U.S. patent application number 17/404842 was filed with the patent office on 2022-02-10 for manipulating plant sensitivity to light.
The applicant listed for this patent is Mendel Biotechnology, Inc., Monsanto Technology, LLC. Invention is credited to Rajnish Khanna, Brent A. Kronmiller, Amanda McClerren, Robert J. Meister, Marie E. Petracek, Sasha Preuss, Qungang Qi, Oliver J. Ratcliffe, T. Lynne Reuber.
Application Number | 20220042029 17/404842 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220042029 |
Kind Code |
A1 |
Khanna; Rajnish ; et
al. |
February 10, 2022 |
MANIPULATING PLANT SENSITIVITY TO LIGHT
Abstract
The present disclosure identifies new genes which have the
potential to increase broad acre yield in crops. This disclosure is
based upon our fundamental knowledge of light signal transduction
and our understanding of tile roles these genes play in regulating
plant growth and development in response to light. Transgenic
plants with gain- or loss-of-function of one of these genes, or in
combination, are expected to show significant improvements in broad
acre yield and stress tolerance.
Inventors: |
Khanna; Rajnish; (Livermore,
CA) ; Kronmiller; Brent A.; (Corvallis, OR) ;
Ratcliffe; Oliver J.; (Hayward, CA) ; Reuber; T.
Lynne; (San Mateo, CA) ; Petracek; Marie E.;
(Glendale, MO) ; Meister; Robert J.; (St. Peters,
MO) ; Preuss; Sasha; (Webster Groves, MO) ;
McClerren; Amanda; (St. Charles, MO) ; Qi;
Qungang; (Chesterfield, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mendel Biotechnology, Inc.
Monsanto Technology, LLC |
Hayward
St. Louis |
CA
MO |
US
US |
|
|
Appl. No.: |
17/404842 |
Filed: |
August 17, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15802397 |
Nov 2, 2017 |
|
|
|
17404842 |
|
|
|
|
14118491 |
Jun 20, 2014 |
|
|
|
PCT/US12/38719 |
May 18, 2012 |
|
|
|
15802397 |
|
|
|
|
61488592 |
May 20, 2011 |
|
|
|
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415 |
Claims
1. A method for modifying a trait in a plant, the method comprising
the steps of: introducing into a target plant at least one
recombinant polynucleotide comprising at least one nucleotide
sequence that encodes a polypeptide, wherein the nucleotide
sequence is selected from the group consisting of: (a) a nucleotide
sequence set forth as 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; 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, or 549;
(b) a nucleotide sequence encoding 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, 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, or any of SEQ ID NO: 235 to 427; and (c) a
nucleotide sequence encoding a polypeptide wherein the polypeptide
has a percentage identity to at least one 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, 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, or any of SEQ ID NO: 235 to 427; wherein
the nucleotide sequence is operably linked to a promoter that is
expressed in a plant cell; wherein the target plant overexpresses
the polypeptide and said overexpression results in the target plant
expressing the modified trait; wherein the percentage 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 100%; wherein the modified trait is selected
from the group consisting of decreased sensitivity to light,
increased yield, greater height, greater stem diameter, greater
resistance to lodging, increased branching, increased secondary
rooting, greater cold tolerance, greater tolerance to water
deprivation, reduced stomatal conductance, altered C/N sensing,
increased tolerance to nitrogen limiting conditions, improved late
season growth and vigor, greater number of primary nodes, greater
late season canopy coverage, increased tolerance to abiotic stress.
increased tolerance to hyperosmotic stress, altered levels of
ureides, altered levels of hexose sugars, altered sucrose phosphate
synthase (SPS) activity, altered levels of starch, and delayed
senescence, as compared to a control plant that does not comprise
the recombinant polynucleotide; and, optionally, selecting a
transgenic plant having at least one of the modified traits as
compared to the control plant.
2. The method of claim 1, wherein the target plant comprises a
second recombinant polynucleotide comprising a trans-acting element
that controls expression of the polypeptide; wherein the target
plant expresses a protein encoded by a nucleic acid selected from
the group consisting of: SEQ ID NO: 429, SEQ ID NO: 431, SEQ ID NO:
433, and SEQ ID NO: 529; or wherein the target plant is stably
transformed with the at least one recombinant polynucleotide.
3-4. (canceled)
5. A method for modifying a trait in a plant, the method comprising
the steps of: introducing into a target plant at least one
recombinant polynucleotide that suppresses the expression of at
least a polypeptide, wherein the polypeptide is encoded by a
polynucleotide comprising a nucleotide sequence selected from the
group consisting of: (a) a nucleotide sequence set forth as 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; 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, or 549; (b) a nucleotide
sequence encoding 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, 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, or
any of SEQ ID NO: 235 to 427; (c) a nucleotide sequence encoding a
polypeptide wherein the polypeptide has a percentage identity to at
least one 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, 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, or any of SEQ
ID NO: 235 to 427; and wherein the percentage 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 100%; wherein the nucleotide sequence is operably
linked to a promoter that is expressed in a plant cell; wherein the
target plant overexpresses the polypeptide and said overexpression
results in the target plant expressing the modified trait wherein
the modified trait is selected from the group consisting of
decreased sensitivity to light, increased yield, greater height,
greater stem diameter, greater resistance to lodging, increased
branching, increased secondary rooting, greater cold tolerance,
greater tolerance to water deprivation, reduced stomatal
conductance, altered C/N sensing, increased tolerance to nitrogen
limiting conditions, improved late season growth and vigor, greater
number of primary nodes, greater late season canopy coverage,
increased tolerance to abiotic stress, increased tolerance to
hyperosmotic stress, altered levels of ureides, altered levels of
hexose sugars, altered SPS activity, altered levels of starch, and
delayed senescence, as compared to a control plant that does not
comprise the recombinant polynucleotide; and optionally, selecting
a transgenic plant having at least one of the modified traits as
compared to the control plant.
6. The method of claim 5, wherein the target plant comprises a
second recombinant polynucleotide comprising a trans-acting element
that controls expression of the polypeptide, wherein the target
plant expresses a protein encoded by a nucleic acid selected from
the group consisting of: SEQ ID NO: 429, SEQ ID NO: 431, SEQ ID NO:
433, and SEQ ID NO: 529; or wherein the target plant is stably
transformed with the at least one recombinant polynucleotide.
7-8. (canceled)
9. The method of claim 1, wherein the promoter is a constitutive,
inducible, tissue-specific, tissue-enhanced, light-regulated,
stress-inducible, diurnally-regulated, chemically-inducible, or
cell-specific promoter.
10. (canceled)
11. The method of claim 1, the method steps further comprising:
crossing the target plant with itself, a second plant from the same
line as the target plant, a non-transgenic plant, a wild-type
plant, or a transgenic plant from a different line of plants, to
produce a transgenic seed.
12. The method of claim 1, wherein the at least one nucleotide
sequence is derived from: the family Pinaceae, cedar, fir, hemlock,
larch, pine, spruce, the family Alliaceae, onion, leek, garlic, the
family Amaranthaceae, spinach, the family Anacardiaceae, mango, the
family Asteraceae, sunflower, endive, lettuce, artichoke, the
family Brassicaceae, Arabidopsis thaliana, rape, oilseed rape,
canola, cauliflower, turnip, radish, broccoli, cabbage,
cauliflower, Brussels sprouts, kohlrabi, the family Bromeliaceae,
pineapple, the family Caricaceae, papaya, the family
Chenopodiaceae, beet, the family Curcurbitaceae, melon, cantaloupe,
squash, watermelon, honeydew, cucumber, pumpkin, the family
Dioscoreaceae, yam, the family Ericaceae, blueberry, the family
Euphorbiaceae, cassava, the family Fabaceae, alfalfa, clover,
peanut, the family Grossulariaceae, currant; the family
Juglandaceae, walnut, the family Lamiaceae, mint, the family
Lauraceae, avocado, the family Leguminosae, soybean, bean, pea, the
family Malvaceae, cotton, the family Marantaceae, arrowroot, the
family Myrtaceae, guava, eucalyptus, the family Rosaceae, peach,
apple, cherry, plum, pear, prune, blackberry, raspberry,
strawberry, the family Rubiaceae, coffee, the family Rutaceae,
citrus, orange, lemon, grapefruit, tangerine, the family
Salicaceae, poplar, willow, the family Solanaceae, potato, sweet
potato, tomato, Capsicum, tobacco, tomatillo, eggplant, Atropa
belladona, Datura stramonium, the family Vitaceae, grape, the
family Umbelliferae, carrot, the family Musaceae, banana, the
family Poaceae, wheat, maize, sweet corn, rice, wild rice, barley,
rye; millet, sorghum, sugar cane, turfgrass, bamboo, oats,
brome-grass, Miscanthus, pampas grass, switchgrass (Panicum),
and/or teosinte.
13. A transgenic plant produced by the method of claim 1, wherein
the transgenic plant comprises the at least one recombinant
polynucleotide.
14. The transgenic plant of claim 13, wherein the transgenic plant
is a dicot or a conifer.
15. The transgenic plant of claim 14, wherein the dicot or conifer
is a: member of the family Pinaceae, cedar plant, fir plant,
hemlock plant, larch plant, pine plant, spruce plant, member of the
family Amaranthaceae, spinach plant, member of the family
Anacardiaceae, mango plant, member of the family Asteraceae,
sunflower plant, endive plant, lettuce plant, artichoke plant,
member of the family Brassicaceae, Arabidopsis thaliana plant, rape
plant, oilseed rape plant, broccoli plant, Brussels sprouts plant,
cabbage plant, canola plant, cauliflower plant, kohlrabi plant,
turnip plant, radish plant, member of the family Bromeliaceae,
pineapple plant, member of the family Caricaceae, papaya plant,
member of the family Chenopodiaceae, beet plant, member of the
family Curcurbitaceae, melon plant, cantaloupe plant, squash plant,
watermelon plant, honeydew plant, cucumber plant, pumpkin plant,
member of the family Dioscoreaceae, yam plant, member of the family
Ericaceae, blueberry plant, member of the family Euphorbiaceae,
cassava plant, member of the family Fabaceae, alfalfa plant, clover
plant, peanut plant, member of the family Grossulariaceae, currant
plant, member of the family Juglandaceae, walnut plant, member of
the family Lamiaceae, mint plant, member of the family Lauraceae,
avocado plant, member of the family Leguminosae, soybean plant,
bean plant, pea plant, member of the family Malvaceae, cotton
plant, member of the family Marantaceae, arrowroot plant, member of
the family Myrtaceae, guava plant, eucalyptus plant, member of the
family Rosaceae, peach plant, apple plant, cherry plant, plum
plant, pear plant, prune plant, blackberry plant, raspberry plant,
strawberry plant, member of the family Rubiaceae, coffee plant,
member of the family Rutaceae, citrus plant, orange plant, lemon
plant, grapefruit plant, tangerine plant, member of the family
Salicaceae, poplar plant, willow plant, member of the family
Solanaceae, potato plant, sweet potato plant, tomato plant,
Capsicum plant, tobacco plant, tomatillo plant, eggplant plant,
Atropa belladona plant, Datura stramonium plant, member of the
family Vitaceae, grape plant, member of the family Umbelliferae,
carrot plant and/or member of the family Musaceae, banana
plant.
16. The transgenic plant of claim 13, wherein the transgenic plant
is a monocot.
17. The transgenic plant of claim 16, wherein the monocot is a:
member of the family Poaceae; maize plant; rice plant; wild rice
plant, wheat plant; barley plant; sorghum plant; rye plant; oat
plant; millet plant; turfgrass plant; sugarcane plant, bamboo
plant, brome-grass plant, pampas grass plant, Miscanthus plant;
switchgrass plant, teosinte plant; and/or member of the family
Alliaceae, onion, leak and garlic.
18. A tissue culture produced from protoplasts or cells from the
transgenic plant of claim 13, wherein said cells or protoplasts are
produced from a plant part selected from the group consisting of
leaf, pollen, ovule, embryo, cotyledon, hypocotyl, meristematic
cell, root, root tip, pistil, anther, flower, seed, shoot, stem,
pod and petiole.
19. A regenerated plant regenerated from the tissue culture of
claim 18.
20. A seed from the transgenic plant of claim 13.
21. A method for producing a transgenic seed, the method comprising
crossing two plants and harvesting resultant seed, wherein at least
one plant is the transgenic plant of claim 13.
22. A transgenic seed produced by the method of claim 1.
23. A transgenic progeny plant, or a part thereof, produced by
growing the seed of claim 22.
24. The transgenic progeny plant of claim 23, wherein the
transgenic progeny plant has a modified trait relative to a control
plant, wherein the modified trait is selected from the group
consisting of decreased sensitivity to light, increased yield,
greater height, greater stem diameter, greater resistance to
lodging, increased branching, increased secondary rooting, greater
cold tolerance, greater tolerance to water deprivation, reduced
stomatal conductance, altered C/N sensing, increased tolerance to
nitrogen limiting conditions, improved late season growth and
vigor, greater number of primary nodes, greater late season canopy
coverage, increased tolerance to abiotic stress, increased
tolerance to hyperosmotic stress, altered levels of ureides,
altered levels of hexose sugars, altered SPS activity, altered
levels of starch, and delayed senescence, as compared to a control
plant that does not comprise the recombinant polynucleotide.
25. A recombinant polynucleotide comprising a nucleotide sequence
that encodes a polypeptide, wherein the nucleotide sequence is
selected from the group consisting of: (a) a nucleotide sequence
set forth as SEQ ID NO: 447, 461, 473, 481, 483, 485, 509; (b) a
nucleotide sequence encoding SEQ ID NO: 448, 462, 474, 482, 484,
486, 510; (c) a nucleotide sequence wherein the nucleotide sequence
has a percentage identity to at least one of SEQ ID NO: 447, 461,
473, 481, 483, 485, 509; and (d) a nucleotide sequence encoding a
polypeptide wherein the polypeptide has a percentage identity to at
least one of SEQ ID NO: 448, 462, 474, 482, 484, 486, 510; wherein
the percentage identity is selected from the group consisting of 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 100%.
26. A recombinant DNA construct comprising a promoter that is
functional in a plant cell and that is operably linked to the
recombinant polynucleotide of claim 25.
27. A plant cell comprising at least one recombinant polynucleotide
of claim 25.
28. A plant cell comprising at least one recombinant polynucleotide
that suppresses the expression of at least a polypeptide, wherein
the polypeptide is encoded by a recombinant polynucleotide of claim
25.
29. A plant comprising the plant cell of claim 27.
30. A seed comprising the plant cell of claim 27.
31. A method for producing a plant comprising the recombinant
polynucleotide of claim 25.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/488,592 (filed on May 20, 2011), which is
incorporated by reference herein in its entirety.
[0002] The claimed invention, in the field of functional genomics
and 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.
RELEVANT FIELD
[0003] The present disclosure relates to plant genomics and plant
improvement
BACKGROUND
[0004] A plant's traits, including its biochemical, developmental,
or phenotypic characteristics that enhance yield or tolerance to
various abiotic stresses, may be controlled through a number of
cellular processes. One important way to manipulate that control is
through transcriptional regulators--proteins that influence the
expression of a particular gene or sets of genes. Transgenic plants
that comprise cells having altered levels of at least one selected
transcriptional regulator, for example, possess advantageous or
desirable traits. Strategies for manipulating traits by altering a
plant cell's transcriptional regulator content can therefore result
in plants and crops with commercially valuable properties.
SUMMARY
[0005] The present disclosure identifies B-box genes that modify
plant sensitivity to light. Manipulating light signaling processes
through altering the expression/activities of these genes in plants
will lead to enhanced agronomic characteristics, for example,
increased crop yield and improved stress tolerance. Altering the,
expression/activity of the B-box genes of the present disclosure
may also provide plants with altered levels of ureides, altered
levels of hexose sugars, altered sucrose phosphate synthase (SPS)
activity, altered levels of starch, and delayed senescence.
[0006] The present disclosure pertains to polynucleotide and
polypeptide sequences provided herein and in the Sequence Listing.
The present disclosure further pertains to nucleic acid constructs
containing and/or expressing or suppressing the polynucleotide
sequences provided herein and in the Sequence Listing, individually
or in combination, either driven by a ubiquitously expressed, a
stress- or a chemical-inducible, a tissue specific, a development
specific or a diurnally-regulated promoter.
[0007] The present disclosure also pertains to transgenic plants
with increased or decreased activities of one or more of the
proteins provided herein and in the Sequence Listing, or encoded by
the nucleic acid constructs containing and/or expressing or
suppressing the polynucleotide sequences provided herein and in the
Sequence Listing. The transgenic plants may exhibit flanges in
light sensitivity, resulting in improved vigor, improved yield and
growth, and improved ability to cope with abiotic stresses compared
to the wild type or other control plants. This approach can be
combined with other growth-promoting factors to expand the
benefits.
[0008] The present disclosure also provides methods for the
selection and production of transgenic plants transformed with the
disclosed B-box sequences or closely-related orthologs or paralogs.
The selection may be made by identification in a plant (that is, a
mature plant, a seed, a plant part, a seedling, plant tissue, plant
cell, etc.) of the presence of a transgene or heterologous sequence
that was transformed into the transgenic plant, or by an altered
trait in the transgenic plant such as improved yield or increased
tolerance to an abiotic stress, such as low nitrogen conditions,
cold, water deprivation or drought, etc.
[0009] The present disclosure is also directed to transgenic seed
produced by any of the transgenic plants of the present disclosure,
including transgenic plants ectopically expressing or suppressing
SEQ ID NO: 2N, Where N=1-117, or 218-275, or ectopically expressing
a polypeptide that regulates transcription and the polypeptide
comprises a conserved domain of any of SEQ ID NO: 235-427, and to
methods for selecting and/or making the disclosed transgenic plants
and transgenic seeds.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS
[0010] The Sequence Listing provides exemplary polynucleotide and
polypeptide sequences. The traits associated with the use of the
sequences are included in the Examples.
[0011] Incorporation of the Sequence Listing. 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 "MBI_0120PCT_ST25". The electronic file
of the Sequence Listing was created on May 16, 2012, and is
(724,396 bytes in size (707 kilobytes in size as measured in
MS-WINDOWS). The Sequence Listing is herein incorporated by
reference in its entirety.
[0012] FIG. 1 shows light-responsiveness of Arabidopsis seedlings
transformed with 35S::AtBBX28 (G1481):cMYC (AtBBX28 is provided as
SEQ ID NO: 2). Several independently transformed lines were grown
in red light and mean hypocotyl lengths of 20-30 seedlings of each
line are plotted with the empty-vector control (pMEN 65) and
seedlings overexpressing BBX32 (G1988 OE; SEQ ID NO: 430). The
dotted line marks the hypocotyl length of the control (pMEN 65; SEQ
ID NO: 428).
[0013] FIG. 2 shows AtBBX28 (G1481, SEQ ID NO: 2) suppressing
transcriptional activity of AtBBX21 (G1482, SEQ ID NO: 56) in
protoplast co-transfection assays. Protoplasts were co-transformed
with prCHS::GUS reporter and either CAT (control) alone, or
HA:AtBBX21 (G1482) with one CAT, HA: BBX32, or HA:AtBBX28 (G1481).
Note that GUS is .beta.-glucuronidase, CAT is chloramphenicol
acetyltransferase, and HA is hemagglutinin protein-fusion tag
(Niman et al., 1983). RGA=relative GUS activity.
[0014] In FIGS. 3 and 4, the light-responsiveness of Arabidopsis
seedlings of independently transformed lines is plotted as measures
of hypocotyl length for the various plant lines. The dotted line
marks the hypocotyl length of the control. Vertical axes are
measured hypocotyl length (HL; in millimeters). An asterisk above
the bars in FIGS. 3 and 4 indicates a p-value<0.05, and double
asterisk a p-value<0.01.
[0015] FIG. 3. Arabidopsis plant lines transformed with AtBBX25
(G1894, SEQ ID NO: 154; FIG. 3A), AtBBX24 (G329, SEQ ED NO: 152;
FIG. 313), AtBBX30 (G1478, SEQ ID NO: 40; FIG. 3C) and AtBBX18
(G1881, SEQ H) NO: 202; FIG. 3D) were grown in red light and mean
hypocotyl lengths (HL) of 20-30 seedlings of each line are plotted
with the control.
[0016] FIG. 4. Arabidopsis plant lines transformed with AtBBX7
(G2440, SEQ ID NO: 104; FIG. 4A), AtBBX26 (G1486, SEQ ID NO: 52;
FIG. 4B)-AtBBX19 (G902, SEQ ID NO: 200; FIG. 4C) and AtBBX20
(G1888, SEQ ID NO: 54; FIG. 4D) were grown in red light and mean
hypocotyl lengths of 20-30 seedlings of each line are plotted with
the control.
[0017] For FIGS. 5A-5E, FIGS. 6A-6D, FIG. 7, FIGS. 8A-8F, FIGS.
9A-9G and FIGS. 10A-10I, the SEQ ID NOs of the sequences appear in
parentheses to the right of the sequence identifiers. In these
Figures, an asterisk below any column indicates amino acid identity
within the column. A colon indicates that the column is comprised
of conservatively-related amino acid residues. A period indicates
semi-conservatively-related amino acid residues.
[0018] FIGS. 5A-5E present an alignment: of AtBBX25 (G1894,
AT2G31380.1; SEQ NO: 154), AtBBX24 (G329, AT1G06040.1; SEQ ID NO:
152) and closely related sequences.
[0019] FIGS. 6A-6D represent an alignment of AtBBX19 (G902,
AT4G38960.1; SEQ ID NO: 200), AtBBX18 (G1881, AT2021320.1; SEQ ID
NO: 202) and closely related sequences.
[0020] FIG. 7 shows an alignment of AtBBX30 (G11478, AT4G15248.1;
SEQ ID NO: 40) and closely related sequences.
[0021] FIGS. 8A-8F present an alignment of AtBBX28 (G1481,
AT4G27310.1; SEQ ID NO: 2) and AtBBX29 (G900, AT5G54470.1.; SEQ ID
NO: 4) and closely related sequences.
[0022] FIGS. 9A-9G displays an alignment of AtBBX20 (G1888,
AT4G39070.1; SEQ ID NO: 54) and AtBBX21 (G1482, AT1G75540.1; SEQ ID
NO: 56) and closely related sequences.
[0023] FIGS. 10A-10I show an alignment of AtBBX7 (G2440,
AT3G07650.1, SEQ ID NO: 104), AtBBX8 (G1479, AT5G482501; SEQ ID NO:
106) and closely related sequences.
[0024] FIG. 11 shows microarray data from field grown soybean
plants. Microarrays performed on tissues sampled throughout the day
from the field demonstrated that 219 genes showed 2-fold or greater
changes in abundance in transgenic events transformed with AtBBX32
relative to the control and that the majority of these changes
occurred around dawn.
