U.S. patent application number 12/863773 was filed with the patent office on 2011-03-10 for nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics in response to cold.
This patent application is currently assigned to CERES, INC.. Invention is credited to Cory Christensen, Bonnie Hund.
Application Number | 20110061122 12/863773 |
Document ID | / |
Family ID | 40901612 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110061122 |
Kind Code |
A1 |
Christensen; Cory ; et
al. |
March 10, 2011 |
NUCLEOTIDE SEQUENCES AND POLYPEPTIDES ENCODED THEREBY USEFUL FOR
MODIFYING PLANT CHARACTERISTICS IN RESPONSE TO COLD
Abstract
Methods and materials for modulating cold tolerance levels in
plants are disclosed. For example, nucleic acids encoding cold
tolerance-modulating polypeptides are disclosed as well as methods
for using such nucleic acids to transform plant cells. Also
disclosed are plants having increased cold tolerance levels and
plant products produced from plants having increased cold tolerance
levels.
Inventors: |
Christensen; Cory;
(Sherwood, OR) ; Hund; Bonnie; (Denver,
CO) |
Assignee: |
CERES, INC.
Thousand Oaks
CA
|
Family ID: |
40901612 |
Appl. No.: |
12/863773 |
Filed: |
January 21, 2009 |
PCT Filed: |
January 21, 2009 |
PCT NO: |
PCT/US09/31609 |
371 Date: |
November 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61022786 |
Jan 22, 2008 |
|
|
|
Current U.S.
Class: |
800/260 ;
435/419; 435/6.13; 536/23.6; 800/289; 800/298; 800/306; 800/312;
800/314; 800/320; 800/320.1; 800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
A01H 1/04 20130101; C07K
14/415 20130101; C12N 15/8273 20130101 |
Class at
Publication: |
800/260 ;
800/289; 435/419; 800/298; 800/320; 800/306; 800/320.1; 800/320.3;
800/312; 800/314; 800/320.2; 800/322; 536/23.6; 435/6 |
International
Class: |
A01H 1/02 20060101
A01H001/02; A01H 1/06 20060101 A01H001/06; C12N 5/10 20060101
C12N005/10; A01H 5/00 20060101 A01H005/00; A01H 5/10 20060101
A01H005/10; C07H 21/04 20060101 C07H021/04 |
Claims
1. A method of producing a plant and/or plant tissue, said method
comprising growing a plant cell comprising an exogenous nucleic
acid, said exogenous nucleic acid comprising a regulatory region
operably linked to a nucleotide sequence encoding a polypeptide,
wherein the HMM bit score of the amino acid sequence of said
polypeptide is greater than about 130, said HMM based on the amino
acid sequences depicted in one of FIG. 1, 2, 3, 4, or 5 and wherein
said plant and/or plant tissue has a difference in the level of
cold tolerance as compared to the corresponding level of cold
tolerance of a control plant that does not comprise said nucleic
acid.
2. The method of claim 1, wherein the HMM bit score of the amino
acid is greater than about 180 based on the amino acid sequences
depicted in FIG. 2.
3. The method of claim 1, wherein the HMM bit score of the amino
acid is greater than about 650 based on the amino acid sequences
depicted in FIG. 3.
4. The method of claim 1, wherein the HMM bit score of the amino
acid is greater than about 310, the HMM is based on the amino acid
sequences depicted in FIG. 4, and wherein the polypeptide is about
150 to 170 amino acids in length.
5. A method of producing a plant and/or plant tissue, said method
comprising growing a plant cell comprising an exogenous nucleic
acid, said exogenous nucleic acid comprising a regulatory region
operably linked to a nucleotide sequence encoding a polypeptide
having 80 percent or greater sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO: 2, 4, 6,
8, 10, 12, 13, 15, 17, 20, 20, 22, 24, 26, 28, 29, 30, 32, 34, 36,
38, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 58,
59, 60, 61, 62, 63, 64, 65, 68, 69, 71, 74, 76, 77, 79, 81, 82, 83,
85, 86, 88, 90, 93, 95, 96, 98, 100, 101, 102, 104, 106, 107, 108,
110, 112, 114, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 130, 131, 132, 134, 136, 137, 138, 139, 141, 143,
170, 172, 173, 175, 176, 178, 180, 181, 182, 183, 185, 186, 187,
189, 190, 192, 193, 194, 195, 196, 198, 199, 201, 203, 204, 205,
206, 207, 208, 210, 211, 212, 213, 214, 216, 218, 219, 220, 221,
223, 163, 164, 165, 167, 169, 157, 158, 159, 160, 161, 225, 226,
227, 229, 231, 233, 235, 237, 239, 241, 242, 244, 246, 247, 249,
250, 252, 254, 255, 256, 258, 259, and 260, wherein a plant and/or
plant tissue produced from said plant cell has a difference in the
level of cold tolerance as compared to the corresponding level of
cold tolerance of a control plant that does not comprise said
nucleic acid.
6. The method of claim 1 or 5, wherein the polypeptide comprises a
B-box zinc finger domain having 60 percent or greater sequence
identity to the B-box zinc finger domain of residues 56 to 103 of
SEQ ID NO: 20 and a CCT motif having 60 percent or greater sequence
identity to the CCT motif of residues 285 to 329 of SEQ ID NO:
20.
7. The method of claim 1 or 5, wherein the polypeptide comprises a
short chain dehydrogenase domain having 60 percent or greater
sequence identity to the short chain dehydrogenase domain of
residues 38 to 173 of SEQ ID NO: 74.
8. The method of claim 1 or 5, wherein the polypeptide comprises a
B3 DNA binding domain having 60 percent or greater sequence
identity to the B3 DNA binding domain of residues 163 to 268 of SEQ
ID NO: 112 and a auxin response factor domain having 60 percent or
greater sequence identity to the auxin response factor domain of
residues 290 to 372 of SEQ ID NO: 112.
9. The method of claim 5, wherein the HMM bit score of the amino
acid sequence of said polypeptide is greater than about 130, said
HMM based on the amino acid sequences depicted in one of FIG. 1, 2,
3, 4, or 5.
10. The method of claim 1 or 5, wherein said polypeptide is
selected from the group consisting of SEQ ID NO: 2, 20, and 93.
11. A method of producing a plant according to claim 1, wherein
said method comprises growing a plant cell comprising an exogenous
nucleic acid, said exogenous nucleic acid comprising a regulatory
region operably linked to a nucleotide sequence or its complement
having 80 percent or greater sequence identity of a nucleotide
sequence selected from the group consisting of SEQ ID NO: 1, 3, 5,
7, 9, 11, 14, 16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41,
55, 57, 66, 66, 67, 67, 70, 72, 73, 75, 78, 80, 84, 87, 89, 91, 92,
94, 97, 99, 103, 105, 109, 111, 113, 115, 129, 133, 135, 140, 142,
144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
162, 166, 168, 171, 174, 177, 179, 184, 188, 191, 197, 200, 202,
209, 215, 217, 222, 224, 228, 230, 232, 234, 236, 238, 240, 243,
245, 248, 251, 253, and 257, or a fragment thereof, wherein a plant
produced from said plant cell has a difference in the level of cold
tolerance as compared to the corresponding level of cold tolerance
of a control plant that does not comprise said nucleic acid.
12. A method of modulating the level of cold tolerance in a plant,
said method comprising introducing into a plant cell an exogenous
nucleic acid, said exogenous nucleic acid comprising a regulatory
region operably linked to a nucleotide sequence encoding a
polypeptide, wherein the HMM bit score of the amino acid sequence
of said polypeptide is greater than about 130, said HMM based on
the amino acid sequences depicted in one of FIG. 1, 2, 3, 4, or 5,
and wherein a plant produced from said plant cell has a difference
in the level of cold tolerance as compared to the corresponding
level of cold tolerance of a control plant that does not comprise
said exogenous nucleic acid.
13. A method of modulating the level of cold tolerance in a plant
according to claim 12 comprising introducing into a plant cell an
exogenous nucleic acid, said exogenous nucleic acid comprising a
regulatory region operably linked to a nucleotide sequence encoding
a polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NO: 2, 4, 6, 8, 10, 12, 13, 15, 17, 20, 20, 22, 24, 26, 28, 29, 30,
32, 34, 36, 38, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 56, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, 71, 74, 76, 77, 79,
81, 82, 83, 85, 86, 88, 90, 93, 95, 96, 98, 100, 101, 102, 104,
106, 107, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121, 122,
123, 124, 125, 126, 127, 128, 130, 131, 132, 134, 136, 137, 138,
139, 141, 143, 170, 172, 173, 175, 176, 178, 180, 181, 182, 183,
185, 186, 187, 189, 190, 192, 193, 194, 195, 196, 198, 199, 201,
203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 216, 218,
219, 220, 221, 223, 163, 164, 165, 167, 169, 157, 158, 159, 160,
161, 225, 226, 227, 229, 231, 233, 235, 237, 239, 241, 242, 244,
246, 247, 249, 250, 252, 254, 255, 256, 258, 259, and 260, wherein
a plant produced from said plant cell has a difference in the level
of cold tolerance as compared to the corresponding level of cold
tolerance of a control plant that does not comprise said nucleic
acid.
14. A method of modulating the level of cold tolerance in a plant
according to claim 12, comprising introducing into a plant cell an
exogenous nucleic acid, said exogenous nucleic acid comprising a
regulatory region operably linked to a nucleotide sequence having
80 percent or greater sequence identity to a nucleotide sequence
selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11,
14, 16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41, 55, 57, 66,
66, 67, 67, 70, 72, 73, 75, 78, 80, 84, 87, 89, 91, 92, 94, 97, 99,
103, 105, 109, 111, 113, 115, 129, 133, 135, 140, 142, 144, 145,
146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 162, 166,
168, 171, 174, 177, 179, 184, 188, 191, 197, 200, 202, 209, 215,
217, 222, 224, 228, 230, 232, 234, 236, 238, 240, 243, 245, 248,
251, 253, and 257, or a fragment thereof, wherein a plant produced
from said plant cell has a difference in the level of cold
tolerance as compared to the corresponding level of cold tolerance
of a control plant that does not comprise said nucleic acid.
15. A plant cell comprising an exogenous nucleic acid said
exogenous nucleic acid comprising a regulatory region operably
linked to a nucleotide sequence encoding a polypeptide, wherein the
HMM bit score of the amino acid sequence of said polypeptide is
greater than about 130, said HMM based on the amino acid sequences
depicted in one of FIG. 1, 2, 3, 4, or 5, and wherein a plant
produced from said plant cell has a difference in the level of cold
tolerance as compared to the corresponding level of cold tolerance
of a control plant that does not comprise said nucleic acid.
16. The plant cell of claim 15, wherein the polypeptide has 80
percent or greater sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10,
12, 13, 15, 17, 20, 20, 22, 24, 26, 28, 29, 30, 32, 34, 36, 38, 40,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 58, 59, 60,
61, 62, 63, 64, 65, 68, 69, 71, 74, 76, 77, 79, 81, 82, 83, 85, 86,
88, 90, 93, 95, 96, 98, 100, 101, 102, 104, 106, 107, 108, 110,
112, 114, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 130, 131, 132, 134, 136, 137, 138, 139, 141, 143, 170,
172, 173, 175, 176, 178, 180, 181, 182, 183, 185, 186, 187, 189,
190, 192, 193, 194, 195, 196, 198, 199, 201, 203, 204, 205, 206,
207, 208, 210, 211, 212, 213, 214, 216, 218, 219, 220, 221, 223,
163, 164, 165, 167, 169, 157, 158, 159, 160, 161, 225, 226, 227,
229, 231, 233, 235, 237, 239, 241, 242, 244, 246, 247, 249, 250,
252, 254, 255, 256, 258, 259, and 260.
