cold germination assays for 6 hours at 8.degree. C. and
in assays conducted with more mature plants conducted with a 6 hour
exposure to 4-8.degree. C., G3393-overexpressing Arabidopsis
seedlings accumulated much less anthocyanin than wild-type
controls. The results of this assay indicate that, similar to other
members of the Glade, the monocot-derived G3393 has the ability to
confer tolerance to cold stress in plants.
[0879] Utilities.
[0880] The phenotypic effects of G3393 overexpression indicates
that the gene or its equivalogs could be used to engineer plants
with increased tolerance to cold stress and low nitrogen
conditions.
G3431 (SEQ ID NO: 359 and 360)
[0881] G3431 is a corn ortholog of G682. G3431 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0882] Experimental Observations.
[0883] Similar to G682 and other homologs of G3431, a number of
G3431-overexpressing lines displayed reduced leaf trichomes and
more root hairs.
[0884] On low nitrogen media, Arabidopsis seedlings overexpressing
G3431 accumulated significantly less anthocyanin than wild-type
control seedlings. G3431 overexpressors also accumulated less
anthocyanin on low nitrogen MS media minus nitrogen and
supplemented with either 3% sucrose, or 3% sucrose and 1 mM
glutamine, indicating an altered C/N sensing phenotype.
[0885] In cold germination assays for 6 hours at 8.degree. C. and
in assays conducted with more mature plants conducted with a 6 hour
exposure to 4-8.degree. C., G3431-overexpressing Arabidopsis
seedlings accumulated much less anthocyanin than wild-type
controls. The results of this assay indicate that, similar to other
members of the Glade, the monocot-derived G3431 has the ability to
confer tolerance to cold stress in plants.
[0886] In osmotic stress assays conducted on MS media containing
9.4% sucrose, G3431-overexpressing Arabidopsis seedlings were
greener and accumulated less anthocyanin than wild-type controls,
indicating osmotic stress tolerance was conferred by overexpressing
G3431.
[0887] Utilities.
[0888] The phenotypic effects of G3431 overexpression indicates
that the gene or its equivalogs could be used to engineer plants
with increased tolerance to cold and osmotic stress and low
nitrogen conditions.
G3444 (SEQ ID NO: 369 and 370)
[0889] G3444 is a corn ortholog of G682. G3444 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0890] Experimental Observations.
[0891] Similar to G682 and other homologs of G3444, a number of
G3444-overexpressing lines had reduced trichomes.
[0892] On low nitrogen media, Arabidopsis seedlings overexpressing
G3444 accumulated less anthocyanin than wild-type control
seedlings. One line of G3444 overexpressors also accumulated less
anthocyanin on low nitrogen MS media minus nitrogen and
supplemented with either 3% sucrose, or 3% sucrose and 1 mM
glutamine, indicating an altered C/N sensing phenotype.
[0893] In heat germination assays and in assays conducted with more
mature plants conducted at 32.degree. C., G3444-overexpressing
Arabidopsis seedlings were greener than wild-type controls. The
results of this assay indicate that, similar to other members of
the clade, the monocot-derived G3444 has the ability to confer
tolerance to abiotic stress in plants.
[0894] Utilities.
[0895] The phenotypic effects of G3444 overexpression indicates
that the gene or its equivalogs could be used to engineer plants
with increased tolerance to heat and low nitrogen conditions.
G3445 (SEQ ID NO: 371 and 372)
[0896] G3445 is a soy ortholog of G682. G3445 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0897] Experimental Observations.
[0898] Similar to G682 and other homologs of G3445, a number of
G3445-overexpressing lines had reduced trichomes.
[0899] In germination assays conducted on media supplemented with
0.3 .mu.M ABA, G3445-overexpressing Arabidopsis seedlings were
larger and greener than wild-type controls.
[0900] Utilities.
[0901] The phenotypic effects of G3445 overexpression indicates
that the gene or its equivalogs could be used to engineer plants
with increased tolerance to osmotic stress conditions.
G3448 (SEQ ID NO: 377 and 378)
[0902] G3448 is a soy ortholog of G682. G3448 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0903] Experimental Observations.
[0904] Similar to G682 and other homologs of G3448, a number of
G3448-overexpressing lines displayed reduced leaf trichomes and
more root hairs.
[0905] On low nitrogen media, Arabidopsis seedlings overexpressing
G3448 accumulated significantly less anthocyanin than wild-type
control seedlings. G3448 overexpressors also accumulated less
anthocyanin on low nitrogen MS media minus nitrogen and
supplemented with either 3% sucrose, or 3% sucrose and 1 mM
glutamine, indicating an altered C/N sensing phenotype.
[0906] In assays conducted with Arabidopsis plants at a 6 hour
exposure to 4-8.degree. C., G3448-overexpressing Arabidopsis
seedlings accumulated less anthocyanin than wild-type controls. The
results of this assay indicate that, similar to other members of
the clade, the dicot-derived G3448 has the ability to confer
tolerance to cold stress in plants.
[0907] Utilities.
[0908] The phenotypic effects of G3448 overexpression indicates
that the gene or its equivalogs could be used to engineer plants
with increased tolerance to cold stress and low nitrogen
conditions.
G3449 (SEQ ID NO: 379 and 380)
[0909] G3449 is a soy ortholog of G682. G3449 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0910] Experimental Observations.
[0911] Similar to G682 and other homologs of G3393, a number of
G3393-overexpressing lines displayed reduced leaf trichomes and
more root hairs.
[0912] On low nitrogen media, Arabidopsis seedlings overexpressing
G3449 accumulated significantly less anthocyanin than wild-type
control seedlings. G3449 overexpressors also accumulated less
anthocyanin on low nitrogen MS media minus nitrogen and
supplemented with either 3% sucrose, or 3% sucrose and 1 mM
glutamine, indicating an altered C/N sensing phenotype.
[0913] In cold germination assays for 6 hours at 8.degree. C.,
G3449-overexpressing Arabidopsis seedlings accumulated much less
anthocyanin than wild-type controls. The results of this assay
indicate that, similar to other members of the clade, the
dicot-derived G3449 has the ability to confer tolerance to cold
stress in plants.
[0914] Utilities.
[0915] The phenotypic effects of G3449 overexpression indicates
that this sequence or its equivalogs could be used to engineer
plants with increased tolerance to cold stress and low nitrogen
conditions.
G3450 (SEQ ID NO: 381 and 382)
[0916] G3450 is a soy ortholog of G682. G3450 and closely-related
clade member sequences each comprise a conserved Myb-related
DNA-binding domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[0917] Experimental Observations
[0918] Similar to G682 and other homologs of G3450, a number of
G3450-overexpressing lines displayed reduced leaf trichomes and
more root hairs.
[0919] On low nitrogen media, Arabidopsis seedlings overexpressing
G3392 accumulated less anthocyanin than wild-type control
seedlings. G3450 overexpressors also accumulated less anthocyanin
on low nitrogen MS media minus nitrogen and supplemented with
either 3% sucrose, or 3% sucrose and 1 mM glutamine, indicating an
altered C/N sensing phenotype.
[0920] In cold germination assays for 6 hours at 8.degree. C. and
in assays conducted with more mature plants conducted with a 6 hour
exposure to 4-8.degree. C., G3450-overexpressing Arabidopsis
seedlings accumulated much less anthocyanin than wild-type
controls. The results of this assay indicate that, similar to other
members of the clade, the dicot-derived G3450 has the ability to
confer tolerance to cold stress in plants.
[0921] In heat germination assays and in assays conducted with more
mature plants conducted at 32.degree. C., G3450-overexpressing
Arabidopsis seedlings were greener than wild-type controls. The
results of this assay indicate that, similar to other members of
the clade, the dicot-derived G3450 has the ability to confer
tolerance to heat stress in plants.
[0922] G3450-overexpressing Arabidopsis plants were also more
tolerant to salt and desiccation than wild-type control plants
grown under similar conditions in plate based-assays, and to
drought conditions in soil-based assays.
[0923] Utilities.
[0924] The phenotypic effects of G3450 overexpression indicates
that this sequence or its equivalogs could be used to engineer
plants with increased tolerance to low nitrogen conditions, salt,
cold stress, heat stress, and low water conditions.
The G24 Clade of Transcription Factor Polypeptides
G24 (SEQ ID NO: 419 and 420)
[0925] G24 corresponds to gene At2g23340 (AAB87098). G24 and
closely-related clade member sequences each comprise a conserved
AP2 DNA-binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0926] Experimental Observations.
[0927] Based on RT-PCR expression analysis, G24 was found to be
ubiquitously expressed at low levels in germinating seedlings. The
function of G24 was studied using transgenic plants in which the
gene was expressed under the control of the 35S promoter. 35S::G24
seedlings often developed black necrotic tissue patches on
cotyledons and leaves, and many died at that stage. Some 35S::G24
seedlings exhibited a weaker phenotype, and although necrotic
patches were visible on the cotyledons, they did not die. These
seedlings developed into plants that were usually small, slow
growing, and poorly fertile in comparison to wild type controls.
The leaves of older 35S::G24 plants were also observed to become
yellow and senesce prematurely compared to wild type. Of the lines
sent for physiological assays, all showed a comparable response to
wild-type. However, 35S::G24 line 2 seedlings became necrotic and
died immediately after germination on MS plates. 35S::G24 line 8
has an intermediate phenotype in which the seedlings develop some
necrotic lesions but survived and 35S::G24 line 11 seedlings
appeared wild-type.
[0928] We have now analyzed 35S::G24 seedlings in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings (Table 14), indicating that
the gene may be involved in the response to low nutrient
conditions.
[0929] Utilities.
[0930] These assays indicate that G24 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G154 Clade of Transcription Factor Polypeptides
G154 (SEQ ID NO: 421 and 422)
[0931] G154 was identified in the sequence of BAC F17K2, from
chromosome 2 (gene At2g45660). It also corresponds to SUPPRESSOR OF
OVEREXPRESSION OF CO (SOC1), and was previously designated AGL20
(Samach et al. (2000) Science. 288: 1613-1616; Lee et al. (2000)
Genes Dev. 14:2366-2376; Borner et al. (2000) Plant J. 24:
591-599). This gene has been isolated several times in genetic and
molecular screens for flowering-time mutants. SOC1/AGL20 was
identified by suppression subtraction hybridization as a direct
target of the zinc finger transcription factor CONSTANS (Samach et
al. (2000) supra), and also genetically as a late flowering mutant
capable of suppressing the phenotype caused by overexpression of CO
(hence the name SOC1) (Onouchi et al. (2000) Plant Cell 12:
885-900). The gene was also identified as a dominant FRIGIDA (FRI)
suppressor in activation tagging mutagenesis (Lee et al. (2000)
supra), as well as a late flowering mutant generated by transposon
tagging (Borner et al. (2000) supra). Genetic and molecular
analyses have allowed the position of this gene within the
flowering-time control network to be determined.
[0932] Samach et al. (Samach et al. (2000) supra) reported that
flowering is triggered by endogenous and environmental signals. CO
promotes flowering of Arabidopsis in response to day length. Early
target genes of co were identified using a steroid-inducible
version of the protein. Two of these genes, SOC1 and FLOWERING
LOCUS T (FT), are required for CO to promote flowering. SOC1 and FT
are also regulated by a second flowering-time pathway that acts
independently of CO. Thus, early target genes of CO define common
components of distinct flowering-time pathways.
[0933] Lee et al. (Lee (2000) supra) reported that the very
late-flowering behavior of Arabidopsis winter-annual ecotypes is
conferred mainly by two genes, FRI and FLOWERING LOCUS C (FLC).
AGL20 was identified as a dominant FRI suppressor in activation
tagging mutagenesis. Overexpression of AGL20 suppresses not only
the late flowering of plants that have functional FRI and FLC
alleles but also the delayed phase transitions during the
vegetative stages of plant development. Interestingly, AGL20
expression is positively regulated not only by the redundant
vernalization and autonomous pathways of flowering but also by the
photoperiod pathway. Our results indicate that AGL20 is an
important integrator of three pathways controlling flowering in
Arabidopsis.
[0934] Borner et al. (Borner et al. (2000) supra) reported that the
flowering time in many plants is triggered by environmental factors
that lead to uniform flowering in plant populations, ensuring
higher reproductive success. So far, several genes have been
identified that are involved in flowering time control. AGL20 is
activated in shoot apical meristems during the transition to
flowering. By transposon tagging we have identified late flowering
agl20 mutants, showing that AGL20 is involved in flowering time
control. In previously described late flowering mutants of the
long-day and constitutive pathways of floral induction the
expression of AGL20 is down-regulated, demonstrating that AGL20
acts downstream to the mutated genes. Moreover, we can show that
AGL20 is also regulated by the gibberellin (GA) pathway, indicating
that AGL20 integrates signals of different pathways of floral
induction and might be a central component for the induction of
flowering. In addition, the constitutive expression of AGL20 in
Arabidopsis is sufficient for photoperiod independent flowering and
the over-expression of the orthologous gene from mustard, MADSA, in
the classical short-day tobacco Maryland Mammoth bypasses the
strict photoperiodic control of flowering.
[0935] G154 and closely-related clade member sequences each
comprise a conserved MADS DNA-binding domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[0936] Experimental Observations.
[0937] The function of G154 was studied using transgenic plants in
which the gene was expressed under the control of the 35S promoter.
Overexpression of G154 produced a range of morphological effects.
Early flowering was noted in a small number of primary
transformants. Additionally, 35S::G154 lines were sometimes small,
spindly and poorly fertile. G154 overexpressing lines behave
similarly to wild-type controls in all physiological and
biochemical assays performed.
[0938] SOC1 has a well-established role in regulation of the onset
of flowering. We have now analyzed 35S::G154 seedlings in a C/N
sensing assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings in all three lines tested
(Table 14). Thus, in addition to its effects on flowering time,
this gene might also influence the response to low nutrient
conditions.
[0939] Utilities.
[0940] These assays indicate that G154 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G384 Clade of Transcription Factor Polypeptides
G384 (SEQ ID NO: 423 and 424)
[0941] G384, also called ATML1 (Lu et al. (1996) Plant Cell
8:2155-2168), belongs to the HD-GL2 class of homeodomain proteins.
It was isolated based on its homology to 039, a homeodomain protein
from orchid. Northern blot analysis indicated that it was floral
bud specific in Arabidopsis and in situ hybridization data showed
that G384 was only expressed in the L1 layer of the shoot meristems
and the protoderms of the pre-torpedo stage embryos. G384 and
closely-related clade member sequences each comprise a conserved
homebox DNA binding domain (or "homeodomain") and a START domain
that are expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[0942] Experimental Observations.
[0943] 35S::G384 lines showed developmental abnormalities including
fused organs. We have now analyzed the function of G384 by
characterizing overexpression lines in C/N sensing assays. These
lines showed increased sensitivity and elevated anthocyanin levels
relative to wild-type (Table 14). It is possible, however, that
G384 is not specifically involved in a C/N sensing response since
addition of glutamine to the growth plates did not alleviate the
phenotype. It should be emphasized that G384 is a member of the
HD-GL2 class of homeodomain proteins. Overexpression lines for two
other HD-GL2 class genes, G1535 and G707, showed comparable
phenotypes to the 35S::G384 lines studied in the present screen.