[0025] FIGS. 12A-12E shows a phylogenetic tree of the Arabidopsis
thaliana and Glycine max B-box family, which contains 61 B-box
sequences from soybean and 32 from Arabidopsis. Phylogenetic
analysis of the B-box sequences shows that the sequences are
clustered into structural groups(St) and clades (C).H refers to
Arabidopsis homolog, N refers to other names.
[0026] FIGS. 13A-13D shows a phylogenetic tree for 61 B-box
sequences from soybean. Phylogenetic analysis of the B-box
sequences shows that these sequences are clustered into structural
groups (St) and chides (C).A Y (yes) under I indicates a punitive
interactor of AtBBX32 (I (At)) or GmBBX52 (I (Gm)) based on Y2H
analyses; T represents tissue of expression where L, I=leaf; C,
c=cotyledon; A, a=all tissues; S, s=seed; F, f=flowers; N,
n=nodules; P, p=pod; R, r=root; NR=not represented in TxP, where
bold upper-case letters represent high expression; upper-case
letters represent moderate expression, and lower-case letters
represent average expression. TP refers to time of peak expression.
SP refers to whether or not a shift in peak expression was observed
in transgenic soybean plants expressing BBX32 (Y for yes and N for
no).CD refers to at least 1.5 fold (except where *.about.1.2-1.4
fold) change in expression at dawn in transgenic soybean plants
expressing BBX32, where I=induced: S=suppressed. The first and
second circles represent the first and the second B-box domain
respectively; the rectangle represents the CCT domain.
[0027] FIG. 14 represents the single B-box clade Arabidopsis
thaliana and Glycine max. The Arabidopsis thaliana genome contains
seven single B-box domain genes white the paleopolyploid Glycine
max genome contains thirteen single B-box genes. Phylogenetic
analysis indicates that GmBBX52 and GmBBX53 are orthologs of the
Arabidopsis thaliana AtBBX32 gene.
[0028] FIG. 15 shows AtBBX32 and GmBBX62 interaction in vivo in
soybean cotyledon protoplasts Luminex co-immunoprecipitation
(co-IP) was performed using antibody against GET (A). The
reciprocal co-IP was conducted using antibody against Flag tag (B).
Components within the co-precipitated protein complexes were probed
with biotinylated anti-Flag (A) and anti-GFP (B) antibodies, and
subsequently detected with a fluorescently labeled R-phycoerythrin.
MFI stands for Median Fluorescence Intensity. **Mean differs
significantly from controls at p<0.05.
[0029] FIG. 16 demonstrates that AtBBX32 interacts directly with
GmBBX62 via in vitro pull-down assay. Pull-down reactions were
carried out using recombinant flag-GmBBX62 and soluble AtBBX32
extracts from wheat germ in vitro translation system. Protein bound
to flag-GmBBX62 was washed, separated on SDS-PAGE and immunoblotted
with anti-flag antibody (A) and anti-AtBBX32 antibody (B)-Lane
flag-GmBBX62; 1a, AtBBX32 wheat germ extract; 2, anti-flag antibody
bound beads only; 3, AtBBX32 only, no flag-GmBBX62; 4,
flag-GmBBX62+AtBBX32.
[0030] FIG. 17 shows AtBBX32 and GmBBX39 interaction in soybean
protoplasts. Luminex co-IP assays were performed on extracts from
soybean cotyledon protoplasts co-expressing AtBBX32 fused to GFP
and GmBBX39 fused to the Myc epitope. A: IP with anti-GFP,
detection with anti-Myc. B: IP with anti-Myc, detection with
anti-GFP. MFI stands for Median Fluorescence Intensity. *denotes
p<0.001.
DETAILED DESCRIPTION
[0031] The present disclosure relates to polynucleotides and
polypeptides for modifying phenotypes of plants, particularly those
associated with increased abiotic stress tolerance and increased
yield with respect to a control plant (for example, a wild-type
plant). 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, used to make and use the disclosed embodiments.
[0032] As used herein and in the appended claims, the singular
forms "an", and "the" include the plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a plant cell" includes a plurality of such plant cells, 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.
[0033] Definitions
[0034] "Polynucleotide" is a nucleic acid molecule comprising a
plurality of polymerized nucleotides, e.g., at least about 15
consecutive polymerized nucleotides. A polynucleotide may he 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, e.g., 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, clement, target, and probe and is
preferably single-stranded.
[0035] A "recombinant polynucleotide" is a polynucleotide that is
not in its native state, e.g., 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, e.g.,
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 riot in proximity For example,
the sequence at issue can be cloned into a vector, or otherwise
recombined with one or more additional nucleic acid.
[0036] 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, e.g., cell lysis,
extraction, centrifugation, precipitation, or the like.
[0037] "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 or folding to obtain a functional protein or
polypeptide. A gene may be isolated, partially isolated, or found
with an organism's genome. By way of example, a transcriptional
regulator gene encodes a transcriptional regulator polypeptide,
which may be functional or require processing to function as an
initiator of transcription.
[0038] 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). 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 sonic
genes that do not have an identifiable promoter). The function of a
gene may also be regulated by enhancers, operators, and other
regulatory elements.
[0039] A "polypeptide" is an amino acid sequence comprising a
plurality of consecutive polymerized amino acid residues e.g., at
least about 15 consecutive polymerized amino acid residues. In many
instances, a polypeptide comprises a polymerized amino acid residue
sequence that is a transcriptional regulator 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; (v) a
protein-protein interaction domain; (vi) 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.
[0040] "Protein" refers to an amino acid sequence, oligopeptide,
peptide, polypeptide>r portions thereof whether naturally
occurring or synthetic.
[0041] 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,
e.g., more than about 5% enriched, more than about 10% enriched, or
more than about 20%, or more than about 50%, or more, enriched,
i.e., alternatively denoted: 105%, 110%, 120%, 150% or more,
enriched 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, e.g., by any of the various protein purification
methods herein.
[0042] "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.
[0043] "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 between 0-100%. 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 or 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.
[0044] "Alignment" refers to a number of nucleotide bases or amino
acid residue sequences aligned by lengthwise comparison so that
components in common (i.e., nucleotide bases or amino acid residues
at corresponding positions) 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 of FIGS. 4A-4F 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 software, 1999 (Accelrys, Inc., San Diego, Calif.).
[0045] 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. A "B-box zinc finger"
domain, such as is found in a polypeptide member of B-box zinc
finger family, is an example of a conserved domain, With respect to
polynucleotides encoding presently disclosed polypeptides, a
conserved domain is preferably at least nine base pairs (bp) in
length. A conserved domain with respect to presently disclosed
polypeptides refers to a domain within a polypeptide family that
exhibits a higher degree of sequence homology, such as at least
about 56%, 57%. 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 96%, 97%, 98%, 99%, or about 100% sequence identity to a
conserved domain of a disclosed polypeptide SEQ ID NOs: 235-427).
Sequences that possess or encode for conserved domains that meet
these criteria of percentage identity, and that have comparable
biological activity to the present polypeptide sequences, thus
being members of the BBX32 clack polypeptides, are encompassed by
the present disclosure. 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 polypeptide 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 transcriptional regulator class, family or sub-family, or the
exact amino acids of a particular transcriptional regulator
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.
[0046] As one of ordinary skill in the art recognize, conserved
domains may be identified as regions or domains of identity to a
specific consensus sequence (see, for example, Riechmann et al.,
2000a, 2000b), Thus, by using alignment methods well known in the
art, the conserved domains of the plant polypeptides, for example,
for the B-box zinc finger proteins (Putterill et al., 1995), may be
determined.
[0047] The conserved domains for many of the disclosed polypeptide
sequences are listed in Table 1. Also, the polypeptides of Table 1
have conserved domains specifically indicated by amino acid
coordinate start and stop sites. A comparison of the regions of
these polypeptides allows one of skill in the art (see, for
example, Reeves and Nissen, 1990, 1995) to identify domains or
conserved domains for any of the polypeptides listed or referred to
in this disclosure.
[0048] "Complementary" refers to the natural hydrogen bonding by
base pair ins, between purines and pyrimidines.sub.-- For example,
the sequence A-L-G-T (5.fwdarw.3') forms hydrogen bonds with its
complements A-C-G-T (5.fwdarw.3') or A-C-C-U (5.fwdarw.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.
[0049] 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 disclosed, orthologous or paralogous
polynucleotides 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; Sambrook et al., 1989; and by
Haymes et al., 1985; which references are incorporated herein by
reference.
[0050] In general, stringency is determined by the temperature,
ionic strength, and concentration of denaturing agents (e.g.,
formamide) used in a hybridization and washing procedure (for a
more detailed description of establishing and determining
stringency, see the section "Identifying Polynucleotides or Nucleic
Acids by Hybridization", 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 related polynucleotide sequences. Numerous variations are
possible in the conditions and means by which nucleic acid
hybridization can be performed to isolate related polynucleotide
sequences having similarity to 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 polynucleotide sequences, such
as, for example, encoded transcriptional regulators having 56% or
greater identity with the conserved domain of disclosed
sequences.
[0051] The terms "paralog" and "ortholog" are defined below in the
section entitled "Orthologs and Paralogs". In brief, orthologs and
paralogs are evolutionarily related genes that have similar
sequences and 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.
[0052] 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".
[0053] In general, the term "variant" refers to molecules with some
differences, generated synthetically or naturally, in their base or
amino acid sequences as compared to a reference (native)
polynucleotide or polypeptide, respectively. These differences
include substitutions, insertions, deletions or any desired
combinations of such changes in a native polynucleotide of amino
acid sequence.
[0054] 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.
[0055] Also within the scope of the disclosed compositions and
methods is a variant of a 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 fisting, 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.
[0056] "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 terms refer to a polypeptide
encoded by an allelic variant of a gene.
[0057] "Splice variant" or "polynucleotide splice variant" as used
herein refers to alternative forms of RNA transcribed from a gene.
Splice variation naturally occurs as a37esult 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.
[0058] 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.
[0059] Differences between presently disclosed polypeptides and
polypeptide variants are limited so that the sequences of the
former and the latter are closely similar overall and, in many
regions, identical. 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 functionally
equivalent polypeptides. 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 disclosed polypeptides and
homolog polypeptides. 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
significant amount of the functional or biological activity of the
polypeptide 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. More rarely, a variant
may have "non-conservative" changes, e.g., 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 wilbout abolishing functional or biological activity may
be found using computer programs well known in the an, for example,
DNASTAR software (see U.S. Pat. No. 5,840,544).
[0060] "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 9 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 polynucleotides listed in the Sequence Listing.
Exemplary fragments also include fragments that comprise a region
that encodes a conserved domain of a polypeptide. Exemplary
fragments also include fragments that comprise a conserved domain
of a polypeptide. Exemplary fragments include fragments that
comprise a conserved domain of a polypeptide, for example, any of
SEQ ID NOs 235-427.
[0061] 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 sonic
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.
[0062] The term "plant" includes whole plants, shoot vegetative
orgauslstructures (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 disclosed methods 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 (sec for example, FIG. 1,
adapted from Daly et al., 2001, FIG. 2, adapted from Ku et al.,
2000; and see also Tudge, 2000).
[0063] A "control plant" as used in the present disclosure 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 disclosure that is expressed in the
transgenic or genetically modified plant being evaluated. A control
plant may in other cases be a progeny of a hemizygous transgenic
plant line that does not contain the recombinant DNA, known as a
negative segregant or negative isoline. 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.
[0064] 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.
[0065] A transgenic plant may contain an expression vector or
cassette. The expression cassette typically comprises a
polypeptide-encoding or polypeptide-suppressing sequence operably
linked to (i.e., under regulatory control of) appropriate
inducible, tissue-enhanced, tissue-specific, diurnally regulated or
constitutive regulatory sequences that allow for the controlled
expression of the 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, e.g., 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.
[0066] A constitutive promoter is active under most environmental
conditions and in most plant parts. Tissue-enhanced (also referred
to as tissue-preferred), tissue-specific, cell type-specific, and
inducible promoters constitute non-constitutive promoters.
Promoters under developmental control include promoters Mat
preferentially initiate transcription in certain tissues, such as
xylem, leaves, roots, or seeds. Such promoters are examples of
tissue-enhanced or tissue-preferred promoters (see U.S. Pat. No.
7,365,186). Tissue-enhanced promoters can be found upstream and
operatively linked to DNA sequences normally transcribed in higher
levels in certain plant tissues or specifically in certain plant
tissues, respectively. "Cell-enhanced", "tissue-enhanced", or
"tissue-specific" regulation thus refer to the control of gene or
protein expression, for example, by a promoter, which drives
expression that is not necessarily totally restricted to a single
type of cell or tissue, but where expression is elevated in
particular cells or tissues to a greater extent than in other cells
or tissues within the organism, and in the case of tissue-specific
regulation, in a manner that is primarily elevated in a specific
tissue. Tissue-enhanced or preferred promoters have been described
in, for example, U.S. Pat. No. 7,365,186, or U.S. Pat. No.
7,619,133.
[0067] A "condition-enhanced" promoter refers to a promoter that
activates a gene in response to a particular environmental
stimulus, for example, an abiotic stress, infection caused by a
pathogen, light treatment, chemical stimulus, etc., and that drives
expression in a unique pattern which may include expression in
specific cell and/or tissue types within the organism (as opposed
to a constitutive expression pattern in all cell types of an
organism at all times).
[0068] A "diurnal promoter" is useful for regulating changes in the
timing of gene expression to a specific time of day. Usually genes
that are diurnally-regulated peak at the same time in the day/night
cycle. Genes that are under the regulation of diurnal promoters
exhibit altered expression profiles under the control of a
circadian oscillator mostly during the switch time of light to dark
but also with diurnal oscillations that persist within a period
close to 24 hour day/night cycle. Diurnal regulation is subject to
environmental inputs such as light and temperature and the
coordination by the circadian clock.
[0069] "Wild type" or "wild-type" as used herein, refers to a plant
cell, seed, plant component, plant part, plant tissue, plant organ
or whole plant that has not been genetically modified or treated in
an experimental sense. Wild-type cells, seeds, components, parts,
tissues, 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 polypeptide's expression is altered, e.g., in that it has
been knocked out, overexpressed, or ectopically expressed.
[0070] 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, e.g. 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, e.g.,
by employing Northern analysis, RT-PCR, microarray gene expression
assays, or reporter gene expression systems, or by agricultural
observations such as hyperosmotic 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. "Trait
modification", "modified trait" or "altered trait" refers to a
detectable difference in a characteristic in a plant ectopically
expressing a polynucleotide or polypeptide of the present
disclosure 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, or an even greater
difference, its an observed trait as compared with a control or
wild-type plant. It is known that there can be natural variations
in the modified trait. Therefore, the trait modification observed
entails a change of the normal distribution and magnitude of the
trait in the plants as compared to control or wild-type plants.
[0071] When two or more plants have "similar morphologies",
"substantially similar morphologies", "a morphology that is
substantially similar", or are "morphologically similar", the
plants have comparable forms or appearances, including analogous
features such as overall dimensions, height, width, mass, root
mass, shape, glossiness, leaf color, stem diameter, leaf size, leaf
dimension, leaf density, internode distance or length, branching,
root branching, number and form of inflorescences, and other
macroscopic characteristics, and the individual plants are not
readily distinguishable based on morphological characteristics
alone.
[0072] "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.
[0073] 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 expression level of a set of genes in a cell in which
a particular polypeptide is overexpressed or suppressed can be
compared to the expression level of the same set of genes in a cell
that has normal levels of that polypeptide. 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.
[0074] With regard to gene knockouts as used herein, the term
"knockout" or "suppression" refers to a plant, plant cell, or plant
tissue having a disruption in at least one gene, where the
disruption results in a reduced or abolished expression or activity
of the polypeptide encoded by that gene compared to a control
plant, cell, or tissue. A knockout or suppression can occur in
different ways, including but not limited to: decreasing levels of
a protein; suppressing a mutation that has resulted in decreased
activity of a protein; suppressing the production of an inhibitory
agent; elevating, reducing or eliminating the level of substrate
that an enzyme requires for activity; producing a new protein; and
activating a normally silent gene, to accumulate a product that
does not normally increase under natural conditions. The suppressor
can he another mutation on a different gene, a suppressor mutation
on the same gene but located some distance from the first
:mutation, or a suppressor in the cytoplasm that has generated due
to a change in non-chromosomal DNA. The knockout can be the result
of, for example, genomic disruptions, including transposous,
tilling, and homologous recombination, antisense constructs, sense
constructs, RNA silencing constructs, or RNA interference. A T-DNA
insertion within a gene is an example of a genotypic: alteration
that may abolish expression of that gene. To knockout or suppress a
gene, a recombinant expression cassette can be made. For example,
the recombinant cassette can comprise a promoter that is functional
in a plant cell and that is operably-linked to a polynucleotide
that when expressed in a plant cell is transcribed into a
polynucleotide molecule that suppresses the level of an endogenous
protein in the plant cell relative to a control. The polynucleotide
molecule can be, for example, a dsRNA that is processed into siRNAs
that targets a messenger RNA encoding the protein; a miRNA that
targets a messenger RNA encoding the protein; a trans-acting small
interfering RNA (ta-siRNA) that is processed into siRNAs and that
targets a messenger RNA encoding the protein; and a cleavage
blocker of a miRNA or a miRNA decoy of a miRNA, which both lead to
decreased activity of a miRNA. Examples of such RNAi-mediated gene
suppression approaches are disclosed in US Patent Application
Publication 2009/61288019, which is incorporated herein by
reference.
[0075] "Ectopic expression or altered expression" in reference to a
polynucleotide indicates that the pattern of expression in, e.g., a
transgenic plant or plant tissue, is different from the expression
pattern in a wild-type plant or plant tissue or a reference plant
or plant tissue of the same species. For example, a. 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.
[0076] 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
polypeptides are under the control of a strong promoter (e.g., the
cauliflower mosaic virus 35S transcription initiation region).
Overexpression may also be under the control of a heterologous
promoter, or an inducible or tissue specific promoter. Thus,
overexpression may occur throughout a plant, in specific tissues of
the plant, or in the presence or absence of particular
environmental signals, depending on the promoter used.
Overexpression may take place in plant cells normally lacking
expression of polypeptides functionally equivalent or identical to
the present polypeptides. Overexpression may also occur in plant
cells where endogenous expression of the present polypeptides or
functionally equivalent molecules normally occurs, but such normal
expression is at a lower level. Overexpression thus results in a
greater titan normal production, or "overproduction" of the
polypeptide in the plant, cell or tissue. The term "transcription
regulating region" refers to a DNA regulatory sequence that
regulates the expression of one or more genes in a plant with a
transcriptional regulator having one or more specific binding
domains that binds to a DNA regulatory sequence. Transcriptional
regulators possess a conserved domain. The transcriptional
regulators 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
transcriptional regulator binds to the regulating region.
[0077] "Increased yield" or "improved yield" or "increased plant
yield" refers to increased plant growth, increased crop growth,
increased biomass, increased grain yield, and/or increased plant
product production, and is dependent to some extent on temperature,
plant size, organ size, planting density, light, water and nutrient
availability, and how the plant copes with various stresses, such
as through temperature acclimation and water or nutrient use
efficiency.
[0078] "Planting density" refers to the number of plants that can
be grown per acre. For crop species, planting or population density
varies from crop to crop, from one growing Legion to another, and
from year to year. Using corn as an example, the average prevailing
density in 2000 was in the range of 20,000-25,000 plants per acre
in Missouri, USA. A desirable higher population density (a measure
of yield) would be at least 22,000 plants per acre, and a more
desirable higher population density would he at least 28,000 plants
per acre, more preferably at least 34,000 plants per acre, and even
more preferably at least 40,000 plants per acre. The average
prevailing densities per acre of a few other examples of crop
plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000;
rice 650,000-900,000; soybean 150,000-200,000, canola
260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000
plants per acre (Cheikh et al., 2003, in U.S. Patent Application
No. 20030101479). A desirable higher population density for each of
these examples, as well as other valuable species of plants, would
be at least 10% higher than the average prevailing density or
yield.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Transcriptional Regulators Modify of Expression of Endogenous
Genes
[0079] A transcriptional regulator 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, transcriptional regulators 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 motif (see, for example, Riechmann et al.,
2000a). The plant transcriptional regulators of the present
disclosure belong to the. B-box zinc linger family (Putterill et
al., 1995) and are putative transcriptional regulators.
[0080] Generally, transcriptional regulators 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 osmotic stresses. The disclosed sequences 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.
[0081] The sequences of the present disclosure and closely related
orthologpus or paralogous sequences 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 disclosed sequences 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.
[0082] In addition to methods for modifying a plant phenotype by
employing one or more of the disclosed polynucleotides arid
polypeptides, the disclosed polynucleotides and polypeptides 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, e.g., mutation reactions, PCR
reactions, or the like; as substrates for cloning e.g., including
digestion or ligation reactions; and for identifying exogenous or
endogenous modulators of the transcriptional regulators. The
polynucleotide can be, e.g., 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.
[0083] Expression of genes that encode polypeptides that modify
expression of endogenous genes, polynucleotides, and proteins are
well known in the art. In addition, transgenic plants comprising
isolated polynucleotides encoding transcriptional regulators may
also modify expression of endogenous genes, polynucleotides, and
proteins. Examples include Pene et al., 1997, and Peng et al.,
1999). In addition, many others have demonstrated that an
Arabidopsis transcriptional regulator expressed in an exogenous
plant species elicits the same or very similar phenotypic response.
See, for example, Fu et al., 2001; Nandi et al., 2000; Coupland,
1995; and Weigel and Nilsson, 1995).
[0084] In another example, Mandel et al., 1992b, and Suzuki et al.,
2001, teach that a transcriptional regulator 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 transcriptional regulators in Arabidopsis (see
Mandel et al., 1992a; Suzuki et al., 2001). Other examples include
Miller et al., 2001; Kim et al., 2001; Kyozuka and Shintamoto,
2002; Boss and Thomas, 2002; He et al., 2000; and Robson et al.,
2001).
[0085] In yet another example, Gilmour et al., 1998, teach an
Arabidopsis AP2 transcriptional regulator, CRF1, which, when
overexpressed in transgenic plants, increased plant freezing
tolerance. Jaglo et al., 2001) further identified sequences in
Brassica napus which encode CBF-like genes and showed 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 which 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).
[0086] Transcriptional regulators mediate cellular responses and
control traits through altered expression of genes containing
cis-acting nucleotide sequences that are targets of the introduced
transcriptional regulator. It is well appreciated in the art that
the effect of a transcriptional regulator on cellular responses or
a cellular trait is determined by the particular genes whose
expression is either directly or indirectly (e.g., by a cascade of
transcriptional regulator binding events and transcriptional
changes) altered by transcriptional regulator binding. In a global
analysis of transcription comparing a standard condition with one
in which a transcriptional regulator is overexpressed, the
resulting transcript profile associated with transcriptional
regulator overexpression is related to the trait or cellular
process controlled by that transcriptional regulator. 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; and Borevitz et al 2000).