17. A plant cell according to claim 15, comprising an exogenous
nucleic acid said exogenous nucleic acid comprising a regulatory
region operably linked to a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NO: 2, 4, 6, 8, 10, 12, 13, 15, 17, 20, 20, 22, 24, 26, 28, 29, 30,
32, 34, 36, 38, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 56, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, 71, 74, 76, 77, 79,
81, 82, 83, 85, 86, 88, 90, 93, 95, 96, 98, 100, 101, 102, 104,
106, 107, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121, 122,
123, 124, 125, 126, 127, 128, 130, 131, 132, 134, 136, 137, 138,
139, 141, 143, 170, 172, 173, 175, 176, 178, 180, 181, 182, 183,
185, 186, 187, 189, 190, 192, 193, 194, 195, 196, 198, 199, 201,
203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 216, 218,
219, 220, 221, 223, 163, 164, 165, 167, 169, 157, 158, 159, 160,
161, 225, 226, 227, 229, 231, 233, 235, 237, 239, 241, 242, 244,
246, 247, 249, 250, 252, 254, 255, 256, 258, 259, and 260, wherein
a plant produced from said plant cell has a difference in the level
of cold tolerance as compared to the corresponding level of cold
tolerance of a control plant that does not comprise said nucleic
acid.
18. A plant cell according to claim 15, comprising an exogenous
nucleic acid said exogenous nucleic acid comprising a regulatory
region operably linked to a nucleotide sequence or its complement
having 80 percent or greater sequence identity of a nucleotide
sequence selected from the group consisting of SEQ ID NO: 1, 3, 5,
7, 9, 11, 14, 16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41,
55, 57, 66, 66, 67, 67, 70, 72, 73, 75, 78, 80, 84, 87, 89, 91, 92,
94, 97, 99, 103, 105, 109, 111, 113, 115, 129, 133, 135, 140, 142,
144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
162, 166, 168, 171, 174, 177, 179, 184, 188, 191, 197, 200, 202,
209, 215, 217, 222, 224, 228, 230, 232, 234, 236, 238, 240, 243,
245, 248, 251, 253, and 257, or a fragment thereof, wherein a plant
produced from said plant cell has a difference in the level of cold
tolerance as compared to the corresponding level of cold tolerance
of a control plant that does not comprise said nucleic acid.
19. A transgenic plant comprising the plant cell of any one of
claims 15-18.
20. The transgenic plant of claim 19, wherein said polypeptide is
selected from the group consisting of SEQ ID NO: 2, 20, and 93.
21. The transgenic plant of claim 19, wherein said plant is a
member of a species selected from the group consisting of Panicum
virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass),
Miscanthus giganteus (miscanthus), Saccharum sp. (energycane),
Populus balsamifera (poplar), Zea mays (corn), Glycine max
(soybean), Brassica napus (canola), Triticum aeslivum (wheat),
Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus
(sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet),
or Pennisetum glaucum (pearl millet).
22. A seed product comprising embryonic tissue from a transgenic
plant according to claim 19.
23. An isolated nucleic acid comprising a nucleotide sequence
having 95% or greater sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 3, 5, 7, 9, 11, 14, 16, 21, 23, 25, 27, 33,
37, 39, 41, 55, 57, 70, 75, 80, 84, 87, 91, 92, 97, 105, 113, 115,
129, 133, or 140.
24. An isolated nucleic acid comprising a nucleotide sequence
encoding a polypeptide having 80% or greater sequence identity to
the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12,
22, 24, 26, 28, 36, 38, 40, 42, 71, 74, 85, 88, 93, 105, 114, 116,
130, 134, 136, 141, or 143.
25. A method of identifying whether a polymorphism is associated
with variation in a trait, said method comprising: a) determining
whether one or more genetic polymorphisms in a population of plants
is associated with the locus for a polypeptide selected from the
group consisting of the polypeptides depicted in FIGS. 1, 2, 3, 4,
or 5 and functional homologs thereof; and b) measuring the
correlation between variation in said trait in plants of said
population and the presence of said one or more genetic
polymorphisms in plants of said population, thereby identifying
whether or not said one or more genetic polymorphisms are
associated with variation in said trait.
26. A method of making a plant line, said method comprising: a)
determining whether one or more genetic polymorphisms in a
population of plants is associated with the locus for a polypeptide
selected from the group consisting of the polypeptides depicted in
FIGS. 1, 2, 3, 4, or 5 and functional homologs thereof; b)
identifying one or more plants in said population in which the
presence of at least one allele at said one or more genetic
polymorphisms is associated with variation in a trait; c) crossing
each said one or more identified plants with itself or a different
plant to produce seed; d) crossing at least one progeny plant grown
from said seed with itself or a different plant; and e) repeating
steps c) and d) for an additional 0-5 generations to make said
plant line, wherein said at least one allele is present in said
plant line.
27. The method of claim 25 or 26, wherein said trait is the level
of cold tolerance.
28. The method of claim 25, wherein said population is a population
of switchgrass, sorghum, sugar cane, or miscanthus plants.
29. A plant cell comprising an exogenous nucleic acid said
exogenous nucleic acid comprising a regulatory region operably
linked to a nucleotide sequence comprising at least a fragment
having 80 percent or greater sequence identity to a nucleic acid
sequence selected from the group consisting of residues 305 to
about 346 of SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO:
66, residues 20 to about 61 of SEQ ID NO: 67, residues 21 to about
62 of SEQ ID NO: 72, residues 21 to about 62 of SEQ ID NO: 73,
residues 77 to about 118 of SEQ ID NO: 144, residues 292 to about
313 of SEQ ID NO: 145, residues 37 to about 78 of SEQ ID NO: 146,
residues 56 to about 97 of SEQ ID NO: 147, residues 37 to about 78
of SEQ ID NO: 148, residues 45 to about 86 of SEQ ID NO: 149,
residues 46 to about 98 of SEQ ID NO: 150, residues 476 to about
497 of SEQ ID NO: 151, residues 21 to about 62 of SEQ ID NO: 152,
residues 21 to about 62 of SEQ ID NO: 153, residues 21 to about 62
of SEQ ID NO: 154, residues 21 to about 62 of SEQ ID NO: 155, and
residues 21 to about 62 of SEQ ID NO: 156, or their complement,
wherein a plant produced from said plant cell has a difference in
the level of cold tolerance as compared to the corresponding level
of cold tolerance of a control plant that does not comprise said
nucleic acid.
30. A transgenic plant comprising the plant cell of claim 29.
31. The transgenic plant of claim 30, wherein said exogenous
nucleic acid comprises a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 66, 67, 72, 73, 111, 144, 145, 146,
147, 148, 149, 150, 151, 152, 153, 154, 155, and 156.
32. A method of producing a plant, said method comprising
introducing into a plant cell an exogenous nucleic acid, said
exogenous nucleic acid comprising a regulatory region operably
linked to a nucleotide sequence encoding at least a fragment having
80 percent or greater sequence identity to a nucleic acid sequence
selected from the group consisting of residues 305 to about 346 of
SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO: 66, residues
20 to about 61 of SEQ ID NO: 67, residues 21 to about 62 of SEQ ID
NO: 72, residues 21 to about 62 of SEQ ID NO: 73, residues 77 to
about 118 of SEQ ID NO: 144, residues 292 to about 313 of SEQ ID
NO: 145, residues 37 to about 78 of SEQ ID NO: 146, residues 56 to
about 97 of SEQ ID NO: 147, residues 37 to about 78 of SEQ ID NO:
148, residues 45 to about 86 of SEQ ID NO: 149, residues 46 to
about 98 of SEQ ID NO: 150, residues 476 to about 497 of SEQ ID NO:
151, residues 21 to about 62 of SEQ ID NO: 152, residues 21 to
about 62 of SEQ ID NO: 153, residues 21 to about 62 of SEQ ID NO:
154, residues 21 to about 62 of SEQ ID NO: 155, and residues 21 to
about 62 of SEQ ID NO: 156, or their complement, wherein a plant
produced from said plant cell has a difference in the level of cold
tolerance as compared to the corresponding level of cold tolerance
of a control plant that does not comprise said nucleic acid.
33. The method of claim 32, wherein expression of a target
polypeptide is suppressed, said target polypeptide having 80
percent or greater sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NO: 112, 114, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130,
131, 132, 134, 136, 137, 138, 139, 141, and 143.
34. The method of claim 33, wherein the HMM bit score of the amino
acid sequence of said polypeptide is greater than about 790, said
HMM based on the amino acid sequences depicted in FIG. 5.
35. The method of claim 32, wherein the nucleotide sequence or its
complement is complementary to RNA transcribed from a gene encoding
said target polypeptide.
36. The method of claim 32, wherein the nucleotide sequence
comprises a microRNA recognition site having 80 percent or greater
sequence identity to a nucleic acid sequence selected from the
group consisting of residues 109 to about 129 of SEQ ID NO: 66,
residues 114 to about 135 of SEQ ID NO: 67, residues 119 to about
139 of SEQ ID NO: 72, residues 108 to about 128 of SEQ ID NO: 73,
residues 234 to about 254 of SEQ ID NO: 144, residues 135 to about
176 of SEQ ID NO: 145, residues 173 to about 189 of SEQ ID NO: 147,
residues 154 to about 170 of SEQ ID NO: 148, residues 134 to about
157 of SEQ ID NO: 149, residues 154 to about 198 of SEQ ID NO: 150,
residues 319 to about 360 of SEQ ID NO: 151, residues 121 to about
141 of SEQ ID NO: 152, residues 120 to about 140 of SEQ ID NO: 153,
residues 121 to about 141 of SEQ ID NO: 154, residues 121 to about
141 of SEQ ID NO: 155, residues 121 to about 141 of SEQ ID NO: 156,
and residues 462 to about 483 of SEQ ID NO: 111.
37. A method of modulating the level of cold tolerance in a plant,
said method comprising introducing into a plant cell an exogenous
nucleic acid, said exogenous nucleic acid comprising a regulatory
region operably linked to a nucleotide sequence or its complement
having 80 percent or greater sequence identity to a nucleic acid
sequence selected from the group consisting of residues 305 to
about 346 of SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO:
66, residues 20 to about 61 of SEQ ID NO: 67, residues 21 to about
62 of SEQ ID NO: 72, residues 21 to about 62 of SEQ ID NO: 73,
residues 77 to about 118 of SEQ ID NO: 144, residues 292 to about
313 of SEQ ID NO: 145, residues 37 to about 78 of SEQ ID NO: 146,
residues 56 to about 97 of SEQ ID NO: 147, residues 37 to about 78
of SEQ ID NO: 148, residues 45 to about 86 of SEQ ID NO: 149,
residues 46 to about 98 of SEQ ID NO: 150, residues 476 to about
497 of SEQ ID NO: 151, residues 21 to about 62 of SEQ ID NO: 152,
residues 21 to about 62 of SEQ ID NO: 153, residues 21 to about 62
of SEQ ID NO: 154, residues 21 to about 62 of SEQ ID NO: 155, and
residues 21 to about 62 of SEQ ID NO: 156, or a fragment thereof,
wherein a plant produced from said plant cell has a difference in
the level of cold tolerance as compared to the corresponding level
of cold tolerance of a control plant that does not comprise said
nucleic acid.
Description
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING OR TABLE
[0001] The material in the accompanying sequence listing is hereby
incorporated by reference into this application. The accompanying
file, named 2008-12-11.sub.--2750-1707WO1_Sequence Listing.txt was
created on Dec. 11, 2008 and is 850 KB. The file can be accessed
using Microsoft Word on a computer that uses Windows OS.
TECHNICAL FIELD
[0002] The present invention relates to methods and materials
involved in modulating cold tolerance in plants, including growth
levels in plants grown under low or chilling temperature stress
conditions (a.k.a. "cold stress"). For example, this invention
provides plants having increased growth rate, vegetative growth,
seedling vigor and/or biomass under cold stress conditions as
compared to wild-type plants grown under similar conditions, as
well as materials and methods for making plants and plant products
having increased growth levels under cold stress conditions.