These findings are of particular interest because GL2 acts in the
genetic pathway through which the CAPRICE (CPC) related genes
regulate root development. The current results indicate that as
well as GL2 itself, other homeodomain proteins from the HD-GL2
class might also act in pathways involving the CAPRICE (CPC)
related genes, given that the CAPRICE (CPC) related genes influence
nutrient limitation responses and anthocyanin production.
[0944] Utilities.
[0945] These assays indicate that G384 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G486 Clade of Transcription Factor Polypeptides
G486 (SEQ ID NO: 293 and 294)
[0946] G486 was identified as a BAC sequence (AC000106) with
homology to CCAAT-like transcription factors. G486 and
closely-related clade member sequences each comprise a conserved
CBFD_NFYB_HMF domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0947] Experimental Observations.
[0948] RT-PCR expression analysis indicated that G486 is expressed
primarily in roots, flowers, cauline leaves and seedlings. The
function of G486 was analyzed through by the generation of
35S::G486 overexpressing plants. 35S::G486 lines were noted to be
somewhat small, rather darker green, and were delayed in the onset
of flowering.
[0949] We have now analyzed 35S::G486 seedlings in a C/N sensing
assay. Anthocyanin accumulation was less than that observed in
control wild-type seedlings in one line of two lines tested,
indicating that overexpression of G486 in Arabidopsis gave a mild
response in overcoming the stress caused by this assay (Table
14).
[0950] Utilities.
[0951] These assays indicate that G486 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G545 Clade of Transcription Factor Polypeptides
G545 (SEQ ID NO: 425 and 426)
[0952] G545 was discovered independently by two groups. Lippuner et
al. (Lippuner et al. (1996) J. Biol. Chem. 271:12859-2866)
identified G545 as an Arabidopsis cDNA (STZ), which increases the
tolerance of yeast to Li+ and Na+. They found that STZ expression
is most abundant in leaves and roots, and that its level of
expression increases slightly upon exposure of the plant to salt.
The second group (Meissner and Michael (1997) Plant Mol. Biol.
33:615-624), identified G545 (ZAT10) in a group of Arabidopsis C2H2
zinc finger protein-encoding cDNAs that they isolated by degenerate
PCR. According to their data, ZAT10 is expressed in roots, shoots
and stems. G545 and closely-related clade member sequences each
comprise a conserved C2H2 DNA-binding zinc finger domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0953] Experimental Observations.
[0954] Plants overexpressing G545 were smaller than wild type
plants, flowered early, and in extreme cases were infertile. G545
overexpression conferred tolerance to phosphate deficiency.
However, the small size of the seedlings made it difficult to make
root growth comparisons with wild type. 35S::G545 lines also
appeared more sensitive to NaCl than wild type plants. Finally,
G545 overexpressing plants appeared to be significantly more
susceptible to pathogens than control plants.
[0955] We have now analyzed the function of G545 by characterizing
35S:G545 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was elevated compared to control wild-type seedlings
(Table 14). Thus, the gene could have a role in the response to
nutrient limitation or abiotic stress.
[0956] Utilities.
[0957] The first useful phenotype G545 overexpressors are
displaying is their tolerance to phosphate deficiency. Young plants
have a rapid intake of phosphorous, so it is important that seed
beds have high enough content in phosphate to sustain their growth.
Also, root crops such as carrot, potato and parsnip will all
decrease in yield if there is insufficient phosphate available.
Phosphate costs represent a relatively small but significant
portion of farmers' operating costs (3-4% of total costs to a corn
farmer in the US, higher to a vegetable grower). Plants that are
tolerant to phosphate deficiency can represent a cost saving for
farmers, especially in areas where soils are very poor in
phosphate.
[0958] Another desirable phenotype, salt tolerance, may arise from
G545 silencing rather than overexpression. Additionally, G545
appears to be induced by cold, drought, salt and osmotic stresses,
which is in agreement with a potential role of the genes in
protecting the plant in such adverse environmental conditions.
[0959] G545 appears to be involved in the control of defense
processes. However, overexpression of G545 made Arabidopsis plants
more susceptible to disease. This negative effect will have to be
corrected before G545 can be used in a crop to induce tolerance to
low phosphate. One example of a method to approach the problem
would be to restrict overexpression of G545 to roots.
[0960] These assays indicate that G545 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G760 Clade of Transcription Factor Polypeptides
G760 (SEQ ID NO: 427 and 428)
[0961] G760 corresponds to the gene NAC2, GenBank accession no.
AF201456. G760 was found to be highly expressed in root meristems.
G760 and closely-related clade member sequences each comprise a
conserved NAC domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[0962] Experimental Observations.
[0963] RT-PCR analysis demonstrated that G760 was uniformly
expressed in all tissues and under all conditions. The function of
G760 gene was analyzed using transgenic plants in which the gene
was expressed under the control of the 35S promoter. Many 35S::G760
primary transformants were small and had rather curled, twisted,
leaves. However, T2 populations all showed a wild-type phenotype,
indicating that activity of the transgene might have been reduced
between the generations. In addition, overexpression of G760 in
Arabidopsis resulted in T2 seedlings that were hypersensitive to
growth on ACC.
[0964] We have now analyzed the function of G760 by characterizing
35S:G760 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was greatly elevated compared to that observed in
control wild-type seedlings in one of three lines tested (Table
14). Thus, G760 could have a role in response to low nutrient
conditions.
[0965] Utilities.
[0966] G760 could be used to manipulate ethylene signal
transduction or response pathways. The gene could be used to
manipulate the processes influenced by ethylene, such as fruit
ripening.
[0967] These assays indicate that G760 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G773 Clade of Transcription Factor Polypeptides
G773 (SEQ ID NO: 429 and 430)
[0968] G773 (AT3G15500) in the sequence of GenBank accession number
AB022218, released by the Arabidopsis Genome Initiative and
corresponds to AtNAC3 (Takada et al. (2001) Development
128:1127-1135). G773 and closely-related clade member sequences
each comprise a conserved NAC domain that is expected to function
in a similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0969] Experimental Observations.
[0970] RT-PCR analysis determined that G773 has the highest levels
of expression in roots, flowers and embryos and is expressed at
medium or low levels in rosettes, siliques and seedlings. RT-PCR
data also indicated a significant induction of G773 transcripts
accumulation upon auxin, heat, osmotic, drought and Fusarium
treatments. The phenotype of the transgenic lines analyzed was wild
type in all assays performed at that time.
[0971] We have now analyzed the function of G773 by characterizing
35S::G773 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was elevated compared to that observed in wild-type
seedlings (Table 14). Thus, G773 could have a role in the response
to nutrient limitation
[0972] Utilities.
[0973] These assays indicate that G773 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G937 Clade of Transcription Factor Polypeptides
G937 (SEQ ID NO: 431 and 432)
[0974] G937 (AT1G49560) was initially identified in the sequence of
BAC F14J22 (GenBank accession AC011807) released by the Arabidopsis
Genome Initiative. G937 and closely-related clade member sequences
each comprise a conserved GARP DNA-binding domain that is expected
to function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[0975] Experimental Observations.
[0976] RT-PCR expression analysis demonstrated that G937 was
expressed at relatively high levels throughout the plant, and was
not induced by any condition tested. The function of this gene was
analyzed using transgenic plants in which G937 was expressed under
the control of the 35S promoter. The majority of 35S::G937 primary
transformants were smaller than wild type, slightly slow
developing, and produced thin inflorescence stems that carried
relatively few siliques.
[0977] We have now analyzed 35S::G937 seedlings in a C/N sensing
assay. Anthocyanin accumulation was less than that observed in
control wild-type seedlings in one of three lines tested (Table
14). Thus, G937 might have a role in the response to nutrient
limitation
[0978] Utilities.
[0979] G937 may be useful for regulation of plant growth and
development.
[0980] These assays indicate that G937 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G971 Clade of Transcription Factor Polypeptides
G971 (SEQ ID NO: 433 and 434)
[0981] G971 (AT3G54990) corresponds to gene F28P10.30 (GenBank
accession CAB41085). G971 and closely-related clade member
sequences each comprise a conserved AP2 DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0982] Experimental Observations.
[0983] RT-PCR expression analysis indicated that G971 is
ubiquitously expressed. The function of G971 was studied using
transgenic plants in which the gene was expressed under the control
of the 35S promoter. Overexpression of G971 produced a marked delay
in the transition to flowering. No obvious phenotype was observed
during that period with 35S::G971 plants in physiological
assays.
[0984] We have now analyzed the function of G971 by characterizing
35S:G971 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was elevated compared to the levels seen in control
wild-type seedlings (Table 14). Thus, G971 could have a role in the
response to low nitrogen conditions.
[0985] Utilities.
[0986] G971 could be used to modify flowering time
characteristics.
[0987] These assays indicate that G971 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G988 Clade of Transcription Factor Polypeptides
G988 (SEQ ID NO: 435 and 436)
[0988] G988 (AT1G55580) corresponds to a protein annotated as
hypothetical in BAC F20N2 (GenBank accession number AC002328) from
chromosome 1 of Arabidopsis. The sequence for G988 is described in
patent application WO 9846759. G988 and closely-related clade
member sequences each comprise a conserved SCR domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[0989] Experimental Observations.
[0990] RT-PCR expression analysis indicated that G988 appears to be
expressed primarily in flower and silique tissue and is induced in
response to heat treatment. The function of this gene was analyzed
using transgenic plants in which G988 was expressed under the
control of the 35S promoter. Plants overexpressing G988 had
multiple morphological phenotypes. The transgenic plants were
generally smaller than wild-type plants, had altered leaf,
inflorescence and flower development, altered plant architecture,
and altered vasculature.
[0991] We have now analyzed the function of G988 by characterizing
35S::G988 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was elevated compared to that observed in control
wild-type seedlings (Table 14). Thus, G988 could have a role in the
response to low nitrogen conditions.
[0992] Utilities.
[0993] Based on the observed morphological phenotypes of the
transgenic plants, it is possible that G988 could be used to create
plants with larger flowers. This could have potential value in the
ornamental horticulture industry. The reduction in the formation of
lateral branches indicates that G988 could have possible utility on
the forestry industry. The Arabidopsis plants overexpressing G988
also had reduced fertility. This could actually be a desirable
trait in some instances, as it could be exploited to prevent or
minimize the escape of GMO pollen into the environment.
[0994] These assays indicate that G988 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G989 Clade of Transcription Factor Polypeptides
G989 (SEQ ID NO: 437 and 438)
[0995] G989 (AT5G41920) corresponds to a predicted SCARECROW gene
regulator-like protein in annotated P1 clone (GenBank accession
AB017067). G989 and closely-related clade member sequences each
comprise a conserved SCR domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[0996] Experimental Observations.
[0997] RT-PCR expression analysis indicated that G989 appeared to
be expressed at highest levels in embryo tissue, and at low levels
in all other tissues tested. Expression of G989 appeared to be
induced in response to treatment with auxin, ABA, heat and drought,
and to a lesser extent in response to salt treatment and osmotic
stress. The function of this gene was also analyzed using
transgenic plants in which G989 was expressed under the control of
the 35S promoter. Plants overexpressing G989 appeared to be
somewhat early flowering, but in other respects appeared normal,
and showed a wild-type response in the physiological assays
performed at that time.
[0998] We have now analyzed 35S::G989 seedlings in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings (Table 14), indicating that
G989 has a role in the response to nutrient limitation.
[0999] Utilities.
[1000] If the early flowering phenotype is reproducible in a larger
number of plants and under a wider range of environmental
conditions, it is possible that G989 could be used to alter
flowering time in other plant species. A number of Arabidopsis
genes have already been shown to accelerate flowering when
constitutively expressed. These include LEAFY, APETALA1 and
CONSTANS. In these cases, however, the early flowering plants
showed undesirable side effects such as extreme dwarfing,
infertility, or premature termination of shoot meristem growth
(Mandel and Yanofsky (1995) Nature 377: 522-524, Weigel and Nilsson
(1995) Nature 377: 495-500, Simon et al. (1996). 384: Nature
59-62). Our initial study indicates that G989 can induce flowering
without these toxic pleiotropic effects.
[1001] These assays indicate that G989 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1073 Clade of Transcription Factor Polypeptides
G1069 (SEQ ID NO: 239 and 240)
[1002] G1069 is a paralog of G1073 from Arabidopsis. G1069
corresponds to AT4G14465 and is a member of the AT-Hook related
proteins in Arabidopsis. G1069 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 76-218) or the DUF296 domain (amino
acids 93-211) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[1003] Experimental Observations.
[1004] G1069 was predominantly expressed in roots, based our
initial analysis of RT-PCR results. The function of G1069 was
analyzed using transgenic plants in which G1069 was expressed under
the control of the 35S promoter. Plants overexpressing G1069 showed
changes in leaf architecture, reduced overall plant size, and
retarded progression through the life cycle. One G1069
overexpressing line showed more tolerance to abiotic stress when
they were germinated in high sucrose plates. This line (line 41)
also showed insensitivity to ABA in a germination assay. Moreover,
seedlings of this line also look smaller and chlorotic in control
germination plates.
[1005] We have now analyzed 35S::G1069 seedlings in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings in one line (line 41)
(Table 14), indicating that overexpression of G1069 in Arabidopsis
gave a very mild response in overcoming the stress caused by this
assay. The other two lines were wild type. Line 41 also gave a
positive stress phenotype when germinated on media containing
sucrose and ABA.
[1006] Utilities.
[1007] Because of its effect on leaf architecture, plant size and
plant development, G1069 may have some utility in modifying plant
growth and development. In addition, the promoter of G1069 may have
some utility as a promoter that is active in roots.
[1008] These assays indicate that G1069 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
G2789 (SEQ ID NO: 247 and 248)
[1009] The sequence of G2789 (AT3G60870) was obtained from the
Arabidopsis genomic sequencing project, GenBank accession number
AL162295, based on its sequence similarity to other AT-hook related
proteins. G2789 is a sequence functionally and structurally related
to G1073 from Arabidopsis. G2789 and closely-related clade member
sequences each comprise a conserved At-hook domain and a second
conserved domain (amino acids 68-208) or the DUF296 domain (amino
acids 86-201) that are expected to function in a similar manner in
each of these related sequences, that is, by playing a central role
in transcriptional regulation and in the conferring of shared
traits.
[1010] Experimental Observations.
[1011] RT-PCR analysis indicated that G2789 is expressed at
moderate levels in roots, flowers, embryos, siliques, and
germinating seeds. At this time, G2789 function was analyzed using
35S::G2789 lines. Overexpression of G2789 in Arabidopsis resulted
in seedlings that were ABA insensitive and abiotic stress tolerant.
Overexpression of G2789 also produced alterations in leaf and
flower development, and caused severe reductions in fertility.
35S::G2789 primary transformants displayed a variety of leaf
abnormalities including; leaf curling, serrations, and changes in
leaf shape and area.
[1012] We have now analyzed 35S::G2789 seedlings in a C/N sensing
assay. Anthocyanin accumulation was significantly less than that
observed in control wild-type seedlings (Table 14). Thus, the gene
might have a role in nutrient limitation responses. However,
because the C/N sensing assay has high levels of sucrose, the
enhanced vigor of seedlings seen in this assay could be related to
the enhanced abiotic stress previously observed. It remains to be
determined whether the effects seen in this assay are related to
the apparent involvement of the gene in shade tolerance
[1013] Utilities.