Consequently, it is evident to one skilled in the art that
similarity of transcript profile upon overexpression of different
transcriptional regulators would indicate similarity of
transcriptional regulator function.
The Disclosed Polypeptides and Polynucleotides
[0087] The present disclosure includes putative transcriptional
regulators, and isolated or recombinant polynucleotides encoding
the polypeptides, or novel sequence variant polypeptides or
polynucleotides encoding novel variants of polypeptides derived
from the specific sequences provided in the Sequence Listing; the
disclosed recombinant polynucleotides may be incorporated in
expression vectors for the purpose of producing transformed plants.
Also provided are methods for modifying yield from a plant by
modifying the mass, size or number of plant organs or seed of a
plant by controlling a number of cellular processes, and for
increasing a plant's resistance to abiotic stresses. These methods
are based on the ability to alter the expression of critical
regulatory molecules that may be conserved between diverse plant
species. Related conserved regulatory molecules may he originally
discovered in a model system such as Arabidopsis and homologous,
functional molecules then discovered in other plant species. The
latter may then be used to confer increased yield or abiotic stress
tolerance in diverse plant species.
[0088] Exemplary polynucleotides encoding the polypeptides provided
in Table 1 and Table 2 were identified in the Arabidopsis thaliana
GenBank database using publicly available sequence analysis
programs and parameters, and an in-house. proprietary database.
Sequences initially identified were then further characterized to
identify sequences comprising specified sequence strings
corresponding to sequence motifs present in families of known
polypeptides. Additional disclosed polynucleotides were identified
by screening Arabidopsis thaliana and/or other plant cDNA libraries
with probes corresponding to known polypeptides 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.
[0089] Many of the sequences in the Sequence Listing, derived from
diverse plant species, have been ectopically expressed in
overexpressor plants. The changes in the characteristic(s) or
trait(s) of the plants were then observed and found to confer
increased yield and/or increased abiotic stress tolerance.
Therefore, the polynucleotides and polypeptides can be used to
improve desirable characteristics of plants.
[0090] The disclosed polynucleotides were also 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 used to change expression
levels of genes, polynucleotides, and/or proteins of plants or
plant cells.
General Considerations Related to improving Plant Yield
[0091] The instant disclosure provides a novel method involving new
gene technologies to improve plant yield. It is expected that
either individually or in a combination with other genes or
promoters, these novel methods will provide significantly higher
improvements compared to previous methods.
[0092] Plants have evolved to sense the direction, color, intensity
and duration of the incumbent light signals. Plants use this
information to redirect their growth and developmental processes to
optimize light absorption by promoting elongation growth under low
light. Several genes have been implicated in playing roles in light
signaling mechanisms regulating plant growth and development. Here,
light responsiveness of young seedlings overexpressing some of the
individual B-box genes to determine their roles in light signaling
was examined. There is precedent that suppressing light signaling
leads to crop improvement (U.S. Pat. No. 7,692,067).
[0093] The instant disclosure identifies several Arabidopsis
thaliana B-box genes which can function negatively in light
signaling and it is expected that either independently or in
combination with other genes (such as with AtBBX32, GmBBX52 or
GmBBX53) or specific promoters, these polynucleotides and their
encoded polypeptides will provide significantly improved yield
benefits and resistance to abiotic stress. It is also expected that
these polynucleotides and their encoded polypeptides will provide
plants with altered levels of ureides, altered levels of hexose
sugars, altered SPS activity, altered levels of starch, and delayed
senescence (International Application No. PCT/US2012/029885). It is
further expected that the functionally similar orthologs or
paralogs of these polynucleotides and their encoded polypeptides,
either expressed individually or in combination with other
polynucleotides and their encoded polypeptides, will lead to plants
with higher yield advantages, improved stress tolerance, altered
levels of ureides, altered levels of hexose sugars, altered SPS
activity, altered levels of starch, and delayed senescence.
[0094] The instant disclosure also identifies several Glycine max
B-box genes. Some of the encoded polypeptides of these Glycine max
genes may interact with AtBBX32, or may be overexpressed or
suppressed, in transgenic soybean plants overexpressing AtBBX32.
Some of the encoded polypeptides of these Glycine max genes may
interact with GmBBX52 in transgenic soybean plants overexpressing
GmBBX52. Since expression of AtBBX32 and its soybean homologs,
GmBBX52 and GmBBX53, modulates a variety of traits including yield,
it is expected that either independently or in combination with
other genes (such as with AtBBX32, GmBBX52 or GmBBX53) or specific
promoters, altering the levels of these polynucleotides and their
encoded polypeptides will provide plants with significantly
improved yield benefits, resistance to abiotic stress, altered
levels of ureides, altered levels of hexose sugars, altered SPS
activity, altered levels of starch, and delayed senescence. It is
also expected that the functionally similar orthologs or paralogs
of these polynucleotides and their encoded polypeptides, either
expressed individually or in combination with other polynucleotides
and their encoded polypeptides, will lead to plants with higher
yield advantages, improved stress tolerance, altered levels of
ureides, altered levels of hexose sugars, altered SPS activity,
altered levels of starch, and delayed senescence.
Background Information for BBX32. the BBX32 Clade, and Related
Sequences
[0095] BBX32 is a B-box zinc finger protein with homology to the
CONSTANS family of transcriptional regulators. BBX32 is expressed
in many tissues and may be diurnally regulated.
[0096] As disclosed below in the Examples, constitutive expression
of BBX32 in Arabidopsis modulates plant growth processes, including
elongation of hypocotyls, extended petioles and upheld leaves,
early flowering; enhanced root and/or shoot growth in
phosphate-limited media; more secondary roots on control media,
enhanced growth and reduced anthocyanin in low nitrogen/high
sucrose media supplemented with glutamine, enhanced root growth on
salt-containing media, and enhanced root growth on polyethylene
glycol-containing media, as compared to control plants. BBX32
overexpression soybean plants has been shown to result in a
statistically significant increase in yield in field trials (see
U.S. Pat. No. 7,692,067) as compared to controls that do not
contain the BBX32 polynucleotide.
[0097] As noted in the Examples presented below, overexpression of
BBX32 in Arabidopsis produced several phenotypes consistent with a
role of this gene in light regulated development and possibly
diurnal or circadian regulated processes. Light exerts a major
influence in the initiation and maintenance (re-setting) of the
plant circadian processes. Early seedling development is light
dependent (de-etiolation) and is marked by inhibition of hypocotyl
cell elongation along with cotyledon expansion and greening. Light
controls morphological changes throughout the life of the plant,
either directly or by regulating circadian gene expression. Some of
these changes include petiole development, control of growth rate
and flowering. It is likely that the ectopic expression of BBX32
product affects light signaling and/or circadian processes. Based
upon the observations described above, it is clear that BBX32 is
involved in photo morphogenesis and regulating plant growth and
development. Hence, its overexpression may improve plant vigor,
thus explaining the yield enhancements seen in 35S::BBX32 soybean
plants.
TABLE-US-00001 TABLE 1 Conserved domains and potentially valuable
morphological traits of B-box sequences Column 7 Column 3 Percent
Column 1 Percent identity of Poly- identity of Column 4 Column 6
domain in peptide polypeptide Amino acid SEQ ID column 5 SEQ Column
2 to clade coordinates Column 5 NO: of (no. identical ID NO:
Species/GID No. identifier of domain Domain sequence domain
residues) AtBBX28 (G1481)clade identifier G1481): 2 At/G1481 100.0%
to 5-41 CDLCNGVARMYCESD 235 100% G1481 QASLCWDCDGKVHGA (37/37)
NFLVAKH 4 At/G900 48.4% to 6-42 CELCCGVARMYCESD 236 95% G1481
QASLCWDCDGKVHGA (37/37) NFLVAKH 6 Zm/ACN33981.1 35.3% to 28-64
CELCGAAARVYCGAD 237 65% G1481 EATLCWGCDAQVHGA (24/37) NFLVARH 8
Pt/Pt_568988 37.1% to 89-125 CELCGSSARMYCESD 238 84% G1481
QASLCWDCDEKVHTA (31/37) NFLVAKH 10 Pt/Pt_571834 26.5% to 4-40
CELCKNPARTYCESD 239 73% G1481 EANLCWNCDTKVHGA (27/37) NFLVARH 12
Pt/Pt_582065 35.4% to 4-40 CELCGSSARMFCESD 240 81% G1481
QASLCWDCDEKVHSA (30/37) NFLVAKH 14 Pt/Pt_758524 28.3% to 4-40
CELCDSFAQMHCESD 241 65% G1481 QAILCSACDAYVHSA (24/37) NFLAAKH 16
Os/Os08g08120.1 34.8% to 24-60 CELCGAAARVYCGAD 242 65% G1481
EATLCWGCDAQVHGA (24/37) NFLVARH 18 Os/Os08g08120.2 36.0% to 24-60
CELCGAAARVYCGAD 243 65% G1481 EATLCWGCDAQVHGA (24/37) NFLVARH 20
Os/Os08g08120.3 36.3% to 24-60 CELCGAAARVYCGAD 244 65% G1481
EATLCWGCDAQVHGA (24/37) VFLVARH 22 Gm/Glyma06g39810.1 41.0% to 8-44
CVLCEKRAMMLCDSD 245 70% G1481 QAKLCWECDEKVHSA (26/37) NFLVAKH 24
Gm/Glyma06g45620.1 38.9% to 4-40 CELCKVPARIFCESD 246 76% G1481
QASLCWDCDAKVHSA (27/37) NFLVARH 26 Gm/G4015 23.7% to 6-42
CELCHQLASLYCPSD 247 59% G1481 SAFLCFHCDAAVHAA (22/37) NFLVARH 28
Gm/Glyma09g35960.1 21.0% to 6-42 CALCKKRAMMLCDSD 248 70% G1481
QAKLCWECDEKVHSA (26/37) NFLVAKH 30 Gm/Glyma12g11230.1 37.1% to 4-40
CELCKVPARIFCESD 249 76% G1481 QASLCWDCDAKVHSA (28/37) NFLVARH 32
Gm/Glyma12g32470.1 41.5% to 4-40 CELCKLPARTFCESD 250 78% G1481
QASLCWDCDAKVHGA (29/37) NFLVARH 34 Gm/G4014 38.1% to 4-40
CELCNSPAKLFCESD 251 73% G1481 QASLCWKCDAKVHSA (27/37) NFLVTKH 36
Gm/G4016 39.6% to 4-40 CELCKLPARTFCESD 252 76% G1481
QASLCWDCDAKVHGA (28/37) NFLVERH 38 Gm/Glyma15g07140.1 37.2% to 4-40
CELCNSPAKLFCESD 253 73% G1481 QASLCWECDAKVHSA (27/37) NFLVTKH
AtBBX25 (G1894) clade (clase identifier G1394): 152 At/G329 69.2%
to 5-42.sup.a CDVCEKAPATVICCA 254 87% G1894 DEAALCPQCDIEIHA (33/38)
ANKLASKH 152 At/G329 69.2% to 57-94.sup.b CDICQEKAAFIFCVE 255 84%
G1894 DRALLCRDCDESIHV (32/38) ANSRSANH 154 At/G1894 100.0% to
5-42.sup.a CDVCEKAPATLICCA 256 100% G1894 DEAALCAKCDVEVHA (38/38)
ANKLASKH 154 At/G1894 100.0% to 57-94.sup.b CDICLEKAAFIFCVE 257
100% G1894 DRALLCRDCDEATHA (38/38) PNTRSANH 156 Zm/ACF84591.1 50.0%
to 5-42.sup.a CDACEGAAATVVCCA 258 82% G1894 DEAALCARCDVEIHA (31/38)
ANKLASKH 156 Zm/ACF84591.1 50.0% to 57-94.sup.b CDVCQEKAAFIFCVE 259
74% G1894 DRALFCQDCDEPIHV (28/38) PGTLSGNH 158 Zm/ACF86823.1 50.4%
to 5-42.sup.a CDACEGAAATVVCCA 260 82% G1894 DEAALCARCDVEIHA (31/38)
ANKLASKH 158 Zm/ACF86823.1 50.4% to 57-94.sup.b CDVCQEKAAFIFCVE 261
76% G1894 DRALFCRDCDEPIHV (29/38) PGTLSGNH 162 Zm/CAW36812.1 50.4%
to 5-42.sup.a CDACEGAAATVVCCA 262 82% G1894 DEAALCARCDVEIHA (31/38)
ANKLASKH 162 Zm/CAW36812.1 50.4% to 57-94.sup.b CDVCQEKAAFIFCVE 263
76% G1894 DRALFCRDCDEPIHV (29/38) PGTLSGNH 176 Pt/Pt_216458 45.4%
to 5-42.sup.a CDVCEKAPATVICCA 264 92% G1894 DEAALCEKCDIEVII (35/38)
AANKLASKH 176 Pt/Pt_216458 45.4% to 57-94.sup.b CDICQEKAAFIFCVE 265
76% G1894 DRALFCRDCDEPIHS (29/38) AGSLSANH 178 Pt/Pt_827145 62.7%
to 5-42.sup.a CDVCEKAPATVICCA 266 95% G1894 DEAALCAKCDIEVHA (36/38)
ANKLASKH 178 Pt/Pt_827145 62.7% to 57-94.sup.b CDICQEKAAFIFCVE 267
76% G1894 DRALFCRDCDEPIHS (29/38) AGSLSANH 180 Os/Os02g39360.1
49.6% to 5-42.sup.a CDACESAAAAVVCCA 268 79% G1894 DEAALCAACDVEVHA
(30/38) ANKLAGKH 180 Os/Os02g39360.1 49.6% to 57-94.sup.b
CDVCQEKAAFIFCVE 269 76% G1894 DRALFCRDCDEPIHV (29/38) PGTLSGNH 182
Os/Os04g41560.2 48.1% to 5-42.sup.a CDACEAAAATVVCCA 270 82% G1894
DEAALCARCDVEIHA (31/38) ANKLASKH 182 Os/Os04g41560.2 48.1% to
58-95.sup.b CDVCQEKAAFIFCVE 271 76% G1894 DRALFCRDCDEPIHV (29/38)
PGTLSGNH 184 Os/Os04g41560.4 41.8% to 5-42.sup.a CEACEAAAATVVCCA
272 82% G1894 DEAALCARCDVEIHA (31/38) ANKLASKH 184 Os/Os04g41560.4
41.8% to 58-95.sup.b CDVCQEKAAFIFCVE 273 76% G1894 DRALFCRDCDEPIHV
(29/38) PGTLSGNH 186 Gm/Glyma11g13570 58.9% to 5-42.sup.a
CDVCEKAPATVICCA 274 97% G1894 DEAALCAKCDVEVHA (37/38) ANKLASKH 186
Gm/Glyma11g13570.1 58.9% to 57-94.sup.b CDICQDKPAFIFCVE 275 68%
G1894 DRALFCKDCDEPIHL (26/38) ASSLSANH 188 Gm/Glyma12g05570.1 58.7%
to 5-42.sup.a CDVCEKAPATVICCA 276 97% G1894 DEAALCAKCDVEVHA (37/38)
ANKLASKH 188 Gm/Glyma12g05570.1 58.7% to 57-94.sup.b
CDICQDKPAFIFCVE 277 68% G1894 DRALFCKDCDEPIHL (26/38) ASSLSANH 190
Gm/Glyma13g41980.1 58.1% to 5-42.sup.a CDVCERAPATVICCA 278 95%
G1894 DEAALCAKCDVEVHA (36/38) ANKLASKH 190 Gm/Glyma13g41980.1 58.1%
to 57-94.sup.b CDICQDKPAFIFCVE 279 68% G1894 DRALFCQDCDEPIHS
(26/38) AGSLSANH 192 Gm/Glyma13g41908.2 39.1% to 24-57.sup.a
QDKPAFIFCVEDRAL 280 35% G1894 FCQDCDEPIHSAGSL (12/34) SANH 192
Gm/Glyma13g41908.2 39.1% to 25-57.sup.b DKPAFIFCVEDRALF 281 67%
G1894 CQDCDEPIHSAGSLS (22/33) ANH 194 Gm/Glyma13g41980.3 39.1% to
24-57.sup.a QDKPAFIFCVEDRAL 282 35% G1894 FCQDCDEPIHSAGSL (12/34)
SANH 194 Gm/Glyma13g41980.3 39.1% to 25-57.sup.b DKPAFIFCVEDRALF
283 67% G1894 CQDCDEPIHSAGSLS (22/33) ANH 196 Gm/Glyma15g03400.1
57.9% to 5-42.sup.a CDVCERAPATVICCA 284 95% G1894 DEAALCAKCDVEVHA
(36/38) ANKLASKII 196 Gm/Glyma15g03400.1 57.9% to 57-94.sup.b
CDICQDKPAFIFCVE 285 68% G1894 DRALFCQDCDEPIHS (26/38) AGSLSANH 198
Sl/AAS67368 58.2% to 5-42.sup.a CDVCEKAQATVICCA 286 92% G1894
DEAALCAKCDIEVHA (35/38) ANKLASKH 198 Sl/AAS67368 58.2% to
57-94.sup.b CDICQDKAAFIFCVE 287 68% G1894 DRALFCKDCDEAIHS (26/38)
ASSLAKNH AtBBX20 (G1888) clade (clade identifier G1888): 54
At/G1888 100.0% to 5-42.sup.a CAVCDKEEASVFCCA 288 100% G1888
DEAALCNGCDRHVHF (38/38) ANKLAGKH 54 At/G1888 100.0% to 58-95.sup.b
CDICGERRALLFCQE 289 100% G1888 DRAILCRECDIPIHQ (38/38) ANEHTKKH 56
At/G1482 37.4% to 5-42.sup.a CDVCDKEEASVFCTA 290 79% G1888
DEASLCGGCDHQVHH (30/38) ANKLASKH 56 At/G1482 37.4% to 60-97.sup.b
CDICQDKKALLFCQQ 291 74% G1888 DRAILCKDCDSSIHA (28/38) ANEHTKKH 58
Zm/ACG44133.1 40.7% to 5-42.sup.a CDVCAAEAASVFCCA 292 76% G1888