BACKGROUND
[0003] Plants are constantly exposed to a variety of biotic (i.e.
pathogen infection and insect herbivory) and abiotic (i.e. high or
low temperature, drought, flood and salinity) stresses. To survive
these challenges to their sessile life, plants have developed
elaborate mechanisms to perceive external signals and to manifest
adaptive responses with proper physiological and morphological
changes (Bohnert et al. 1995). Plants exposed to cold or chilling
conditions typically have low yields of biomass, seeds, fruit and
other edible products. The term "chilling sensitivity" is used for
the description of physiological and developmental damages in the
plant caused by low, but above freezing, temperatures. Important
agricultural crop plants such as corn, soybean, rice and cotton
have tropical ancestors that make them chilling sensitive. In some
countries or agricultural regions of the world chilling
temperatures are a significant cause of crop losses and a primary
factor limiting the geographical range and growing season of many
crop species. Another example is that chilling conditions can cause
significant concern in early spring planting of corn or canola.
Poor germination and reduced growth of chilling sensitive crops in
the spring results in less ground coverage, more erosion and
increased occurrence of weeds leading to less nutrient supply for
the crop.
[0004] Typically, chilling damage includes wilting, necrosis or ion
leakage from cell membranes, especially calcium leakage, and
decreased membrane fluidity, which consequently impacts membrane
dependent processes such as: photosynthesis, protein synthesis,
ATPase activity, uptake of nitrogen, etc. (see Levitt J (1980)
Chilling injury and resistance. In Chilling, Freezing, and High
Temperature Stresses: Responses of Plant to Environmental Stresses,
Vol 1., T T Kozlowsky, ed, Academic Press, New York, pp 23-64;
Graham and Patterson (1982) Annu Rev Plant Physiol 33: 347-372; Guy
(1990) Annu Rev Plant Physiol Plant Mol Biol 41: 187-223; and
Nishida and Murata (1996) Annu Rev Plant Physiol Plant Mol Biol 47:
541-568.). In addition, cold temperatures are often associated with
wet conditions. The combination of cold and wet can result in
hypoxic stress on the roots, causing an even more severe reduction
of growth rate but, more critically, can be lethal to the plants,
especially sensitive plant species such as corn and cotton.
[0005] Yet it has been observed that environmental factors, such as
low temperature, can serve as triggers to induce cold acclimation
processes allowing plants responding thereto to survive and thrive
in low temperature environments. It would, therefore, be of great
interest and importance to be able to identify genes that regulate
or confer improved cold acclimation characteristics to enable one
to create transformed plants (such as crop plants) with improved
cold tolerance characteristics such as faster germination and/or
growth and/or improved nitrogen uptake under cold conditions to
improve survival or performance under low or chilling
temperatures.
[0006] In the fields of agriculture and forestry, efforts are
constantly being made to produce plants with an increased growth
potential in order to feed the ever-increasing world population and
to guarantee the supply of reproducible raw materials. This is done
conventionally through plant breeding. The breeding process is,
however, both time-consuming and labor-intensive. Furthermore,
appropriate breeding programs must be performed for each relevant
plant species.
[0007] Progress has been made in part by the genetic manipulation
of plants; that is by introducing and expressing recombinant
nucleic acid molecules in plants. Such approaches have the
advantage of not usually being limited to one plant species, but
instead being transferable among plant species. There is a need for
generally applicable processes that improve forest or agricultural
plant growth potential. Therefore, the present invention relates to
a method for increasing growth potential, decreasing chilling
damage, and/or increasing levels of cold acclimation in plants
under low temperature, chilling or cold conditions, characterized
by expression of recombinant DNA molecules stably integrated into
the plant genome.
SUMMARY
[0008] The present invention provides methods and materials related
to plants having modulated levels of cold tolerance. For example,
this invention provides transgenic plants and plant cells having
increased levels of cold tolerance, nucleic acids (i.e. isolated
polynucleotides), polypeptides encoded thereby used to generate
transgenic plants and plant cells having increased levels of cold
tolerance, and methods for making plants and plant cells having
increased levels of cold tolerance. Such plants and plant cells
having increased cold tolerance will produce biomass under cold
stress conditions that may be useful in producing biomass for
conversion to a liquid fuel or other chemicals, or may be useful as
a thermochemical fuel.
[0009] Methods of producing a plant and/or plant tissue are
provided herein. In one aspect, a method comprises growing a plant
cell comprising an exogenous nucleic acid. The exogenous nucleic
acid comprises a regulatory region operably linked to a nucleotide
sequence encoding a polypeptide. The Hidden Markov Model (HMM) bit
score of the amino acid sequence of the polypeptide is greater than
about 130, 180, 650, or 315, using an HMM generated from the amino
acid sequences depicted in one of FIG. 1, 2, 3, 4, or 5,
respectively. The plant and/or plant tissue has a difference in the
level of cold tolerance as compared to the corresponding level of
cold tolerance of a control plant that does not comprise the
exogenous nucleic acid.
[0010] In another aspect, a method comprises growing a plant cell
comprising an exogenous nucleic acid. The exogenous nucleic acid
comprises a regulatory region operably linked to a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence set forth in SEQ ID
NOs: 2, 20, 74, or 93. A plant and/or plant tissue produced from
the plant cell has a difference in the level of cold tolerance as
compared to the corresponding level of cold tolerance of a control
plant that does not comprise the exogenous nucleic acid.
[0011] In another aspect, a method comprises growing a plant cell
comprising an exogenous nucleic acid. The exogenous nucleic acid
comprises a regulatory region operably linked to a nucleotide
sequence having 80 percent or greater sequence identity to a
nucleotide sequence or at a fragment thereof set forth in SEQ ID
NO: 1, 19, 92, 97, or 111. A plant and/or plant tissue produced
from the plant cell has a difference in the level of cold tolerance
as compared to the corresponding level of cold tolerance of a
control plant that does not comprise the exogenous nucleic
acid.
[0012] Methods of modulating the level of cold tolerance in a plant
are provided herein. In one aspect, a method comprises introducing
into a plant cell an exogenous nucleic acid, that comprises a
regulatory region operably linked to a nucleotide sequence encoding
a polypeptide. The HMM bit score of the amino acid sequence of the
polypeptide is greater than about 130, 180, 650, 315, or 790 using
an HMM generated from the amino acid sequences depicted in any one
of FIG. 1, 2, 3, 4 or 5, respectively. A plant and/or plant tissue
produced from the plant cell has a difference in the level of cold
tolerance as compared to the corresponding level of cold tolerance
of a control plant that does not comprise the exogenous nucleic
acid.
[0013] In certain embodiments, the amino acid sequence of the
polypeptide has an HMM score greater than about 180, using an HMM
generated from the amino acid sequences depicted in FIG. 2, wherein
the polypeptide comprises an CCT motif domain having 80 percent or
greater sequence identity to amino acid residues 285 to 329 of SEQ
ID NO: 20, residues 291 to 335 of SEQ ID NO: 22, residues 235 to
279 of SEQ ID NO: 24, residues 217 to 261 of SEQ ID NO: 26,
residues 311 to 355 of SEQ ID NO: 28, residues 285 to 329 of SEQ ID
NO: 29, residues 281 to 325 of SEQ ID NO: 30, residues 302 to 346
of SEQ ID NO: 32, residues 289 to 333 of SEQ ID NO: 34, residues
295 to 339 of SEQ ID NO: 36, residues 261 to 305 of SEQ ID NO: 38,
residues 284 to 328 of SEQ ID NO: 40, residues 288 to 332 of SEQ ID
NO: 42, residues 261 to 305 of SEQ ID NO: 43, residues 239 to 283
of SEQ ID NO: 44, residues 294 to 338 of SEQ ID NO: 45, residues
279 to 323 of SEQ ID NO: 46, residues 261 to 305 of SEQ ID NO: 47,
residues 239 to 283 of SEQ ID NO: 48, residues 294 to 338 of SEQ ID
NO: 49, residues 261 to 305 of SEQ ID NO: 50, residues 298 to 342
of SEQ ID NO: 51, residues 241 to 285 of SEQ ID NO: 52, residues
268 to 312 of SEQ ID NO: 53, residues 245 to 289 of SEQ ID NO: 54,
residues 238 to 282 of SEQ ID NO: 56, residues 245 to 289 of SEQ ID
NO: 58, residues 279 to 323 of SEQ ID NO: 59, residues 236 to 280
of SEQ ID NO: 60, residues 250 to 294 of SEQ ID NO: 61, residues
322 to 366 of SEQ ID NO: 62, residues 297 to 341 of SEQ ID NO: 63,
residues 348 to 392 of SEQ ID NO: 64, residues 312 to 356 of SEQ ID
NO: 65, residues 340 to 384 of SEQ ID NO: 68, residues 307 to 351
of SEQ ID NO: 69, or residues 253 to 297 of SEQ ID NO: 71, or CCT
motifs identified in the sequence listing.
[0014] In certain embodiments, the amino acid sequence of the
polypeptide has an HMM score greater than about 180, using an HMM
generated from the amino acid sequences depicted in FIG. 2, wherein
the polypeptide comprises a B-box zinc finger domain having 80
percent or greater sequence identity to amino acid residues 56 to
103 of SEQ ID NO: 20, residues 62 to 109 of SEQ ID NO: 22, residues
64 to 106 of SEQ ID NO: 24, residues 34 to 81 of SEQ ID NO: 26,
residues 63 to 110 of SEQ ID NO: 28, residues 56 to 103 of SEQ ID
NO: 29, residues 56 to 103 of SEQ ID NO: 30, residues 60 to 107 of
SEQ ID NO: 32, residues 56 to 103 of SEQ ID NO: 34, residues 51 to
98 of SEQ ID NO: 36, residues 70 to 112 of SEQ ID NO: 38, residues
51 to 98 of SEQ ID NO: 40, residues 52 to 99 of SEQ ID NO: 42,
residues 72 to 114 of SEQ ID NO: 43, residues 62 to 104 of SEQ ID
NO: 44, residues 50 to 97 of SEQ ID NO: 45, residues 55 to 102 of
SEQ ID NO: 46, residues 72 to 114 of SEQ ID NO: 47, residues 62 to
104 of SEQ ID NO: 48, residues 50 to 97 of SEQ ID NO: 49, residues
27 to 71 of SEQ ID NO: 50, residues 60 to 107 of SEQ ID NO: 51,
residues 1 to 48 of SEQ ID NO: 52, residues 1 to 48 of SEQ ID NO:
53, residues 1 to 48 of SEQ ID NO: 54, residues 62 to 104 of SEQ ID
NO: 56, residues 64 to 106 of SEQ ID NO: 58, residues 1 to 48 of
SEQ ID NO: 59, residues 61 to 103 of SEQ ID NO: 60, residues 70 to
112 of SEQ ID NO: 61, residues 52 to 99 of SEQ ID NO: 62, residues
51 to 98 of SEQ ID NO: 63, residues 77 to 119 of SEQ ID NO: 64,
residues 59 to 106 of SEQ ID NO: 65, residues 59 to 106 of SEQ ID
NO: 68, residues 34 to 66 of SEQ ID NO: 69, or residues 64 to 106
of SEQ ID NO: 71, or a B-box zinc finger domain identified in the
sequence listing.