[1014] G2789 could be used to alter a plant's response to water
deficit conditions and therefore, could be used to engineer plants
with enhanced tolerance to drought, salt stress, and freezing.
[1015] These assays also indicate that G2789 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1090 Clade of Transcription Factor Polypeptides
G1090 (SEQ ID NO: 439 and 440)
[1016] Experimental Observations.
[1017] In a C/N sensing assay anthocyanin accumulation was slightly
less in G1090 seedlings than that observed in control wild-type
seedlings in one of three lines tested (Table 14), indicating that
overexpression of G1090 in Arabidopsis gives a mild response in
overcoming the stress caused by this assay. G1090 and
closely-related clade member sequences each comprise a conserved
AP2 DNA binding domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[1018] Utilities.
[1019] These assays indicate that G1090 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1322 Clade of Transcription Factor Polypeptides
G1322 (SEQ ID NO: 441 and 442)
[1020] G1322 is a member of the (R1)R2R3 subfamily of myb
transcription factors. G1322 corresponds to Myb57, a gene
identified by Kranz et al. (1998) Plant J. 16: 263-276). The
authors used a reverse-Northern blot technique to study the
expression of this gene in a variety of tissues and under a variety
of environmental conditions. They were unable to detect the
expression of G1322 in any tissue or treatments tested (Kranz et al
(1998) supra). G1322 and closely-related clade member sequences
each comprise a conserved MYB_related DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[1021] Experimental Observations.
[1022] RT-PCR analysis indicated that G1322 is expressed primarily
in flower tissue and is not induced in response to any
environmental stress-related condition tested. At that time, the
function of G1322 was analyzed using transgenic plants in which the
gene was expressed under the control of the 35S promoter.
35S::G1322 transgenic plants had changes in overall plant size and
leaf development. 35S::G1322 plants were distinctly smaller than
controls and developed curled dark-green leaves. Following the
switch to flowering, the plants formed relatively thin
inflorescence stems and had a rather poor seed yield. In addition,
overexpression of G1322 resulted in plants with an altered
etiolation response as well as enhanced tolerance to germination
under chilling conditions.
[1023] We have now analyzed 35S::G1322 seedlings in a C/N sensing
assay. Anthocyanin accumulation was significantly less than that
observed in control wild-type seedlings in one of three lines
examined (Table 14), indicating that the gene may play a role in
the response to low nutrient conditions.
[1024] Utilities.
[1025] The potential utilities of G1322 include altering a plant's
chilling sensitivity and altering a plant's light response.
[1026] These assays indicate that G1322 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1587 Clade of Transcription Factor Polypeptides
G1587 (SEQ ID NO: 443 and 444)
[1027] G1587 (AT2G01500) was originally identified as a novel
homeobox gene within BAC F2I9 (GenBank accession AC005560). G1587
and closely-related clade member sequences each comprise a
conserved homeobox domain that is expected to function in a similar
manner in each of these related sequences, that is, by playing a
central role in transcriptional regulation and in the conferring of
shared traits.
[1028] Experimental Observations.
[1029] RT-PCR experiments revealed that the gene is predominantly
expressed in flowers. At that time, the function of G1587 was
assessed by analysis of transgenic Arabidopsis lines in which the
cDNA was constitutively expressed from the 35S CaMV promoter.
However, overexpression of G1587 produced deleterious effects on
growth and development. The most severely affected 35S::G1587
primary transformants died at very early stages of development.
Other seedlings, however, displayed rather contorted cotyledons,
long hypocotyls, and produced small narrow dark green leaves.
Following the switch to flowering, such plants formed rather thin
inflorescence stems that carried somewhat small numbers of flowers.
Floral organs were often contorted or poorly developed, and as a
result, seed yield was poor. The three lines used for physiological
analysis showed a relatively weak phenotype.
[1030] We have now analyzed 35S::G1587 seedlings in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings in all three of the lines
examined (Table 14), indicating that the gene may play a role in
the response to low nutrient conditions.
[1031] Utilities.
[1032] The RT-PCR data indicates that the G1587 promoter might be
of utility for driving expression of transgenes within flowers.
Additionally, if future studies confirm that G1587 has a function
in the regulation of flower development, the gene might be used to
manipulate those structures.
[1033] These assays indicate that G1587 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1666 Clade of Transcription Factor Polypeptides
G1666 (SEQ ID NO: 445 and 446)
[1034] The sequence of G1666 was obtained from the Arabidopsis
genome sequencing project, GenBank accession number AL049482, based
on its sequence similarity within the conserved domain to other
HLH/MYC related proteins in Arabidopsis. G1666 has been recently
identified as TT8 from a T-DNA mutagenized Arabidopsis collection
(Nesi et al. (2000) Plant Cell. 12:1863-1878). G1666 and
closely-related clade member sequences each comprise a conserved
HLH DNA-binding and dimerization domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[1035] It has been shown that G1666/TT8 is involved in the
regulation of flavonoid biosynthesis in the Arabidopsis seed coat.
The protein is required for normal expression of two flavonoid
biosynthetic genes, DFR and BAN. G1666 transcripts accumulate more
in developing siliques and in young seedlings compared to other
tissues.
[1036] Experimental Observations,
[1037] RT-PCR expression analysis indicated that G1666 was
predominantly expressed in reproductive tissue such as embryo,
siliques and flowers. At that time, the function of G1666 was
analyzed using a line homozygous for a T-DNA insertion in the gene
and transgenic plants in which the gene was expressed under the
control of the 35S promoter. Plants homozygous for a T-DNA
insertion within G1666 produced yellow seed. However, at all other
developmental stages, these plants appeared wild type. G1666
knockout mutant seedlings responded differently in an ethylene
insensitivity assay compared to the wild-type controls. Seedlings
germinated in the dark on ACC-containing media are more severely
stunted than the wild-type controls. 35S::G1666 plants were wild
type in all assays performed.
[1038] We have now analyzed the function of G1666 by characterizing
a line homozygous for a T-DNA insertion in G1666 in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings in knocked-out G1666 lines
(Table 14), indicating that the gene might have a role in the
response to nutrient limitation. However the lack of anthocyanin
production observed in this assay could be related to the block in
flavonoid biosynthesis caused by the T-DNA insertion within
G1666.
[1039] Utilities.
[1040] Because expression of G1666 is flower, embryo and silique
specific, its promoter could be useful for targeted gene expression
in these organs.
[1041] Co-overexpression of G1666 with G669, and G663 could be used
to increase the production of flavonoid compounds, including
anthocyanins and condensed tannins, in Arabidopsis. The potential
utilities of this gene include alterations in pigment production
for horticultural purposes, and possibly increasing stress
resistance in combination with another transcription factor.
Flavonoids have antimicrobial activity and could be used to
engineer pathogen resistance. Several flavonoid compounds have
health promoting effects such as the inhibition of tumor growth and
cancer, prevention of bone loss and the prevention of the oxidation
of lipids. Increasing levels of condensed tannins, whose
biosynthetic pathway is shared with anthocyanin biosynthesis, in
forage legumes is an important agronomic trait because they prevent
pasture bloat by collapsing protein foams within the rumen.
[1042] These assays indicate that G1666 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1700 Clade of Transcription Factor Polypeptides
G1700 (SEQ ID NO: 447 and 448)
[1043] G1700 (AT4G10150), a member of the RING C3H2C3 gene family,
was identified in the sequence of BAC T9A4 (GenBank accession
AF096373), released by the Arabidopsis Genome Initiative. G1700 and
closely-related clade member sequences each comprise a conserved
homeobox and RING finger domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[1044] Experimental Observations.
[1045] RT-PCR expression analysis indicated that G1700 was highly
expressed in embryos. No expression in any other tissue was
detected at that time. A line homozygous for a T-DNA insertion in
G1700 was used to determine the function of this gene. The
phenotype of G1700 knock-out plants was wild type in all assays
performed.
[1046] We have now analyzed the function of G1700 by characterizing
a line homozygous for a T-DNA insertion in G1700 in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings (Table 14), indicating that
the gene might have a role in the response to low nutrient
conditions.
[1047] Utilities.
[1048] The strong expression in embryos indicates that the promoter
of G1700 could be used to drive embryo specific expression.
[1049] These assays indicate that G1700 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
HAP5 Transcription Factor Polypeptides
G1818 (SEQ ID NO: 449 and 450)
[1050] G1818 (AT5G50490), a member of the Hap5-like subfamily of
CCAAT-box binding transcription factors, was identified in the
sequence of P1 clone MBA10 (GenBank accession AB025619). G1818 and
closely-related clade member sequences each comprise a conserved
CCAAT binding factor domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[1051] Experimental Observations.
[1052] RT-PCR expression analysis indicated that G1818 expression
was detected in embryos, flowers and siliques. Expression of G1818
could also be detected in leaf tissue following cold and auxin
treatments. At that time, the function of this gene was analyzed
using transgenic plants in which G1818 was expressed under the
control of the 35S promoter. With the exception of delayed
flowering and subtle changes in leaf morphology (flatter leaves),
the phenotype of these transgenic plants was wild-type in all
assays performed.
[1053] We have now analyzed 35S::G1818 seedlings in a C/N sensing
assay. Anthocyanin accumulation was substantially lower than that
observed in control wild-type seedlings (Table 14), indicating that
G1818 plays a role in the response to low nutrient conditions.
[1054] Utilities.
[1055] G1818 could be used to delay flowering in plants, which may
extend vegetative development and bring about larger yields.
[1056] These assays indicate that G1818 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1868 Clade of Transcription Factor Polypeptides
G1868 (SEQ ID NO: 451 and 452)
[1057] G1868 (AT4G37740) was found in the sequence of BAC clone
T28119 (GenBank accession AL035709) based on its amino acid
sequence similarity to the rice Growth-regulating-factor1 (GRF1).
G1868 and closely-related clade member sequences each comprise a
QRQ and WRC conserved domain that are expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[1058] Experimental Observations.
[1059] RT-PCR expression analysis revealed a constitutive
expression in all tissues except roots. At this time, the function
of G1868 was analyzed through its ectopic overexpression in plants.
No apparent changes were apparent when compared to control
plants.
[1060] We have now analyzed 35S::G1868 seedlings in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings in two lines (Table 14),
indicating that G1868 might have a minor role in the response to
low nutrient conditions.
[1061] Utilities.
[1062] These assays indicate that G1868 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G1888 Clade of Transcription Factor Polypeptides
G1888 (SEQ ID NO: 453 and 454)
[1063] G1888 (AT4G39070) was identified in the sequence of BAC
accession number AL035679, released by the Arabidopsis Genome
Initiative and is a member of the Z-CO-like transcription factor
family. G1888 and closely-related clade member sequences each
comprise at least one conserved B-Box-type zinc finger domain that
is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[1064] Experimental Observations.
[1065] G1888 was found to be constitutively expressed in all
tissues and environmental conditions tested based on RT-PCR
expression analysis. The function of this gene was analyzed using
transgenic plants in which G1888 was expressed under the control of
the 35S promoter at that time. Overexpression of G1888 produced
plants with dark green leaves, markedly slowed development (bolting
and senescing late), and reduced overall plant size. When grown on
MS agar plates, increased leaf anthocyanin levels and chlorosis
were noted.
[1066] We have now analyzed the function of G1888 by characterizing
35S:G1888 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was strikingly elevated compared to that observed in
control wild-type seedlings (Table 14). It should be noted that the
higher levels of anthocyanin seen in these assays could be related
to the generally darker coloration of 35S::G1888 lines that we
observed previously. It is interesting that the gene is most
closely related to G1482, which also causes elevation of
anthocyanin levels in seedlings when overexpressed. Thus, this pair
of genes might represent transcriptional regulators of the
phenylpropanoid pathway and as such might be used to impact a
variety of additional traits such as disease responses, lignin
composition, and nutritional quality.
[1067] Utilities.
[1068] These assays indicate that G1888 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G2117 Clade of Transcription Factor Polypeptides
G2117 (SEQ ID NO: 455 and 456)
[1069] G2117 (AT1G68880) was identified in the sequence of BAC T6L1
(GenBank accession AC011665) released by the Arabidopsis Genome
Initiative and is a member of the bZIP transcription factor family.
It has also been described as AtbZIP8 (GenBank accession number
AF400621). G2117 and closely-related clade member sequences each
comprise a conserved basic region leucin zipper (bZIP) domain that
is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[1070] Experimental Observations.
[1071] RT-PCR expression analysis indicated that G2117 was highly
expressed in roots compared to all other tissues tested. The
function of G2117 was analyzed using transgenic plants in which the
gene was expressed under the control of the 35S promoter. Plants
overexpressing G2117 had altered leaf morphology, coloration, and
smaller overall plant size and were generally small with short,
rounded, dark green leaves that became curled later in development.
These plants generated thin inflorescence stems developed a rather
bushy appearance, and had reduced fertility.
[1072] We have now analyzed the function of G2117 by characterizing
35S:G2117 overexpressing lines in a C/N sensing assay. Anthocyanin
accumulation was elevated compared to the levels observed in
control wild-type seedlings (Table 14). Thus, G2117 could have a
role in the response to nutrient limitation. However, given that
increased anthocyanin levels were seen on control plates, the
phenotype is possibly an aspect of darker coloration seen in these
lines, rather than an indicator of a C/N sensing response.
[1073] Utilities.
[1074] These assays indicate that G2117 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G2131 Clade of Transcription Factor Polypeptides
G2131 (SEQ ID NO: 457 and 458)
[1075] G2131 (AT1G79700) corresponds to gene F20B17.12 (GenBank
accession AAF68121) and is a member of the AP2 transcription factor
family. G2131 and closely-related clade member sequences each
comprise a conserved AP2 DNA binding domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits. G2131 and closely-related
clade member sequences each comprise a conserved domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[1076] Experimental Observations.
[1077] RT-PCR expression analysis indicated that G2131 is
ubiquitously expressed and was not significantly induced by any of
the environmental conditions tested. At that time, the function of
G2131 was studied using transgenic plants in which the gene was
expressed under the control of the 35S promoter. 35S::G2131 plants
did not show consistent alterations in morphology and development,
and were essentially wild type in the physiological analyses that
were performed.
[1078] G2131 overexpressing plants showed elevated levels of
campesterol in leaves.
[1079] We have now analyzed 35S::G2131 seedlings in a C/N sensing
assay. Anthocyanin accumulation was significantly less than that
observed in control wild-type seedlings (Table 14). Thus, this gene
is indicated as having a role in responses to nutrient
limitation.
[1080] Utilities.
[1081] Phytosterols are an important source of precursors for the
manufacture of human steroid hormones by semisynthesis. Sitosterols
and stigmasterols, not campesterol, are the preferred sources from
seed crops. However, it is conceivable that proper regulation of
G2131 expression or activity could lead to elevated levels of the
important human steroid precursors. Phytosterols and their
hydrogenated derivatives phytostanols also have proven
cholesterol-lowering properties. However, it is unclear what the
relative efficacies of sitosterol and campesterol are for lowering
blood cholesterol levels. If G2131 could be used to increase total
phytosterol levels in leaves, it would be very useful for both
types of applications.