DEAALCDACDDRVHR (29/38) ANKLAGKH 58 Zm/ACG44133.1 40.7% to
61-98.sup.b CDICQERRGFLFCKE 293 71% G1888 DRAILCRECDAPVHS (27/38)
ANDMTRRH 60 Zm/CAW36814.1 44.4% to 5-42.sup.a CDVCAAEAASVFCCA 294
76% G1888 DEAALCDACDDRVHR (29/38) ANKLAGKH 60 Zm/CAW36814.1 44.4%
to 61-98.sup.b CDICQERRGFLFCKE 295 71% G1888 DRAILCRECDAPVHS
(27/38) ANDMTRRH 68 Pt/Pt_195637 51.0% to 5-42.sup.a
CDVCDNVEATVFCCA 296 76% G1888 DEAALCDGCDHRVHH (29/38) ANKLASKH 68
Pt/Pt_195637 51.0% to 58-95.sup.b CDICQERRALLFCQE 297 89% G1888
DRAILCRECDLPIHK (34/38) ANEHTQKH 70 Pt/Pt_550941 39.5% to
5-42.sup.a CDVCNKEEASVFCTA 298 76% G1888 DEAALCDTCDHRVHH (29/38)
ANKLASKH 70 Pt/Pt_550941 39.5% to 58-95.sup.b CDICQEKRAFLFCQQ 299
82% G1888 DRAILCRECDGPIHT (31/38) ANEHTQKH 72 Pt/Pt_801530 38.2% to
5-42.sup.a CDVCSKEEASVFCTA 300 76% G1888 DEAALCDTCDHRVHH (29/38)
ANKLASKH 72 Pt/Pt_801530 38.2% to 58-95.sup.b CDICQDKRAFLFCQQ 301
76% G1888 DRAILCRDCDGPIHT (29/38) ANEHTQKH 74 Pt/Pt_804174 54.5% to
5-42.sup.a CDVCDKCRATVFCCA 302 76% G1888 DEAALCDGCDHRVHH (29/38)
ANTLASKH 74 Pt/Pt_804174 54.5% to 58-95.sup.b CDICQERRAVLFCQE 303
84% G1888 DRAILCRECDLPIHK (32/38) VNEHTQKH 76 Os/Os02g43170.1 37.5%
to 5-42.sup.a CDVCAAEAASVFCCA 304 76% G1888 DEAALCDACDIIRVI (29/38)
IRANKLAGKH 76 Os/Os02g43170.1 37.5% to 65-102.sup.b CDICQEKRGFLFCKE
305 68% G1888 DRAILCRECDVPVHT (26/38) ASELTMRH 78 Os/Os02g43170.2
43.4% to 5-42.sup.a CDVCAAEAASVFCCA 306 76% G1888 DEAALCDACDHRVHR
(29/38) ANKLAGKH 78 Os/Os02g43170.2 43.4% to 65-102.sup.b
CDICQEKRGFLFCKE 307 68% G1888 DRAILCRECDVPVHT (26/38) ASELTMRH 80
Os/Os04g45690.1 37.7% to 5-42.sup.a CDVCAAEAASVFCCA 308 79% G1888
DEAALCDACDRRVHS (30/38) ANKLAGKH 80 Os/Os04g45690.1 37.7% to
63-100.sup.b CDICQEKRGFLFCKE 309 63% G1888 DRAILCRECDVTVHT (24/38)
TSRLTRRH 82 Os/G5159 37.7% to 5-42.sup.a CDVCAAEAASVFCCA 310 79%
G1888 DEAALCDACDRRVHS (30/38) ANKLAGKH 82 Os/G5159 37.7% to
63-100.sup.b CDICQEKRGFLFCKE 311 63% G1888 DRAILCRECDVTVHT (24/38)
TSELTRRH 84 Os/Os06g49880.1 31.8% to 5-42.sup.a CDVCAAEPAAVLCCA 312
68%
G1888 DEAALCSACDRRVHR (26/38) ANRLASKH 84 Os/Os06g49880.1 31.8% to
66-103.sup.b CDVCREKRGLVFCVE 313 66% G1888 DRAILCADCDEPIHS (25/38)
ANDLTAKH 86 Os/Os06g49880.2 31.9% to 5-42.sup.a CDVCAAEPAAVLCCA 314
68% G1888 DEAALCSACDRRVHR (26/38) ANRLASKH 86 Os/Os06g49880.2 31.9%
to 66-103.sup.b CDVCREKRGLVFCVE 315 66% G1888 DRAILCADCDEPIHS
(25/38) ANDLTAKH 88 G/mGlyma04g01120.1 40.1% to 5-42.sup.a
CAVCDKVEASVFCSA 316 79% G1888 DEAALCHSCDRTIHH (30/38) ANKLATKH 88
G/mGlyma04g01120.1 40.1% to 58-95.sup.b CDICQERRAYLFCQE 317 84%
G1888 DRALLCRECDVPIHR (32/38) ANEHTQKH 90 Gm/G5365 39.4% to
5-42.sup.a CDVCNKQASFFCTAD 318 71% G1888 EAALCDGCDHRVHHA (27/38)
NKLASKH 90 Gm/G5365 58-95.sup.b CDVCQERRAFVFCQQ 319 DRAILCKECDVPIHS
ANDLTKNH 92 Gm/Glyma04g02960.2 41.7% to 5-42.sup.a CDVCNKHQASFFCTA
320 71% G1888 DEAALCDGCDHRVHH (27/38) ANKLASKH 92
Gm/Glyma04g02960.2 41.7% to 58-95.sup.b CDVCQERRAFVFCQQ 321 71%
G1888 DRAILCKECDVPIHS (27/38) ANDLTKNH 94 Gm/Glyma06g01140.1 49.4%
to 5-42.sup.a CDVCDKVEASVFCPA 322 76% G1888 DEAALCHSCDRTIHR (29/38)
ANKLATKH 94 Gm/Glyma06g01140.1 49.4% to 58-95.sup.b CDICQERRAYLFCQE
323 84% G1888 DRALLCRECDVPIHR (32/38) ANEHTQKH 96 Gm/G5367 39.1% to
5-42.sup.a CDVCNKQQASLFCTA 324 71% G1888 DEAALCDGCDHRVHH (27/38)
ANKLASKH 96 Gm/G5367 39.1% to 58-95.sup.b CDVCQERRAFVFCQQ 325 68%
G1888 DRAILCKECDVPVHS (26/38) ANDLTKNH 98 Gm/Glyma11g12060.1 40.8%
to 5-42.sup.a CDVCHNEVASFFCPS 326 63% G1888 DEASLCHACDRTIHH (24/38)
ANKLADKH 98 Gm/Glyma11g12060.1 40.8% to 58-95.sup.b CDICHERRAYLFCKE
327 82% G1888 DRAILCRECDLSIHG (31/38) VNEHTKKH 100 Gm/G5396 43.0%
to 5-42.sup.a CDVCNKHEASVFCTA 328 76% G1888 DEAALCDGCDHRVHH (29/38)
ANKLASKH 100 Gm/G5396 43.0% to 58-95.sup.b CDICQERRAFTFCQQ 329 76%
G1888 DRAILCKECDVSIHS (29/38) ANEHTLKH 102 Gm/G5400 43.0% to
5-42.sup.a CDVCNKHEASVFCTA 330 76% G1888 DEAALCDGCDHRVHH (29/38)
ANKLASKH 102 Gm/G5400 43.0% to 58-95.sup.b CDICQERRAFTFCQQ 331 76%
G1888 DRAILCKECDVSIHS (29/38) ANEHTLKH AtBBX26 (G1486) clase (clade
identifier G1486): 52 At/G1486 100.0% to 5-41 CHTCRHVTAVIHCVT 332
100% G1486 EALNFCLTCDNLRHH (37/37) NNIHAEH AtBBX30 (G1478) or
AtBBX31 (G1929) clade (clade identifier G1478): 40 At/G1478 100.0%
to 32-68 CELCGENATVYCEAD 333 100% G1478 AAFLCRKCDRWVHSA (37/37)
NFLARRH 42 At/G1929 62.3% to 31-67 CELCDGDASVFCEAD 334 78% G1478
SAFLCRKCDRWVHGA (29/37) NFLAWRII 44 Pt/Pt_562313 43.2% to 32-68
CELCGSRASLYCQAD 335 73% G1478 DAFLCQKCDKWVHGA (27/37) NFLAQRH 46
Pt/Pt_594447 44.1% to 31-67 CELCGSRATLYCQAD 336 76% G1478
HAFLCQKCDGWVHGA (28/37) NFLALRH 48 Gm/Glyma12g32230.1 43.8% to
30-66 CELCGLQASLYCQAD 337 68% G1478 DAYLCKKCDKRVHEA (25/37) NFLALRH
50 Gm/G4019 40.6% to 30-66 CELCGLQASLYCQAD 338 70% G1478
DAYLCRKCDKRVHEA (26/37) NFLALRH AtBBX7 (G2440) or AtBBX8 (G1479)
clase (clade identifier G2440): 104 At/G2440 100.0% to 5-42.sup.a
CDFCGEQRSMVYCRS 339 100% G2440 DAACLCLSCDRSVHS (38/38) ANALSKRH 104
At/G2440 100.0% to 48-94.sup.b CERCNAQPATVRCVE 340 96% G2440
ERVSLCQNCDWSGHN (45/47) NSNNNNSSSSTSTQQ H 104 At/G2440 100.0% to
315-356.sup.c RNNAVMRYKEKKKAR 341 100% G2440 KFDKRVRYASRKARA
(42/42) DVRRRVKGRFVK 106 At/G1479 70.1% to 5-42.sup.a
CDFCGEQRSMVYCRS 342 97% G2440 DAACLCLSCDRNVHS (37/38) ANALSKRH 106
At/G1479 70.1% to 48-87.sup.b CERCNAQPASVRCSD 343 80% G2440
ERVSLCQNCDWSGHD (32/40) GKNSTTTSHH 106 At/G1479 70.1% to
316-357.sup.c RNNAVMRYKEKKKAR 344 95% G2440 KFDKRVRYVSRKERA (40/42)
DVRRRVKGRFVK 108 Zm/ACF80403.1 49.6% to 5-42.sup.a CGFCGKQRSMIYCRS
345 87% G2440 DAASLCLSCDRSVHS (33/38) ANALSRRH 108 Zm/ACF80403.1
49.6% to 48-85.sup.b CDRCGLQPASVRCLE 346 55% G2440 DNTSLCQNCDWNGHD
(21/38) AASGASGH 108 Zm/ACF80403.1 49.6% to 349-390.sup.c
RDSALTRYKEKKKKR 347 76% G2440 MFDKKIRYASRKARA (32/42) DVRKRVKGRFIK
110 Zm/ACG34885.1 50.4% to 5-42.sup.a CDFCGEQRSMVYCRS 348 92% G2440
DAASLCLSCDRNVHS (35/38) ANALSRRH 110 Zm/ACG34885.1 50.4% to
48-85.sup.b CERCASQPAMVRCLA 349 58% G2440 ENASLCQNCDWNGHI (22/38)
AGSSSAGH 110 Zm/ACG34885.1 50.4% to 350-391.sup.c RDSALTRYKEKKMRR
350 79% G2440 KFDKKIRYASRKARA (33/42) DVRKRVKGRFVK 114
Zm/ACN25813.1 47.1% to 5-42.sup.a CDFCGKQRSMIYCRS 351 87% G2440
DAASLCLSCDRNVHS (33/38) ANALSRRH 114 Zm/ACN25813.1 47.1% to
48-85.sup.b CDRCGSQPASVRCLE 352 55% G2440 DNASLCQNCDWNGHD (21/38)
AESGASGH 114 Zm/ACN25813.1 47.1% to 372-391.sup.c YASRKARADVRKRVK
353 90% G2440 GRFIK (18/20) 120 Pt/Pt_247140 59.7% to 5-42.sup.a
CDFCGEQRSMVYCRS 354 95% G2440 DAASLCLSCDRNVHS (36/38) ANALSKRH 120
Pt/Pt_247140 59.7% to 48-85.sup.b CERCNSQPALVRCAE 355 74% G2440
ERISLCQNCDWIGHG (28/38) TSTSASTH 120 Pt/Pt_247140 59.7% to
322-363.sup.c RSDAVMRYKEKKKTR 356 86% G2440 MFEKKVRYASRKARA (36/42)
DVRRRVKGRFVK 122 Pt/Pt_409151 59.9% to 5-42.sup.a CDFCGEQRSMVYCRS
357 95% G2440 DAACLCLSCDQIVHS (36/38) ANALSKRH 122 Pt/Pt_409151
59.9% to 48-85.sup.b CERCNSQPALVRRVE 358 71% G2440 ERISLCQNCDWMGYG
(27/38) SSTSASTH 122 Pt/Pt_409151 59.9% to 372-391.sup.c
RSDAVKRYMEKKKTR 359 83% G2440 KFEKKVRYASRKARA (35/42) DVRRRVKGRFVK
124 Os/Os02g49230.1 51.7% to 5-42.sup.a CDFCREQRSMVYCRS 360 89%
G2440 DAASLCLSCDRNVHS (34/38) ANALSRRH 124 Os/Os02g49230.1 51.7% to
48-85.sup.b CDRCVGQPAAVRCLE 361 61% G2440 ENTSLCQNCDWNGHG (23/38)
AASSAAGH 124 Os/Os02g49230.1 51.7% to 350-391.sup.c RDNALTRYKEKKKRR
362 83% G2440 KFDKKIRYASRKARA (35/42) DVRKRVKGRFVK 126
Os/Os02g49230.2 44.2% to 78-115.sup.a CDFCREQRSMVYCRS 363 89% G2440
DAASLCLSCDRNVHS (34/38) ANALSRRH 126 Os/Os02g49230.2 44.2% to
121-158.sup.b CDRCVGQPAAVRCLE 364 61% G2440 ENTSLCQNCDWNGHG (23/38)
AASSAAGH 126 Os/Os02g49230.2 44.2% to 423-464.sup.c RDNALTRYKEKKKRR
365 83% G2440 KFDKKIRYASRKARA (35/42) DVRKRVKGRFVK 128
Os/Os02g49230.3 51.7% to 5-42.sup.a CDFCREQRSMVYCRS 366 89% G2440
DAASLCLSCDRNVHS (34/38) ANALSRRH 128 Os/Os02g49230.3 51.7% to
48-85.sup.b CDRCVGQPAAVRCLE 367 61% G2440 ENTSLCQNCDWNGHG (23/38)
AASSAAGH 128 Os/Os02g49230.3 51.7% to 350-391.sup.c RDNALTRYKEKKKRR
368 83% G2440 KFDKKIRYASRKARA (35/42) DVRKRVKGRFVK 130
Os/Os02g49230.4 51.7% to 5-42.sup.a CDFCREQRSMVYCRS 369 89% G2440
DAASLCLSCDRNVHS (34/38) ANALSRRH 130 Os/Os02g49230.4 51.7% to
48-85.sup.b CDRCVGQPAAVRCLE 370 61% G2440 ENTSLCQNCDWNGHG (23/38)
AASSAAGH 130 Os/Os02g49230.4 51.7% to 350-391.sup.c RDNALTRYKEKKKRR
371 83% G2440 KFDKKIRYASRKARA (35/42) DVRKRVKGRFVK 132
Os/Os02g49230.5 51.8% to 5-42.sup.a CDFCREQRSMVYCRS 372 89% G2440
DAASLCLSCDRNVHS (34/38) ANALSRRH 132 Os/Os02g49230.5 51.8% to
48-85.sup.b CDRCVGQPAAVRCLE 373 61% G2440 ENTSLCQNCDWNGHG (23/38)
AASSAAGH 132 Os/Os02g49230.5 51.8% to 349-390.sup.c RDNALTRYKEKKKRR
374 83% G2440 KFDKKIRYASRKARA (35/42) DVRKRVKGRFVK 134
Os/Os02g49230.6 51.7% to 5-42.sup.a CDFCREQRSMVYCRS 375 89% G2440
DAASLCLSCDRNVHS (34/38) ANALSRRH 134 Os/Os02g49230.6 51.7% to
48-85.sup.b CDRCVGQPAAVRCLE 376 61% G2440 ENTSLCQNCDWNGHG (23/38)
AASSAAGH 134 Os/Os02g49230.6 51.7% to 350-391.sup.c RDNALTRYKEKKKRR
377 83% G2440 KFDKKIRYASRKARA (35/42) DVRKRVKGRFVK 136
Os/Os06g19444.1 51.7% to 5-42.sup.a CDFCGEQRSMVYCRS 378 92% G2440
DAASLCLSCDRNVHS (35/38) ANALSRRH 136 Os/Os06g19444.1 51.7% to
48-85.sup.b CDRCASQPAMVRCLV 379 58% G2440 ENASLCQNCDWNGHS (22/38)
AGSSAAGH 136 Os/Os06g19444.1 51.7% to 373-392.sup.c YASRKARADVRKRVK
380 95% G2440 GRFVK (19/20) 138 Os/Os06g19444.2 51.8% to 5-42.sup.a
CDFCGEQRSMVYCRS 381 92% G2440 DAASLCLSCDRNVHS (35/38) ANALSRRH 138
Os/Os06g19444.2 51.8% to 48-85.sup.b CDRCASQPAMVRCLV 382 58% G2440
ENASLCQNCDWNGHS (22/38) AGSSAAGH 138 Os/Os06g19444.2 51.8% to
372-391.sup.c YASRKARADVRKRVK 383 95% G2440 GRFVK (19/20) 140
Gm/Glyma02g38870.1 56.3% to 5-42.sup.a CDFCGDQRSLVYCRS 384 87%
G2440 DSACLCLSCDRNVHS (33/38) ANALSRRH 140 Gm/Glyma02g38870.1 56.3%
to 48-85.sup.b CERCNSQPAFVRSVE 385 68% G2440 EKISLCQNCDWLGHG
(26/38) TSPSSSMH 140 Gm/Glyma02g38870.1 56.3% to 348-389.sup.c
RSNAVMRYKEKKKTR 386 88% G2440 KFEKKVRYASRKARA (37/42) DVRKRVKGRFVK
142 Gm/Glyma13g1590.1 36.6% to G2440 142 Gm/Glyma13g1590.1 36.6% to
G2440 142 Gm/Glyma13g1590.1 36.6% to 224-365.sup.c RSNAVMRYKEKKKTR
387 90% G2440 KFDKKVRYASRKARA (38/42) DVRRRVKGRFVK 144
Gm/Glyma14g36930.1 55.3% to 5-42.sup.a CDFCGDQRSLVYCRS 388 89%
G2440 DAACLCLSCDRNVHS (34/38) ANALSRRH 144 Gm/Glyma14g36930.1 55.3%
to 48-85.sup.b CERCNSQPAFVRCVD 389 68% G2440 EKISLCQNCDWLGHG
(26/38) TSPSSSTH 144 Gm/Glyma14g36930.1 55.3% to 354-395.sup.c
RSNAVMRYKEKKKTR 390 88% G2440 KFEKKVRYASRKARA (37/42) DVRKRVKGRFVK
146 Gm/Glyma14g36930.2 55.3% to 5-42.sup.a CDFCGDQRSLVYCRS 391 89%
G2440 DAACLCLSCDRNVHS (34/38) ANALSRRH 146 Gm/Glyma14g36930.2 55.3%
to 48-85.sup.b CERCNSQPAFVRCVD 392 68% G2440 EKISLCQNCDWLGHG
(26/38) TSPSSSTH 146 Gm/Glyma14g36930.2 55.3% to 354-395.sup.c
RSNAVMRYKEKKKTR 393 88%
G2440 KFEKKVRYASRKARA (37/42) KVRKRVKGRFVK 148 Gm/Glyma20g07050.1
37.4% to G2440 148 Gm/Glyma20g07050.1 37.4% to G2440 148
Gm/Glyma20g07050.1 37.4% to 226-267.sup.c RSNAVMRYKEKKKTR 394 90%
G2440 MFDKKVRYASRKARA (38/42) DVRRRVKGRFVK 150 Gm/Glyma20g07050.2
37.4% to G2440 150 Gm/Glyma20g07050.2 37.4% to G2440 150
Gm/Glyma20g07050.2 37.4% to 226-267.sup.c RSNAVMRYKEKKKTR 395 90%
G2440 MFDKKVRYASRKARA (38/42) DVRRRVKGRFVK AtBBX19 (G902) or
AtBBX18 (G1881) clase (clade identifier G902): 200 At/G902 100.0%
to 5-42.sup.a CDACENAAAHFCAAD 396 100% G902 EAALCRPCDEKVHMC (38/38)
NKLASRH 200 At/G902 100.0% to 56-91.sup.b CDICENAPAFFYCEI 397 100%
G902 DGSSLCLQCDMVVHV (36/36) GGKRTH 202 At/G1881 70.1% to
5-42.sup.a CDACESAAAIVFCAA 398 87% G902 DEAALCCSCDEKVHK (33/38)
CNKLASRH 202 At/G1881 70.1% to 56-91.sup.b CDICENAPAFFYCEI 399 100%
G902 DGSSLCLQCDMVVHV (36/36) GGKRTH 204 Zm/ACF85933.1 57.5% to
5-42.sup.a CDVCESAPAVLFCAA 400 87% G902 DEAALCRPCDEKVHM (33/38)
CNKLASRH 204 Zm/ACF85933.1 57.5% to 56-91.sup.b CDICENSPAFFYCEI 401
89% G902 DGTSLCLSCDMTVHV (32/36) GGKRTH 208 Zm/ACL52378.1 57.8% to
5-42.sup.a CDVCESAPAVLFCAA 402 87% G902 DEAALCRPCDEKVHM (33/38)
CNKLASRH 208 Zm/ACL52378.1 57.8% to 56-91.sup.b CDICENSPAFFYCEI 403
89% G902 DGTSLCLSCDMTVHV (32/36) GGKRTH 212 Pt/Pt_648693 61.4% to
5-42.sup.a CDVCESAAAILFCAA 404 89% G902 DEAALCRSCDEKVHM (34/38)
CNKLASRH 212 Pt/Pt_648693 61.4% to 56-91.sup.b CDICEKAPAFFYCEI 405
94% G902 DGSSLCLQCDMIVHV (34/36) GGKRTH 214 Pt/Pt_721866 61.0% to
5-42.sup.a CDVCESAAAILFCAA 406 87% G902 DEAALCRSCDEKVHL (33/38)
CNKLASRH 214 Pt/Pt_721866 61.0% to 56-91.sup.b CDICENAPAFFYCEI 407
97% G902 DGSSLCLQCDMIVHV (35/36) GGKRTH 216 Pt/Pt_741179 54.5% to
5-42.sup.a CDACESAFAIVFCAA 408 84% G902 DEAALCLACDKKVHM (32/38)
CNKLASRH 216 Pt/Pt_741179 54.5% to 56-91.sup.b CDICENAPAFFYCET 409
94% G902 DGSSLCLQCDMTVHV (34/36) GGKRTH 218 Pt/Pt_820080 56.2% to
5-42.sup.a CDACESAAAIVFCAA 410 89% G902 DEAALCLACDEKVHM (34/38)
CMKLASRH 218 Pt/Pt_820080 56.2% to 56-91.sup.b CDICENAPAFFYCET 411
94% G902 DGSSLCLQCDMTVHV (34/36) GGKRTH 220 Os/Os09g35880.1 57.1%
to 5-42.sup.a CDVCESAPAVLFCVA 412 79% G902 DEAALCRSCDEKVHM (30/38)
CNKLARRH 220 Os/Os09g35880.1 57.1% to 56-91.sup.b CDICENAPAFFYCEI
413 92% G902 DGTSLCLSCDMTVHV (33/36) GGKRTH 222 Gm/Glyma01g37370.1
55.2% to 5-42.sup.a CDACESAAAIVFCAA 414 92% G902 DEAALCRACDEKVHM
(35/38) CNKLASRH 222 Gm/Glyma01g37370.1 55.2% to 56-91.sup.b
CDICENAPAFFYCET 415 94% G902 DGSSLCLQCDMIVHV (34/36) GGKRTH 224
Gm/Glyma11g07930.1 56.7% to 5-42.sup.a CDACESAAAIVFCAA 416 92% G902
DEAALCRACDEKVHM (35/38) CNKLASRH 224 Gm/Glyma11g07930.1 56.7% to
56-91.sup.b CDICENAPAFFYCET 417 94% G902 DGSSLCLQCDMIVHV (34/36)
GGKRTH 226 Gm/Glyma11g07930.2 55.7% to 5-42.sup.a CDACESAAAIVFCAA
418 92% G902 DEAALCRACDEKVHM (35/38) CNKLASRH 226
Gm/Glyma11g07930.2 55.7% to 56-91.sup.b CDICENAPAFFYCET 419 94%
G902 DGSSLCLQCDMIVHV (34/36) GGKRTH 228 Gm/Glyma11g07930.3 55.7% to
5-42.sup.a CDACESAAAIVFCAA 420 92% G902 DEAALCRACDEKVHM (35/38)
CNKLASRH 228 Gm/Glyma11g07930.3 55.7% to 56-91.sup.b
CDICENAPAFFYCET 421 94% G902 DGSSLCLQCDMIVHV (34/36) GGKRTH 230
Gm/Glyma11g07930.4 56.2% to 5-42.sup.a CDACESAAAIVFCAA 422 92% G902
DEAALCRACDEKVHM (35/38) CMKLASRH 230 Gm/Glyma11g07930.4 56.2% to
56-91.sup.b CDICENAPAFFYCET 423 94% G902 DGSSLCLQCDMIVHV (34/36)
GGKRTH 232 Gm/Glyma11g11850.1 58.0% to 5-42.sup.a CDVCESAAAILFCAA
424 82% G902 DEAALCSACDHKIHM (31/38) CNKLASRH 232
Gm/Glyma11g11850.1 58.0% to 56-91.sup.b CDICENAPAFFYCEI 425 97%
G902 DGSSLCLQCDMIVHV (35/36) GGKRTH 234 Gm/Glyma12g04130.1 62.2% to
5-42.sup.a CDVCESAAAIVFCAA 426 82% G902 DEAALCSACDHKIHM (31/38)
CNKLASRH 234 Gm/Glyma12g04130.1 62.2% to 56-91.sup.b
CDICENAPAFFYCEI 427 97% G902 DGSSLCLQCDMIVHV (35/36) GGKRTH Species
abbreviations for Table 1: At-Arabidopsis thaliana; Gm-Glycine max;
Os-Oryza sativa; Pt-Populus trichocarpa; S1-Solanum lycopersicum;
Zm-Zea mays. .sup.afirst B-box type ZF domain .sup.bsecond B-box
type ZF domain .sup.cputative CCT nuclear localization domain
[0098] Exemplary polynucleotides encoding the polypeptides of
soybean B-box proteins (GmBBX) as represented in Table 2 were
identified from yeast two hybrid screens and from phylogenetic
analysis.