[0015] In certain embodiments, the amino acid sequence of the
polypeptide has an HMM score greater than about 650, using an HMM
generated from the amino acid sequences depicted in FIG. 3, wherein
the polypeptide comprises an short-chain dehydrogenase domain
having 80 percent or greater sequence identity to amino acid
residues 38 to 173 of SEQ ID NO: 74, residues 37 to 174 of SEQ ID
NO: 76, residues 23 to 160 of SEQ ID NO: 77, residues 7 to 168 of
SEQ ID NO: 79, residues 43 to 179 of SEQ ID NO: 81, residues 49 to
188 of SEQ ID NO: 82, residues 48 to 187 of SEQ ID NO: 83, residues
37 to 172 of SEQ ID NO: 85, residues 35 to 170 of SEQ ID NO: 86,
residues 20 to 160 of SEQ ID NO: 88, or residues 37 to 174 of SEQ
ID NO: 90, or a short-chain dehydrogenase domain identified in the
sequence listing.
[0016] In certain embodiments, the amino acid sequence of the
polypeptide has an HMM score greater than about 790, using an HMM
generated from the amino acid sequences depicted in FIG. 5, wherein
the polypeptide comprises a B3 DNA binding domain and an auxin
response factor.
[0017] In another aspect, a method comprises introducing into a
plant cell an exogenous nucleic acid that comprises a regulatory
region operably linked to a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence set forth in SEQ ID NO: 2, 20, 74, 93 or 112. A
plant and/or plant tissue produced from the plant cell has a
difference in the level of cold tolerance as compared to the
corresponding level of cold tolerance of a control plant that does
not comprise the exogenous nucleic acid.
[0018] In another aspect, a method comprises introducing into a
plant cell an exogenous nucleic acid, that comprises a regulatory
region operably linked to a nucleotide sequence having 80 percent
or greater sequence identity to a nucleotide sequence set forth in
SEQ ID NO: 1, 19, 92, 97, or 111, or a fragment thereof. A plant
and/or plant tissue produced from the plant cell has a difference
in the level of cold tolerance as compared to the corresponding
level of cold tolerance of a control plant that does not comprise
the exogenous nucleic acid.
[0019] Methods of modulating the level of cold tolerance in a plant
are provided herein. In one aspect, a method comprises introducing
into a plant cell an exogenous nucleic acid, that comprises a
regulatory region operably linked to a nucleotide sequence encoding
a trans-activating small-interfering RNA (tasiRNA) that acts upon,
e.g. suppresses expression of, an auxin responsive factor (ARF)
polypeptide. The HMM bit score of the amino acid sequence of the
ARF polypeptide is greater than about 790, using an HMM generated
from the amino acid sequences depicted in FIG. 5. A plant and/or
plant tissue produced from the plant cell has a difference in the
level of cold tolerance as compared to the corresponding level of
cold tolerance of a control plant that does not comprise the
exogenous nucleic acid.
[0020] Plant cells comprising an exogenous nucleic acid are
provided herein. In one aspect, the exogenous nucleic acid
comprises a regulatory region operably linked to a nucleotide
sequence encoding a tasiRNA. In some embodiments, the nucleotide
sequence comprises a tasiRNA coding region having 80 percent or
greater sequence identity to a nucleic acid sequence selected from
the group consisting of residues 305 to about 346 of SEQ ID NO:
111, residues 21 to about 62 of SEQ ID NO: 66, residues 20 to about
61 of SEQ ID NO: 67, residues 21 to about 62 of SEQ ID NO: 72,
residues 21 to about 62 of SEQ ID NO: 73, residues 77 to about 118
of SEQ ID NO: 144, residues 292 to about 313 of SEQ ID NO: 145,
residues 37 to about 78 of SEQ ID NO: 146, residues 56 to about 97
of SEQ ID NO: 147, residues 37 to about 78 of SEQ ID NO: 148,
residues 45 to about 86 of SEQ ID NO: 149, residues 46 to about 98
of SEQ ID NO: 150, residues 476 to about 497 of SEQ ID NO: 151,
residues 21 to about 62 of SEQ ID NO: 152, residues 21 to about 62
of SEQ ID NO: 153, residues 21 to about 62 of SEQ ID NO: 154,
residues 21 to about 62 of SEQ ID NO: 155, and residues 21 to about
62 of SEQ ID NO: 156, wherein a plant produced from said plant cell
has a difference in the level of cold tolerance as compared to the
corresponding level of cold tolerance of a control plant that does
not comprise said nucleic acid. Transgenic plants comprising such
plant cells are provided herein. In some embodiments, the
transgenic plant comprises an exogenous nucleic acid having a
sequence selected from the group consisting of SEQ ID NO: 66, 67,
72, 73, 111, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, and 156.
[0021] Methods of producing a plant and/or plant tissue are
provided herein. In one aspect, a method comprises introducing into
a plant cell an exogenous nucleic acid, said exogenous nucleic acid
comprising a regulatory region operably linked to a nucleotide
sequence encoding a gene suppressing tasiRNA, said nucleotide
sequence comprising a tasiRNA coding region having 80 percent or
greater sequence identity to a nucleic acid sequence selected from
the group consisting of residues 305 to about 346 of SEQ ID NO:
111, residues 21 to about 62 of SEQ ID NO: 66, residues 20 to about
61 of SEQ ID NO: 67, residues 21 to about 62 of SEQ ID NO: 72,
residues 21 to about 62 of SEQ ID NO: 73, residues 77 to about 118
of SEQ ID NO: 144, residues 292 to about 313 of SEQ ID NO: 145,
residues 37 to about 78 of SEQ ID NO: 146, residues 56 to about 97
of SEQ ID NO: 147, residues 37 to about 78 of SEQ ID NO: 148,
residues 45 to about 86 of SEQ ID NO: 149, residues 46 to about 98
of SEQ ID NO: 150, residues 476 to about 497 of SEQ ID NO: 151,
residues 21 to about 62 of SEQ ID NO: 152, residues 21 to about 62
of SEQ ID NO: 153, residues 21 to about 62 of SEQ ID NO: 154,
residues 21 to about 62 of SEQ ID NO: 155, and residues 21 to about
62 of SEQ ID NO: 156, wherein a plant produced from said plant cell
has a difference in the level of cold tolerance as compared to the
corresponding level of cold tolerance of a control plant that does
not comprise said nucleic acid. In certain embodiments, the
expression of a target ARF gene is suppressed in a plant. In some
embodiments, the ARF gene encodes a polypeptide having 80 percent
or greater sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NO: 112, 114, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132,
134, 136, 137, 138, 139, 141, and 143. In other embodiments, the
ARF gene encodes a polypeptide and the HMM bit score of the amino
acid sequence of said polypeptide is greater than about 790, said
HMM based on the amino acid sequences depicted in FIG. 5. In other
embodiments, the gene suppressing tasiRNA or its complement is
complementary to RNA transcribed from said target ARF gene. In
other embodiments, the nucleotide sequence encoding a gene
suppressing tasiRNA comprises a microRNA recognition site having 80
percent or greater sequence identity to a nucleic acid sequence
selected from the group consisting of residues 109 to about 129 of
SEQ ID NO: 66, residues 114 to about 135 of SEQ ID NO: 67, residues
119 to about 139 of SEQ ID NO: 72, residues 108 to about 128 of SEQ
ID NO: 73, residues 234 to about 254 of SEQ ID NO: 144, residues
135 to about 176 of SEQ ID NO: 145, residues 173 to about 189 of
SEQ ID NO: 147, residues 154 to about 170 of SEQ ID NO: 148,
residues 134 to about 157 of SEQ ID NO: 149, residues 154 to about
198 of SEQ ID NO: 150, residues 319 to about 360 of SEQ ID NO: 151,
residues 121 to about 141 of SEQ ID NO: 152, residues 120 to about
140 of SEQ ID NO: 153, residues 121 to about 141 of SEQ ID NO: 154,
residues 121 to about 141 of SEQ ID NO: 155, residues 121 to about
141 of SEQ ID NO: 156, and residues 462 to about 483 of SEQ ID NO:
111.
[0022] Plant cells comprising an exogenous nucleic acid are
provided herein. In one aspect, the exogenous nucleic acid
comprises a regulatory region operably linked to a nucleotide
sequence encoding a polypeptide. The HMM bit score of the amino
acid sequence of the polypeptide is greater than about 130, using
an HMM based on the amino acid sequences depicted in one of FIG. 1,
2, 3, 4, or 5. The plant and/or plant cells has a difference in the
level of cold tolerance as compared to the corresponding level of
cold tolerance of a control plant that does not comprise the
exogenous nucleic acid. In another aspect, the exogenous nucleic
acid comprises a regulatory region operably linked to a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO: 2, 20, 93, or 74. A plant and/or plant
tissue produced from the plant cell has a difference in the level
of cold tolerance as compared to the corresponding level of cold
tolerance of a control plant that does not comprise the exogenous
nucleic acid. In another aspect, the exogenous nucleic acid
comprises a regulatory region operably linked to a nucleotide
sequence having 80 percent or greater sequence identity to a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 1, 19, 92, 97, or 111. A plant and/or plant tissue produced
from the plant cell has a difference in the level of cold tolerance
as compared to the corresponding level of cold tolerance of a
control plant that does not comprise the exogenous nucleic acid. A
transgenic plant comprising such a plant cell is also provided.
Also provided is a seed product. The product comprises embryonic
tissue from a transgenic plant.
[0023] Isolated nucleic acids are also provided. In one aspect, an
isolated nucleic acid comprises a nucleotide sequence having 80% or
greater sequence identity to the nucleotide sequence set forth in
SEQ ID NO: 3, 5, 7, 9, 11, 14, 16, 21, 23, 25, 27, 33, 35, 37, 39,
41, 55, 57, 70, 75, 80, 84, 87, 91, 92, 97, 105, 113, 115, 129,
133, 140, or 142.
[0024] In another aspect, an isolated nucleic acid comprises a
nucleotide sequence encoding a polypeptide having 80% or greater
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 4, 6, 8, 10, 12, 22, 24, 26, 28, 36, 38, 40, 42, 71, 74, 85,
88, 93, 105, 114, 116, 130, 134, 136, 141, or 143.
[0025] In another aspect, methods of identifying a genetic
polymorphism associated with variation in the level of cold
tolerance are provided. The methods include providing a population
of plants, and determining whether one or more genetic
polymorphisms in the population are genetically linked to the locus
for a polypeptide selected from the group consisting of the
polypeptides depicted in FIGS. 1, 2, 3, 4, and 5 and functional
homologs thereof, such as, but not limited to, those identified in
the Sequence Listing. The correlation between variation in the
level of cold tolerance in a tissue in plants of the population and
the presence of the one or more genetic polymorphisms in plants of
the population is measured, thereby permitting identification of
whether or not the one or more genetic polymorphisms are associated
with such variation.
[0026] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0027] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is an alignment of Ceres SEEDLINE ID no.ME00327 with
homologous and/or orthologous amino acid sequences Ceres SEEDLINE
ID no.ME00327 (SEQ ID NO: 2), Ceres CLONE ID no.1915941 (SEQ ID NO:
8), Ceres ANNOT ID no.1461830 (SEQ ID NO: 10), Ceres CLONE ID
no.1080942 (SEQ ID NO: 15), and Ceres CLONE ID no.1073190 (SEQ ID
NO: 17). In all the alignment figures shown herein, a dash in an
aligned sequence represents a gap, i.e., a lack of an amino acid at
that position. Identical amino acids or conserved amino acid
substitutions among aligned sequences are identified by boxes. FIG.
1 and the other alignment figures provided herein were generated
using the program MUSCLE version 3.52.
[0029] FIG. 2 is an alignment of Ceres SEEDLINE ID no.ME04315 (SEQ
ID NO: 20) with homologous and/or orthologous amino acid sequences
Ceres CLONE ID no.1842825 (SEQ ID NO: 22), Ceres ANNOT ID
no.1482536 (SEQ ID NO: 28), Ceres CLONE ID no.463157 (SEQ ID NO:
32), Ceres CLONE ID no.1674443 (SEQ ID NO:40), GI ID no.116310719
(SEQ ID NO: 44), Ceres CLONE ID no.907473 (SEQ ID NO: 58), and
Ceres CLONE ID no.1755065 (SEQ ID NO: 71).