[1082] These assays indicate that G2131 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
The G2520 Clade of Transcription Factor Polypeptides
G2520 (SEQ ID NO: 459 and 460)
[1083] The sequence of G2520 (AT1G59640) was originally obtained
from Arabidopsis genomic sequencing project, GenBank accession
number AC009317, based on its sequence similarity within the
conserved domain to other bHLH related proteins. G2520 and
closely-related clade member sequences each comprise a conserved
HLH DNA-binding and dimerization domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[1084] Experimental Observations.
[1085] RT-PCR expression analysis indicated that G2520 was
expressed ubiquitously. At that time, the function of G2520 was
analyzed using transgenic plants in which G2520 was expressed under
the control of the 35S promoter. At early stages, 35S::G2520
transformants displayed abnormal curled cotyledons, long
hypocotyls, and rather short roots. During the vegetative phase,
these plants were formed somewhat small flat leaves. Following the
switch to reproductive growth, 35S::G2520 inflorescences were
typically very spindly, slightly pale colored, and stems often
split open at late stages. Flowers were frequently small with
narrow organs and showed poor pollen production. Because of these
defects, seed yield from 35S::G2520 plants was low compared to
wild-type controls.
[1086] We have now analyzed 35S::G2520 seedlings in a C/N sensing
assay. Anthocyanin accumulation was significantly less than that
observed in control wild-type seedlings (Table 14), indicating that
this gene might have a role in the response to nutrient
limitation.
[1087] Interestingly, we previously observed that overexpression
lines showed light response phenotypes such as long hypocotyls, and
a pale coloration (reduced levels of pigmentation). However, it
remains to be determined whether the response to low nutrient
conditions is related to these effects. However, it is interesting
compare the strikingly similar effects on pigment production
observed between 35S::G2520 lines and overexpression lines from the
G682 subclade reports. Given that genes from the MYB family are in
some cases known to have partners in the HLH/MYC family, it is
possible that the G2520 might act in the same pathway.
[1088] Utilities.
[1089] In addition to the observed shade avoidance phenotype, these
assays indicate that G2520 and its equivalogs are potential
regulators of a plant's response to low nutrient conditions. For
further analysis, see the discussion above: "Potential Applications
of Polynucleotides and Polypeptides that Regulate C/N sensing".
The G2522 Clade of Transcription Factor Polypeptides
G2522 (SEQ ID NO: 461 and 462)
[1090] The sequence of G2522 (AT3G61310) was initially obtained
from the Arabidopsis genomic sequencing project (GenBank accession
AL137898) based on its sequence similarity within the conserved
domain to other AT-hook related proteins. G2522 and closely-related
clade member sequences each comprise a conserved domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits. G2522 and
closely-related clade member sequences each comprise a conserved
At-hook domain and a second conserved domain (amino acids 143-291)
or the DUF296 domain (amino acids 164-284) that are expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits
[1091] Experimental Observations.
[1092] RT-PCR expression analysis indicated that G2522 is expressed
at moderate levels in flowers, embryos, and siliques, and is found
at significantly lower levels throughout the rest of the plant. The
gene was not significantly induced by any environmental condition
tested. The function of G2522 was also analyzed using transgenic
plants in which G2522 was expressed under the control of the 35S
promoter. Overexpression of G2522 did not produce any consistent
phenotypic alteration in any assay performed when compared to
wild-type control plants.
[1093] We have now analyzed 35S::G2522 seedlings in a C/N sensing
assay. Anthocyanin accumulation was slightly less than that
observed in control wild-type seedlings (Table 14), indicating that
the gene might have a role in the response to low nutrient
conditions.
[1094] Utilities.
[1095] These assays indicate that G2522 and its equivalogs are
potential regulators of a plant's response to low nutrient
conditions. For further analysis, see the discussion above:
"Potential Applications of Polynucleotides and Polypeptides that
Regulate C/N sensing".
[1096] Table 14 lists the results obtained with transgenic
seedlings germinated on two different media for the purpose of
differentiating plants with altered C/N sensing. The first column
lists the Gene Identification Number (GID), the second column
identifies the gene family of the corresponding sequence, the third
column identifies whether the gene was overexpressed or knocked
out, and the fourth and fifth columns list the results obtained
with high sucrose media lacking a nitrogen source and high sucrose
media with glutamine as a nitrogen source, respectively. Generally,
increased tolerance was measured as lower anthocyanin accumulation
than controls, and increased sensitivity as greater anthocyanin
accumulation than controls. The plants' responses as they appear in
the fourth and fifth columns were given one of four scores:
[1097] ++ markedly enhanced tolerance;
[1098] + mild/moderately enhanced tolerance;
[1099] wt comparable tolerance to wild-type controls, and
[1100] - mild to moderately increased sensitivity.
TABLE-US-00015 TABLE 14 Sequences identified as modifying the
response to nutrient limitation in C/N sensing assays Response of
Response of transgenic transgenic plants on high plants on high
sucrose without sucrose plus GID Gene family OE/KO a nitrogen
source glutamine G24 AP2 OE + + G154 MADS OE + + CPC MYB-related OE
++ ++ G226 MYB-related OE ++ ++ G384 HB OE - - G486 CAAT OE + +
G545 Z-C2H2 OE - - G682 MYB-related OE ++ + G760 NAC OE - - G773
NAC OE - - G937 GARP OE + + G971 AP2 OE - - G988 SCR OE - - G989
SCR OE + + G1069 AT-hook OE + + G1090 AP2 OE + + G1322 MYB-(R1)R2R3
OE ++ ++ G1587 HB OE + + G1666 HLH/MYC KO + + G1700 RING/C3H2C3 KO
+ + G1816 MYB-related OE ++ + G1818 CAAT OE + + G1868 GRF-like OE +
+ G1888 Z-CO-like OE - - G2117 bZIP OE - - G2131 AP2 OE ++ ++ G2520
HLH/MYC OE ++ ++ G2522 AT-hook OE + + G2718 MYB-related OE ++ +
G2789 AT-hook OE ++ ++ G8 AP2 OE - - G27 AP2 OE wt - G156 MADS OE +
+ G161 MADS OE - - G168 MADS OE - wt G183 WRKY OE + + G189 WRKY OE
+ + G200 MYB-(R1)R2R3 KO - - G234 MYB-(R1)R2R3 OE + + G237
MYB-(R1)R2R3 OE + + G275 AKR OE + + G326 Z-CO-like OE - - G347
Z-LSD-like OE wt + G427 HB OE + + G505 NAC OE - - G590 HLH/MYC OE +
+ G602 DBP OE + + G618 TEO OE + + G635 TH OE + + G643 TH OE + +
G653 Z-LIM OE + + G657 MYB-(R1)R2R3 OE wt + G837 AKR OE wt + G866
WRKY OE + + G872 AP2 OE + + G904 RING/C3H2C3 OE + + G912 AP2 OE + +
G932 MYB-(R1)R2R3 OE wt + G958 NAC OE + wt G964 HB KO ++ + G975 AP2
OE + + G979 AP2 OE wt + G1049 bZIP OE + + G1246 MYB-(R1)R2R3 OE + +
G1255 Z-CO-like OE + + G1266 AP2 OE wt + G1331 MYB-(R1)R2R3 OE + +
G1332 MYB-(R1)R2R3 OE + + G1494 HLH/MYC OE + + G1535 HB KO wt +
G1649 HLH/MYC OE + + G1750 AP2 OE + + G1773 RING/C3HC4 KO + + G1835
GATA/Zn OE wt - G1930 AP2 OE + + G2053 NAC OE wt + G2057 TEO OE + +
G2133 AP2 OE wt + G2144 HLH/MYC OE + + G2145 HLH/MYC OE + + G2295
MADS OE + + G2512 AP2 OE + + G2531 NAC OE + wt G2535 NAC OE - -
G2590 MADS OE + + G2719 MYB-(R1)R2R3 OE + + G1792 AP2 OE + +
Abbreviations and symbols: wt wild-type response + increased growth
and/or vigor relative to wild-type - decreased growth and/or vigor
relative to wild-type
Example X: Results of Shade Tolerance Assays
[1101] This example provides experimental evidence for increased
shade tolerance controlled by transcription factor polypeptides and
polypeptides of the invention.
[1102] The twelve shade avoidance-inducing sequences that were most
extensively scrutinized spanned a range of diverse gene families:
TH (G634), bZIP (G1048), RING/C3H2C3 (G1100), NAC (G1412, G2505),
AP2 (G1796), Z-C2H2 (G1995), HS (G2467), HB (G2550), SRS (G2640),
WRKY (G2686), and AT-hook (G2789). Experimental data are provided
for each of these sequences in Example VIII, but a number of the
genes warrant special mention here.
[1103] G634 is of particular interest since we have determined that
35S::G634 Arabidopsis lines also show enhanced drought tolerance in
addition to shade tolerance. This gene could therefore confer yield
savings via multiple different traits.
[1104] The same ability to confer enhanced performance and yield by
improving multiple traits is true for G2789. Overexpressors of
G2789 were shown to be insensitive to ABA, had altered
carbon:nitrogen balance sensing (and thus overexpressors of this
gene may thrive better than wild type under low nutrient
conditions), were osmotic stress tolerant, and recovered better
from drought than wild-type plants in a soil-based drought
assay.
[1105] A G1412 homozygous T-DNA insertion mutant line for this gene
showed shade tolerance. Thus, G1412 might be a target for obtaining
shade tolerance via a non-transgenic strategy by screening for
mutant lines of crops that carry a lesion within the ortholog(s) of
G1412.
[1106] A number of the top lead genes, particularly G2550 and
G2640, produced Arabidopsis plants with a compact shoot morphology
when overexpressed, which may represent a constitutive shade
avoidance phenotype. Such features are similar to those seen in the
high-yielding dwarf varieties of cereals that facilitated the
so-called "green revolution." The effects of G2550 and G2640
overexpression on yield will be examined in target crop
species.
The G634 Clade of Transcription Factor Polypeptides
G634 (SEQ ID NO: 231 and 232)
[1107] G634 (AT1G33240) was initially identified as two public
partial cDNAs sequences (GTL1 and GTL2) which are splice variants
of the same gene (Smalle et al. (1998) Proc. Natl. Acad. Sci. USA
95: 3318-3322). The published expression pattern shows that G634 is
highly expressed in siliques and not expressed in leaves, stems,
flowers or roots. G634 and closely-related clade member sequences
each comprise at least one conserved TH domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[1108] Experimental Observations.
[1109] Three constructs were initially made for G634: P324 (SEQ ID
NO: 1015), P1374 (SEQ ID NO: 1013) and P1717 (SEQ ID NO: 1017).
P324 was found to encode a shortened version of the G634 protein
(SEQ ID NO: 1016). P1374 and P1717 represent longer splice variants
of G634 (SEQ ID NOs: 1014 and 1018, respectively). Overexpression
lines for P1717 were never analyzed. However lines for P324 showed
some variable effects on size, but otherwise appeared normal.
Plants overexpressing G634 had a dramatic increase the density of
trichomes. The trichomes were also larger in size than those of
wild-type plants. The increase in trichome density was most
noticeable on later arising rosette leaves, cauline leaves,
inflorescence stems and sepals with the stem trichomes being more
highly branched than controls. Approximately half of the primary
transformants and two of three T2 lines showed the phenotype.
[1110] G634 overexpressing Arabidopsis lines did not exhibit a
shade avoidance phenotype when grown under light deficient in the
red region of the visible spectrum; in experiments comparing
35S::G634 plants with wild type controls, individual seedlings were
examined after being grown under light deficient in red wavelengths
(b/FR). The G634 overexpressors did not exhibit a shade avoidance
phenotype, as indicated by their short hypocotyls produced under
these conditions.
[1111] We recently tested lines for P324 and P1374 in a soil
drought assay and found that they showed an enhanced performance
versus wild type; 6634 overexpressors recovered from the effects of
a drought treatment significantly better than wild-type control
plants. Additionally, our recent array experiments on plants
undergoing a soil-drought experiment, indicated that G634 shows a
small but significant up-regulation specifically in the recovery
phase, following re-watering at the end of the drought (see patent
application Ser. No. 10/714,887).
[1112] Utilities.
[1113] We have now analyzed 35S::G634 lines (containing P1374, SEQ
ID NO: 1013, which encodes SEQ ID NO: 1014) under white light
versus light deficient in red wavelengths. All three lines tested
did not exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation.
The G1048 Clade of Transcription Factor Polypeptides
G1048 (SEQ ID NO: 807 and 808)
[1114] G1048 (AT1G42990) was initially identified as public partial
EST T88194 and in BAC F13A11 (GenBank accession AC068324) released
by the Arabidopsis Genome Initiative. G1048 and closely-related
Glade member sequences each comprise a conserved basic region
leucin zipper (bZIP) domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[1115] Experimental Observations.
[1116] During the genomics program, RT-PCR expression analysis
indicated that G1048 was constitutively expressed and not induced
by any condition tested. At that time, the function of G1048 was
investigated by constitutively expressing G1048 using the 35S
promoter. Plants overexpressing G1048 were not significantly
different to controls in any assay performed.
[1117] G1048 overexpressing lines did not exhibit a shade avoidance
phenotype when grown under light deficient in the red region of the
visible spectrum; individual seedlings grown on light deficient in
red wavelengths (b/FR) were compared with wild-type control
seedlings. This effect was seen in two repeat experiments on a
batch of mixed seed from three independent lines.
[1118] Utilities.
[1119] We have now analyzed 35S::G1048 lines grown under white
light versus light deficient in red light. A shade tolerance
phenotype was observed, indicating that G1048 might be involved in
the transcriptional regulation of response to shade or light
quality. As yet, though, the phenotype observed in a mixed batch of
35S::G1048 lines has not been confirmed by testing of individual
lines. However, this gene was given an "A" ranking because the
phenotype seen in the screen on mixed lines was moderately strong,
and because G1048 is potentially related to HY5 (Oyama et al.
(1997) Genes Dev. 11:2983-2995), a gene that is well established to
be involved in light regulated development.
The G1100 Clade of Transcription Factor Polypeptides
G1100 (SEQ ID NO: 809 and 810)
[1120] G1100 was identified in the sequence of BACs T29F13, F1913
and T4C15 based on its sequence similarity within the conserved
domain to other RING C3H2C3 related proteins in Arabidopsis. G1100
and closely-related clade member sequences each comprise a
conserved RING finger domain that is expected to function in a
similar manner in each of these related sequences, that is, by
playing a central role in transcriptional regulation and in the
conferring of shared traits.
[1121] Experimental Observations.
[1122] In our earlier genomics program, the function of G1100 was
analyzed by disrupting the gene with a T-DNA insertion. Homozygotes
for this insertion appeared wild type in all assays performed. For
the present experiments, the function of G1100 was studied using
transgenic plants in which the gene was expressed under the control
of the 35S promoter. Overexpression of G1100 resulted in plants
that were small, dark green, and slow developing. These effects
were most prominent at later stages. Flowers were also small, had
defects in organ formation and pollen production, and set few
seeds. RT-PCR analysis indicated that G1100 is strongly and
specifically induced by drought and salicylic acid, and is not
detectable under normal conditions.