TABLE-US-00002 TABLE 2 Glycine max B-box polynucleotide and
polypeptide sequences (GmBBX). Column 1 Column 2 Polynucleotide
Polypeptide Column 3 Column 4 SEQ ID NO: SEQ ID NO: Gene Name
Domains 435 436 GmBBX1 zf-B_box[2]::CCT 437 438 GmBBX2
zf-B_box[2]::CCT 439 440 GmBBX3 zf-B_box[2]::CCT 441 442 GmBBX4
zf-B_box[2]::CCT 443 444 GmBBX5 zf-B_box[2]::CCT 445 446 GmBBX6
zf-B_box[2]::CCT 447 448 GmBBX7 zf-B_box[2] 449 450 GmBBX11
zf-B_box[2]::CCT 451 452 GmBBX12 zf-B_box[2]::CCT 453 454 GmBBX13
zf-B_box[1]::CCT 455 456 GmBBX14 zf-B_box[1]::CCT 457 458 GmBBX15
zf-B_box[1]::CCT 459 460 GmBBX16 zf-B_box[1]::CCT 461 462 GmBBX17
zf-B_box[1]::CCT 463 464 GmBBX18 zf-B_box[1]::CCT 465 466 GmBBX19
zf-B_box[2]::CCT 467 468 GmBBX20 zf-B_box[2]::CCT 469 470 GmBBX21
zf-B_box[2]::CCT 471 472 GmBBX22 zf-B_box[2]::CCT 473 474 GmBBX23
zf-B_box[2] 475 476 GmBBX24 zf-B_box[1]::CCT 477 478 GmBBX25
zf-B_box[2]::CCT 479 480 GmBBX26 zf-B_box[2]::CCT 481 482 GmBBX27
zf-B_box[2] 483 484 GmBBX28 zf-B_box[2] 485 486 GmBBX29
zf-B_box[2]::CCT 487 488 GmBBX30 zf-B_box[2]::CCT 489 490 GmBBX31
zf-B_box[2] 491 492 GmBBX33 zf-B_box[2] 493 494 GmBBX34 zf-B_box[2]
495 496 GmBBX35 zf-B_box[2] 497 498 GmBBX36 zf-B_box[2] 499 500
GmBBX37 zf-B_box[2] 501 502 GmBBX38 zf-B_box[2] 503 504 GmBBX39
zf-B_box[2] 505 506 GmBBX40 zf-B_box[2] 507 508 GmBBX41 zf-B_box[2]
509 510 GmBBX42 zf-B_box[2] 511 512 GmBBX43 zf-B_box[2] 513 514
GmBBX44 zf-B_box[2] 515 516 GmBBX45 zf-B_box[2] 517 518 GmBBX46
zf-B_box[2] 519 520 GmBBX47 zf-B_box[2] 521 522 GmBBX48 zf-B_box[2]
523 524 GmBBX49 zf-B_box[2] 525 526 GmBBX50 zf-B_box[1] 527 528
GmBBX51 zf-B_box[1] 529 530 GmBBX52 zf-B_box[1] 531 532 GmBBX53
zf-B_box[1] 533 534 GmBBX54 zf-B_box[1] 535 536 GmBBX55 zf-B_box[1]
537 538 GmBBX56 zf-B_box[1] 539 540 GmBBX57 zf-B_box[1] 541 542
GmBBX58 zf-B_box[1] 543 544 GmBBX59 zf-B_box[1] 545 546 GmBBX60
zf-B_box[1] 547 548 GmBBX61 zf-B_box[1] 549 550 GmBBX62 zf-B_box[1]
Abbreviations for Table 2 zf-B_box[1]: first B-box type zinc finger
domain zf-B_box[2]: second B-box type zinc finger domain CCT:
nuclear localization domain Gm: Glycine max
Orthologs and Paralogs
[0099] 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. General methods for
identifying orthologs and paralogs, including phylogenetic methods,
sequence similarity and hybridization methods, are described
herein; an ortholog or paralog, including equivalogs, may be
identified by one or more of the methods described below.
[0100] As described by Eisen, 1998, evolutionary information may be
used to predict gene function. It is common for groups of genes
that are homologous in sequence to have diverse, although. usually
related, functions. However, in many cases, the identification of
homologs is not sufficient to make specific predictions because not
all homologs have the same function. Thus, an initial analysis of
functional relatedness based on sequence similarity alone may not
provide one with a means to determine where similarity ends and
functional relatedness begins. Fortunately, it is well known in the
art that protein function can be classified using phylogenetic
analysis of gene trees combined with the corresponding species.
Functional predictions can be greatly improved by focusing on how
the genes became similar in sequence (i.e., by evolutionary
processes) rather than on the sequence similarity itself (Eliseo,
1998). in fact, many specific examples exist in which gene function
has been shown to correlate well with gene phylogeny (Eisen, 1998).
Thus, "[t]he first step in making functional predictions is the
generation of a phylogenetic tree representing, the evolutionary
history of the gene of interest and its homologs. Such trees are
distinct from clusters and other means of characterizing sequence
similarity because they are inferred by techniques that help
convert patterns of similarity into evolutionary relationships . .
. After the gene tree is inferred, biologically determined
functions of the various homologs are overlaid onto the tree.
Finally, the structure of the tree and the relative phylogenetic
positions of genes of different functions are used to trace the
history of functional changes, which is then used to predict
functions of [as yet] uncharacterized genes" (Eisen, supra).
[0101] 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 chide (a group of similar genes) when a gene family phylogeny
is analyzed using programs such as CLUSTAL, (Thompson et al., 1994;
Higgins et al., 1996). Groups of similar genes can also be
identified with pair-wise BLAST analysis (Feng and Doolittle,
1987). For example, a clade of very similar MADS domain
transcriptional regulators from Arabidopsis all share a common
function in flowering time (Ratcliffe et al., 2001), and a group of
very similar AP2 domain transcriptional regulators from Arabidopsis
are involved in tolerance of plants to freezing (Gilmour et al.,
1998). Analysis of groups of similar genes with similar function
that fall within one elude 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
Glade may contain paralogous sequences, or orthologous sequences
that share the same function (see also, for example, Mount,
2001).
[0102] Transcriptional regulator gene sequences are conserved
across diverse eukaryotic species lines (Goodrich et al., 1993; Lin
et al., 1991; Sadowski et al., 1988). Plants are no exception to
this observation; diverse plant species possess transcriptional
regulators that have similar sequences and functions. Speciation,
the production of new species from a parental species, gives 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;
Higgins et al., 1996) 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 he deduced from the identified function of the
reference sequence.
[0103] By using a phylogenetic analysis, one skilled in the art
would recognize that the ability to predict similar functions
conferred by closely-related polypeptides is predictable. This
predictability has been confirmed by our own many studies in which
we have found that a wide variety of polypeptides have orthologous
or closely-related homologous sequences that function as does the
first, closely-related reference sequence. For example, distinct
transcriptional regulators, including:
[0104] (i) AP2 family Arabidopsis G47 (found in U.S. Pat. No.
7,135,616), a phylogenetically-related sequence from soybean, and
two phylogenetically-related homologs from rice all can confer
greater tolerance to drought, hyperosmotic stress, or delayed
flowering as compared to control plants;
[0105] (ii) CAAT family Arabidopsis G481 (found in PCT patent
publication WO2004076638), and numerous phylogenetically-related
sequences from dicots and monocots can confer greater tolerance to
drought-related stress as compared to control plants;
[0106] (iii) Myb-related Arabidopsis G682 (found in PCT patent
publication WO2004076638) and numerous phylogenetically-related
sequences from dicots and monocots can confer greater tolerance to
heat, drought-related stress, cold , and salt as compared to
control plants;
[0107] (iv) WRKY family Arabidopsis G1274 (found in U.S. Pat. No.
7,196,245) and numerous closely-related sequences from dicots and
monocots have been shown to confer increased water deprivation
tolerance, and
[0108] (v) AT-hook family soy sequence G3456 (found in US Patent
publication US20040128712A1) and numerous phylogenetically-related
sequences from dicots and monocots, increased biomass compared to
control plants when these sequences are overexpressed in
plants.
[0109] The polypeptides sequences belong to distinct clades of
polypeptides that include members from diverse species. In each
case, most or all of the clade member sequences derived from both
dicots and monocots have been shown to confer increased yield or
tolerance to one or more abiotic stresses when the sequences were
overexpressed. These studies each demonstrate that evolutionarily
conserved genes from diverse species are likely to function
similarly (i.e., by regulating similar target sequences and
controlling the same traits), and that polynucleotides from one
species may be transformed into closely-related or
distantly-related plant species to confer or improve traits.
[0110] As shown in Table 1 and Table 2, polypeptides that are
phylogenetically related to disclosed polypeptides, including SEQ
ID NOs: 2n, where n=1 to 117 or 218 to 275, may have conserved
domains that share at least 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about
100% amino acid sequence identity to the, listed sequences, for
example, SEQ ID NOs: 235-427, and have similar functions in that
the disclosed and closely related polypeptides may, when
overexpressed, confer at least one trait l5 selected from the group
consisting of decreased sensitivity to light, increased yield,
greater height, greater stem diameter, greater resistance to
lodging, increased secondary rooting, greater cold tolerance,
greater tolerance to water deprivation, reduced stomatal
conductance, altered C/N sensing, increased low nitrogen tolerance,
greater late season growth and vigor, greater number of primary
nodes, greater late season canopy coverage, increased tolerance to
hyperosmotic stress, altered levels of ureides, altered levels of
hexose sugars, altered SPS activity, altered levels of starch, and
delayed senescence, as compared to a control plant.
[0111] At the nucleotide level, sequences that are closely related
to the disclosed polynucleotide sequences will typically share at
least about 30% or at least 40%, at least 41%, at least 42%, at
least 43%, at least 44%, at least 45%, at least 46%, at least 4'7%,
at least 48%, or at least 49% nucleotide sequence identity,
preferably at least about 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%, or at least 59% nucleotide sequence identity, or at
least 60%, or at least 61%, or at least 62%, or at least 63%, or at
least 64%, or at least 65%, or at least 66%, or at least 67%, or at
least 68%, or at least 69%, or at least 70%, or at least 71%, or at
least 72%, or at least 73%, or at least 74%, or at least 75%, or at
least 76%, or at least 77%, or at least 78%, or at least 79%, or at
least 80%, or at least 81%, or at least 82%, or at least 83%, or at
least 84%, or at least 85%, or at least 86%, or at least 87%, or at
least 88%, or at least 89%, or at least 90%, or at least 91%, or at
least 92%, or at least 93%, or at least 94%, or at least 95%, or at
least 96%, or at least 9'7%, or at least 98%, or at least 99%, or
about 100% sequence identity to one or more of the listed
full-length polynucleotide sequences, or to a polynucleotide
sequence encoding a conserved domain of a listed polypeptide
sequence. 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.
Polynucleotides that encode polypeptides that are closely-related,
that is, orthologous or paralogous, to the disclosed polypeptide
sequences will be identifiable by having at least any of the
percentage identities provided in this paragraph.
[0112] At the polypeptide level, sequences that are closely related
to the disclosed polypeptide sequences will typically share at
least about 30% or at least 40%, at least 41%, at least 42%, at
least 43%, at least 44%, at least 48%, at least 46%, at least 47%,
at least 48%, or at least 49% amino acid sequence identity,
preferably at least about 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%, or at least 59';',) nucleotide sequence identity, or at
least 60%, or at least 61%, or at least 62%, or at least 63%, or at
least 64%, or at least 65%, or at least 66%, or at least 67%, or at
least 68%, or at least 69%, or at least 70%, or at least 71%, or at
least 72%, or at least 73%, or at least 74%, or at least 75%, or at
least 76%, or at least 77%, or at least 78%, or at least 79%, or at
least 80%, or at least 81%, or at least 82%, or at least 83%, or at
least 84%, or at least 85%, or at least 86%, or at least 87%, or at
least 88%, or at least 89%, or at least 90%, or at least 91%, or at
least 92%, or at least 93%, or at least 94%, or at least 95%, or at
least 96%, or at least 97%, or at least 98%, or at least 99%, or
about 100% sequence identity to one or more of the listed
full-length polypeptide sequences, or to conserved domain of a
listed full length sequence, or to a listed conserved domain, or to
a listed polypeptide sequence but excluding or outside of the
region(s) encoding, a known consensus sequence or consensus
DNA-binding site, or outside of the region(s) encoding one or all
conserved domains. Polypeptides, or their domains, that are
closely-related, that is, orthologous or paralogous, to the
disclosed polypeptide sequences, or their domains, will be
identifiable by having at least any of the percentage identities
provided in this paragraph.
[0113] Percent identity can be determined electronically, e.g., 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 (see, for example, Higgins and Sharp, 1988). 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 he 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, e.g., each amino acid gap is weighted as if it were a single
amino acid or nucleotide mismatch between the two sequences (see
U.S. Pat. No. 6,262,333).
[0114] Software for performing BLAST analyses is publicly
available, e.g., through the National Center for Biotechnology
information (see Internet website at www.ncbi.nlm.nih.gov/). 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, 1990; Altschul et al., 1993). 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 (see Henikoff & Henikoff, 1989). Unless
otherwise indicated for comparisons of predicted polynucleotides,
"sequence identity" 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"
(see, for example, internet website at www.ncbi.nlm.nih.gov/).
[0115] Other techniques for alignment are described by Doolittle,
1996. 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 (see
Shpaer, 1997). 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.
[0116] The percentage similarity between two polypeptide sequences,
e.g., 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, e.g., the
Jotun Hein method (see, for example, Hein, 1990). identity between
sequences can also be determined by other methods known in the art,
e.g., by varying hybridization conditions (see US Patent
Application No. 20010010913).
[0117] Thus, the present disclosure 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.
[0118] In addition, one or more polynucleotide sequences or one or
more polypeptides encoded by polynucleotide sequences :may be used
to search against a BLOCKS (Bairoch et al., 1997), 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) as well as algorithms such as Basic Local Alignment
Search Tool (BLAST; Altschul, 1990; Altschul et al., 1993), BLOCKS
(Henikoff and Henikoff, 1991), Hidden Markov Models (HMM; Eddy,
1996; Sonnhammer et al., 1997), 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
at, 1997, and in Meyers, 1995.
[0119] 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 polypeptides. 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 (e.g., with greater than 50% regulated transcripts in
common, or with greater than 70% regulated transcripts in common,
or with greater than 90% regulated transcripts in common) will have
highly similar functions. Fowler and Thoniashow, 2002, have shown
that three paralogous AP2 family genes (CBF1, CBF2 and CBF3) are
induced upon cold treatment, and each of which can condition
improved freezing tolerance, and all have highly similar transcript
profiles. Once a polypeptide has been shown to provide a specific
function, its transcript profile becomes a diagnostic tool to
determine whether paralogs or orthologs have the same function.
[0120] 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 B-box zinc finger
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 and a polypeptide
sequence encoded by a polynucleotide sequence that 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.
[0121] Orthologs and paralogs of presently disclosed polypeptides
may be cloned using compositions provided by the present disclosure
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 sequences. Appropriate mRNA sources may be identified by
interrogating Northern blots with probes designed from the present
sequences, after which a library is prepared from the mRNA obtained
from a positive cell or tissue. Polypeptide-encoding cDNA is then
isolated using, for example, PCR, using primers designed from a
presently disclosed 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, microarrays, Northern blots,
quantitative PCR, or any other technique for monitoring changes in
expression. Genomic clones may be isolated using similar techniques
to those.
[0122] Examples of orthologs of the Arabidopsis polypeptide
sequences and their functionally similar orthologs are listed in
Tables 1 and 2 and in the Sequence Listing. In addition to the
sequences in Table 1, Table 2, and the Sequence Listing, the
present disclosure encompasses isolated nucleotide sequences that
are phylogenetically and structurally similar to sequences listed
in the Sequence Listing and can function in a plant by increasing
yield and/or and abiotic stress tolerance when ectopically
expressed in a plant.
[0123] Since a significant number of these sequences are
phylogenetically and sequentially related to each other and have
been shown to increase yield from a plant and/or abiotic stress
tolerance, one skilled in the art would predict that other similar,
phylogenetically related sequences falling within the present
chides of polypeptides would also perform similar functions when
ectopically expressed.
Identifying Polynucleotides or Nucleic Acids by Hybridization
[0124] Polynucleotides homologous to the sequences illustrated in
the Sequence Listing and tables can be identified, e.g., by
hybridization to each other under stringent or under highly
stringent conditions.
[0125] Polynucleotides that encode polypeptides that are
closely-related, that is, orthologous or paralogous, to the
disclosed polypeptide sequences may be identifiable by their
ability to hybridize to the disclosed polynucleotides under high
stringency conditions, including the high stringency conditions
provided herein.
[0126] 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
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 below (e.g., Sambrook et al., 1989; Berger
and Kimmel, 1987; and Anderson and Young, 1985).
[0127] Encompassed by the present disclosure are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, including any of the polynucleotides
within the Sequence Listing, and fragments thereof under various
conditions of stringency (see, for example, Wahl and Berger, 1987;
and Kimmel, 1987). In addition to the nucleotide sequences listed
in the Sequence Listing, full length cDNA, orthologs, and paralogs
of the present nucleotide sequences may be identified and isolated
using well-known methods. The cDNA libraries, orthologs, and
paralogs of the present nucleotide sequences may be screened using
hybridization methods to determine their utility as hybridization
target or amplification probes.
[0128] With regard to hybridization, conditions that are highly
stringent, and means for achieving them, are well known in the art.
See, for example, Sambrook et al., 1989; Berger, 1987. pages
467-469; and Anderson and Young, 1985).
[0129] 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:
T.sub.m(.degree. C.)=81.5+16.6(log [Na+])+0.41(% G+C)-0.62(%
formamide)-500/L (I) DNA-DNA:
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 (II) DNA-DNA:
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 (III) DNA-DNA:
[0130] 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.
[0131] Hybridization experiments are generally conducted in a
buffer of pH between 0.8 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). 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. Dextral 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.
[0132] 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.
[0133] 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
inure than 100 complementary residues is about 5.degree. C. to
2.0.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
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,
e.g., to a unique subsequence, of the DNA.
[0134] 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, e.g., forniamide, 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, 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.
[0135] 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.
[0136] 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 polypeptides include, for example:
[0137] 6.times.SSC at 65.degree. C.;
[0138] 50% formamide, 4.times.SSC at 42.degree. C.; or
[0139] 0.5.times.SSC, 0.1% SDS at 65.degree. C.;
[0140] 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.
[0141] A person of skill in the art would not expect substantial
variation among polynucleotide species encompassed within the scope
of the present disclosure because the highly stringent conditions
set forth in the above formulae yield structurally similar
polynucleotides.
[0142] 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 minutes, or
about 0.1.times.SSC, 0.1% SDS at 65.degree. C. and washing twice
for 30 minutes. 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 temperanire, 50.degree. C.
[0143] 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 minutes. 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 minutes. 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 (see, for example, US Patent
Application No. 20010010913).
[0144] 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 polypeptide known as
of the filing date of the application. It may he 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, e.g., 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.
[0145] Encompassed by the present disclosure are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, including any of the polynucleotides
within the. Sequence Listing, and fragments thereof under various
conditions of stringency (see, for example, Wahl and Berger, 1987,
pages 399-407; and Kimmel, 1987). In addition to the nucleotide
sequences in the Sequence Listing, full length cDNA, orthologs, and
paralogs of the present nucleotide sequences may be identified and
isolated using well-known methods. The cDNA libraries, orthologs,
and paralogs of the present nucleotide sequences may be screened
using hybridization methods to determine their utility as
hybridization target or amplification probes.
Sequence Variations
[0146] It will readily be appreciated by those of skill in the art
that the instant disclosure includes any of a variety of
polynucleotide sequences provided in the Sequence Listing or
capable of encoding polypeptides that function similarly to those
provided in the Sequence Listing. 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 (that is, 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 instant
disclosure and claims.
[0147] 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.
[0148] 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 tables are a feature of the disclosure.
[0149] In addition to silent variations, other conservative
variations that alter one, or a few amino acids in the encoded
polypeptide, can be made without altering the function of the
polypeptide. For example, substitutions, deletions and insertions
introduced into the sequences provided in the Sequence Listing are
also envisioned. Such sequence modifications can be engineered into
a sequence by site-directed mutagenesis (for example, Olson et al.,
Smith et al., Zhao et al., and other articles in Wu, 1993), or the
other methods known in the art or noted herein. 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 preferred
embodiments, 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 transcriptional
regulator 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.
[0150] 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 3 when it is desired to maintain
the activity of the protein. Table 3 shows amino acids which can be
substituted for an amino acid in a protein and which are typically
regarded as conservative substitutions.
TABLE-US-00003 TABLE 3 Possible conservative amino acid
substitutions Amino Acid Residue Conservative substitutions 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
[0151] 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
EXAMPLES
[0152] It is to be understood that the present disclosure is not
limited to the particular devices, machines, materials and methods
described. Although particular embodiments are described,
equivalent embodiments may be used to produce the instant
compositions and practice the instant methods.
[0153] The disclosure, 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 disclosure and are not intended to
limit claims to the instant compositions or methods. It will be
recognized by one of skill in the art that a polypeptide 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.
Example I
Vector and Cloning Information for Arabidopsis
[0154] A number of constructs were used to modulate the activity of
the disclosed sequences. An individual project was defined as the
analysis of lines for a particular construct (for example, this
might include any of the disclosed lines that constitutively
overexpress a disclosed B-box polypeptide sequence). In the present
study, a full-length wild-type version of a gene was directly fused
to a promoter that drove its expression in transgenic plants. Such
a promoter could be the native promoter of that gene, or the CaMV
35S promoter. Alternatively, a promoter that drives tissue specific
or conditional expression could be used in similar studies.
[0155] In the present study, expression of a given polynucleotide
from a particular promoter was achieved by either a direct-promoter
fusion construct in which that sequence was cloned directly behind
the promoter of interest, or by a two-component expression system.
A direct fusion approach has the advantage of allowing for simple
genetic analysis if a given promoter-polynucleotide line is to be
crossed into different genetic backgrounds at a later date. The
two-component method, on the other hand, potentially allows for
greater versatility in the creation of various promoter-gene
combinations, and has the potential for generating stronger
expression to be obtained via an amplification of
transcription.
[0156] A list of constructs (PIDs) used to transform plants,
indicating the promoter fragment that was used to drive the
transgene and the cloning vector backbone, is provided in Table 4.
Compilations of the sequences of promoter fragments and the
expressed transgene sequences within the PIDs are provided in the
Sequence Listing. The list of constructs presented in Table 4 is
not intended to limit the instant description; other constructs
that may be used to transform plants, and which may comprise useful
promoter-gene combinations that are active in various tissues or in
response to various or particular environmental conditions may also
be envisioned.
TABLE-US-00004 TABLE 4 Sequences of promoter fragments and the
expressed transgene sequences SEQ ID Plant from NO: of which Gene
Gene sequence Construct Promoter:: Expression Identifier Identifier
derived (PID) Leader construct(s) AT4G27310; 1 Arabidopsis
pMON125158 35S::Hsp70L Direct promoter-fusion or AtBBX28 (G1481)
AT2G31380; 153 Arabidopsis pMON125664 35S::Hsp70L Direct
promoter-fusion or AtBBX25 (G1894) AT1G06040 151 Arabidopsis
pMON125195 35S::Hsp70L Direct promoter-fusion or AtBBX24 (G329)
AT4G15248; 39 Arabidopsis pMON125199 35S::Hsp70L Direct
promoter-fusion or AtBBX30 (G1478) At2G21320; 201 Arabidopsis
pMON125196 35S::Hsp70L Direct promoter-fusion or AtBBX18 (G1881)
AT3G07650 103 Arabidopsis pMON125230 35S::Hsp70L Direct
promoter-fusion or AtBBX7 (G2440) AT1G60250 51 Arabidopsis
pMON125732 35S::Hsp70L Direct promoter-fusion or AtBBX26 (G1486)
At4G38960 199 Arabidopsis pMON125730 35S::Hsp70L Direct
promoter-fusion or AtBBX19 (G9902) AT4G39070 53 Arabidopsis
pMON125197 35S::Hsp70L Direct promoter-fusion or AtBBX20
(G1888)
Example II
Transformation
[0157] Gruber et al., 1993, described several expression vectors
and culture methods that may be used for cell or tissue
transformation and subsequent regeneration.