[0030] FIG. 3 is an alignment of full length homologous and/or
orthologous amino acid sequences of Ceres SEEDLINE ID no.ME17294
(SEQ ID NO: 93), including Ceres CLONE ID no.473040 (SEQ ID NO:
79), Ceres CLONE ID no.922223 (SEQ ID NO: 81), GI ID no.125528967
(SEQ ID NO: 82), GI ID no.125573200 (SEQ ID NO: 83), Ceres ANNOT ID
no.1527409 (SEQ ID NO: 85), GI ID no.92871098 (SEQ ID NO: 86),
Ceres CLONE ID no.1831117 (SEQ ID NO: 88), and Ceres ANNOT ID
no.857222 (SEQ ID NO: 90).
[0031] FIG. 4 is an alignment of a truncated version of Ceres
SEEDLINE ID no.ME17294 (SEQ ID NO: 93) with homologous and/or
orthologous amino acid truncated sequences, Ceres CLONE ID
no.1831117 (SEQ ID NO: 95), Ceres CLONE ID no.1844076 (SEQ ID NO:
98), Ceres CLONE ID no.473040 (SEQ ID NO:104), Ceres CLONE ID
no.922223 (SEQ ID NO: 106), and GI ID no.125528967 (SEQ ID
NO:107).
[0032] FIG. 5 is an alignment of functional homologs of the ARF
(Auxin Response Factor) genes ARF2, ARF3, and ARF4, including LOCUS
ID no. AT5G62000 (SEQ ID NO: 112), Ceres ANNOT ID no.1527370 (SEQ
ID NO: 114), Ceres ANNOT ID no.1473961 (SEQ ID NO: 116), GI ID
no.62319853 (SEQ ID NO: 117), GI ID no.62319903 (SEQ ID NO:118), GI
ID no.47716275 (SEQ ID NO: 119), GI ID no.125534572 (SEQ ID
NO:120), GI ID no.26251300(SEQ ID NO:121), GI ID no.115441981 (SEQ
ID NO:123), GI ID no.23893346 (SEQ ID NO:124), GI ID no.115485689
(SEQ ID NO:125), GI ID no.108864435 (SEQ ID NO:126), GI ID
no.50511471 (SEQ ID NO:127), LOCUS ID no. At2g33860 (SEQ ID
NO:128), GI ID no.2245390 (SEQ ID NO:131), GI ID no.3228517 (SEQ ID
NO:132), Ceres CLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE ID
no.1598488 (SEQ ID NO: 136), GI ID no.125553314 (SEQ ID NO:138),
and Ceres CLONE ID no.462443 (SEQ ID NO:143).
DETAILED DESCRIPTION
[0033] The invention features methods and materials related to
modulating cold tolerance levels in plants. In some embodiments,
the cold tolerance plants of the invention, under cold stress
and/or cold flux conditions, have modulated levels of growth, cold
acclimation, and/or cold damage. The methods can include
transforming a plant cell with a nucleic acid encoding a cold
tolerance modulating polypeptide, wherein expression of the
polypeptide results in a modulated level of cold tolerance. Plant
cells produced using such methods can be grown to produce plants
having an increased cold tolerance. Such plants, and the seeds of
such plants, may be used to produce, for example, plants and/or
plant tissues having increased biomass.
I. Definitions
[0034] "Amino acid" refers to one of the twenty biologically
occurring amino acids and to synthetic amino acids, including D/L
optical isomers.
[0035] "Cell type-preferential promoter" or "tissue-preferential
promoter" refers to a promoter that drives expression
preferentially in a target cell type or tissue, respectively, but
may also lead to some transcription in other cell types or tissues
as well.
[0036] "Cold." Plant species vary in their capacity to tolerate low
temperatures. Chilling-sensitive plant species, including many
agronomically important species, can be injured by cold,
above-freezing temperatures. At temperatures below the
freezing-point of water most plant species will be damaged. Thus,
"cold" can be defined as the temperature at which a given plant
species will be adversely affected as evidenced by symptoms such as
decreased photosynthesis and membrane damage (measured by
electrolyte leakage). Since plant species vary in their capacity to
tolerate cold, the precise environmental conditions that cause cold
stress cannot be generalized. However, cold tolerant plants are
characterized by their ability to retain their normal appearance,
recover quickly from low temperature conditions, exhibit normal or
increased growth under low temperature conditions, and/or have
improved cold acclimation. Such cold tolerant plants produce higher
biomass and/or yield than plants that are not cold tolerant.
Differences in physical appearance, recovery and yield can be
quantified and statistically analyzed using well known measurement
and analysis methods.
[0037] "Control plant" refers to a plant that does not contain the
exogenous nucleic acid present in a transgenic plant of interest,
but otherwise has the same or similar genetic background as such a
transgenic plant. A suitable control plant can be a non-transgenic
wild type plant, a non-transgenic segregant from a transformation
experiment, or a transgenic plant that contains an exogenous
nucleic acid other than the exogenous nucleic acid of interest.
[0038] "Domains" are groups of substantially contiguous amino acids
in a polypeptide that can be used to characterize protein families
and/or parts of proteins. Such domains have a "fingerprint" or
"signature" that can comprise conserved primary sequence, secondary
structure, and/or three-dimensional conformation. Generally,
domains are correlated with specific in vitro and/or in vivo
activities. A domain can have a length of from 10 amino acids to
400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino
acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to
60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino
acids.
[0039] "Down-regulation" refers to regulation that decreases
production of expression products (mRNA, polypeptide, or both)
relative to basal or native states.
[0040] "Exogenous" with respect to a nucleic acid indicates that
the nucleic acid is part of a recombinant nucleic acid construct,
or is not in its natural environment. For example, an exogenous
nucleic acid can be a sequence from one species introduced into
another species, i.e., a heterologous nucleic acid. Typically, such
an exogenous nucleic acid is introduced into the other species via
a recombinant nucleic acid construct. An exogenous nucleic acid can
also be a sequence that is native to an organism and that has been
reintroduced into cells of that organism. An exogenous nucleic acid
that includes a native sequence can often be distinguished from the
naturally occurring sequence by the presence of non-natural
sequences linked to the exogenous nucleic acid, e.g., non-native
regulatory sequences flanking a native sequence in a recombinant
nucleic acid construct. In addition, stably transformed exogenous
nucleic acids typically are integrated at positions other than the
position where the native sequence is found. It will be appreciated
that an exogenous nucleic acid may have been introduced into a
progenitor and not into the cell under consideration. For example,
a transgenic plant containing an exogenous nucleic acid can be the
progeny of a cross between a stably transformed plant and a
non-transgenic plant. Such progeny are considered to contain the
exogenous nucleic acid.
[0041] "Expression" refers to the process of converting genetic
information of a polynucleotide into RNA through transcription,
which is catalyzed by an enzyme, RNA polymerase, and into protein,
through translation of mRNA on ribosomes.
[0042] "Heterologous polypeptide" as used herein refers to a
polypeptide that is not a naturally occurring polypeptide in a
plant cell, e.g., a transgenic Panicum virgatum plant transformed
with and expressing the coding sequence for a nitrogen transporter
polypeptide from a Zea mays plant.
[0043] "Isolated nucleic acid" as used herein includes a
naturally-occurring nucleic acid, provided one or both of the
sequences immediately flanking that nucleic acid in its
naturally-occurring genome is removed or absent. Thus, an isolated
nucleic acid includes, without limitation, a nucleic acid that
exists as a purified molecule or a nucleic acid molecule that is
incorporated into a vector or a virus. A nucleic acid existing
among hundreds to millions of other nucleic acids within, for
example, cDNA libraries, genomic libraries, or gel slices
containing a genomic DNA restriction digest, is not to be
considered an isolated nucleic acid.
[0044] "Modulation" of the level of a cold tolerance refers to the
change in the level of the indicated compound or constituent that
is observed as a result of expression of, or transcription from, an
exogenous nucleic acid in a plant cell. The change in level is
measured relative to the corresponding level in control plants.
[0045] "Nucleic acid" and "polynucleotide" are used interchangeably
herein, and refer to both RNA and DNA, including cDNA, genomic DNA,
synthetic DNA, and DNA or RNA containing nucleic acid analogs.
Polynucleotides can have any three-dimensional structure. A nucleic
acid can be double-stranded or single-stranded (i.e., a sense
strand or an antisense strand). Non-limiting examples of
polynucleotides include genes, gene fragments, exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA,
micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, nucleic acid probes and nucleic acid primers. A
polynucleotide may contain unconventional or modified
nucleotides.
[0046] "Operably linked" refers to the positioning of a regulatory
region and a sequence to be transcribed in a nucleic acid so that
the regulatory region is effective for regulating transcription or
translation of the sequence. For example, to operably link a coding
sequence and a regulatory region, the translation initiation site
of the translational reading frame of the coding sequence is
typically positioned between one and about fifty nucleotides
downstream of the regulatory region. A regulatory region can,
however, be positioned as much as about 5,000 nucleotides upstream
of the translation initiation site, or about 2,000 nucleotides
upstream of the transcription start site.
[0047] "Polypeptide" as used herein refers to a compound of two or
more subunit amino acids, amino acid analogs, or other
peptidomimetics, regardless of post-translational modification,
e.g., phosphorylation or glycosylation. The subunits may be linked
by peptide bonds or other bonds such as, for example, ester or
ether bonds. Full-length polypeptides, truncated polypeptides,
point mutants, insertion mutants, splice variants, chimeric
proteins, and fragments thereof are encompassed by this
definition.
[0048] "Progeny" includes descendants of a particular plant or
plant line. Progeny of an instant plant include seeds formed on
F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5, F.sub.6 and subsequent
generation plants, or seeds formed on BC.sub.1, BC.sub.2, BC.sub.3,
and subsequent generation plants, or seeds formed on
F.sub.1BC.sub.1, F.sub.1BC.sub.2, F.sub.1BC.sub.3, and subsequent
generation plants. The designation F.sub.1 refers to the progeny of
a cross between two parents that are genetically distinct. The
designations F.sub.2, F.sub.3, F.sub.4, F.sub.5 and F.sub.6 refer
to subsequent generations of self- or sib-pollinated progeny of an
F.sub.1 plant.
[0049] "Regulatory region" refers to a nucleic acid having
nucleotide sequences that influence transcription or translation
initiation and rate, and stability and/or mobility of a
transcription or translation product. Regulatory regions include,
without limitation, promoter sequences, enhancer sequences,
response elements, protein recognition sites, inducible elements,
protein binding sequences, 5' and 3' untranslated regions (UTRs),
transcriptional start sites, termination sequences, polyadenylation
sequences, introns, and combinations thereof. A regulatory region
typically comprises at least a core (basal) promoter. A regulatory
region also may include at least one control element, such as an
enhancer sequence, an upstream element or an upstream activation
region (UAR). For example, a suitable enhancer is a cis-regulatory
element (-212 to -154) from the upstream region of the octopine
synthase (ocs) gene. Fromm et al., The Plant Cell, 1:977-984
(1989).
[0050] "Up-regulation" refers to regulation that increases the
level of an expression product (mRNA, polypeptide, or both)
relative to basal or native states.
[0051] "Vector" refers to a replicon, such as a plasmid, phage, or
cosmid, into which another DNA segment may be inserted so as to
bring about the replication of the inserted segment. Generally, a
vector is capable of replication when associated with the proper
control elements. The term "vector" includes cloning and expression
vectors, as well as viral vectors and integrating vectors. An
"expression vector" is a vector that includes a regulatory
region.