[1123] G1100 overexpressing lines did not exhibit a shade avoidance
phenotype when grown under light deficient in red region of the
visible spectrum; individual seedlings grown on light deficient in
red wavelengths (b/FR) were compared with wild-type control
seedlings. When the assay was repeated on individual lines, all
three lines analyzed showed the phenotype. 35S::G1100 seedlings had
short hypocotyls compared with wild-type seedlings.
[1124] Utilities.
[1125] We have now analyzed 35S::G1100 lines grown under white
light or deficient in red light. All three lines did not exhibit a
shade avoidance phenotype under conditions where wild-type
seedlings had enhanced hypocotyl elongation.
The G1412 Clade of Transcription Factor Polypeptides
G1412 (SEQ ID NO: 657 and 658)
[1126] G1412 is a member of the NAC family of transcription
factors. G1412 corresponds to gene At4g27410 and to sequence 1543
from Harper (2002) Patent Application WO 0216655-A. In this
application, G1412 was reported to be cold, osmotic and salt
responsive in their microarray analysis (WO 0216655-A). G1412 and
closely-related clade member sequences each comprise a conserved
NAC domain that is expected to function in a similar manner in each
of these related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[1127] Experimental Results.
[1128] In our original genomics screens, G1412 appeared to be
constitutively expressed in all tissues tested using RT-PCR
analysis. Induction of G1412 in leaf tissue was observed in
response to ABA, heat, drought, and mannitol. This result was
confirmed by microarray experiments, which showed that G1412 is
induced by a variety of drought related treatments.
[1129] In our earlier studies, a T-DNA insertion mutant for G1412
was not shown to be morphologically different from wild type.
35S::G1412 transgenic plants showed normal morphology but were
insensitive to ABA, and were significantly more tolerant to osmotic
stress in a germination assay on media containing high
concentrations of sucrose.
[1130] In our most recent experiments, T-DNA insertion mutants for
G1412 did not exhibit a shade avoidance phenotype when grown under
light deficient in red region of the visible spectrum. Individual
seedlings grown on light deficient in red wavelengths (b/FR) were
compared with wild-type control seedlings; The G1412 knock-out
seedlings had short hypocotyls compared with wild-type
seedlings.
[1131] Utilities.
[1132] We have now analyzed KO.G1412 seedlings grown under white
light versus light deficient in the red wavelengths. KO.G1412
seedlings did not exhibit a shade avoidance phenotype under
conditions where wild-type seedlings had enhanced hypocotyl
elongation. Thus, G1412 might be required to mediate the shade
avoidance response. However, 35S::G1412 lines were not observed to
show alterations in light-regulated development, suggesting that
this gene is not sufficient to trigger a shade response.
The G1796 Clade of Transcription Factor Polypeptides
G1796 (SEQ ID NO: 811 and 812)
[1133] G1796 (At1g12980) is found in the sequence of GenBank
accession number AC007357. G1796 was identified by Banno et al.
(Banno et al. (2001) Plant Cell 13: 2609-2618) as ESR1 (ENHANCER OF
SHOOT REGENERATION) in a screen for Arabidopsis cDNAs that could
confer cytokinin-independent shoot formation from root cultures
when overexpressed. G1796 was found to be included in Patent
Application W00200903. G1796 and closely-related clade member
sequences each comprise a conserved AP2 DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[1134] Experimental Results.
[1135] In our earlier genomics program, overexpression of G1796 was
shown to cause growth defects: seedlings were generally small and
formed dark curled leaves. Portions of the flower and overall
structure of the inflorescence were also affected. Flowers had
poorly developed outer whorl organs and formed thickened club-like
carpels. RT-PCR expression analysis indicated that G1796 was
expressed at low levels in root, flower and rosette, but not in
stems, siliques, embryos or germinating seeds.
[1136] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G1796 overexpressing lines
did not exhibit a shade avoidance phenotype.
[1137] Utilities.
[1138] We have now analyzed 35S::G1796 seedlings grown under white
light or white light deficient in wavelengths corresponding to the
red region of the visible spectrum. 35S::G1796 seedlings did not
exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation. This gene
was given an "A" ranking because the phenotype seen in the screen
on mixed lines was moderately strong.
[1139] 35S::G1796 seedlings were also dark green in color compared
to wild type, confirming a result seen earlier in the earlier
genomics program.
The G1995 Clade of Transcription Factor Polypeptides
G1995 (SEQ ID NO: 813 and 814)
[1140] G1995 (At3g58070) was identified in the sequence of BAC
T10K17 (GenBank accession number AL132977) based on its sequence
similarity within the conserved domain to other Z-C2H2 related
proteins in Arabidopsis. G1995 and closely-related clade member
sequences each comprise a conserved C2H2 DNA-binding zinc finger
domain that is expected to function in a similar manner in each of
these related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[1141] Experimental Results.
[1142] The function of G1995 was studied using transgenic plants in
which the gene was expressed under the control of the 35S promoter.
Overexpression of G1995 resulted in plants that were rather small,
slow growing, and that had flowers with increased trichome density
on sepals and ectopic trichomes on carpels. The flowers also had
rather poor pollen production and many of the lines yielded only
relatively small quantities of seed. Additionally a single extreme
line displayed aerial rosette like structures and had floral organs
that were converted towards a bract-like identity. Interestingly,
in the strongest lines, the plants failed to undergo a clear
transition to reproductive growth and formed leafy floral organs
with vegetative characteristics. Thus, G1995 might regulate this
developmental transition.
[1143] In physiological analyses G1995 overexpressors showed size
segregation and a slight increase in sensitivity to nutrient
limitation.
[1144] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G1995 overexpressing lines
did not exhibit a shade avoidance phenotype.
[1145] Utilities.
[1146] We have now analyzed 35S::G1995 seedlings grown under white
light or white light deficient in wavelengths corresponding to the
red region of the visible spectrum. 35S::G1995 seedlings did not
exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation. Two out of
three lines did not exhibit a shade avoidance phenotype.
[1147] It should be noted that G1995 is closely related to five
other Z-C2H2 genes we have previously analyzed: G370, G2826, G361,
G362, and G2838, which produced broadly similar phenotypes when
overexpressed, such as ectopic trichomes on flowers, aerial
rosettes, and various other morphological defects. Importantly,
these genes all produced a general failure in the vegetative to
reproductive transition and showed floral organs that were
leaf-like. This effect, and the absence of hypocotyl elongation
seen in 35S::G1995 lines in this assay, could indicate that this
group of TFs is involved in mediating a range of phytochrome
regulated responses. However, we did not observe any effect on
hypocotyl elongation when these other Z-C2H2 overexpressing plants
were examined in our shade avoidance screen. Nevertheless, it
should be noted that the lines were generally of very poor
fertility and strongly affected lines set insufficient seed for
inclusion in the shade tolerance assay (i.e. only lines with a
relatively weak morphological phenotype could be tested).
The G2467 Clade of Transcription Factor Polypeptides
G2467 (SEQ ID NO: 815 and 816)
[1148] G2467 is a member of the class-A heat shock transcription
factor family characterized by an extended HR-A/B oligomerization
domain. G2467 is found in the sequence of the P1 clone MAA21
(GenBank accession AL163818) released by the Arabidopsis Genome
Initiative. G2467 and closely-related clade member sequences each
comprise a conserved HSF-type DNA-binding domain (or HS domain)
that is expected to function in a similar manner in each of these
related sequences, that is, by playing a central role in
transcriptional regulation and in the conferring of shared
traits.
[1149] Experimental Observations.
[1150] In studies performed during the earlier genomics program,
35S::G2467 transformants were generally smaller than wild type, and
formed rather thin inflorescence stems that carried flowers that
sometimes displayed abnormal, poorly developed organs.
Additionally, rosette leaf senescence appeared to occur
prematurely.
[1151] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G2467 overexpressing lines
did not exhibit a shade avoidance phenotype. When individual lines
were retested, one line did not exhibit a shade avoidance phenotype
whereas two lines were wild type in their response.
[1152] Utilities.
[1153] We have now analyzed 35S::G2467 seedlings grown under white
light or white light deficient in wavelengths corresponding to the
red region of the visible spectrum. 35S::G2467 seedlings did not
exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation.
The G2505 Clade of Transcription Factor Polypeptides
G2505 (SEQ ID NO: 817 and 818)
[1154] G2505 (AT4G10350) is a novel member of the NAC family of
transcription factors. G2505 and closely-related clade member
sequences each comprise a conserved NAC domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[1155] Experimental Observations.
[1156] During our earlier genomics program, RT-PCR expression
analysis indicated that G2505 was expressed at low or
non-detectable levels in most tissue types. However, higher levels
of transcript were found in roots compared to other tissues. No
induction of G2505 expression in leaf tissue was detected in
response to environmental stress related conditions. At that time,
it was extremely hard to obtain 35S::G2505 transformants. A few
lines were obtained and these were distinctly small and dark in
coloration. Only two of these lines produced sufficient seed for
physiology assays to be performed. However, both of those lines
displayed enhanced performance in a severe drought assay.
[1157] G2505 overexpressing lines (from a mixed seed lot comprised
of two independent transgenic lines) and wild-type controls were
grown on light deficient in red wavelengths (b/FR) and compared.
Under these conditions, the G2505 overexpressing lines did not
exhibit a shade avoidance phenotype.
[1158] Utilities.
[1159] We have now analyzed 35S::G2505 lines grown under white
light versus light deficient in red light. 35S::G2505 seedlings
exhibited a shade tolerant phenotype, suggesting that this gene
might be involved in light regulated development. However, it
should be noted that as yet, the phenotype observed in a mixed
batch of 35S::G2505 lines has not been confirmed by testing of
individual lines. Nevertheless, this gene was given an "A" ranking
because the phenotype seen in the screen on mixed lines was
moderately strong.
The G2550 Clade of Transcription Factor Polypeptides
G2550 (SEQ ID NO: 819 and 820)
[1160] We initially identified G2550 within sequence released by
the Arabidopsis Genome Initiative (GenBank accession AC023754) as a
gene encoding a novel homeodomain protein of the BEL1 class. G2550
and closely-related clade member sequences each comprise a
conserved PDX domain and a homeodomain domain that is expected to
function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[1161] Experimental Observations.
[1162] During our genomics program, 35S::G2550 transgenic plants
exhibited a wild-type response to physiological assays, but
displayed a number of morphological phenotypes. Initially,
35S::G2550 seedlings appeared wild type at early stages. However,
at the mid rosette stage, 35S::G2550 lines were dark in coloration,
displayed alterations in leaf shape, and formed shorter more
compact inflorescences than controls. Following the switch to
flowering, 35S::G2550 transformants formed short, compact, bushy
inflorescences, which had reduced internode elongation, and flowers
bunched together at the tips. Fertility also appeared reduced,
silique set was rather poor, and senescence was somewhat delayed
compared to wild type.
[1163] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G2550 overexpressing lines
did not exhibit a shade avoidance phenotype.
[1164] Utilities.
[1165] We have now analyzed 35S::G2550 seedlings grown under white
light or white light deficient in wavelengths corresponding to the
red region of the visible spectrum. 35S::G2550 seedlings did not
exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation. However, it
be should noted that the 35S::G2550 lines had short internodes and
were short and compact at adult stages. Thus, the shade tolerance
phenotype (reduced hypocotyl elongation) observed in the current
study could be part of the general dwarf phenotype seen in these
lines.
The G2640 Clade of Transcription Factor Polypeptides
G2640 (SEQ ID NO: 821 and 822)
[1166] G2640, a member of the SRS (SHORT INTERNODES, SHI)
transcription factor family, corresponds to AT3G51060 as annotated
by the Arabidopsis Genome Initiative. The founding member of the
SRS family has been implicated in the suppression of GA induced
cell elongation. G2640 and closely-related clade member sequences
each comprise a conserved DUF702 domain comprising at least one
zinc finger domain that is expected to function in a similar manner
in each of these related sequences, that is, by playing a central
role in transcriptional regulation and in the conferring of shared
traits.
[1167] Experimental Observations.
[1168] The function of G2640 was analyzed in our genomics program
using transgenic plants in which a cDNA clone of the gene was
expressed under the control of the 35S promoter. While 35S::G2640
lines displayed a wild-type response in all of the physiological
assays, several developmental alterations were observed during
morphological analysis. 35S::G2640 transformants were smaller than
wild type controls and produced leaves with short petioles.
Inflorescences from these plants were compact and had very short
internodes. Flowers displayed a variety of non-specific
abnormalities with organs often being poorly developed. As a result
of such defects, the seed yield from most of the lines was very
low.
[1169] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G2640 overexpressing lines
did not exhibit a shade avoidance phenotype. When individual lines
were tested, two lines did not exhibit a shade avoidance phenotype,
and were observed to have long narrow leaves.
[1170] Utilities.
[1171] We have now analyzed 35S::G2640 seedlings grown under white
light or white light deficient in wavelengths corresponding to the
red region of the visible spectrum. 35S::G2640 seedlings did not
exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation. This
phenotype was only seen in two lines (an individual line repeat
could not be performed using the third line because seed was not
available).
[1172] It should be should noted that during our initial genomics
program, we observed that 35S::G2640 lines were rather short and
compact at adult stages. Thus, the shade tolerance phenotype
(reduced hypocotyl elongation) observed in the current study could
be part of the general short internode phenotype seen in these
lines.
The G2686 Clade of Transcription Factor Polypeptides
G2686 (SEQ ID NO: 823 and 824)
[1173] G2686 corresponds to gene At1g66600, and it has also been
described as WRKY63. G2686 and closely-related clade member
sequences each comprise a conserved WRKY DNA-binding domain that is
expected to function in a similar manner in each of these related
sequences, that is, by playing a central role in transcriptional
regulation and in the conferring of shared traits.
[1174] Experimental Observations.
[1175] We had previously studied the function of the gene was using
transgenic plants in which the gene was expressed under the control
of the 35S promoter. G2686 overexpressing lines behaved similarly
to the wild-type controls in all physiological assays performed.
However, in morphological examinations, 35S::G2686 plants were
observed to be generally smaller than wild-type controls. Some
lines also had short rounded leaves.
[1176] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G2686 overexpressing lines
did not exhibit a shade avoidance phenotype. When individual lines
were retested, two of three lines did not exhibit a shade avoidance
phenotype.
[1177] Utilities.
[1178] We have now analyzed 35S::G2686 seedlings grown under white
light or white light deficient in wavelengths corresponding to the
red region of the visible spectrum. 35S::G2686 seedlings did not
exhibit a shade avoidance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation. However, the
shade tolerance phenotype (reduced hypocotyl elongation) observed
in the current study could be part of the general small size
phenotype that was earlier seen in these lines.
The G1073 Clade of Transcription Factor Polypeptides
G2789 (SEQ ID NO: 247 and 248)
[1179] The sequence of G2789 (AT3G60870) was obtained from the
Arabidopsis genome sequencing project (GenBank accession AL162295)
based on its sequence similarity to other AT-hook related proteins.
G2789 and closely-related clade member sequences each comprise a
conserved At-hook domain and a second conserved domain (amino acids
68-208) or the DUF296 domain (amino acids 86-201) that are expected
to function in a similar manner in each of these related sequences,
that is, by playing a central role in transcriptional regulation
and in the conferring of shared traits.
[1180] Experimental Observations.