Dicot and Monocot Expression Vectors
[0158] Transformation of plants and plant parts by
Agrobacterium-mediated transformation can be practiced using a
recombinant DNA construct, vector or cassette with the genetic
elements as shown in Table 5 for dicots and Table 6 for monocots.
The disclosed recombinant polynucleotides in the Sequence Listing
"MBI_0120PCT_ST25" may be incorporated in such recombinant DNA
constructs. The elements in a recombinant DNA construct provided in
Tables 5 and 6 are only examples of different elements that can be
used for the purpose of producing transgenic plants that have
incorporated the polynucleotides. Other elements can be used as
well in combination with the polynucleotides of the present
disclosure to achieve trait modification.
[0159] A base recombinant DNA construct specifically useful for
inserting a recombinant expression cassette into a chromosome in a
nucleus in a dicot plant by Argrobacierium-mediated transformation
is provided in Table 5. In Table 5, column 1 describes the function
of the segment of the vector, column 2 provides a short name of a
discrete genetic element and column 3 provides a more detailed
description of the element.
TABLE-US-00005 TABLE 5 Example of an expression vector useful for
transformation of dicot plants and plant parts. Function Name
Element Description Agrobacterium T-DNA B-AGRtu.left border Agro
left border sequence, essential for transfer of T- transfer DNA.
Plant selectable marker P-At.Act7 Promoter from the Arabidopsis
actin 7 gene expression cassette L-At.Act7 5'UTR of Arabidopsis
Act7 gene I-At.Act7 Intron from the Arabidopsis actin7 gene
TS-At.ShkG-CTP2 Transit peptide region of Arabidopsis EPSPS
CR-AGRtu.aroA- Codon modified CP4 coding region CP4.nno_At
T-AGRtu.nos A 3' non-translated region of the nopaline synthase
gene of Agrobacterium tumefaciens Ti plasmid which functions to
direct polyadenylation of the mRNA. Gene of interest P-CaMV.35S-cnh
Promoter for 35S RNA from CaMV containg a expression cassette
duplication of the -90 to -350 region. GOI A polynucleodtide or
polypeptide-coding sequence in Tables 1 or 2 T-Gb.E6-3b 3'
untranslated region from the fiber protein E6 gene of sea-island
cotton. Agrobacterium T-DNA B-AGRtu.right border Agro right border
sequence, essential for transfer of T- transfer DNA. Maintenance in
E. coli OR-Ec.oriV-RK2 The vegetative origin of replication from
plasmid RK2. CR-Ec.rop Coding region for repressor of primer from
the ColE1 plasmid. Expression of this gene product interferes with
primer binding at the origin of replication, keeping plasmid copy
number low. OR-Ec.ori-ColE1 The minimal origin of replication from
the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7
adenylytransferase (AAD (3'')) CR-Ec.aadA-SPC/STR Coding region for
Tn7 adenylyltransferase (AAD (3')) conferring spectinomycin and
streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR from the Tn7
adenylyltransferase (AAD (3'')) gene of E. coli.
[0160] An example of an expression vector that may be used to
transform a monocot plant by Agrobacterium-mediated transformation
is provided in Table 6. In Table 6, column 1 describes the function
of the segment of the vector, column 2 provides a short name of the
discrete genetic elements and column 3 provides a more detailed
description of the elements.
TABLE-US-00006 TABLE 6 Example of an expression vector useful for
transformation of monocot plants and plant parts. Function Name
Element Description Agrobacterium T- B-AGRtu.right border
Agrobacterium right border sequence essential for DNA trasfer
transfer of T-DNA. Gene of interest E-Os.Act1 Upstream promoter
region of the rice actin 1 gene expression cassette
E-CaMV.35S.2xA1-B3 Duplicated35S A1-B3 domain without TATA box
P-Os.Act1 Promoter region of the rice actin 1 gene L-Ta.Lhcb1 5'
untranslated leader of wheat major chlorophyll a/b binding protein
I-Os.Act1 First intron and flanking UTR exon sequences from the
rice actin 1 gene GOI A polynucleotide or polypeptide-coding
sequence in Table 1 and Table 2 T-St.Pis4 3' non-translated region
of the potato proteinase inhibitor II gene which functions to
direct polyadenylation of the mRNA Plant selectable P-Os.Act1
Promoter from the rice actin 1 gene marker expression L-Os.Act1
First exon of the rice actin 1 gene cassette I-Os.Act1 First intron
and flanking UTR exon sequences from the rice actin 1 gene
TS-At.ShkG-CTP2 Transit peptide region of Arabidopsis EPSPS
CR-AGRtu.aroA-CP4.nat Coding region for bacterial strain CP4 native
aroA gene. T-AGRtu.nos A 3' non-trranslated region of the nopaline
synthase gene of Argrobacterium tumefaciens Ti plasmid which
functions to direct polyadenylation of the mRNA. Agrobacterium T-
B-AGRtu.left border Agro left border sequence, essential for
transger of T- DNA transfer DNA. Maintenance in E. OR-Ec.oriV-RK2
The vegetative origin of replication from plasmid RK2. coli
CR-Ec.rop Coding region for repressor of primer from the ColE1
plasmid. Expression of this gene product interferes with primer
binding at the origin of replication, keeping plasmid copy number
low. OR-Ec.ori-ColE1 The minimal origin of replication from the E.
coli plasmid ColE1. P.Ec.aadA-SPC/STR Promoter for Tn7
adenylyltransferase (AAD (3'')) CR-Ec.aadA-SPC/STR Coding region
for Tn7 adenylyltransferase (AAD (3'')) conferring spectinomycin
and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR from the Tn7
adenylyltransferase (AAD (3'')) gene of E. coli.
[0161] To construct expression vectors for expressing a protein
identified in Table 1 and Table 2, primers for PER amplification of
the protein coding nucleotides are designed at or near the start
and stop codons of the coding sequence, in order to eliminate most
of the 5' and 3' untranslated regions. The protein coding
nucleotides are inserted into the base vector in the gene of
interest expression cassette at an insertion site, i.e. between the
intron element and the polyadenylation element (Table 5 or Table 6,
depending on whether the plant to be transformed is a dicot or
monocot, respectively).
[0162] To construct expression vectors for suppressing a protein
identified in Tables 1 and 2, the amplified protein coding
nucleotides may be assembled in a sense and antisense arrangement
and inserted into the base expression vector at the insertion site
in the gene of interest expression cassette (Table 5 or Table 6,
depending on whether the plant to be transformed is a dicot or
monocot, respectively) to provide transcribed RNA that will form a
double-stranded RNA for RNA interference suppression of the
protein. Expression vectors for suppressing a protein identified in
Tables 1 and 2 may also be designed to insert an expression
cassette that expresses (a) a mRNA that targets the gene for
suppression, (b) a messenger RNA that is translated to the target
protein and has a synthetic miRNA recognition site that would
result in down-modulation of the target protein, (c) an RNA that
forms a dsRNA and that is processed into siRNAs that effect down
regulation of the target protein, (d) a ssRNA that forms a ta-siRNA
which results in the production of siRNAs that effect
down-modulation of the target protein.
Transformation of Dicots or Eudicots to Produce Increased Yield
and/or Abiotic Stress Tolerance
[0163] Crop species that overexpress or suppress the disclosed and
closely related polypeptides may produce plants with increased
water deprivation or drought tolerance, cold and/or nutrient
tolerance and/or yield in both stressed and non-stressed
conditions. Thus, polynucleotide sequences listed in the Sequence
Listing recombined into, for example, one of the disclosed
expression vector, or another suitable expression vector such as
one comprising a sequence that is closely related to the instantly
disclosed sequences, may be transformed into a plant for the
purpose of modifying plant traits such as improved yield and/or
quality. The expression vector may contain a constitutive promoter,
an inducible promoter, a diurnally-regulated promoter, a
tissue-enhanced promoter, a tissue-preferred promoter, or a
tissue-specific promoter operably linked to the polynucleotide.
[0164] The expression vector may be introduced into a variety of
plants by means well known in the art such as, for example, direct
DNA transfer or Agrobacterium tumefaciens-mediated transformation.
It is now a routine to produce transgenic plants from most dicot
plants (see Weissbach and Weissbach, 1989; Gelvin et al., 1990;
Herrera-Estrella et al., 1983; Bevan, 1984; and Klee, 1985).
Methods for analysis of traits are routine in the art and examples
are disclosed above. There are a substantial number of alternatives
to Agrobacterium-mediated transformation protocols. 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;
Christou et al., 1992; Sanford, 1993; Klein et al., 1987; U.S. Pat.
No. 5,015,580 (Christou et al, issued May 14, 1991); and U.S. Pat.
No. 5,322,783 (Tonics et al., issued Jun. 21, 1994)).
Alternatively, sonication methods (see, for example, Zhang et al.,
1991); direct uptake of DNA into protoplasts using CaCl.sub.2
precipitation, polyvinyl alcohol or poly-L-ornithine (see, for
example, Hain et al., 1985; Draper et al., 1982); liposome or
spheroplast fusion (see, for example, Deshayes et al., 1985;
Christou et al., 1987); and electroporation of protoplasts and
whole cells and tissues (see, for example, Donn et al., 1990;
D'Halluin et al,, 1992; and Spencer et al., 1994) have been used to
introduce foreign DNA and expression vectors into plants.
Transformation of Arahidopsis5
[0165] Transformation of Arabidopsis was performed by an
Agrobacterium-mediated protocol based on the method of Bechtold
Sand Pelletier, 1998. Unless otherwise specified, all experimental
work was done using the Columbia ecotype.
[0166] Plant preparation. Arabidopsis seeds were sown on mesh
covered pots. The seedlings were thinned so that 6-10 evenly spaced
plants remained on each pot 10 days after planting. The primary
bolts were cut off a week before transformation to break apical
dominance and encourage auxiliary shoots to form. Transformation
was typically performed at 4-5 weeks after sowing.
[0167] Bacterial culture preparation. Agrobacterium stocks were
inoculated from single colony plates or from glycerol stocks and
grown with the appropriate antibiotics until saturation. On the
morning of transformation, the saturated cultures were centrifuged
and bacterial pellets were re-suspended in Infiltration Media (0.5X
MS, IX B5 Vitamins, 5% sucrose, 1 mg/ml benzylaminopurine riboside,
200 .mu.l/L Silwet L77) until an A600 reading of 0.8 was
reached.
[0168] Transformation and seed harvest. The Agrobacterium solution
was poured into dipping containers. All flower buds and rosette
leaves of the plants were immersed in this solution for 30 Seconds.
The plants were laid on then side and wrapped to keep the humidity
high. The plants were kept this way overnight at 4.degree. C. and
their the pots were turned upright, unwrapped, and moved to the
growth racks.
[0169] The plants were maintained on the growth rack under 24-hour
light until seeds were ready to be harvested. Seeds were harvested
when 80% of the siliques of the transformed plants were ripe
(approximately 5 weeks after the initial transformation). This seed
was deemed T0 seed, since it was obtained from the T0 generation,
and was later plated on selection plates (with either kanamycin or
sulfonamide, see Example VI). Resistant plants that were identified
on such selection plates comprised the T1 generation.
[0170] Several independently transformed lines with each gene were
grown on MS-agar plates without. sucrose. After stratification, a 3
h white light treatment was used to synchronize germination,
followed by 21 h of dark treatment, before transferring the plates
to continuous red light (30-40 .mu.moles/m.sup.2/s). Hypocotyl
lengths were measured from digital images of 20-30 seedlings per
line and compared to appropriate controls. Mean values of hypocotyl
length were plotted with standard errors to determine whether
increased expression of each of the B-box genes resulted in
elongated hypocotyls, suggesting a negative role in light
signaling.
Transformation of Tomato Plants
[0171] Transformation of tomato plants may be conducted using the
protocols of Koornneef et al, 1986, and in U.S. Pat. No. 6,613,962.
The latter method is described briefly 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 disclosed or orthologous or
paralogous polynucleotide for 5-10 minutes, blotted dry on sterile
filter paper and cocultured for 48 hours on the original feeder
layer plates. 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.
[0172] 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.
Transformation of Soybean Plants
[0173] Transformation of soybean plants may be conducted using the
methods found in, for example, Miki et al., 1993, and U.S. Pat.
Nos. 5,914,451, 5,824,877 and 6,384,301.
[0174] For Agrobacterium mediated transformation, soybean seeds are
imbibed overnight and the meristem explants excised. Soybean
explants are mixed with induced Agrobacterium cells from a strain
containing plasmid DNA with the gene of interest cassette and a
plant selectable marker cassette no later than 14 hours from the
time of initiation of seed inhibition, and wounded using
sonication. Following wounding, explants are placed in co-culture
for 2-5 days at which point they are transferred to selection media
for 6-8 weeks to allow selection and growth of transgenic shoots.
Resistant shoots are harvested approximately 6-8 weeks and placed
into selective rooting media for 2-3 weeks. Shoots producing roots
are transferred to the greenhouse and potted in soil. Shoots that
remain healthy on selection, but do not produce roots are
transferred to non-selective rooting media for an additional two
weeks. Roots from any shoots that produce roots off selection are
tested for expression of the plant selectable marker before they
are transferred to the greenhouse and potted in soil.
Transformation of Cotton Plants
[0175] Transformation of cotton plants may be performed as
generally described in WO0036911, U.S. Pat. Nos. 5,846,797,
7,790,460 and 7,947,869.
Transformation of Canola Plants
[0176] Transformation of rapeseed/canola plants may be performed as
described in U.S. Patent Publication No. 2010/0218271.
Alternatively, transformation of cotyledonary petioles and
hypocotyls of 5-6 day old young canola seedlings are used as
explants for tissue culture and transformed according to Babic et
al., 1998.
[0177] Tissues from in vitro grown canola seedlings are prepared
and inoculated with overnight-grown. Agrobacterium cells containing
plasmid DNA with the gene of interest cassette and a plant
selectable marker cassette. Following co-cultivation with
Agrobacterium, the infected tissues are allowed to grow on
selection to promote growth of transgenic shoots, followed by
growth of roots from the transgenic shoots. The selected plantlets
are then transferred to the greenhouse and potted in soil.
Molecular characterizations are performed to confirm the presence
of the gene of interest, and its expression in transgenic plants
and progenies. Progeny transgenic plants are selected from a
population of transgenic canola events under specified growing
conditions and are compared with control canola plants. Control
canola plants are substantially the same canola genotype but
without the recombinant DNA, for example, either a parental canola
plant of the same genotype that is not transformed with the
identical recombinant DNA or a negative isoline of the transformed
plant.
Obtaining and Screening Events
[0178] The above process is repeated to produce multiple events of
transgenic soybean, cotton and canola plant cells that are
transformed with recombinant DNA of the present disclosure. Progeny
transgenic plants and seed of the transformed plant cells are
screened for decreased sensitivity to light, increased yield,
greater height, greater stem diameter, greater resistance to
lodging, increased secondary rooting, greater cold tolerance,
greater tolerance to water deprivation or drought, reduced stomatal
conductance, altered C/N sensing, increased tolerance to nitrogen
limited conditions, greater late season growth and vigor, greater
number of primary nodes, greater late season canopy coverage,
increased tolerance to hyperosmotic stress, altered levels of
ureides, altered levels of hexose sugars, altered SPS activity,
altered levels starch, and delayed senescence.
[0179] 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 front a different
transgenic line of plains. Crossing provides the advantages of
producing new and often stable transgenic varieties. Genes and
their conferred traits may be moved into distinct lines of plants
using traditional backcrossing techniques well known in the
art.
Transformation of Monocots to Produce Increased Yield or Ablotie
Stress Tolerance
[0180] Cereal plants such as, but not limited to, corn, wheat,
rice, sorghum, sugarcane, or barley, may be transformed with the
present polynucleotide sequences, including monocot or
dicot-derived sequences such as those presented in the present
Tables, cloned into an expression vector such as the one described
in Table 6 or pGA643 containing a kanamycin-resistance marker or a
herbicide-tolerance marker such as a glyphosate-tolerance marker,
and expressed constitutively under, for example, the CaMV 35S or
COR15 promoters, or with tissue-specific, tissue-enhanced, tissue
preferred, diurnally-regulated or inducible promoters. The
expression vectors may be one found in the Sequence Listing (for
example, pMEN65, SEQ ID NO: 428), or any other suitable expression
vector may he similarly used.
[0181] The expression vector may he 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 the
dedifferentiating tissue with the Agrobacterium containing the
expression vector.
[0182] The sample tissues are immersed in a suspension of
3.times.10.sup.-9 cells of Agrobacterium containing the expression
vector for 3-10 minutes. The callus material is cultured on solid
medium at 25.degree. C. in the dark for several days. The calla
grown on this medium are transferred to fresh Regeneration medium
at 2-3 week intervals(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.
[0183] For Agrobacterium-mediated transformation of corn embryo
cells, corn plants of a readily transformable line are grown in the
greenhouse and ears are harvested when the embryos are 1.5 to 2.0
mm in length. Ears are surface sterilized by spraying or soaking
the ears in 80% ethanol, followed by air drying. Immature embryos
are isolated from individual kernels on surface sterilized ears.
Prior to inoculation of maize cells, Agrobacterium cells are grown
overnight at room temperature. Immature maize embryo cells are
inoculated with Agrobacterium shortly after excision, and incubated
at room temperature with Agrobacterium for 5-20 minutes. Immature.
embryo plant cells are then co-cultured with Agrobacterium for 1 to
3 days at 23.degree. C. in the dark. Co-cultured embryos are
transferred to selection media and cultured for approximately two
weeks to allow embryogenic callus to develop. Embryogenic callus is
transferred to culture medium containing a selective agent and
subcultured at about two week intervals. Transformed plant cells
are recovered 6 to 8 weeks after initiation of selection.
[0184] To regenerate transgenic corn plants a callus of transgenic
plant cells resulting from. transformation and selection is placed
on media to initiate shoot development into plantlets which are
transferred to potting soil for initial growth in a growth chamber
at 26.degree. C. followed by a mist bench before transplanting to 5
inch pots where plants are grown to maturity. The regenerated
plants are self-fertilized and seed is harvested for use in one or
more methods to select seeds, seedlings or progeny second
generation transgenic plants (R2 plants) or hybrids, e.g. by
selecting transgenic plants exhibiting an enhanced trait as
compared to a control plant.
[0185] The transformed plants are analyzed for the presence of the
NPTII or other marker protein by ELISA, using the ELISA NPTII kit
from 5Prime-3Prime (Boulder, Colo.), or for the presence of the
transgene by PCR.
[0186] Examples of wheat transformation are illustrated in U.S.
Pat. No. 6,153,812 (microprojectile bombardment method) and U.S.
Pat. No. 7,026,528 (Agrobacterium-mediated method).
[0187] It is also routine to use other methods to produce
transgenic plants of most cereal crops (Vasil, 1994) such as corn,
wheat, rice, sorghum (Cassas et al., 1993), and barley (Wan and
Lemeaux, 1994). DNA transfer methods such as the microprojectile
method can be used for corn (Fromm et al., 1990; Gorden-Kamm et al,
1990; Ishida, 1990), wheat (Vasil et al., 1992; Vasil et al., 1993;
Weeks et al., 1993), and rice (Christou, 1991; Hei et al., 1994;
Aldernita and Hodges, 1996; and Hiei et al., 1997). For most cereal
plants, embryogenic cells derived from immature scutellum tissues
are the preferred cellular targets for transformation (Hiei et al.,
1997; Vasil, 1994). After microprojectile bombardment the tissues
are selected on selection medium to identify the transgenic
embryogenic cells (Gordon-Kamm et al., 1990). Transgenic plants are
regenerated by standard corn regeneration techniques (Fromm et al.,
1990; Gordon-Kamm et al., 1990).
Example III
Stacking of the Disclosed Polynucleotides with Other Sequences of
Interest, Including Other Disclosed Polynucleotides
[0188] It is envisioned that one or more of the polynucleotides in
the instant sequence listing can be introduced into a plant in
combination with other disclosed polynucleotide sequences, or with
other polynucleotide sequences of interest (that is, the
polynucleotides may be "stacked" within the plant) for the purpose
of producing plants with enhanced or multiple useful traits. A
single plant may thus comprise. and ectopically express,
overexpress, or suppress one or more of the disclosed
polynucleotide sequences and/or their encoded polypeptides. For
example, since it was shown that AtBBX32 was physically associated
with GmBBX62 in vivo (Example V), introduction and co-expression of
both AtBBX32 and GmBBX62 in a transgenic plant may provide the
transgenic plant with enhanced traits. Therefore, stacking of the
disclosed polynucleotide sequences may be used to create one or
more transgenic plants with at least one useful trait, said traits
including, for example (but not limited to), decreased sensitivity
to light, increased yield, greater height, greater stem diameter,
greater resistance to lodging, increased secondary rooting, greater
cold tolerance, greater tolerance to water deprivation or drought,
reduced stomatal conductance, altered C/N sensing, increased
tolerance to nitrogen limiting conditions, improved late season
growth and vigor, greater number of nodes, greater late season
canopy coverage, increased tolerance to hyperosmotic stress,
altered levels of ureides, altered levels of hexose sugars, altered
SPS activity, altered levels of starch, and delayed senescence, as
compared to a control plant that does not ectopically express or
overexpress the disclosed polynucleotide sequences.
[0189] The combinations of polynucleotides introduced into target
plants may also include multiple copies of any one of the disclosed
polynucleotides. The disclosed polynucleotides may also be stacked
with any other polynucleotide or combination of polynucleotide to
produce transgenic plants with one or more useful trait
combinations, including, for example (but not limited to), one or
more of the traits listed in the previous paragraph, and possibly
herbicide resistance (e.g., glyphosate resistance), disease
resistance or tolerance, increased tolerance to heat, increased
tolerance to oxidative stress, increased oil content, male
sterility, lodging resistance, early flowering and/or development,
delayed flowering and/or development, extended flowering and/or
development time, traits desirable for chemical processing, and/or
improved digestibility.
[0190] The combinations of polynucleotides within an individual
plant can be introduced through a variety of means. This may
include, for example, transforming a target plant with two or more
nucleic acid sequences of interest at the same time or at different
times, or introducing the two or more nucleic acid sequences of
interest by different means such as a combination of transformation
and breeding, where the nucleic acid sequences may be integrated
into one or more loci. The two sequence cassettes can be contained
in separate expression vectors (trans), where one of the expression
vectors comprises a trans-acting element. A trans-acting element is
a DNA sequence, that controls transcriptional activity of a target
gene through a diffusible gene product such as a protein, microRNA,
or other diffusible repressor or activator. The regulated target
gene may include a polynucleotide of the instant Sequence Listing.