II. Polypeptides
[0052] Polypeptides described herein include cold
tolerance-modulating polypeptides. Cold tolerance-modulating
polypeptides can be effective to modulate cold tolerance levels
when expressed in a plant or plant cell. Such polypeptides
typically contain at least one domain indicative of cold
tolerance-modulating polypeptides, as described in more detail
herein. Cold tolerance-modulating polypeptides typically have an
HMM bit score that is greater than 130, as described in more detail
herein. In some embodiments, cold tolerance-modulating polypeptides
have greater than 80% identity to SEQ ID NOs: 2, 20, 74, 93 or 112,
as described in more detail herein.
A. Domains Indicative of Cold Tolerance-Modulating Polypeptides
[0053] A cold tolerance-modulating polypeptide can contain a B-box
zinc finger domain. The B-box zinc finger domain is often found
associated with CCT motif. SEQ ID NO: 20 sets forth the amino acid
sequence of an Arabidopsis clone, identified herein as Ceres
SEEDLINE ID no.ME04315 (SEQ ID NO: 20), that is predicted to encode
a polypeptide containing a CCT motif and a B-box zinc finger
domain. A B-box zinc finger domain is typically around 40 amino
acids in length. This motif is generally associated with a finger.
It is found, for example, in transcription factors,
ribonucleoproteins and protooncoproteins. It has been shown to be
essential but not sufficient to localize the PML protein in a
punctate pattern in interphase nuclei. Among the 7 possible ligands
for the zinc atom contained in a B-box, only 4 are used and bind
one zinc atom in a Cys2-His2 tetrahedral arrangement. The NMR
analysis reveals that the B-box structure comprises two
beta-strands, two helical turns and three extended loop regions
different from any other zinc binding motif. A CCT motif can be
found in a number of plant proteins. It is rich in basic amino
acids and has been called a CCT motif after Co, Co1 and Toc1. The
CCT motif is about 45 amino acids long and contains a putative
nuclear localization signal within the second half of the CCT
motif. Toc1 mutants have been identified in this region. The CCT
(CONSTANS, CO-like, and TOC1) domain is a highly conserved basic
module of .about.43 amino acids, which is found near the C-terminus
of plant proteins. The CCT domain is often found in association
with other domains, such as the B-box zinc finger, the GATA-type
zinc finger, the ZIM motif or the response regulatory domain. The
CCT domain contains a putative nuclear localization signal within
the second half of the CCT motif and has been shown to be involved
in nuclear localization and probably also has a role in
protein-protein interaction.
[0054] In embodiments of the invention, a cold tolerance-modulating
polypeptide can comprise a CCT motif having 80% or greater sequence
identity to amino acid residues 285 to 329 of SEQ ID NO: 20,
residues 291 to 335 of SEQ ID NO: 22, residues 235 to 279 of SEQ ID
NO: 24, residues 217 to 261 of SEQ ID NO: 26, residues 311 to 355
of SEQ ID NO: 28, residues 285 to 329 of SEQ ID NO: 29, residues
281 to 325 of SEQ ID NO: 30, residues 302 to 346 of SEQ ID NO: 32,
residues 289 to 333 of SEQ ID NO: 34, residues 295 to 339 of SEQ ID
NO: 36, residues 261 to 305 of SEQ ID NO: 38, residues 284 to 328
of SEQ ID NO: 40, residues 288 to 332 of SEQ ID NO: 42, residues
261 to 305 of SEQ ID NO: 43, residues 239 to 283 of SEQ ID NO: 44,
residues 294 to 338 of SEQ ID NO: 45, residues 279 to 323 of SEQ ID
NO: 46, residues 261 to 305 of SEQ ID NO: 47, residues 239 to 283
of SEQ ID NO: 48, residues 294 to 338 of SEQ ID NO: 49, residues
261 to 305 of SEQ ID NO: 50, residues 298 to 342 of SEQ ID NO: 51,
residues 241 to 285 of SEQ ID NO: 52, residues 268 to 312 of SEQ ID
NO: 53, residues 245 to 289 of SEQ ID NO: 54, residues 238 to 282
of SEQ ID NO: 56, residues 245 to 289 of SEQ ID NO: 58, residues
279 to 323 of SEQ ID NO: 59, residues 236 to 280 of SEQ ID NO: 60,
residues 250 to 294 of SEQ ID NO: 61, residues 322 to 366 of SEQ ID
NO: 62, residues 297 to 341 of SEQ ID NO: 63, residues 348 to 392
of SEQ ID NO: 64, residues 312 to 356 of SEQ ID NO: 65, residues
340 to 384 of SEQ ID NO: 68, residues 307 to 351 of SEQ ID NO: 69,
or residues 253 to 297 of SEQ ID NO: 71, or a CCT motif identified
in the sequence listing.
[0055] In embodiments of the invention, a cold tolerance-modulating
polypeptide can comprise a B-box zinc finger domain having 80% or
greater sequence identity to amino acid residues 56 to 103 of SEQ
ID NO: 20, residues 62 to 109 of SEQ ID NO: 22, residues 64 to 106
of SEQ ID NO: 24, residues 34 to 81 of SEQ ID NO: 26, residues 63
to 110 of SEQ ID NO: 28, residues 56 to 103 of SEQ ID NO: 29,
residues 56 to 103 of SEQ ID NO: 30, residues 60 to 107 of SEQ ID
NO: 32, residues 56 to 103 of SEQ ID NO: 34, residues 51 to 98 of
SEQ ID NO: 36, residues 70 to 112 of SEQ ID NO: 38, residues 51 to
98 of SEQ ID NO: 40, residues 52 to 99 of SEQ ID NO: 42, residues
72 to 114 of SEQ ID NO: 43, residues 62 to 104 of SEQ ID NO: 44,
residues 50 to 97 of SEQ ID NO: 45, residues 55 to 102 of SEQ ID
NO: 46, residues 72 to 114 of SEQ ID NO: 47, residues 62 to 104 of
SEQ ID NO: 48, residues 50 to 97 of SEQ ID NO: 49, residues 27 to
71 of SEQ ID NO: 50, residues 60 to 107 of SEQ ID NO: 51, residues
1 to 48 of SEQ ID NO: 52, residues 1 to 48 of SEQ ID NO: 53,
residues 1 to 48 of SEQ ID NO: 54, residues 62 to 104 of SEQ ID NO:
56, residues 64 to 106 of SEQ ID NO: 58, residues 1 to 48 of SEQ ID
NO: 59, residues 61 to 103 of SEQ ID NO: 60, residues 70 to 112 of
SEQ ID NO: 61, residues 52 to 99 of SEQ ID NO: 62, residues 51 to
98 of SEQ ID NO: 63, residues 77 to 119 of SEQ ID NO: 64, residues
59 to 106 of SEQ ID NO: 65, residues 59 to 106 of SEQ ID NO: 68,
residues 34 to 66 of SEQ ID NO: 69, or residues 64 to 106 of SEQ ID
NO: 71, or a B-box zinc finger domain identified in the sequence
listing.
[0056] A cold tolerance-modulating polypeptide can contain a
short-chain dehydrogenase domain The motif is present in SEQ ID NO:
93, which sets forth the amino acid sequence of an Arabidopsis
clone, identified herein as Ceres SEEDLINE ID no.ME17294 (SEQ ID
NO: 93), that is predicted to encode a polypeptide containing a
short-chain dehydrogenase domain. The short-chain
dehydrogenases/reductases family (SDR) is a very large family of
enzymes, most of which are known to be NAD- or NADP-dependent
oxidoreductases. As the first member of this family to be
characterized was Drosophila alcohol dehydrogenase, this family
used to be called `insect-type`, or `short-chain` alcohol
dehydrogenases. Most members of this family are proteins of about
250 to 300 amino acid residues.
[0057] In embodiments of the invention, a cold tolerance-modulating
polypeptide can comprise a short-chain dehydrogenase domain having
80% or greater identity to amino acid residues 38 to 173 of SEQ ID
NO: 74, residues 37 to 174 of SEQ ID NO: 76, residues 23 to 160 of
SEQ ID NO: 77, residues 7 to 168 of SEQ ID NO: 79, residues 43 to
179 of SEQ ID NO: 81, residues 49 to 188 of SEQ ID NO: 82, residues
48 to 187 of SEQ ID NO: 83, residues 37 to 172 of SEQ ID NO: 85,
residues 35 to 170 of SEQ ID NO: 86, residues 20 to 160 of SEQ ID
NO: 88, or residues 37 to 174 of SEQ ID NO: 90, or a short-chain
dehydrogenase domain identified in the sequence listing.
[0058] In some embodiments, a cold tolerance-modulating polypeptide
is truncated at the amino- or carboxy-terminal end of a naturally
occurring polypeptide. A truncated polypeptide may retain certain
domains of the naturally occurring polypeptide while lacking
others. Thus, length variants that are up to 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 amino
acids shorter or longer typically exhibit the cold
tolerance-modulating activity of a truncated polypeptide. In some
embodiments, a truncated polypeptide is a dominant negative
polypeptide. SEQ ID NO: 93 sets forth the amino sequence of a cold
tolerance-modulating polypeptide that is truncated at the
amino-terminal end relative to a naturally occurring polypeptide.
Expression in a plant and/or plant tissue of such a truncated
polypeptide confers a difference in the level of cold tolerance in
a plant and/or tissue of the plant as compared to the corresponding
level in tissue of a control plant.
B. Functional Homologs Identified by Reciprocal BLAST
[0059] In some embodiments, one or more functional homologs of a
reference cold tolerance-modulating polypeptide defined by one or
more of the Pfam descriptions indicated above are suitable for use
as cold tolerance-modulating polypeptides. A functional homolog is
a polypeptide that has sequence similarity to a reference
polypeptide, and that carries out one or more of the biochemical or
physiological function(s) of the reference polypeptide. A
functional homolog and the reference polypeptide may be natural
occurring polypeptides, and the sequence similarity may be due to
convergent or divergent evolutionary events. As such, functional
homologs are sometimes designated in the literature as homologs, or
orthologs, or paralogs. Variants of a naturally occurring
functional homolog, such as polypeptides encoded by mutants of a
wild type coding sequence, may themselves be functional homologs.
Functional homologs can also be created via site-directed
mutagenesis of the coding sequence for a cold tolerance-modulating
polypeptide, or by combining domains from the coding sequences for
different naturally-occurring cold tolerance-modulating
polypeptides ("domain swapping"). The term "functional homolog" is
sometimes applied to the nucleic acid that encodes a functionally
homologous polypeptide.
[0060] Functional homologs can be identified by analysis of
nucleotide and polypeptide sequence alignments. For example,
performing a query on a database of nucleotide or polypeptide
sequences can identify homologs of cold tolerance-modulating
polypeptides. Sequence analysis can involve BLAST, Reciprocal
BLAST, or PSI-BLAST analysis of nonredundant databases using a cold
tolerance-modulating polypeptide amino acid sequence as the
reference sequence. Amino acid sequence is, in some instances,
deduced from the nucleotide sequence. Those polypeptides in the
database that have greater than 40% sequence identity are
candidates for further evaluation for suitability as a cold
tolerance-modulating polypeptide Amino acid sequence similarity
allows for conservative amino acid substitutions, such as
substitution of one hydrophobic residue for another or substitution
of one polar residue for another. If desired, manual inspection of
such candidates can be carried out in order to narrow the number of
candidates to be further evaluated. Manual inspection can be
performed by selecting those candidates that appear to have domains
present in cold tolerance-modulating polypeptides, e.g., conserved
functional domains.
[0061] Conserved regions can be identified by locating a region
within the primary amino acid sequence of a cold
tolerance-modulating polypeptide that is a repeated sequence, forms
some secondary structure (e.g., helices and beta sheets),
establishes positively or negatively charged domains, or represents
a protein motif or domain. See, e.g., the Pfam web site describing
consensus sequences for a variety of protein motifs and domains on
the World Wide Web at sanger.ac.uk/Software/Pfam/ and
pfam.janelia.org/. A description of the information included at the
Pfam database is described in Sonnhammer et al., Nucl. Acids Res.,
26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997);
and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved
regions also can be determined by aligning sequences of the same or
related polypeptides from closely related species. Closely related
species preferably are from the same family. In some embodiments,
alignment of sequences from two different species is adequate.