[1181] During earlier studies, RT-PCR analysis indicated that G2789
was expressed at moderate levels in roots, flowers, embryos,
siliques, and germinating seeds. It was not detectable in rosette
leaves or shoots. No significant induction of G2789 was observed in
rosette leaves by any condition tested. At this time, the function
of this gene was analyzed using transgenic plants in which G2789
was expressed under the control of the 35S promoter. Overexpression
of G2789 in Arabidopsis resulted in seedlings that were ABA
insensitive, had significantly more osmotic stress tolerance than
wild-type plants, had altered carbon:nitrogen balance sensing, were
osmotic stress tolerant, and recovered better from drought that
wild-type plants in soil-based drought assays.
[1182] Overexpression of G2789 also produced alterations in leaf
and flower development, and caused severe reductions in
fertility.
[1183] Seedlings of overexpressing lines and wild-type controls
were grown on light deficient in red wavelengths (b/FR) and
compared. Under these conditions, the G2789 overexpressing lines
did not exhibit a shade avoidance phenotype. When the assay was
repeated on individual lines, two of three lines analyzed showed a
shade tolerant phenotype and had short hypocotyls compared with
wild-type seedlings. One line was wild type.
[1184] Utilities.
[1185] We have now analyzed 35S::G2789 lines grown under white
light versus light deficient in red light. Two of three lines
tested exhibited a shade tolerance phenotype under conditions where
wild-type seedlings had enhanced hypocotyl elongation. Thus, G2789
might be involved in the modulation of light regulated development.
It remains to be determined whether this function is related to the
apparent involvement of the gene in conferring abiotic stress
tolerance and tolerance to low nutrient availability.
Summary of Results for Above GIDs and Others Tested
TABLE-US-00016 [1186] TABLE 15 GIDs identified as conferring shade
tolerance under low R:FR conditions PID of Shade Lines OEX
tolerance Growth under Priority GID Gene family OE/KO tested
construct phenotype.sup.1 white light.sup.2 Ranking.sup.3 G634 TH
OE 5, 6, 8 P1374 + - A G1048 bZIP OE 23, 24, 28 P1257 + - A G1100
RING/C3H2C3 OE 27, 31, 38 P1353 ++ wt A G1412 NAC KO KO NA + wt A
G1796 AP2 OE 5, 28, 32 P2053 + - A G1995 Z-C2H2 OE 22, 37, 38 P2360
++ wt A G2467 HS OE 7, 9, 10 P2744 + wt A G2505 NAC OE 86, 81 P2776
+ wt A G2550 HB OE 1, 3, 4 P16180 ++ wt A G2640 SRS OE 26, 30, 31
P2675 ++ + A G2686 WRKY OE 5, 6, 10 P2095 + wt A G2789 AT-hook OE
5, 9, 19 P2058 + - A G24 AP2 OE 2, 8, 11 P969 + wt B G38 AP2 OE 3,
6, 10 P179 + wt B G44 AP2 OE 4, 5, 6 P182 + wt B G230 MYB-(R1)R2R3
OE 61, 63, 67 P810 + - B G234 MYB-(R1)R2R3 OE 1, 2, 3 P201 + wt B
G261 HS OE 1, 2, 3 P206 + + B G271 AKR OE 3, 4, 5 P209 + - B G303
HLH/MYC OE 3, 8, 18 P1410 + wt B G359 Z-C2H2 OE 4, 5, 7 P2379 + wt
B G377 RING/C3H2C3 OE 7, 9, 20 P1354 + wt B G388 HB KO KO NA + - B
G435 HB OE 4, 8, 16 P30 + - B G442 AP2 OE 6, 7, 8 P909 + wt B G468
IAA OE 1, 22, 24 P2466 + wt B G571 bZIP OE 22, 26, 27 P1557 + wt B
G652 Z-CLDSH KO KO NA + - B G664 MYB-(R1)R2R3 OE 2, 3, 7 P98 + - B
G772 NAC OE 4, 15, 19 P868 + wt B G798 Z-Dof OE 1 P132 + wt B G818
HS OE 12, 16, 19 P1786 + wt B G971 AP2 OE 1, 14, 18 P1247 + wt B
G974 AP2 OE 3, 4, 8 P1510 + wt B G988 SCR OE 21, 23, 25 P1475 + - B
G1062 HLH/MYC KO KO NA + wt B G1069 AT-hook OE 41, 42, 64 P1178 +
wt B G1129 HLH/MYC OE 2, 10, 16 P1298 + - B G1137 HLH/MYC OE 1, 14,
15 P938 + wt B G1425 NAC OE 22, 27, 28 P1361 + - B G1517 RING/C3HC4
OE 1, 2, 3 P1096 + - B G1655 HLH/MYC OE 10, 14, 19 P1008 + - B
G1743 RING/C3H2C3 OE 1, 5, 7 P15028 + - B G1789 MYB-related OE 5,
11, 19 P1562 + wt B G1806 bZIP OE 3, 6, 8 P1559 + wt B G1911
MYB-related OE 4, 5, 6 P989 + - B G2011 HS OE 5, 12, 18 P1813 + - B
G2155 AT-hook OE 2, 8, 12 Pl742 + wt B G2215 bZIP-NIN OE 3, 5, 7
P1948 + wt B G2452 MYB-related OE 7, 11, 16 P2023 + wt B G2455
YABBY OE 8, 11, 17 P2584 + wt B G2510 AP2 OE 12, 14, 20 P2038 + wt
B G2515 MADS OE 5, 41, 45 P13372 + wt B G2571 AP2 OE 1, 5, 8 P1998
+ wt B G2702 MYB-(R1)R2R3 OE 23, 29, 31 P13807 + wt B G2763 HLH/MYC
OE 1, 2, 5 P2387 + - B G2774 HLH/MYC OE 12, 15, 18 P16177 + wt B
G2888 Z-C2H2 OE 21, 24, 27 P2656 + - B G2958 IAA OE 22, 26, 30
P15168 + wt B Table 15 Notes. All scores presented in Table 15
(other than wild type) were based on data from two independent
experiments on the seed batches (assuming sufficient seed was
available to repeat the experiment twice). .sup.1Shade tolerance
phenotype" column. Score of "++" indicates a strong suppression of
shade responses; the phenotype was very consistent and growth was
significantly above the normal levels of variability observed for
the assay. Score of "+" in the indicates a mild/moderate
suppression of shade responses; the response was consistent but was
only moderately above the normal levels of variability observed for
the assay .sup.2"Growth under white light" column. A score of "-"
indicates that the seedlings from that line were generally smaller
than wild-type controls under normal, white light conditions. A
score of "+" indicates that the seedlings from that line were
generally slightly larger than controls under normal conditions. A
score of "wt" indicates that the seedlings were normal under such
conditions. .sup.3GIDs which are considered top hits and have been
confirmed in multiple experiments are given an A ranking. GIDs that
are considered potential leads but require confirmation in
follow-up studies are given a B ranking. The "A" and "B" rankings
are not meant to be construed as an indication of the relative
value and potential utility of these candidate sequences, but
represent the degree of testing completeness.
Example XI: Identification of Homologous Sequences
[1187] This example describes identification of genes that are
orthologous to Arabidopsis thaliana transcription factors from a
computer homology search.
[1188] Homologous sequences, including those of paralogs and
orthologs from Arabidopsis and other plant species, were identified
using database sequence search tools, such as the Basic Local
Alignment Search Tool (BLAST) (Altschul et al. (1990) supra; and
Altschul et al. (1997) Nucleic Acid Res. 25: 3389-3402). The
tblastx sequence analysis programs were employed using the
BLOSUM-62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl.
Acad. Sci. 89: 10915-10919). The entire NCBI GenBank database was
filtered for sequences from all plants except Arabidopsis thaliana
by selecting all entries in the NCBI GenBank database associated
with NCBI taxonomic ID 33090 (Viridiplantae; all plants) and
excluding entries associated with taxonomic ID 3701 (Arabidopsis
thaliana).
[1189] These sequences are compared to sequences representing genes
of the invention, for example, polynucleotides found in the
Sequence Listing, using the Washington University TBLASTX algorithm
(version 2.0a19MP) at the default settings using gapped alignments
with the filter "off". For each polynucleotide sequence found in
the Sequence Listing, individual comparisons were ordered by
probability score (P-value), where the score reflects the
probability that a particular alignment occurred by chance. For
example, a score of 3.6E-40 is 3.6.times.10-40. In addition to
P-values, comparisons were also scored by percentage identity.
Percentage identity reflects the degree to which two segments of
DNA or protein are identical over a particular length. Examples of
sequences so identified are presented in Tables 8 and 9. The
percent sequence identity among these sequences can be as low as
47%, or even lower sequence identity.
[1190] Candidate paralogous sequences were identified among
Arabidopsis transcription factors through alignment, identity, and
phylogenic relationships. Candidate orthologous sequences were
identified from proprietary unigene sets of plant gene sequences in
Zea mays, Glycine max and Oryza sativa based on significant
homology to Arabidopsis transcription factors. These candidates
were reciprocally compared to the set of Arabidopsis transcription
factors. If the candidate showed maximal similarity in the protein
domain to the eliciting transcription factor or to a paralog of the
eliciting transcription factor, then it was considered to be an
ortholog. Identified non-Arabidopsis sequences that were shown in
this manner to be orthologous to the Arabidopsis sequences are
provided in Tables 8 and 9.
Example XII: Screen of Plant cDNA Library for Sequence Encoding a
Transcription Factor DNA Binding Domain that Binds to a
Transcription Factor Binding Promoter Element and Demonstration of
Protein Transcription Regulation Activity
[1191] The "one-hybrid" strategy (Li and Herskowitz (1993) Science
262: 1870-1874) is used to screen for plant cDNA clones encoding a
polypeptide comprising a transcription factor DNA binding domain, a
conserved domain. In brief, yeast strains are constructed that
contain a lacZ reporter gene with either wild-type or mutant
transcription factor binding promoter element sequences in place of
the normal UAS (upstream activator sequence) of the GAL4 promoter.
Yeast reporter strains are constructed that carry transcription
factor binding promoter element sequences as UAS elements are
operably linked upstream (5') of a lacZ reporter gene with a
minimal GAL4 promoter. The strains are transformed with a plant
expression library that contains random cDNA inserts fused to the
GAL4 activation domain (GAL4-ACT) and screened for blue colony
formation on X-gal-treated filters (X-gal:
5-bromo-4-chloro-3-indolyl- -D-galactoside; Invitrogen Corporation,
Carlsbad Calif.). Alternatively, the strains are transformed with a
cDNA polynucleotide encoding a known transcription factor DNA
binding domain polypeptide sequence.
[1192] Yeast strains carrying these reporter constructs produce low
levels of .beta.-galactosidase and form white colonies on filters
containing X-gal. The reporter strains carrying wild-type
transcription factor binding promoter element sequences are
transformed with a polynucleotide that encodes a polypeptide
comprising a plant transcription factor DNA binding domain operably
linked to the acidic activator domain of the yeast GAL4
transcription factor, "GAL4-ACT". The clones that contain a
polynucleotide encoding a transcription factor DNA binding domain
operably linked to GAL4-ACT can bind upstream of the lacZ reporter
genes carrying the wild-type transcription factor binding promoter
element sequence, activate transcription of the lacZ gene and
result in yeast forming blue colonies on X-gal-treated filters.
[1193] Upon screening about 2.times.10.sup.6 yeast transformants,
positive cDNA clones are isolated; i.e., clones that cause yeast
strains carrying lacZ reporters operably linked to wild-type
transcription factor binding promoter elements to form blue
colonies on X-gal-treated filters. The cDNA clones do not cause a
yeast strain carrying a mutant type transcription factor binding
promoter elements fused to LacZ to turn blue. Thus, a
polynucleotide encoding transcription factor DNA binding domain, a
conserved domain, is shown to activate transcription of a gene.
Example XIII: Gel Shift Assays
[1194] The presence of a transcription factor comprising a DNA
binding domain which binds to a DNA transcription factor binding
element is evaluated using the following gel shift assay. The
transcription factor is recombinantly expressed and isolated from
E. coli or isolated from plant material. Total soluble protein,
including transcription factor, (40 ng) is incubated at room
temperature in 10 .mu.l of 1.times. binding buffer (15 mM HEPES (pH
7.9), 1 mM EDTA, 30 mM KCl, 5% glycerol, 5% bovine serum albumin, 1
mM DTT) plus 50 ng poly(dl-dC):poly(dl-dC) (Pharmacia, Piscataway
N.J.) with or without 100 ng competitor DNA. After 10 minutes
incubation, probe DNA comprising a DNA transcription factor binding
element (1 ng) that has been .sup.32P-labeled by end-filling
(Sambrook et al. supra) is added and the mixture incubated for an
additional 10 minutes. Samples are loaded onto polyacrylamide gels
(4% w/v) and fractionated by electrophoresis at 150V for 2h
(Sambrook et al. supra). The degree of transcription factor-probe
DNA binding is visualized using autoradiography. Probes and
competitor DNAs are prepared from oligonucleotide inserts ligated
into the BamHI site of pUC118 (Vieira et al. (1987) Methods
Enzymol. 153: 3-11). Orientation and concatenation number of the
inserts are determined by dideoxy DNA sequence analysis (Sambrook
et al. supra). Inserts are recovered after restriction digestion
with EcoRI and HindIII and fractionation on polyacrylamide gels
(12% w/v) (Sambrook et al. supra).
Example XIV. Transformation of Dicots
[1195] Crop species overexpressing members of the G1792 clade of
transcription factor polypeptides have been shown experimentally to
produce plants with increased tolerance to disease. This
observation indicates that these genes, when overexpressed, will
result in larger yields of various plant species, particularly
during conditions of biotic stress.
[1196] Thus, transcription factor sequences listed in the Sequence
Listing recombined into pMEN20 or pMEN65 expression vectors may be
transformed into a plant for the purpose of modifying plant traits.
The cloning vector may be introduced into a variety of cereal
plants by means well known in the art such as, for example, direct
DNA transfer or Agrobacterium tumefaciens-mediated transformation.
It is now routine to produce transgenic plants using most dicot
plants (see Weissbach and Weissbach, (1989) supra; Gelvin et al.
(1990) supra; Herrera-Estrella et al. (1983) supra; Bevan (1984)
supra; and Klee (1985) supra). Methods for analysis of traits are
routine in the art and examples are disclosed above.
[1197] Methods for transforming cotton may be found in U.S. Pat.
Nos. 5,004,863, 5,159,135 and 5,518,908; for transforming Brassica
species may be found in U.S. Pat. No. 5,463,174; for transforming
peanut plants may be found in Cheng et al. (1996) Plant Cell Rep.
15: 653-657, and McKently et al. (1995) Plant Cell Rep. 14:
699-703; and for transforming pea may be found in Grant et al.
(1995) Plant Cell Rep. 15: 254-258.
[1198] Numerous protocols for the transformation of tomato and soy
plants have been previously described, and are well known in the
art. Gruber et al. ((1993) in Methods in Plant Molecular Biology
and Biotechnology, p. 89-119, Glick and Thompson, eds., CRC Press,
Inc., Boca Raton) describe several expression vectors and culture
methods that may be used for cell or tissue transformation and
subsequent regeneration. For soybean transformation, methods are
described by Mild et al. (1993) in Methods in Plant Molecular
Biology and Biotechnology, p. 67-88, Glick and Thompson, eds., CRC
Press, Inc., Boca Raton; and U.S. Pat. No. 5,563,055, (Townsend and
Thomas), issued Oct. 8, 1996.