Alternatively, the two sequence cassettes can be contained in the
same expression vector (cis). Introducing the sequences at the same
time into a plant may be referred to as co-transformation,
simultaneous transformation, or parallel transformation.
Combinations of recombinant polynucleotides can also be introduced
into a plant through subsequent, serial or super-transformation or
re-transformation, that is, by providing a transgenic plant
previously transformed with one polynucleotide of interest, and
transforming the same plant with one or more distinct recombinant
polynucleotides. In this method, a transgenic plant exhibiting one
or more desired traits can be used as a target plant to introduce
additional traits by super-transformation.
[0191] Thus, if the polynucleotide sequences (including one or more
of the disclosed polynucleotide sequences) are introduced into
plants by genetic transformation, the polynucleotide sequences can
be transformed into the plant at any time and in any order.
[0192] Another method of stacking genes in a plant combines
transformation with breeding methods. A plant that has been
transformed with at least one polynucleotide sequence of interest
(including one or more of the disclosed polynucleotide sequences)
may be cross-bred with a transgenic or non-transformed plant in
order to stack more than one gene in the plant.
[0193] Expression of the polynucleotide sequences can be regulated
by the same promoter or by different promoters.
[0194] It may also be desirable to introduce an expression vector
or cassette that will suppress the expression of a polynucleotide
of interest. This may be combined with any combination of other
suppression vectors or cassettes or overexpression vectors or
cassettes to generate a desirable combination of traits in the
plant, including traits disclosed in this Example.
Example IV
Expression and Analysis of Increased Yield and/or Abiotic Stress
Tolerance in non-Arabidopsis Species or Crop Plants
[0195] It has been shown that overexpression of a B-box
polypeptide, BBX32 (AtBBX32; SEQ ID NO: 430), decreases light
sensitivity of plants when the polypeptide is overexpressed in the
plants, including in plants other than Arabidopsis (e.g., crop
plants). BBX32 has been shown to improve crop performance and acts
to repress the transcriptional activity of another B-box protein,
AtBBX21 (G1482, SEQ ID NO: 56) on its native target promoter
(prCHS) sequences (unpublished data). The instantly disclosed
results provide evidence that HA:AtBBX28 (G1481) inhibited the
transcriptional activity of HA:AtBBX21 to levels similar to the
repression by HA:BBX32 (FIG. 2), suggesting that AtBBX28 is able to
act as a repressor like BBX32. AtBBX28 may have common targets with
BBX32 (FIG. 2) or it may act on specific targets independently of
BBX32.
[0196] In fact, a number of the disclosed polypeptide sequences,
including those found in the Sequence Listing, including 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,
234,436, 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 or 550 or orthologous sequences (as
defined or claimed herein) that function in the same manner, may
alter light sensitivity of plants when the sequences are
overexpressed in the plants. It is expected that structurally
similar orthologs and paralogs of the disclosed polypeptide
sequences, which may be derived from diverse plant species, will
also alter light sensitivity of plants when these orthologous and
paralogous sequences are overexpressed. Furthermore, it is expected
that the disclosed B-box polypeptide sequences, and structurally
similar orthologs and paralogs of the disclosed B-box polypeptide
sequences, can, at least in part and as a result of their altering
light sensitivity, confer increased yield or increased tolerance to
a number of abiotic stresses, including water deprivation or
drought, cold, and nitrogen-limiting conditions, altered levels of
ureides, altered levels of hexose sugars, altered SPS activity,
altered levels of starch, and delayed senescence, relative to
control plants, and thus may increase yield of crop or other
commercially important plant species.
[0197] Relationships between the disclosed sequences in the present
disclosure may be recognized by comparing structural similarity,
for example, determined by a disclosed percentage identity to full
length protein or their conserved domains, or by hybridization to a
disclosed polynucleotide sequence.
[0198] Northern blot analysis, RT-PCR or microarray analysis of the
regenerated, transformed plants may be used to show expression of a
polypeptide of the present disclosure, or related orthologous or
paralogous sequences that are capable of inducing increased yield
or increased tolerance to abiotic stresses, including water
deprivation, cold, and low nitrogen conditions.
[0199] After a eudicot plant, dicot plant, monocot plant or plant
cell has been transformed (and the latter regenerated into a plant)
and shown to have increased tolerance to abiotic stresses,
including water deprivation or drought, cold, and nitrogen-limiting
conditions, that is, able to tolerate greater planting density with
a coincident increase in yield, or tolerance to abiotic stress, or
produce greater yield relative to a control plant in the absence of
stress conditions or under stress conditions, the transformed plant
may be crossed with itself or a plant from time same line, a
non-transformed or wild-type plant, or another transformed plant
from a different transgenic line of plants.
[0200] The functions of specific polypeptides, including
closely-related orthologs, may be analyzed and may be further
characterized and incorporated into crop plants. The ectopic
overexpression of these sequences may he regulated using
constitutive, inducible, or tissue specific regulatory elements.
Genes that have been examined and have been shown to modify plant
traits (including altered light sensitivity with predicted
increased yield and/or abiotic stress tolerance) encode
polypeptides found in the Sequence Listing. In addition to these
sequences, it is expected that newly discovered polynucleotide and
polypeptide sequences closely related to polynucleotide and
polypeptide sequences found in the Sequence Listing can also confer
alteration of traits in a similar manner to the sequences found in
the Sequence Listing, when transformed into any of a considerable
variety of plants of different species, and including dicots and
monocots. The polynucleotide and polypeptide sequences derived from
monocots (e.g., the rice sequences) may be used to transform both
monocot and dicot plants, and those derived from dicots (e.g., the
Arabidopsis and soy genes) may be used to transform either group,
although it is expected that some of these sequences will function
best if the gene is transformed into a plant from the same group
from which the sequence is derived.
[0201] As an example of a first step to determine water
deprivation-related tolerance, seeds of these transgenic plants may
be subjected to germination assays to measure sucrose sensing,
severe desiccation or drought. Methods that may be used to measure
relative sucrose sensing, severe desiccation tolerance or drought
tolerance in genetically altered (for example, over-expressing) and
control plants are as follows.
[0202] For sucrose sensing, germination assays may be conducted in
growth media containing 9.4% sucrose with Arabidopsis
overexpressors of BBX32 and closely-related sequences. Growing the
plants under controlled temperature and humidity on sterile medium
produces uniform plant material that has not been exposed to
additional stresses (such as water stress) which could cause
variability in the results obtained. Where possible, assay
conditions are originally tested in a blind experiment with
controls that have phenotypes related to the condition tested.
[0203] Prior to plating, seed for all experiments are surface
sterilized in the following manner: (1) 5 minute incubation with
mixing in 70% ethanol, (2) 20 minute incubation with mixing in 30%
bleach. 0.01% triton-X 100, (3) 5.times. rinses with sterile water,
(4) Seeds are re-suspended in 0.1% sterile agarose and stratified
at 4.degree. C. for 3-4 days. Germination assays follow
modifications of the same basic protocol. Sterile seeds are sown on
the conditional media that has a basal composition of 80%
MS+Vitamins. Plates are incubated at C under 24-hour light (120-130
.mu.E m.sup.-2 s.sup.-1) in a growth chamber. Evaluation of
germination and seedling vigor is performed five days after
planting. For severe desiccation (plate-based water deprivation)
assays, seedlings are grown for 14 days on MS+Vitamins+1% Sucrose
at 22.degree. C. Plates are opened in a sterile hood for 3 hr for
hardening and then seedlings are removed from the media and dried
for two hours in the sterile hood. After this time the plants are
transferred back to plates and incubated at 22.degree. C. for
recovery. The plants are then evaluated after five days.
[0204] The soil drought assay (performed in clay pots) is based on
that described by Haake et al., 2002.
[0205] Previously, we have performed clay-pot assays on segregating
T2 populations, sown directly to soil. However, in the current
procedure, seedlings are first germinated on selection plates
containing either kanamycin or sulfonamide.
[0206] Seeds are sterilized by a 2 minute ethanol treatment
followed by 20 minutes in 30% bleach/0.01% Tween and five washes in
distilled water. Seeds were sown to MS agar in 0.1% agarose and
stratified for three days at 4.degree. C., before transfer to
growth cabinets with a temperature of 22.degree. C. After seven
days of growth on selection plates, seedlings are transplanted to
3.5 inch diameter clay pots containing 80 g of a 50:50 mix of
vermiculite:perlite topped with 80g, of ProMix. Typically, each pot
contains 14 seedlings, and plants of the transgenic line being
tested are in separate pots to the wild-type controls. Pots
containing the transgenic line versus control pots are interspersed
in the growth room, maintained under 24-hour light conditions
(18-23.degree. C., and 90-100 .mu.E m.sup.-2 s.sup.-1) and watered
for a period of 14 days. Water is then withheld and pots are placed
on absorbent paper for a period of 8-10 days in to apply a drought
treatment. After this period, a visual qualitative "drought score"
from 0-6 is assigned to record the extent of visible drought stress
symptoms. A score of "6" corresponds to no visible symptoms whereas
a scare of "0" corresponds to extreme wilting and the leaves having
a "crispy" texture. At the end of the drought period, pots are
re-watered and scored after 5-6 days the number of surviving plants
in each pot is counted, and the proportion of the total plants in
the pot that survived is calculated.
[0207] In a given experiment, six or more pots of a transgenic line
are compared with six or more pots of the appropriate control. The
mean drought score and mean proportion of plants surviving(survival
rate) are calculated for both the transgenic line and the wild-type
pots. In each case a p-value* is calculated, which indicates the
significance of the difference between the two mean values. The
results for each transgenic line across each planting for a
particular project are then presented in a results table.
[0208] Calculation of p-values For the assays where control and
experimental plants were in separate pots, survival is analyzed
with a logistic regression to account for the fact that the random
variable is a 3.5 proportion between 0 and 1. The reported p-value
is the significance of the experimental proportion contrasted to
the control, based upon regressing the logit-transformed data.
[0209] Drought score, being an ordered factor with no real numeric
meaning, is analyzed with a non-parametric test between the
experimental and control groups. The p-value is calculated with a
Mann-Whitney rank-sum test.
[0210] Plants overexpressing the disclosed, orthologous or
paralogous sequences may he found to be more tolerant to high
sucrose by having better germination, longer radicles, and more
cotyledon expansion.
[0211] Disclosed or orthologous or paralogous sequences, or other
sequences closely related to the disclosed B-box polynucleotides
and B-box polypeptides, may also be used to generate transgenic
plants that are more tolerant to nitrogen-limiting conditions or
cold than control plants.
[0212] All of these abiotic stress tolerances conferred by
disclosed B-box polynucleotides and B-box polypeptides may
contribute to increased yield of commercially available plants.
BBX32 overexpressors have been shown to increase yield of plants in
the apparent absence of significantly obvious abiotic stress, as
evidenced by including increased height, increased early season
vigor and estimated stand count, and increased late season canopy
coverage observed in soy plants overexpressing BBX32. Thus, it is
expected that disclosed B-box polynucleotides and B-box
polypeptides, and closely related orthologs and paralogs, will also
improve yield in plants relative to control plants, including in
leguminous species, even in the absence of overt abiotic
stresses.
[0213] It is expected that the disclosed methods may be applied to
identify other useful and valuable sequences of the present
polypeptide clacks, and the sequences may be derived from a diverse
range of plant species. These sequences and/or the sequences
provided in the Sequence Listing may be introduced ditto plants
other than Arabidopsis (e.g., crop plants), and by expressing said
sequences may thus be made more tolerant than controls to water
deprivation assays, nitrogen-limiting conditions or cold, and/or
produce greater yield. It is expected that said plants under these
conditions are greener, more vigorous, will have better survival
rates than controls, or will recover better from these treatments
than control plants.
Example V
Results
[0214] The data presented herein present the results obtained in
experiments with polynucleotides and polypeptides that may be
expressed in plants in order to obtain advantageous traits in said
plants including improved yield under non-stressed or low stress
conditions, or for reduced yield losses under biotic and abiotic
stress conditions.
[0215] We have previously demonstrated that under a variety of
conditions, including unstressed, nutrient stress, or osmotic
stress conditions, Arabidopsis seedlings ectopically expressing
AtBBX32 showed increased hypocotyl growth rates and altered
transcription of genes associated with light regulated signal
transduction and photosynthesis. Soybean plants ectopically
expressing AtBBX32 were taller, tended to have more nodes, pods,
and flowers, greater late season canopy coverage, improved late
season vigor, in some cases had thicker stems, and ultimately had
significantly higher broad acre yield than wild-type soybean plants
did (e.g., see Creelinan et al., U.S. Pat. No. 7,692,067). Based on
a phylogenetic analysis of the soybean B-box family, G4004 (also
referred to as Glycine max BBX53 or GmBBX53, and listed as SEQ ID
NO: 432 or 532), GmBBX52 variant (SEQ ID NO: 434) and GmBBX52 (SEQ
ID NO: 530) were identified as the soybean orthologs of the
Arabidopsis AtBBX32 gene. Like AtBBX32, both soybean genes contain
a single N-terminus B-box domain. Both soybean genes had diurnal
oscillations in transcript abundance. Arabidopsis plants
overexpressing either GmBBX52 or GmBBX53 displayed increased
hypocotyl growth rates and altered transcription of genes
associated with light regulated signal transduction and
photosynthesis, similar to plant lines overexpressing AtBBX32. In
order to better understand the biological role of GmBBX52 and
GmBBX53 genes on soybean yield, we generated constructs that would
constitutively express either GmBBX52 or GmBBX53 or knockdown boat
GmBBX52 and GmBBX53 transcript levels. Eight independently
generated plants from both the GmBBX52 constitutive over-expression
construct and the GmBBX52/GmBBX53-miRNA constructs and four
independent events from the GmBBX53 over-expression construct were
tested in broad acre yield trials. Lines ectopically
over-expressing GmBBX52 yielded, on average, 6.1% more bushels per
acre than did wild-type control plants, while the top performing
line yielded 9.0% more bushels per acre. Lines ectopically
over-expressing GmBBX53 yielded 4.1% higher than the wild-type
control, while the top event improved yield by 6.7% over the
wild-type control. In contrast, miRNA mediated suppression of
GmBBX52 and GmBBX53 transcript levels led to a significant decrease
in GmBBX52 RNA levels and decreased yield. GmBBX52/GmBBX53-miRNA
lines yielded, on average, 5.5% fewer bushels per acre than control
lines while the lowest yielding line across the eight produced
11.8% fewer bushels per acre relative to controls. These data
demonstrate that the ectopic expression of the soybean homologs of
AtBBX32, GmBBX52 and GmBBX53, led to yield improvements similar to
that of soybean plants expressing the Arabidopsis gene.
[0216] We then examined several independently transformed
Arabidopsis lines overexpressing other disclosed B-box
polynucleotides. The level of gene expression is expected to vary
in each line, which is likely to result in a range of hypocotyl
lengths. Lines with hypocotyl lengths similar to the controls are
likely to lack any significant expression from the transgene. On
the other hand, an increase in hypocotyl length may be attributed
to the transgene, either through an indirect effect or directly
through overexpression of the specific B-box transgene. Increased
hypocotyl lengths of multiple independent transformants of the same
gene can be used to correlate the reduced light responsiveness to
increased gene expression. Using this method, a number of
independent Arabidopsis transgenic lines carrying 35S::AtBBX28:eMYC
were found to have longer hypocotyls than control lines transformed
with an empty vector control (pMEN 65), and some of the
35S::AtBBX28:cMYC lines had hypocotyl lengths similar to
BBX32-over-expressing seedlings (FIG. 1). These data suggest that
seedlings overexpressing AtBBX28 are hyposensitive to red light
implying that AtBBX28 functions negatively in light signal
transduction.
[0217] BBX32 improves crop performance and is thought to repress
the transcriptional activity of another B-box protein, AtBBX21 (SEQ
ID NO: 56) on its native target promoter (prCHS) sequences
(unpublished data), To test whether AtBBX28 (SEQ ID NO: 2) can also
repress gene expression, we co-transformed prCHS:GUS reporter
constructs with HA:AtBBX21 or in combination with HA:AtBBX28, HA:
BBX32 or CAT (used as control) in Arabidopsis. Our results show
that HA:AtBBX28 inhibited the transcriptional activity of
HA:AtBBX21 to levels similar to the repression by HA: BBX32 (FIG.
2). These data suggest that AtBBX28 is able to act as a repressor
like BBX32. AtBBX28 may have common targets with BBX32 (FIG. 2) or
it may act on specific targets independently of BBX32. Other B-box
proteins were analyzed for their potential roles in light
signaling. Seedlings of several independently transformed
Arabidopsis lines transformed with B-box genes were grown under red
light to examine the effects on hypocotyl length, As seen in FIGS.
3 and 4, we identified 8 additional B-box genes which, when
constitutively expressed, produced longer hypocotyls in light. The
levels of expression of these genes in the transgenic lines are not
yet known, but as expected the magnitude of the hypocotyl length
phenotype varied greatly between independent transformants of the
same gene. These results indicate that AtBBX25 (G1894, SEQ ID NO:
154), AtBBX26 (G1486, SEQ ID NO: 52), AtBBX30 (G1478, SEQ ID NO:
40), AtBBX7 (G2440, SEQ ID NO: 104), AtBBX24 (G329, SEQ ID NO:
152), AtBBX20 (G1888, SEQ ID NO: 54), AtBBX19 (G902, SEQ ID NO:
200), and AtBBX18 (G1881, SEQ ID NO: 202) function to suppress
light signaling. One or more of these genes can be used to alter
light sensitivity leading to increased crop yield and stress
tolerance.
AtBBX32 Modulates Expression of Soybean BBX Proteins
[0218] A microarray analysis was performed to examine the
expression of AtBBX32 on the modulation of gene expression in field
grown soybean sampled at five time points: 3:00 am, 6:00 am (dawn),
9:00 am, noon, and 3:00 pm. The expression of AtBBX32 in soybean
affected the abundance of specific gene transcripts with the
majority of these changes in gene expression occurring at dawn as
shown in FIG. 11.
[0219] Phylogenetic analyses of the Arabidopsis thaliana and
Glycine max B-box gene families were conducted for the purpose of
identifying functional similarities of proteins in the B-box family
(FIGS. 12, 13 and 14). The phyogenetic trees were generated using
an in-house proprietary database and clustalW analysis clusters to
group sequences based on the domain structure of the proteins. The
genomes of both species contain large families of B-box domain
genes; the Arabidopsis genome contains 32 predicted B-box domain
proteins, while the paleopolyploid soybean genome contains 61 B-box
containing genes, The phylogenetic analysis conducted using 32
B-box sequences from Arabidopsis thaliana and 61 B-box sequences
from Glycine max grouped the Arabidopsis and soybean sequences into
five structural groups and seventeen chides (FIG. 12). A
phylogenetic tree specific for the 61 B-box sequences from soybean
is shown in FIG. 13. Two of the soybean Bbox sequences showed a
shift in the time of peak expression. Three sequences were induced
significantly at dawn and thirteen were down-regulated in the
At.BBX32 transgenic soybean plants (FIG. 13). A single B-box clade
is further represented in FIG. 14 for Arabidopsis thaliana and
Glycine max sequences. Within the phylogenetic grouping of
AtBBX32-like single B-box domain containing genes, thirteen B-box
genes from Glycine max grouped with seven Arabidopsis single B-box
genes. Phylogenetic analysis indicates that GmBBX52 and GmBBX53 are
orthologs of the Arabidopsis thaliana AtBBX32 gene.
Utilities of B-box Polynucleotides, Polypeptides and
Phylogenetically-Related Sequences
[0220] Based on the data obtained in the instantly disclosed
Examples, a total of nine Arabidopsis B-box genes and several GmBBX
genes that function negatively in light signaling have been
identified, and it is expected that either independently or in
combination with other genes or specific promoters (for example,
heterologous promoters, or constitutive, inducible,
diurnally-regulated, tissue-enhanced, tissue-preferred, or
tissue-specific promoters), these genes will provide significantly
improved yield benefits and resistance to abiotic stress.
[0221] Sequences that are orthologous, i.e., full length sequences
or modified sequences that share significant sequence identity or
similarity, to those provided in the Sequence Listing, may be
derived from any of a wide variety of plants, including but not
limited to, the plant families Myrtaceae, Pinacece, Salicaceae,
Leguminosae, Umbelliferae, Cruciferae, Curcurbitaceae, Solanaceae,
Brassicaceae, or from the plants Arabidopsis thaliana, soybean,
potato, cotton, rape, oilseed rape (including canola), sunflower,
alfalfa, clover; or fruits and vegetables, such as banana,
blackberry, blueberry, strawberry, and raspberry, cantaloupe,
carrot, cauliflower, coffee, cucumber, eggplant, mint, grapes,
honeydew, lettuce, mango, melon, onion, papaya, peas, beans,
peppers, pineapple, pumpkin, spinach, squash, tobacco, tomato,
tomatillo, watermelon, rosaceous fruits (such as apple, peach,
pear, cherry and plum), vegetable brassicas (such as broccoli,
cabbage, cauliflower, Brussels sprouts, and kohlrabi), 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, sweet potato, wheat, corn or
sweet corn (maize), rice, wild rice, sugarcane, bamboo, oats,
turfgrass, brome-grass, Miscanthus, pampas grass, or switchgrass
(Panicum). 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).
[0222] Transgenic plants (or plant cells, or plant explants, or
plant tissues) incorporating the disclosed polynucleotides and
expressing the disclosed polypeptides or orthologs of the disclosed
polypeptides can be produced by a variety of well-established
techniques as described in the Examples. After construction of a
nucleic acid vector, including an expression vector or cassette,
including one or more polynucleotides encoding one or more B-box
transcriptional regulators or B-box transcriptional regulator
orthologs, standard techniques can be used to introduce the vector
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.
[0223] 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), Poaceae (formerly Gramineae, including wheat, corn,
rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco,
peppers, etc.). and various other crops. See protocols described in
Aramirato et al., 1984; Shimamoto et al. 1989; Fromm et al., 1990;
and Vasil et al., 1990).
Identification of Interacting Partners with AtBBX32
[0224] Genetic and biochemical approaches were employed to identify
proteins that may interact with AtBBX32 in transgenic soybean
plants. Engineering of crop plants with polynucleotides that encode
or suppress such proteins may improve yield and other agronomic
traits.
Yeast 2-Hybrid Screen
[0225] In order to identify BBX32 binding partners that may be
involved in the regulation of AtBBX32 in overexpressing transgenic
soybean, a yeast 2-hybrid (Y2H) screen was carried out using full
length and truncatedAtBBX32 and the soy orthologGmBBX52 as baits in
a total of 6 screens against a soybean cDNA library (Table 7). The
six BBX32 baits from Table 7 were subcloned into pUC vectors and
then recloned as LexA fusion constructs for use in Y2H screens.