[0062] Typically, polypeptides that exhibit at least about 40%
amino acid sequence identity are useful to identify conserved
regions. Conserved regions of related polypeptides exhibit at least
45% amino acid sequence identity (e.g., at least 50%, at least 60%,
at least 70%, at least 80%, or at least 90% amino acid sequence
identity). In some embodiments, a conserved region exhibits at
least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
[0063] Examples of amino acid sequences of functional homologs of
the polypeptide set forth in SEQ ID NO: 2 are provided in FIG. 1
and in the Sequence Listing. Such exemplary functional homologs
include Ceres CLONE ID no.1897908 (SEQ ID NO:4), Ceres CLONE ID
no.1938030 (SEQ ID NO: 6), Ceres CLONE ID no.1915941 (SEQ ID NO:
8), Ceres ANNOT ID no.1461830 (SEQ ID NO: 10), Ceres ANNOT ID
no.1439985 (SEQ ID NO: 12), GI ID no.15241794 (SEQ ID NO: 13),
Ceres CLONE ID no.1080942 (SEQ ID NO:15), and Ceres CLONE ID
no.1073190 (SEQ ID NO:17). In some cases, a functional homolog of
SEQ ID NO: 2 has an amino acid sequence with at least 20% sequence
identity, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%, 61%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence
identity, to the amino acid sequence set forth in SEQ ID NO: 2, 4,
6, 8, 10, 12, 13, 15, or 17.
[0064] Examples of amino acid sequences of functional homologs of
the polypeptide set forth in SEQ ID NO: 20 are provided in FIG. 2
and in the Sequence Listing. Such exemplary functional homologs
include Ceres CLONE ID no.1842825 (SEQ ID NO: 22), Ceres CLONE ID
no.1834027 (SEQ ID NO: 24), Ceres CLONE ID no.1837064 (SEQ ID NO:
26), Ceres ANNOT ID no.1482536 (SEQ ID NO: 28), GI ID no.18424009
(SEQ ID NO: 29), GI ID no.9759262 (SEQ ID NO: 30), Ceres CLONE ID
no.463157 (SEQ ID NO: 32), Ceres CLONE ID no.685991 (SEQ ID NO:
34), Ceres CLONE ID no.702632 (SEQ ID NO: 36), Ceres CLONE ID
no.1559496 (SEQ ID NO: 38), Ceres CLONE ID no.1674443 (SEQ ID NO:
40), Ceres CLONE ID no.1828897 (SEQ ID NO: 42), GI ID no.125540249
(SEQ ID NO: 43), GI ID no.116310719 (SEQ ID NO: 44), GI ID
no.125556324 (SEQ ID NO: 45), GI ID no.125538317 (SEQ ID NO: 46),
GI ID no.115447239 (SEQ ID NO: 47), GI ID no.115459216 (SEQ ID NO:
48), GI ID no.115469296 (SEQ ID NO: 49), GI ID no.125582846 (SEQ ID
NO: 50), GI ID no.92875402 (SEQ ID NO: 51), GI ID no.3341723 (SEQ
ID NO: 52), GI ID no.4091806 (SEQ ID NO: 53), GI ID no.60459257
(SEQ ID NO: 54), Ceres CLONE ID no.1756710 (SEQ ID NO: 56), Ceres
CLONE ID no.907473 (SEQ ID NO: 58), GI ID no.4091804 (SEQ ID NO:
59), GI ID no.21667487 (SEQ ID NO: 60), GI ID no.21655154 (SEQ ID
NO: 61), GI ID no.45544883 (SEQ ID NO: 62), GI ID no.21655166 (SEQ
ID NO: 63), GI ID no.10946337 (SEQ ID NO: 64), GI ID no.90657642
(SEQ ID NO: 65), GI ID no.45544887 (SEQ ID NO: 68), GI ID
no.47606678 (SEQ ID NO: 69), or Ceres CLONE ID no.1755065 (SEQ ID
NO: 71). In some cases, a functional homolog of SEQ ID NO: 20 has
an amino acid sequence with at least 20% sequence identity, e.g.,
25%, 30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO: 20, 22, 24, 26, 28, 29,
30, 32, 34, 36, 38, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 56, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, or 71.
[0065] Examples of amino acid sequences of full length functional
homologs of the truncated polypeptide set forth in SEQ ID NO: 93
are provided in FIG. 3 and in the Sequence Listing. Such exemplary
functional homologs include Ceres CLONE ID no.1844076 (SEQ ID NO:
74), Ceres CLONE ID no.35974 (SEQ ID NO: 76), GI ID no.10176876
(SEQ ID NO: 77), Ceres CLONE ID no.473040 (SEQ ID NO: 79), Ceres
CLONE ID no.922223 (SEQ ID NO: 81), GI ID no.125528967 (SEQ ID NO:
82), GI ID no.125573200 (SEQ ID NO: 83), Ceres ANNOT ID no.1527409
(SEQ ID NO: 85), GI ID no.92871098 (SEQ ID NO: 86), Ceres CLONE ID
no.1831117 (SEQ ID NO: 88), and Ceres ANNOT ID no.857222 (SEQ ID
NO: 90). In some cases, a full length functional homolog of SEQ ID
NO: 93 has an amino acid sequence with at least 20% sequence
identity, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%, 61%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence
identity, to the amino acid sequence set forth in SEQ ID NO: 74,
76, 77, 79, 81, 82, 83, 85, 86, 88, 90, or 93.
[0066] Examples of amino acid sequences of functional homologs of
the polypeptide set forth in SEQ ID NO: 93 are provided in FIG. 4
and in the Sequence Listing. Such exemplary functional homologs
include Ceres ANNOT ID no.857222 (SEQ ID NO: 110), Ceres CLONE ID
no.1831117 (SEQ ID NO: 95), GI ID no.92871098 (SEQ ID NO: 96),
Ceres CLONE ID no.1844076 (SEQ ID NO: 98), Ceres CLONE ID no.35974
(SEQ ID NO: 100), GI ID no.110737329 (SEQ ID NO: 101), GI ID
no.10176876 (SEQ ID NO: 102), Ceres CLONE ID no.473040 (SEQ ID NO:
104), Ceres CLONE ID no.922223 (SEQ ID NO: 106), GI ID no.125528967
(SEQ ID NO: 107), and GI ID no.115442007 (SEQ ID NO: 108). In some
cases, a functional homolog of SEQ ID NO: 93 has an amino acid
sequence with at least 20% sequence identity, e.g., 25%, 30%, 35%,
40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence
set forth in SEQ ID NO: 93, 110, 95, 96, 98, 100, 101, 102, 104,
106, 107, or 108.
[0067] Examples of amino acid sequences of functional homologs of
the polypeptide set forth in SEQ ID NO: 116 are provided in FIG. 5
and in the Sequence Listing. Such exemplary functional homologs
include LOCUS ID no. AT5G62000 (SEQ ID NO: 112), Ceres ANNOT ID
no.1527370 (SEQ ID NO: 114), GI ID no.62319853 (SEQ ID NO: 117), GI
ID no.62319903 (SEQ ID NO: 118), GI ID no.47716275 (SEQ ID NO:
119), GI ID no.125534572 (SEQ ID NO: 120), GI ID no.26251300 (SEQ
ID NO: 121), GI ID no.125528952 (SEQ ID NO: 122), GI ID
no.115441981 (SEQ ID NO: 123), GI ID no.23893346 (SEQ ID NO: 124),
GI ID no.115485689 (SEQ ID NO: 125), GI ID no.108864435 (SEQ ID NO:
126), GI ID no.50511471 (SEQ ID NO: 127), LOCUS ID no. At2g33860
(SEQ ID NO: 128), Ceres ANNOT ID no.1536494 (SEQ ID NO: 130), GI ID
no.2245390 (SEQ ID NO: 131), GI ID no.3228517 (SEQ ID NO: 132),
Ceres CLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE ID
no.1598488 (SEQ ID NO: 136), GI ID no.125527740 (SEQ ID NO: 137),
GI ID no.125553314 (SEQ ID NO: 138), LOCUS ID no. At5g60450 (SEQ ID
NO: 139), Ceres ANNOT ID no.1515383 (SEQ ID NO: 141), and Ceres
CLONE ID no.462443 (SEQ ID NO: 143). In some cases, a functional
homolog of SEQ ID NO: 116 has an amino acid sequence with at least
20% sequence identity, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 52%,
56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%
sequence identity, to the amino acid sequence set forth in SEQ ID
NO: 116, 112, 114, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 130, 131, 132, 134, 136, 137, 138, 139, 141, or
143.
[0068] Examples of nucleic acid sequences of functional homologs of
the tasiRNA encoding nucleic acid sequence set forth in SEQ ID NO:
111 are found in the Sequence Listing. Such exemplary functional
homologs include 66, 67, 72, 73, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, and 156. In some cases, a functional
homolog of SEQ ID NO: 111 has an nucleic acid sequence with at
least 20% sequence identity, e.g., 25%, 30%, 35%, 40%, 45%, 50%,
52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or
99% sequence identity, to the nucleic acid sequence set forth in
SEQ ID NO: 111, 66, 67, 72, 73, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, or 156.
[0069] The identification of conserved regions in a cold
tolerance-modulating polypeptide facilitates production of variants
of cold tolerance-modulating polypeptides. Variants of cold
tolerance-modulating polypeptides typically have 10 or fewer
conservative amino acid substitutions within the primary amino acid
sequence, e.g., 7 or fewer conservative amino acid substitutions, 5
or fewer conservative amino acid substitutions, or between 1 and 5
conservative substitutions. A useful variant polypeptide can be
constructed based on one of the alignments set forth in FIG. 1,
FIG. 2, FIG. 3, FIG. 4, or FIG. 5, and/or homologs identified in
the Sequence Listing. Such a polypeptide includes the conserved
regions, arranged in the order depicted in the Figures from
amino-terminal end to carboxy-terminal end. Such a polypeptide may
also include zero, one, or more than one amino acid in positions
marked by dashes. When no amino acids are present at positions
marked by dashes, the length of such a polypeptide is the sum of
the amino acid residues in all conserved regions. When amino acids
are present at all positions marked by dashes, such a polypeptide
has a length that is the sum of the amino acid residues in all
conserved regions and all dashes.
C. Functional Homologs Identified by HMMER
[0070] In some embodiments, useful cold tolerance-modulating
polypeptides include those that fit a Hidden Markov Model based on
the polypeptides set forth in any one of FIGS. 1-4 or ARFs that are
acted upon by tasiRNA (FIG. 5). A Hidden Markov Model (HMM) is a
statistical model of a consensus sequence for a group of functional
homologs. See, Durbin et al., Biological Sequence Analysis:
Probabilistic Models of Proteins and Nucleic Acids, Cambridge
University Press, Cambridge, UK (1998). An HMM is generated by the
program HMMER 2.3.2 with default program parameters, using the
sequences of the group of functional homologs as input. The
multiple sequence alignment is generated by ProbCons (Do et al.,
Genome Res., 15(2):330-40 (2005)) version 1.11 using a set of
default parameters: -c, --consistency REPS of 2; -ir,
--iterative-refinement REPS of 100; -pre, --pre-training REPS of 0.
ProbCons is a public domain software program provided by Stanford
University.
[0071] The default parameters for building an HMM (hmmbuild) are as
follows: the default "architecture prior" (archpri) used by MAP
architecture construction is 0.85, and the default cutoff threshold
(idlevel) used to determine the effective sequence number is 0.62.
HMMER 2.3.2 was released October 3, 2003 under a GNU general public
license, and is available from various sources on the World Wide
Web such as hmmer janelia.org; hmmer wustl.edu; and
fr.com/hmmer232/. Hmmbuild outputs the model as a text file.