[1199] There are a substantial number of alternatives to
Agrobacterium-mediated transformation protocols, other methods for
the purpose of transferring exogenous genes into soybeans or
tomatoes. One such method is microprojectile-mediated
transformation, in which DNA on the surface of microprojectile
particles is driven into plant tissues with a biolistic device
(see, for example, Sanford et al., (1987) Part. Sci. Technol.
5:27-37; Christou et al. (1992) Plant. J. 2: 275-281; Sanford
(1993) Methods Enzymol. 217: 483-509; Klein et al. (1987) Nature
327: 70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May
14, 1991; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun.
21, 1994.
[1200] Alternatively, sonication methods (see, for example, Zhang
et al. (1991) Bio/Technology 9: 996-997); direct uptake of DNA into
protoplasts using CaCl2 precipitation, polyvinyl alcohol or
poly-L-ornithine (see, for example, Hain et al. (1985) Mol. Gen.
Genet. 199: 161-168; Draper et al., Plant Cell Physiol. 23: 451-458
(1982)); liposome or spheroplast fusion (see, for example, Deshayes
et al. (1985) EMBO J., 4: 2731-2737; Christou et al. (1987) Proc.
Natl. Acad. Sci. USA 84: 3962-3966); and electroporation of
protoplasts and whole cells and tissues (see, for example, Donn et
al. (1990) in Abstracts of VIIth International Congress on Plant
Cell and Tissue Culture IAPTC, A2-38: 53; D'Halluin et al. (1992)
Plant Cell 4: 1495-1505; and Spencer et al. (1994) Plant Mol. Biol.
24: 51-61) have been used to introduce foreign DNA and expression
vectors into plants.
[1201] After a plant or plant cell is transformed (and the latter
regenerated into a plant), the transformed plant may be crossed
with itself or a plant from the same line, a non-transformed or
wild-type plant, or another transformed plant from a different
transgenic line of plants. Crossing provides the advantages of
producing new and often stable transgenic varieties. Genes and the
traits they confer that have been introduced into a tomato or
soybean line may be moved into distinct line of plants using
traditional backcrossing techniques well known in the art.
Transformation of tomato plants may be conducted using the
protocols of Koornneef et al (1986) In Tomato Biotechnology: Alan
R. Liss, Inc., 169-178, and in U.S. Pat. No. 6,613,962, the latter
method described in brief here. Eight day old cotyledon explants
are precultured for 24 hours in Petri dishes containing a feeder
layer of Petunia hybrida suspension cells plated on MS medium with
2% (w/v) sucrose and 0.8% agar supplemented with 10 .mu.M
.alpha.-naphthalene acetic acid and 4.4 .mu.M 6-benzylaminopurine.
The explants are then infected with a diluted overnight culture of
Agrobacterium tumefaciens containing an expression vector
comprising a polynucleotide of the invention for 5-10 minutes,
blotted dry on sterile filter paper and cocultured for 48 hours on
the original feeder layer plates. Culture conditions are as
described above. Overnight cultures of Agrobacterium tumefaciens
are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7) to
an OD.sub.600 of 0.8.
[1202] Following cocultivation, the cotyledon explants are
transferred to Petri dishes with selective medium comprising MS
medium with 4.56 .mu.M zeatin, 67.3 .mu.M vancomycin, 418.9 .mu.M
cefotaxime and 171.6 .mu.M kanamycin sulfate, and cultured under
the culture conditions described above. The explants are
subcultured every three weeks onto fresh medium. Emerging shoots
are dissected from the underlying callus and transferred to glass
jars with selective medium without zeatin to form roots. The
formation of roots in a kanamycin sulfate-containing medium is a
positive indication of a successful transformation.
[1203] Transformation of soybean plants may be conducted using the
methods found in, for example, U.S. Pat. No. 5,563,055. In this
method, soybean seed is surface sterilized by exposure to chlorine
gas evolved in a glass bell jar. Seeds are germinated by plating on
1/10 strength agar solidified medium without plant growth
regulators and culturing at 28.degree. C. with a 16 hour day
length. After three or four days, seed may be prepared for
cocultivation. The seedcoat is removed and the elongating radicle
removed 3-4 mm below the cotyledons.
[1204] Overnight cultures of Agrobacterium tumefaciens harboring
the expression vector comprising a polynucleotide of the invention
are grown to log phase, pooled, and concentrated by centrifugation.
Inoculations are conducted in batches such that each plate of seed
was treated with a newly resuspended pellet of Agrobacterium. The
pellets are resuspended in 20 ml inoculation medium. The inoculum
is poured into a Petri dish containing prepared seed and the
cotyledonary nodes are macerated with a surgical blade. After 30
minutes the explants are transferred to plates of the same medium
that has been solidified. Explants are embedded with the adaxial
side up and level with the surface of the medium and cultured at
22.degree. C. for three days under white fluorescent light. These
plants may then be regenerated according to methods well
established in the art, such as by moving the explants after three
days to a liquid counter-selection medium (see U.S. Pat. No.
5,563,055).
[1205] The explants may then be picked, embedded and cultured in
solidified selection medium. After one month on selective media
transformed tissue becomes visible as green sectors of regenerating
tissue against a background of bleached, less healthy tissue.
Explants with green sectors are transferred to an elongation
medium. Culture is continued on this medium with transfers to fresh
plates every two weeks. When shoots are 0.5 cm in length they may
be excised at the base and placed in a rooting medium.
Example XV: Altered C/N Sensing and Increased Shade and Abiotic
Stress Tolerance in Monocots
[1206] Cereal plants such as, but not limited to, corn, wheat,
rice, sorghum, or barley, may be transformed with the present
polynucleotide sequences, including monocot or dicot-derived
sequences such as those presented in Tables 1, 3, 8 or 9, cloned
into a vector such as pGA643 and containing a kanamycin-resistance
marker, and expressed constitutively under, for example, the CaMV
35S or COR15 promoters. pMEN20 or pMEN65 and other expression
vectors may also be used for the purpose of modifying plant traits.
For example, pMEN020 may be modified to replace the NptII coding
region with the BAR gene of Streptomyces hygroscopicus that confers
resistance to phosphinothricin. The KpnI and BglII sites of the Bar
gene are removed by site-directed mutagenesis with silent codon
changes.
[1207] The cloning vector may be introduced into a variety of
cereal plants by means well known in the art including direct DNA
transfer or Agrobacterium tumefaciens-mediated transformation. The
latter approach may be accomplished by a variety of means,
including, for example, that of U.S. Pat. No. 5,591,616, in which
monocotyledon callus is transformed by contacting dedifferentiating
tissue with the Agrobacterium containing the cloning vector.
[1208] The sample tissues are immersed in a suspension of
3.times.10.sup.-9 cells of Agrobacterium containing the cloning
vector for 3-10 minutes. The callus material is cultured on solid
medium at 25.degree. C. in the dark for several days. The calli
grown on this medium are transferred to Regeneration medium.
Transfers are continued every 2-3 weeks (2 or 3 times) until shoots
develop. Shoots are then transferred to Shoot-Elongation medium
every 2-3 weeks. Healthy looking shoots are transferred to rooting
medium and after roots have developed, the plants are placed into
moist potting soil.
[1209] The transformed plants are then analyzed for the presence of
the NPTII gene/kanamycin resistance by ELISA, using the ELISA NPTII
kit from 5Prime-3Prime Inc. (Boulder, Colo.).
[1210] It is also routine to use other methods to produce
transgenic plants of most cereal crops (Vasil (1994) Plant Mol.
Biol. 25: 925-937) such as corn, wheat, rice, sorghum (Cassas et
al. (1993) Proc. Natl. Acad. Sci. USA 90: 11212-11216, and barley
(Wan and Lemeaux (1994) Plant Physiol. 104:37-48). DNA transfer
methods such as the microprojectile method can be used for corn
(Fromm et al. (1990) Bio/Technol. 8: 833-839); Gordon-Kamm et al.
(1990) Plant Cell 2: 603-618; Ishida (1990) Nature Biotechnol.
14:745-750), wheat (Vasil et al. (1992) Bio/Technol. 10:667-674;
Vasil et al. (1993) Bio/Technol. 11:1553-1558; Weeks et al. (1993)
Plant Physiol. 102:1077-1084), and rice (Christou (1991)
Bio/Technol. 9:957-962; Hiei et al. (1994) Plant J. 6:271-282;
Aldemita and Hodges (1996) Planta 199:612-617; and Hiei et al.
(1997) Plant Mol. Biol. 35:205-218). For most cereal plants,
embryogenic cells derived from immature scutellum tissues are the
preferred cellular targets for transformation (Hiei et al. (1997)
Plant Mol. Biol. 35:205-218; Vasil (1994) Plant Mol. Biol. 25:
925-937). For transforming corn embryogenic cells derived from
immature scutellar tissue using microprojectile bombardment, the
A188XB73 genotype is the preferred genotype (Fromm et al. (1990)
Bio/Technol. 8: 833-839; Gordon-Kamm et al. (1990) Plant Cell 2:
603-618). After microprojectile bombardment the tissues are
selected on phosphinothricin to identify the transgenic embryogenic
cells (Gordon-Kamm et al. (1990) Plant Cell 2: 603-618). Transgenic
plants are regenerated by standard corn regeneration techniques
(Fromm et al. (1990) Bio/Technol. 8: 833-839; Gordon-Kamm et al.
(1990) Plant Cell 2: 603-618).
[1211] Northern blot analysis, RT-PCR or microarray analysis of the
regenerated, transformed plants may be used to show expression of
G1792 and related genes that are capable of conferring tolerance to
biotic or abiotic stress.
[1212] To verify the ability to confer abiotic stress tolerance,
mature plants overexpressing a transcription factor of the
invention, or alternatively, seedling progeny of these plants, may
be challenged in an abiotic stress assay, such as a drought, heat,
high salt, or freezing assay, in an osmotic stress condition that
may also measure altered sugar sensing, such as a high sugar
condition, in a shade tolerance assay, or in a C/N sensing assay to
identify plants with altered stress or shade tolerance of altered
C/N sensing. By comparing wild type and transgenic plants similarly
treated, the transgenic plants may be shown to have greater
tolerance to abiotic stress.
[1213] After a monocot plant or plant cell has been transformed
(and the latter regenerated into a plant) and shown to have greater
tolerance to biotic or abiotic stress, or produce greater yield
relative to a control plant under the stress conditions, the
transformed monocot plant may be crossed with itself or a plant
from the same line, a non-transformed or wild-type monocot plant,
or another transformed monocot plant from a different transgenic
line of plants.
Example XVI: Genes that Confer Significant Improvements to
Non-Arabidopsis Species
[1214] The function of specific orthologs of transcription factors
of the invention has been analyzed and may be further characterized
by incorporation into crop plants. The function of specific
orthologs of the sequences in the Sequence Listing may be analyzed
through their altered expression (e.g., ectopic overexpression, or
knocking out) in plants, using constitutive, inducible, or tissue
specific regulatory elements, as disclosed above. These sequences
include polynucleotide sequences found in the Sequence Listing such
as, for example:
[1215] (i) those sequences conferring drought tolerance found in
Arabidopsis thaliana SEQ ID NO: 2 (G47) and SEQ ID NO: 12 (G2133);
Oryza sativa (japonica cultivar-group) SEQ ID NO: 98 (G3649), SEQ
ID NO: 100 (G3651), and SEQ ID NO: 90 (G3644); Glycine max SEQ ID
NO: 88 (G3643); Zinnia elegans SEQ ID NO: 96 (G3647); Brassica rapa
subsp. Pekinensis SEQ ID NO: 92 (G3645); and Brassica oleracea SEQ
ID NO: 94 (G3646);
[1216] (ii) those sequences conferring altered C/N sensing found in
Arabidopsis thaliana SEQ ID NO: 234, 286, 312, and 32 (G682, G226,
G1816, and G2718; Oryza sativa SEQ ID NO: 326 and 328 (G3392 and
G3393); Glycine max SEQ ID NO: 372, 374, 376, 378, 380, and 382
(G3445, G3446, G3447, G3448, G3449, and G3450); and Zea mays SEQ ID
NO: 360 and 370 (G3431 and G3444); and
[1217] (iii) those sequences conferring shade tolerance found in
Arabidopsis thaliana SEQ ID NO: 232 (G634), SEQ ID NO: 818 (G2505),
and SEQ ID NO: 248 (G2789), and G2789 orthologs in Glycine max
(Gma_54935598) and Pinus taeda (Pta_515799222, Pta_516786360,
Pta_516788492, and Pta_516802054).
[1218] The polynucleotide and polypeptide sequences derived from
monocots may be used to transform both monocot and dicot plants,
and those derived from dicots may be used to transform either
group, although some of these sequences will function best if the
gene is transformed into a plant from the same group as that from
which the sequence is derived.
[1219] Transformation procedures are provided in these Examples,
and may employ the use of an expression vector. After the vector is
introduced into a plant cell, a plant may be regenerated from the
cell, after which the plant is allowed to overexpress one of the
polypeptides of the invention that have the property of increasing
abiotic stress tolerance, shade tolerance, or altered C/N sensing
in the transgenic plant. Plants with these altered traits may be
identified by comparison with wild-type or non-transformed plants
that do not overexpress the polypeptide, after which one or more
plant with a desirable degree of one or more improved traits may be
selected. In this manner, plants with enhanced shade tolerance,
increased abiotic stress tolerance, altered C/N sensing, or more
than one of these altered traits may be selected.
[1220] For drought tolerance-related analysis, seeds of these
transgenic plants are subjected to germination assays to measure
sucrose sensing. Sterile monocot seeds, including, but not limited
to, corn, rice, wheat, rye and sorghum, as well as dicots
including, but not limited to soybean and alfalfa, are sown on 80%
MS medium plus vitamins with 9.4% sucrose; control media lack
sucrose. All assay plates are then incubated at 22.degree. C. under
24-hour light, 120-130 .mu.Ein/m.sup.2/s, in a growth chamber.
Evaluation of germination and seedling vigor is then conducted
three days after planting. Overexpressors of these sequences may be
found to be more tolerant to high sucrose by having better
germination, longer radicles, and more cotyledon expansion. These
results would indicate that overexpressors of the orthologs in the
Sequence Listing are involved in sucrose-specific sugar
sensing.
[1221] Plants overexpressing these orthologs may also be subjected
to soil-based drought assays to identify those lines that are more
tolerant to water deprivation than wild-type control plants.
Generally, ortholog overexpressing plants will appear significantly
larger and greener, with less wilting or desiccation, than
wild-type controls plants, particularly after a period of water
deprivation is followed by rewatering and a subsequent incubation
period.
[1222] For C/N sensing-related analysis, seeds of these transgenic
plants are subjected to germination or growth assays to measure C/N
sensing or tolerance to low nitrogen. Sterilized monocot seeds,
including, but not limited to, corn, rice, wheat, rye and sorghum,
as well as dicots including, but not limited to soybean and
alfalfa, are sown on basal media comprising 80% MS+Vitamins.