Most bait constructs were prepared in the pB27 background, which
placed the LexA gene at the N terminus of the bait sequence. Other
vector backbones include pB29 for C terminal LexA fusions and pB6
for N terminal Gal4 fusions. Two control screens were also
performed using GmCOP1-like protein and GmPABP-like (Poly A binding
protein) protein as baits (data not shown).
[0226] A random primed library was made from total RNA isolated
from wild-type non-transgenic V5 soy leaves harvested at 4 hr
intervals during a 14 hr diurnal cycle as well as from dark
germinated soy seedlings. The two uppermost leaf tri-foliates,
shoot, and meristein tissues were harvested from V5 stage. soy
plants at 3 am, 7 am, 11 am, 3 pm, 7 pm, and 11 pm. Whole seedlings
were collected at a single time point. All tissues were harvested
from plants grown in the growth chamber where lights were turned on
at 5 am and off at 7 pm. RNA was isolated from all tissues for
preparation of a cDNA library in the appropriate prey vector. The
two-Hybrid screens were perforated by Hybrigenics Services.
[0227] Interacting clones were isolated and sequenced. Combining
all six screens, approximately 471 interacting contigs were
identified. Contigs are defined as an interacting fragment of a
particular sequence. Separate contigs may represent overlapping
portions of the same mRNA. Furthermore, some contigs identified in
separate screens may be identical, thus reducing the total number
of contigs identified. Translated BLAST searches against the public
database revealed approximately 45 groups of contigs and 57 single
contigs (including 333 different contigs) with similarity to
Arabidopsis genes. An additional three contig groups did not show
significant homology to publicly available gene sequences. Six
contig groups did not show significant homology to genes within the
Arabidopsis genome nor to the map to known soy genes. Of the
remaining single contigs that map to soy gene sequences,
approximately 107 showed no matches to known plant genes.
TABLE-US-00007 TABLE 7 Bait insert sequences tested against the soy
DNA library described above. Italicized sequence in N and C
terminal baits contained overlap regions pesent in both bait
constructs. Bait Sequence Nucleotides N-terminal
MVSFCELCGAEADLHCAADSAFLCRSCDAKFHASNFLFARHFRRVICPNC 1-222 At.BBX32
KSLTQNFVSGPLLPWPPRTTCCSE C-terminal
TQNFVSGPLLPWPPRTTCCSESSSSCCSSLDCVSSSELSSTTRDVNRARG 160-678 At.BBX32
RENRVNAKAVAVTVADGIFVNWCGKLGLNRDLTNAVVSYASLALAVETRP
RATKRVFLAAAFWFGVKNTTTWQNLKKVEDVTGVSAGMIRAVESKLARAM
TQQLRRWRVDSEEGWAENDNV Full length
MVSFCELCGAEADLHCAASSAFLCRSCDAKFHASNFLFARHFRRVICPNC 1-678 At.BBX32
KSLTQNFVSGPLLPWPPRTTCCSESSSSSCCSSLDCVSSSELSSTTRDVN
RARGRENRVNAKAVAVTVADGIFVNWCGKLGLNRDLTNAVVSYASLALAV
ETRPRATKRVFLAAAFWFGVKNTTTWQNLKKVEDVTGVSAGMIRAVESKL
ARAMTQQLRRWRVDSEEGWAENDNV N-terminal
MKPKTCELCHQLASLYCPSDSAFLCFHCDAAVHAANFLVARHLRRLLCSK 1-228 Gm.BBX52
CNRFAAIHISGAISRHLSSTCTSCSL C-terminal
AAIHISGAISRHLSSTCTSCSLEIPSADSDSLPSSSTCVSSSESCSTNQI 163-732 Gm.BBX52
KAEKKRRRRRRSFSSSSVTDDASPAAKKRRRNGGSVAEVFEKWSREIGLG
LGVNGNRVASNALSVCLGKWRSLPFRVAAATSFWLGLRFCGDRGLATCQN
LARLEAISGVPAKLILGAHANLARVFTHRRELQEGWGES Full length
MKPKTCELCHQLASLYCPSDSAFLCFHCDAAVHAANFLVARHLRRLLCSK 1-732 Gm.BBX52
CNRFAAIHISGAISRHLSSTCTSCSLEIPSADSDSLPSSSTCVSSSESCS
TNQIKAEKKRRRRRRSFSSSSVTDDASPAAKKRRRNGGSVAEVFEKWSRE
IGLGLGVNGNRVASNALSVCLGKWRSLPFRVAAATSFWLGLRFCGDRGLA
TCQNLARLEAISGVPAKLILGAHANLARVFTHRRELQEGWGES
[0228] Analysis of Y2H prey identified in each screen revealed
several consistent patterns of potential interaction. In general,
the N-terminal, B-Box containing region of both the AtBBX32and Gm
BBX32 proteins showed potential interaction with other B-Box
proteins. Many of the potentially interacting proteins are
expressed during a timeframe in which AtBBX32 overexpression has
been shown to influence expression of the most mRNAs in growth
chamber experiments (2 hr predawn and dawn). At a molecular level,
AtBBX32 overexpression resulted in marked changes in mRNA
expression for a number of nuclear genes, suggesting that AtBBX32
plays a role in transcriptional regulation (FIG. 11). As a putative
component of the transcriptional network, it is likely that BBX32
functions through interactions with other proteins. Taken together,
these data indicate that, like BBX32, these genes may modulate a
variety of traits, including yield, when overexpressed or
suppressed in plants.
[0229] AtBBX32 putative interacting proteins from the yeast 2
hybrid screens were identified and are further described in the
phylogenetic tree in FIGS. 12 and 13. Several possible interacting
proteins of GmBBX52 were also identified from yeast 2 hybrid
screens. For example, GmG1481(GmBBX62) was identified and is
proposed to function in protein-protein stabilization and could
facilitate light signaling in leaves, roots and nodules. The
interaction of BBX32 with B-box proteins may also help in
stabilizing the nuclear complex. Together such a complex could lead
to altered gene expression of key regulatory and metabolic genes
resulting in enhanced vigor, growth and yield.
AtBBX32 and GmBBX62 are Physically Associated In Vivo
[0230] To confirm the interaction between AtBBX32 and GmBBX62
identified through Y2H screening, the ability of AtBBX32 to
associate with GmBBX62 in vivo was tested. AtBBX32::GFP and
Flag::GmBBX62 fusion proteins were transiently co-expressed in
soybean protoplasts. Soybean protoplasts were isolated from 4-6 mm
cotyledons and transformed as described by Abel and Theologis,
1994, Approximately 1.times.10.sup.6 protoplasts were transformed
in 15 mL Falcon conical tubes with 90 .mu.g of Qiagen prepped
plasmid DNA. Protoplasts were then incubated at 22.degree. C. for
18 to 24 hours, and harvested at 150.times.g for 3 minutes and
lysed lysis buffer (50 mM Tris 7.8, 150 mM NaCl, 1% Triton-X 100
and Complete Protease Inhibitor (Roche) for 1 hr on ice with vortex
mixing every 15 min. The lysate was centrifuged for 5 min at
3000.times.g and soluble fractions were retained for use in
Luminex-based co-immunoprecipitation assays.
[0231] Luminex co-immunoprecipitation (co-IP) assay was carried out
using the miniaturized sandwich immunoassay and co-1P method with
modifications, Poetz et al., 2009. This combination of co-IP and
sandwich immunoassay allows the relative quantification of
components of the complex. Briefly, antibodies for GFP (MBL
International, D153-3) and FLAG (Bethyl Laboratories-A190-101A)
were covalently coupled to carboxylated fluorescent microspheres
(Luminex) according to the manufacturer's protocol. Fifty .mu.l of
protoplast cell lysate (above) was aliquoted to three different
wells in a 96 well clear fiat-bottom plate (Bio-Rad) for Luminex
assays. To each sample, 50 .mu.l of conjugated beads was added to
each appropriate well and incubated for 30 minutes with shaking.
Forward and reverse co-IPs were performed with anti-GFP conjugated
beads and anti-FLAG conjugated beads. Beads were captured for 2
minutes and washed three times with PBST (137 mM NaCl, 8.1 mM
Na.sub.2HPO.sub.4, 2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, and 0.05%
Tween-20). Biotinylated GFP (ab6658, 1:1000; Abcam) and FLAG
(A190-101B, 1:1000; Bethyl) antibodies were added to appropriate
wells. The plate was incubated on a shaker for 30 minutes. After
washing, 100 .mu.l of reporter NeutrAvidin R-phycoerythrin at a
1:1000 dilution was added to each well and incubated for 30 minutes
with subsequent washing. Each well was resuspended in 100 .mu.l of
PBST buffer before plates were analyzed using FLEX MAP 3D system
(Luminex Corp, Austin, Tex.). Approximately 100 beads were measured
per sample to determine the median fluorescence intensity
(MFI).
[0232] A co-immunoprecipitation (co-IP) experiment was performed
with the co-expressed soybean protoplast extracts using an antibody
against GFP. The protein complex was captured via the immobilized
GFP antibody and the associated components within the complex were
detected via anti-FLAG antibody and visualized using
phycoerythrin-conjugated reporter molecules, As shown in FIG. 15A,
the median fluorescence intensity (MFI) from cell extracts
co-expressing AtBBX32::GFP and Flag::GmBBX62 is significantly
higher (6-fold) than the control samples, including non-transfected
cells and cell extracts lacking either AtBBX32::GFP or
FLAGAimBBX62. As expected, low fluorescence signals for the single
gene construct samples did not show a significant difference from
non-transfected cell extract, suggesting that these signals
represent non-specific binding. A reciprocal co-IP experiment with
anti-FLAG antibody-immobilized beads was also performed. The
presence of AtBBX32::GFP within the complex was detected by
anti-GFP antibody. Similarly, the fluorescence intensity for the
co-expressed cell extract exhibited a 5-fold increase compared to
the controls (FIG. 15B). The results indicate that AtBBX32 and
GmBBX62 physically interact in soybean cells, further demonstrating
the biological relevance of GmBBX62 as a partner of AtBBX32.
AtBBX32 Binds GmBBX62 In Vitro
[0233] In vitro pull-down and ELISA-based protein-protein
interaction assays were also conducted to confirm the binding of
AtBBX32 with GmBBX62, Purified FLAG::GmBBX62 was bound to anti-FLAG
M2 agarose resin and incubated with soluble protein extracts from
wheat germ lysate expressing AtBBX32. The beads were pelleted and
washed, and the bound protein complexes were eluted using SDS-PAGE
loading buffer. The eluted protein complexes were identified by
western blotting analysis. As expected, FLAG::GmBBX62 was detected
with anti-Flag antibody from FLAG-GmBBX62-bound beads (FIG. 16A),
confirming the presence of the GmBBX62 protein in reactions.
AtBBX32 within the protein complex was detected with anti-AtBBX32
antibody from FLAG::GmBBX62 beads but not from the negative
controls (FIG. 16B), demonstrating that FLAG::GmBBX62 can bind to
AtBBX32 in vitro.
Co-Immunoprecipitation of AtBBX32 with GmBBX39 and Bead-Based
Fluorescence Detection
[0234] Bead-based co-IP assays with Luminex on-bead detection
method was used to confirm the eraction between the AtBBX32 and
GmBBX39 proteins. Pairs of potential interacting proteins were
co-expressed in soybean cotyledon protoplasts as described
above.
[0235] Capture antibodies for GFP (MBL, International, D153-3), Myc
(Bethyl Laboratories cat #A190-104A)and FLAG (Bethyl Laboratories,
A 190-101A) were covalently coupled to carhoxylated fluorescent
microspheres (Luminex) according to the manufacturer's protocol.
Luminex two-step Carbodimide coupling protocol was lodified as
follows: 3.5.times.10.sup.6 beads were conjugated to 15 .mu.g of
antibody and resuspended in coupling buffer containing 100 mM MES
pH6. Biotinylated antibodies for GFP (Abecm, ab6658), Myc (Betbyl
Laboratories cat #A190-104B) and FLAG (Bethyl laboratories,
A190-101B) were used for detection of interacting proteins in the
miniaturized sandwich immunoassay. NeutrAvidin R-phycoerythrin was
used as the reporter assay reagent (Invitrogen, A2660).
[0236] Protein input for BBX32::GFP and interactions were detected
using the miniaturized sandwich munoassay and co-IP method (Poetz,
et al., 2009). Protein complexes were captured from extracts on
antibody-conjugated beads, and the bound partner proteins were
detected with biotinylated antibodies. NeutrAvidin R-phycoerythrin
provided a fluorescence read-out for the quantity of bound
detection antibody. Interacting proteins were defined as pairs of
potential interacting proteins that yielded a greater median
fluorescence intensity (MFI) than any of the controls (single
protein expressed or untransform.ed cells) with a p-value<0.001
and a change in MFI that was 4-fold or greater when compared to
each of the controls.
[0237] To assess the potential interaction between AtBBX32 and
GmBBX39, two experiments were conducted consisting of a total of
three repetitions (exp. 1, n=1; exp. 2, n=2). BBX32:;GFP was
co-expressed with FLAG:GmBBX39 in soybean cotyledon protoplasts.
Expression analyses on one replicate of each treatment using either
minex (BBX32::GFP) or Western blot (FLAG::BBX39) detection
confirmed that each protein was expressed at detectable levels
(data not shown). When co-expressed in protoplasts, BBX32::GFP and
FLAG::BBX39 co-precipitated with either anti-GFP or anti-FLAG heads
(FIG. 17). BBX32::GFP did not co-IP with anti-FLAG beads in the
absence of FLAG::BBX39 and FLAG::BBX39 did not co-IP with anti-GFP
beads in the absence of BBX32::GFP These results demonstrate that
AtBBX32 and GmBBX39 physically interact in soybean protoplasts.
Similar experiments also demonstrated physical interactions between
GaiBBX39 and GroBBX62, between GmBBX52 and GmBBX39, and between
GmBBX52 and GmBBX62 (data not shown).
REFERENCES CITED
[0238] Abel and Theologis (1994) Plant J. 5: 421-427 [0239]
Aldemita and Hodges (1996) Planta 199: 612-617 [0240] Altschul
(1990) J. Mol. Biol. 215: 403-410 [0241] Altschul (1993) Mol. Evol.
36: 290-300 [0242] Ammirato et al., eds. (1984), Handbook of Plant
Culture--Crop Species, Macmillan Publ. Co., New York, N.Y. [0243]
Anderson and Young (1985) "Quantitative Filter Hybridisation", In:
Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical
Approach, Oxford, IRL Press, 73-111 [0244] Ausubel et al. (1997)
Short Protocols in Molecular Biolocy, John Wiley &. Sons, New
York, N.Y., unit 7.7 [0245] Babic et al. (1998) Plant Cell Reports
17: 183-188 [0246] Bairoch et al. (1997) Nucleic Acids Res. 25:
217-221 [0247] Bechtold and Pelletier (1998) Methods Mol. Biol. 82:
259-266 [0248] Berger and Kimmel (1987), "Guide to Molecular
Cloning Techniques", in Methods in Enzymology, vol. 152, Academic
Press, Inc., San Diego, Calif. [0249] Bevan (1984) Nucleic Acids
Res. 12: 8711-8721 [0250] Borevitz et al. (2000) Plant Cell 12:
2383-2393 [0251] Boss and Thomas (2002) Nature, 416: 847-850 [0252]
Bruce et al. (2000) Plant Cell 12: 65-79 [0253] Cassas et al.
(1993) Proc. Natl. Acad., Sci. USA 90:11212-11216 [0254] Cheikh et
al. (2003) U.S. Patent Application No. US20030101479 [0255]
Christou et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3962-3966
[0256] Christou (1991) Bio/Technol. 9:957-962 [0257] Christou et
al. (1992) Plant. J. 2: 27.5-281 [0258] D'Halluin et al. (1992)
Plant Cell 4: 1495-1505 [0259] Daly et al. (2001) Plant Physiol.
127: 1328-1333 [0260] Deshayes et al. (1985) EMBO J., 4: 2731-2737
[0261] Donn et al,(1990) in Abstracts of VIIth International
Congress on Plant Cell and Tissue Culture IAPTC, A2-38: 53 [0262]
Doolittle, ed. (1996) Methods in Enzymology, vol. 266: "Computer
Methods For Macromolecular Sequence Analysis" Academic Press, Inc.,
San Diego, Calif., USA [0263] Draper et al. (1982) Plant Cell
Physiol. 2.3: 451-458 [0264] Eddy (1996) Curr. Opin. Str. Biol. 6:
361-365 [0265] Eisen (1998) Genome Res. 8: 163-167 [0266] Peng and
Doolittle (1987) J. Mol. Evol, 25: 351-360 [0267] Fowler and
Thomashow (2.002) Plant Cell 14: 1675-1690 [0268] Fromm et al.
(1990) Bio/Technol, 8: 833-839 [0269] Fu et al. (2001) Plant Cell
13: 1791-1802 [0270] Gelvin et al. (1990) Plant Molecular Biology
Manual, Kluwer Academic Publishers [0271] Gilmour et al, (1998)
Plant J. 433-442 [0272] Goodrich et al. (1993) Cell 75: 519-530
[0273] Gordon-Kamm et al. (1990) Plant Cell 2: 603-618 [0274]
Gruber et al., in Glick and Thompson (1993) Methods in Plant
Molecular Biology and Biotechnology. eds., CRC Press, Inc., Boca
Raton [0275] Haake et ad. (2002) Plant Physiol. 130: 639-648 [0276]
Hain et al. (1985) Mol. Gen. Genet. 199: 161-168 [0277] Haymes et
al. (1985) Nucleic Acid Hybridization: A Practical Approach, IRL
Press, Washington, D.C. [0278] He et al. (2000) Transgenic Res. 9:
223-227 [0279] Hein (1990) Methods Enzymol. 183: 626-645 [0280]
Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915
[0281] Henikoff and Henikoff (1991) Nucleic Acids Res. 19:
6565-6572 [0282] Herrera-Estrella et al. (1983) Nature 303: 209
[0283] Hiei et al. (1994) Plant J. 6:271-282 [0284] Hiei et al.
(1997) Plant Mol. Biol. 35:205-218 [0285] Higgins and Sharp (1988)
Gene 73: 237-244 [0286] Higgins et al. (1996) Methods Enzymol. 266:
383-402 [0287] Ishida (1990) Nature Biotechnol. 14:745-750 [0288]
Jaglo et al. (2001) Plant Physiol. 127: 910-917 [0289] Kashima et
al. (1985) Nature 313: 402-404 [0290] Khanna et al. (2006) Plant
Cell 18, 2157-2171 [0291] Kim et al. (2001) Plant 25: 247-259
[0292] Kimmel (1987) Methods Enzymol. 152: 507-511 [0293] Klee
(1985) Bio/Technology 3: 637-642
[0294] Klein et al. (1987) Nature 327: 70-73 [0295] Koornneef et al
(1986) In Tomato Biotechnology: Alan R. Liss, Inc., 169-178 [0296]
Ku et al. (2000) Proc. Natl. Acad. Sci. USA 97: 9121-9126 [0297]
Kyozuka and Shimamoto (2002) Plant Cell Physiol. 43: 130-135 [0298]
Lin et al. (1991) Nature 353: 569-571 [0299] Mandel (1992a) Nature
360: 273-277 [0300] Mandel et al. (1992b) Cell 71-133-143 [0301]
Meyers (1995) Molecular Biology and Biotechnology, Wiley VCR New
York, N.Y., p 856-853 [0302] Miki et al. (1993) in Methods in Plant
Molecular Biology and Blotechnology, p. 67-88, Glick and Thompson,
eds., CRC Press, Inc., Boca Raton [0303] Mount (2001), in
Bioinformaties: Sequence and Genome Analysis, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., p. 543 [0304] Miller et
al. (2001) Plant J. 28: 169-179 [0305] Nandi et al, (2000) Curr.
Biol. 10: 215-218 [0306] Niman et al. (1983). Proc. Natl. Acad.
Sci. U.S.A. 80: 4949-4953 [0307] Peng et al. (1997) Genes
Development 11: 3194-3205 [0308] Peng et at. (1999) Nature 400:
256-261 [0309] Poets, O., K. Luckert, et al. (2009) Anal. Blochem
195:244-248 [0310] Putterill et al, (1995) Cell 80: 847-857 [0311]
Ratcliffe et al. (2001) Plant Phy,siol. 126: 122-132 [0312] Reeves
and Nissen (1990) J. Biol. Chem. 265, 8573-8582 [0313] Reeves and
Nissen (1995). Prog. Cell Cycle Res, 1: 339-349 [0314] Riechniann
et al. (2000a) Science 290, 21105-2110 [0315] Riechmann and
Ratcliffe (2000b) Curr. Opin. Plant Biol. 3, 423-434 [0316] Rieger
et at. (1976) Glossary of Genetics and Cytogenetics: Classical and
Molecular, 4th ed,, Springer Verlag, Berlin [0317] Robson et al.
(2001) Plant J. 28: 619-631 [0318] Sadowski et al. (1988) Nature
335: 563-564 [0319] Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. [0320] Sanford et al, (1987) Part. Sci.
Technol. 5:27-37 [0321] Sanford (1993) Methods Enzymol. 217:
483-509 [0322] Shiniamoto at al. 1989; Nature 338: 274-276 [0323]
Shpaer(1997) Methods Mol. Biol. 70: 173-187 [0324] Smith et al.
(1992) Protein Engineering 5: 35-51 [0325] Sonnhammer et al. (1997)
Proteins 28: 405-420 [0326] Spencer et at. (1994) Plant Mot. Biol.
24: .51-61 [0327] Suzuki et al. (2001) Plant J. 28: 409-418 [0328]
Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680 [0329]
Tudge (2000) in The Variety of Life, Oxford University Press, New
York, N.Y. pp. 547-606 [0330] Vasil et al. (1990) Bio/Technol. 8:
429-434 [0331] Vasil et al. (1992) Bio/Technol. 10:667-674 [0332]
Vasil et al. (1993) Bio/Technol. 11:1553-1558 [0333] Vasil (1994)
Plant Mol. Biol. 25: 925-937 [0334] Wahl and Berger (1987) Methods
Enzymol. 152: 399-407 [0335] Wan and Lemeaux (1994) Plant Phvsiol.
104: 37-48 [0336] Weeks et al. (1993) Plant Physiol. 102:1077-1084
[0337] Weigel and Nilsson (1995) Nature 377: 482-500 [0338]
Weissbach and Weissbach (1989) Methods for Plant Molecular Biology,
Academic Press [0339] Wu (ed.) Meth. Enzymol. (1993) vol. 217,
Academic Press [0340] Zhang et al. (1991) Bio/Technology 9:
996-997
[0341] 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.
[0342] The present disclosure is not limited by the specific
embodiments described herein. The disclosure 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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220042029A1).
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220042029A1).
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