[1223] The sterile seeds sown onto plates containing media based on
80% MS without a nitrogen source. For C/N assays, the media
contains 3% sucrose. The -N/+Gln media the same media, supplemented
with 1 mM glutamine, is used. Plates are incubated in a 24-hour
light C (120-130 .mu.Eins.sup.-2 m.sup.-1) growth chamber at
22.degree. C. Evaluation of germination and seedling vigor is
performed five days after planting. Overexpressors of these genes
that are more tolerant to low nitrogen than control plants have
better germination, longer radicles, more cotyledon expansion, more
root hairs, greater root mass, more vegetative growth, a greener
appearance, or less anthocyanin. The latter (production of less
anthocyanin on these media) is generally associated with increased
tolerance to nitrogen limitation.
[1224] A transgene responsible for the altered response is likely
involved in the plant's ability to perceive their carbon and
nitrogen status.
[1225] For shade tolerance-related analysis, seeds of these
transgenic plants are subjected to germination or growth assays to
measure shade tolerance. Sterilized monocot seeds, including, but
not limited to, corn, rice, wheat, rye and sorghum, as well as
dicots including, but not limited to soybean and alfalfa, are sown
on 80% MS medium plus vitamins. Plates are incubated at 22.degree.
C. under 24-hour light (about 50 .mu.Einsteins.sup.-2 m.sup.-1)
under both white light (control) and under light depleted in red
wavelengths. Seedlings are then assessed for shade tolerance at 7
days, and shade tolerance is scored by visually observing
differences in hypocotyl length compared with control seedlings
grown under white light and grown under light lacking the red
wavelengths.
[1226] Overexpressors of these sequences may be found to be more
tolerant to low light by having altered morphological
characteristics associated with a shade tolerant phenotype, or
improved growth or yield in conditions of low light. Overexpressors
of these genes may also be found to be more tolerant to shade or
abiotic stresses, and may show altered cotyledon, altered
hypocotyl, altered leaf orientation, altered petiole, and/or
constitutive photomorphogenesis, better germination, longer
radicles, more cotyledon expansion, more vegetative growth, a
greener appearance, or less anthocyanin in stress conditions. These
results would indicate that overexpressors of the orthologs in the
Sequence Listing are involved in shade tolerance responses.
[1227] Plants overexpressing these orthologs may also be subjected
to low light or abiotic stress assays to identify those lines that
are more tolerant to low light conditions or abiotic stresses than
wild-type control plants in these conditions. Generally, ortholog
overexpressing plants will show morphological features that are
associated with a shade avoidance phenotype (e.g., altered
cotyledon, altered hypocotyl, altered leaf orientation, altered
petiole, and/or constitutive photomorphogenesis), and may also
appear larger, greener, and healthier than wild-type controls
plants.
Example XVII: Identification of Orthologous and Paralogous
Sequences
[1228] Orthologs to Arabidopsis genes may identified by several
methods, including hybridization, amplification, or
bioinformatically. This example describes how one may identify
homologs to the Arabidopsis AP2 family transcription factor CBF1,
which confers tolerance to abiotic stresses (Thomashow et al.
(2002) U.S. Pat. No. 6,417,428), and an example to confirm the
function of homologous sequences. In this example, orthologs to
CBF1 were found in canola (Brassica napus) using polymerase chain
reaction (PCR).
[1229] Degenerate primers were designed for regions of AP2 binding
domain and outside of the AP2 (carboxyl terminal domain; U.S. Pat.
No. 6,417,428):
TABLE-US-00017 Mol 368 (reverse) (SEQ ID NO: 1437) 5'-CAY CCN ATH
TAY MGN GGN GT-3' Mol 378 (forward) (SEQ ID NO: 1438) 5'-GGN ARN
ARC ATN CCY TCN GCC-3' (Y: C/T, N: A/C/G/T, H: A/C/T, M: A/C, R:
A/G)
[1230] Primer Mol 368 is in the AP2 binding domain of CBF1 (amino
acid sequence: His-Pro-Ile-Tyr-Arg-Gly-Val; SEQ ID NO: 1439) while
primer Mol 378 is outside the AP2 domain (carboxyl terminal domain)
(amino acid sequence: Met-Ala-Glu-Gly-Met-Leu-Leu-Pro); SEQ ID NO:
1440).
[1231] The genomic DNA isolated from B. napus was PCR-amplified by
using these primers following these conditions: an initial
denaturation step of 2 min at 93.degree. C.; 35 cycles of
93.degree. C. for 1 min, 55.degree. C. for 1 min, and 72.degree. C.
for 1 min; and a final incubation of 7 min at 72.degree. C. at the
end of cycling.
[1232] The PCR products were separated by electrophoresis on a 1.2%
agarose gel and transferred to nylon membrane and hybridized with
the AT CBF1 probe prepared from Arabidopsis genomic DNA by PCR
amplification. The hybridized products were visualized by
colorimetric detection system (Boehringer Mannheim) and the
corresponding bands from a similar agarose gel were isolated using
the Qiagen Extraction Kit (Qiagen). The DNA fragments were ligated
into the TA clone vector from TOPO TA Cloning Kit (Invitrogen) and
transformed into E. coli strain TOP10 (Invitrogen).
[1233] Seven colonies were picked and the inserts were sequenced on
an ABI 377 machine from both strands of sense and antisense after
plasmid DNA isolation. The DNA sequence was edited by sequencer and
aligned with the AtCBF1 by GCG software and NCBI blast
searching.
[1234] The nucleic acid sequence and amino acid sequence of one
canola ortholog found in this manner (bnCBF1; U.S. Pat. No.
6,417,428) identified by this process is shown in the Sequence
Listing.
[1235] The aligned amino acid sequences show that the bnCBF1 gene
has 88% identity with the Arabidopsis sequence in the AP2 domain
region and 85% identity with the Arabidopsis sequence outside the
AP2 domain when aligned for two insertion sequences that are
outside the AP2 domain.
[1236] Similarly, paralogous sequences to Arabidopsis genes, such
as CBF1, may also be identified.
[1237] Two paralogs of CBF1 from Arabidopsis thaliana: CBF2 and
CBF3. CBF2 and CBF3 have been cloned and sequenced as described
below. The sequences of the DNA and encoded proteins are set forth
in U.S. Pat. No. 6,417,428.
[1238] A lambda cDNA library prepared from RNA isolated from
Arabidopsis thaliana ecotype Columbia (Lin and Thomashow (1992)
Plant Physiol. 99: 519-525) was screened for recombinant clones
that carried inserts related to the CBF1 gene (Stockinger et al.
(1997) Proc. Natl. Acad. Sci. 94:1035-1040). CBF1 was
.sup.32P-radiolabeled by random priming (Sambrook et al. supra) and
used to screen the library by the plaque-lift technique using
standard stringent hybridization and wash conditions (Hajela et al.
(1990) Plant Physiol. 93:1246-1252; Sambrook et al. supra)
6.times.SSPE buffer, 60.degree. C. for hybridization and
0.1.times.SSPE buffer and 60.degree. C. for washes). Twelve
positively hybridizing clones were obtained and the DNA sequences
of the cDNA inserts were determined. The results indicated that the
clones fell into three classes. One class carried inserts
corresponding to CBF1. The two other classes carried sequences
corresponding to two different homologs of CBF1, designated CBF2
and CBF3. The nucleic acid sequences and predicted protein coding
sequences for Arabidopsis CBF1, CBF2, CBF3, and the Brassica napus
CBF ortholog are set forth in U.S. Pat. No. 6,417,428.
[1239] A comparison of the nucleic acid sequences of Arabidopsis
CBF1, CBF2 and CBF3 indicate that they are 83 to 85% identical as
shown in Table 16.
TABLE-US-00018 TABLE 16 Percent identity.sup.a DNA.sup.b
Polypeptide cbf1/cbf2 85 86 cbf1/cbf3 83 84 cbf2/cbf3 84 85
.sup.aPercent identity was determined using the Clustal algorithm
from the MEGALIGN program (DNASTAR, Inc.). .sup.bComparisons of the
nucleic acid sequences of the open reading frames are shown.
[1240] Similarly, the amino acid sequences of the three CBF
polypeptides range from 84 to 86% identity. An alignment of the
three amino acid sequences reveals that most of the differences in
amino acid sequence occur in the acidic C-terminal half of the
polypeptide. This region of CBF1 serves as an activation domain in
both yeast and Arabidopsis (not shown).
[1241] Residues 47 to 106 of CBF1 correspond to the AP2 domain of
the protein, a DNA binding motif that to date, has only been found
in plant proteins. A comparison of the AP2 domains of CBF1, CBF2
and CBF3 indicates that there are a few differences in amino acid
sequence. These differences in amino acid sequence might have an
effect on DNA binding specificity.
Example XVIII: Transformation of Canola with a Plasmid Containing
CBF1, CBF2, or CBF3
[1242] After identifying homologous genes to CBF1, canola was
transformed with a plasmid containing the Arabidopsis CBF1, CBF2,
or CBF3 genes cloned into the vector pGA643 (An (1987) Methods
Enzymol. 253: 292). In these constructs the CBF genes were
expressed constitutively under the CaMV 35S promoter. In addition,
the CBF1 gene was cloned under the control of the Arabidopsis COR15
promoter in the same vector pGA643. Each construct was transformed
into Agrobacterium strain GV3101. Transformed Agrobacteria were
grown for 2 days in minimal AB medium containing appropriate
antibiotics.
[1243] Spring canola (B. napus cv. Westar) was transformed using
the protocol of Moloney et al. ((1989) Plant Cell Reports 8: 238)
with some modifications as described. Briefly, seeds were
sterilized and plated on half strength MS medium, containing 1%
sucrose. Plates were incubated at 24.degree. C. under 60-80
.mu.E/m.sup.2s light using a 16 hour light/8 hour dark photoperiod.
Cotyledons from 4-5 day old seedlings were collected, the petioles
cut and dipped into the Agrobacterium solution. The dipped
cotyledons were placed on co-cultivation medium at a density of 20
cotyledons/plate and incubated as described above for 3 days.
Explants were transferred to the same media, but containing 300
mg/l timentin (SmithKline Beecham, Pa.) and thinned to 10
cotyledons/plate. After 7 days explants were transferred to
Selection/Regeneration medium. Transfers were continued every 2-3
weeks (2 or 3 times) until shoots had developed. Shoots were
transferred to Shoot-Elongation medium every 2-3 weeks. Healthy
looking shoots were transferred to rooting medium. Once good roots
had developed, the plants were placed into moist potting soil.
[1244] The transformed plants were then analyzed for the presence
of the NPTII gene/kanamycin resistance by ELISA, using the ELISA
NPTII kit from 5Prime-3Prime Inc. (Boulder, Colo.). Approximately
70% of the screened plants were NPTII positive. Only those plants
were further analyzed.
[1245] From Northern blot analysis of the plants that were
transformed with the constitutively expressing constructs, showed
expression of the CBF genes and all CBF genes were capable of
inducing the Brassica napus cold-regulated gene BN115 (homolog of
the Arabidopsis COR15 gene). Most of the transgenic plants appear
to exhibit a normal growth phenotype. As expected, the transgenic
plants are more freezing tolerant than the wild-type plants. Using
the electrolyte leakage of leaves test, the control showed a 50%
leakage at -2 to -3.degree. C. Spring canola transformed with
either CBF1 or CBF2 showed a 50% leakage at -6 to -7.degree. C.
Spring canola transformed with CBF3 shows a 50% leakage at about
-10 to -15.degree. C. Winter canola transformed with CBF3 may show
a 50% leakage at about -16 to -20.degree. C. Furthermore, if the
spring or winter canola are cold acclimated the transformed plants
may exhibit a further increase in freezing tolerance of at least
-2.degree. C.
[1246] To test salinity tolerance of the transformed plants, plants
were watered with 150 mM NaCl. Plants overexpressing CBF1, CBF2 or
CBF3 grew better compared with plants that had not been transformed
with CBF1, CBF2 or CBF3.
[1247] These results demonstrate that homologs of Arabidopsis
transcription factors can be identified and shown to confer similar
functions in non-Arabidopsis plant species.
Example IXX: Cloning of Transcription Factor Promoters
[1248] Promoters are isolated from transcription factor genes that
have gene expression patterns useful for a range of applications,
as determined by methods well known in the art (including
transcript profile analysis with cDNA or oligonucleotide
microarrays, Northern blot analysis, semi-quantitative or
quantitative RT-PCR). Interesting gene expression profiles are
revealed by determining transcript abundance for a selected
transcription factor gene after exposure of plants to a range of
different experimental conditions, and in a range of different
tissue or organ types, or developmental stages. Experimental
conditions to which plants are exposed for this purpose includes
cold, heat, drought, osmotic challenge, varied hormone
concentrations (ABA, GA, auxin, cytokinin, salicylic acid,
brassinosteroid), pathogen and pest challenge. The tissue types and
developmental stages include stem, root, flower, rosette leaves,
cauline leaves, siliques, germinating seed, and meristematic
tissue. The set of expression levels provides a pattern that is
determined by the regulatory elements of the gene promoter.
[1249] Transcription factor promoters for the genes disclosed
herein are obtained by cloning 1.5 kb to 2.0 kb of genomic sequence
immediately upstream of the translation start codon for the coding
sequence of the encoded transcription factor protein. This region
includes the 5'-UTR of the transcription factor gene, which can
comprise regulatory elements. The 1.5 kb to 2.0 kb region is cloned
through PCR methods, using primers that include one in the 3'
direction located at the translation start codon (including
appropriate adaptor sequence), and one in the 5' direction located
from 1.5 kb to 2.0 kb upstream of the translation start codon
(including appropriate adaptor sequence). The desired fragments are
PCR-amplified from Arabidopsis Col-0 genomic DNA using
high-fidelity Taq DNA polymerase to minimize the incorporation of
point mutation(s). The cloning primers incorporate two rare
restriction sites, such as NotI and Sfi1, found at low frequency
throughout the Arabidopsis genome. Additional restriction sites are
used in the instances where a NotI or Sfi1 restriction site is
present within the promoter.
[1250] The 1.5-2.0 kb fragment upstream from the translation start
codon, including the 5'-untranslated region of the transcription
factor, is cloned in a binary transformation vector immediately
upstream of a suitable reporter gene, or a transactivator gene that
is capable of programming expression of a reporter gene in a second
gene construct. Reporter genes used include green fluorescent
protein (and related fluorescent protein color variants),
.beta.-glucuronidase, and luciferase. Suitable transactivator genes
include LexA-GAL4, along with a transactivatable reporter in a
second binary plasmid (as disclosed in U.S. patent application Ser.
No. 09/958,131, incorporated herein by reference). The binary
plasmid(s) is transferred into Agrobacterium and the structure of
the plasmid confirmed by PCR. These strains are introduced into
Arabidopsis plants as described in other examples, and gene
expression patterns determined according to standard methods know
to one skilled in the art for monitoring GFP fluorescence,
.beta.-glucuronidase activity, or luminescence.
[1251] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[1252] The present invention is not limited by the specific
embodiments described herein. The invention now being fully
described, it will be apparent to one of ordinary skill in the art
that many changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.
Modifications that become apparent from the foregoing description
and accompanying figures fall within the scope of the claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190367565A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190367565A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References