[0072] The HMM for a group of functional homologs can be used to
determine the likelihood that a candidate cold tolerance-modulating
polypeptide sequence is a better fit to that particular HMM than to
a null HMM generated using a group of sequences that are not
structurally or functionally related. The likelihood that a
candidate polypeptide sequence is a better fit to an HMM than to a
null HMM is indicated by the HMM bit score, a number generated when
the candidate sequence is fitted to the HMM profile using the HMMER
hmmsearch program. The following default parameters are used when
running hmmsearch: the default E-value cutoff (E) is 10.0, the
default bit score cutoff (T) is negative infinity, the default
number of sequences in a database (Z) is the real number of
sequences in the database, the default E-value cutoff for the
per-domain ranked hit list (domE) is infinity, and the default bit
score cutoff for the per-domain ranked hit list (domT) is negative
infinity. A high HMM bit score indicates a greater likelihood that
the candidate sequence carries out one or more of the biochemical
or physiological function(s) of the polypeptides used to generate
the HMM. A high HMM bit score is at least 20, and often is higher.
Slight variations in the HMM bit score of a particular sequence can
occur due to factors such as the order in which sequences are
processed for alignment by multiple sequence alignment algorithms
such as the ProbCons program. Nevertheless, such HMM bit score
variation is minor
[0073] The cold tolerance-modulating polypeptides discussed below
fit the indicated HMM with an HMM bit score greater than 20 (e.g.,
greater than 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or
500). In some embodiments, the HMM bit score of a cold
tolerance-modulating polypeptide discussed below is about 50%, 60%,
70%, 80%, 90%, or 95% of the HMM bit score of a functional homolog
provided in the Sequence Listing of this application. In some
embodiments, a cold tolerance-modulating polypeptide discussed
below fits the indicated HMM with an HMM bit score greater than 20,
and has a domain indicative of a cold tolerance-modulating
polypeptide. In some embodiments, a cold tolerance-modulating
polypeptide discussed below fits the indicated HMM with an HMM bit
score greater than 20, and has 70% or greater sequence identity
(e.g., 75%, 80%, 85%, 90%, 95%, or 100% sequence identity) to an
amino acid sequence shown in any one of FIGS. 1-5.
[0074] Examples of polypeptides are shown in the Sequence Listing
that have HMM bit scores greater than 130 when fitted to an HMM
generated from the amino acid sequences set forth in FIG. 1 are
identified in the Sequence Listing of this application. Such
polypeptides include Ceres CLONE ID no.1915941 (SEQ ID NO: 8),
Ceres ANNOT ID no.1461830 (SEQ ID NO: 10), Ceres CLONE ID
no.1080942 (SEQ ID NO: 15), and Ceres CLONE ID no.1073190 (SEQ ID
NO: 17).
[0075] Examples of polypeptides are shown in the Sequence Listing
that have HMM bit scores greater than 185 when fitted to an HMM
generated from the amino acid sequences set forth in FIG. 2 are
identified in the Sequence Listing of this application. Such
polypeptides include Ceres CLONE ID no.1842825 (SEQ ID NO:22),
Ceres ANNOT ID no.1482536 (SEQ ID NO: 28), Ceres CLONE ID no.463157
(SEQ ID NO: 32), Ceres CLONE ID no.1674443 (SEQ ID NO: 40), GI ID
no.116310719 (SEQ ID NO: 44), Ceres CLONE ID no.907473 (SEQ ID NO:
58), and Ceres CLONE ID no.1755065 (SEQ ID NO:71).
[0076] Examples of polypeptides are shown in the Sequence Listing
that have HMM bit scores greater than 655 when fitted to an HMM
generated from the amino acid sequences set forth in FIG. 3 are
identified in the Sequence Listing of this application. Such
polypeptides include Ceres CLONE ID no.473040 (SEQ ID NO: 79),
Ceres CLONE ID no.922223 (SEQ ID NO: 81), GI ID no.125528967 (SEQ
ID NO: 82), GI ID no.125573200 (SEQ ID NO: 83), Ceres ANNOT ID
no.1527409 (SEQ ID NO: 85), GI ID no.92871098 (SEQ ID NO: 86),
Ceres CLONE ID no.1831117 (SEQ ID NO: 88), and Ceres ANNOT ID
no.857222 (SEQ ID NO: 90).
[0077] Examples of polypeptides are shown in the Sequence Listing
that have HMM bit scores greater than 315 when fitted to an HMM
generated from the amino acid sequences set forth in FIG. 4 are
identified in the Sequence Listing of this application. Such
polypeptides include Ceres SEEDLINE ID no.ME17294 (SEQ ID NO: 93),
Ceres CLONE ID no.1831117 (SEQ ID NO: 95), Ceres CLONE ID
no.1844076 (SEQ ID NO: 98), Ceres CLONE ID no.473040 (SEQ ID NO:
104), Ceres CLONE ID no.922223 (SEQ ID NO: 106), and GI ID
no.125528967 (SEQ ID NO: 107).
[0078] Examples of polypeptides are shown in the Sequence Listing
that have HMM bit scores greater than 790 when fitted to an HMM
generated from the amino acid sequences set forth in FIG. 5 are
identified in the Sequence Listing of this application. Such
polypeptides include LOCUS ID no. AT5G62000 (SEQ ID NO: 112), Ceres
ANNOT ID no.1527370 (SEQ ID NO: 114), Ceres ANNOT ID no.1473961
(SEQ ID NO: 116), GI ID no.62319853 (SEQ ID NO: 117), GI ID
no.62319903 (SEQ ID NO:118), GI ID no.47716275 (SEQ ID NO: 119), GI
ID no.125534572 (SEQ ID NO:120), GI ID no.26251300(SEQ ID NO:121),
GI ID no.115441981 (SEQ ID NO:123), GI ID no.23893346 (SEQ ID
NO:124), GI ID no.115485689 (SEQ ID NO:125), GI ID no.108864435
(SEQ ID NO:126), GI ID no.50511471 (SEQ ID NO:127), LOCUS ID no.
At2g33860 (SEQ ID NO:128), GI ID no.2245390 (SEQ ID NO:131), GI ID
no.3228517 (SEQ ID NO:132), Ceres CLONE ID no.827306 (SEQ ID NO:
134), Ceres CLONE ID no.1598488 (SEQ ID NO: 136), GI ID
no.125553314 (SEQ ID NO: 138), and Ceres CLONE ID no.462443 (SEQ ID
NO:143).
D. Percent Identity
[0079] In some embodiments, a cold tolerance-modulating polypeptide
has an amino acid sequence with at least 45% sequence identity,
e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98%, or 99% sequence identity, to one of the amino acid
sequences set forth in SEQ ID NOs: 2, 20, 93, and 74.
[0080] Polypeptides having such a percent sequence identity often
have a domain indicative of a cold tolerance-modulating polypeptide
and/or have an HMM bit score that is greater than 130, as discussed
above Amino acid sequences of cold tolerance-modulating
polypeptides having at least 80% sequence identity to one of the
amino acid sequences set forth in SEQ ID NOs: 2, 20, 93, and 74 are
provided in FIGS. 1-5 and in the Sequence Listing.
[0081] "Percent sequence identity" refers to the degree of sequence
identity between any given reference sequence, e.g., SEQ ID NO: 2,
and a candidate cold tolerance-modulating sequence. A candidate
sequence typically has a length that is from 80 percent to 200
percent of the length of the reference sequence, e.g., 82, 85, 87,
89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150,
160, 170, 180, 190, or 200 percent of the length of the reference
sequence. A percent identity for any candidate nucleic acid or
polypeptide relative to a reference nucleic acid or polypeptide can
be determined as follows. A reference sequence (e.g., a nucleic
acid sequence or an amino acid sequence) is aligned to one or more
candidate sequences using the computer program ClustalW (version
1.83, default parameters), which allows alignments of nucleic acid
or polypeptide sequences to be carried out across their entire
length (global alignment). Chenna et al., Nucleic Acids Res.,
31(13):3497-500 (2003).
[0082] ClustalW calculates the best match between a reference and
one or more candidate sequences, and aligns them so that
identities, similarities and differences can be determined. Gaps of
one or more residues can be inserted into a reference sequence, a
candidate sequence, or both, to maximize sequence alignments. For
fast pairwise alignment of nucleic acid sequences, the following
default parameters are used: word size: 2; window size: 4; scoring
method: percentage; number of top diagonals: 4; and gap penalty: 5.
For multiple alignment of nucleic acid sequences, the following
parameters are used: gap opening penalty: 10.0; gap extension
penalty: 5.0; and weight transitions: yes. For fast pairwise
alignment of protein sequences, the following parameters are used:
word size: 1; window size: 5; scoring method: percentage; number of
top diagonals: 5; gap penalty: 3. For multiple alignment of protein
sequences, the following parameters are used: weight matrix:
blosum; gap opening penalty: 10.0; gap extension penalty: 0.05;
hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn,
Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on.
The ClustalW output is a sequence alignment that reflects the
relationship between sequences. ClustalW can be run, for example,
at the Baylor College of Medicine Search Launcher site
(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at
the European Bioinformatics Institute site on the World Wide Web
(ebi.ac.uk/clustalw).
[0083] To determine percent identity of a candidate nucleic acid or
amino acid sequence to a reference sequence, the sequences are
aligned using ClustalW, the number of identical matches in the
alignment is divided by the length of the reference sequence, and
the result is multiplied by 100. It is noted that the percent
identity value can be rounded to the nearest tenth. For example,
78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while
78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[0084] In some cases, a cold tolerance-modulating polypeptide has
an amino acid sequence with at least 45% sequence identity, e.g.,
50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO: 2. Amino acid sequences of polypeptides having
greater than 45% sequence identity to the polypeptide set forth in
SEQ ID NO: 2 are provided in FIG. 1 and in the Sequence Listing.
Examples of such polypeptides include Ceres CLONE ID no.1915941
(SEQ ID NO: 8), Ceres ANNOT ID no.1461830 (SEQ ID NO: 10), Ceres
CLONE ID no.1080942 (SEQ ID NO: 15), and Ceres CLONE ID no.1073190
(SEQ ID NO: 17).
[0085] In some cases, a cold tolerance-modulating polypeptide has
an amino acid sequence with at least 45% sequence identity, e.g.,
50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO: 20 Amino acid sequences of polypeptides having
greater than 45% sequence identity to the polypeptide set forth in
SEQ ID NO: 20 are provided in FIG. 2 and in the Sequence Listing.
Examples of such polypeptides include Ceres CLONE ID no.1842825
(SEQ ID NO: 22), Ceres ANNOT ID no.1482536 (SEQ ID NO: 28), Ceres
CLONE ID no.463157 (SEQ ID NO: 32), Ceres CLONE ID no.1674443 (SEQ
ID NO:40), GI ID no.116310719 (SEQ ID NO: 44), Ceres CLONE ID
no.907473 (SEQ ID NO: 58), and Ceres CLONE ID no.1755065 (SEQ ID
NO: 71).
[0086] In some cases, a cold tolerance-modulating polypeptide has
an amino acid sequence with at least 45% sequence identity, e.g.,
50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO: 93 Amino acid sequences of polypeptides having
greater than 45% sequence identity to the polypeptide set forth in
SEQ ID NO: 93 are provided in FIG. 3 and in the Sequence Listing.
Examples of such polypeptides include Ceres CLONE ID no.473040 (SEQ
ID NO: 79), Ceres CLONE ID no.922223 (SEQ ID NO: 81), GI ID
no.125528967, (SEQ ID NO: 82), GI ID no.125573200 (SEQ ID NO: 83),
Ceres ANNOT ID no.1527409 (SEQ ID NO: 85), GI ID no.92871098 (SEQ
ID NO: 86), Ceres CLONE ID no.1831117 (SEQ ID NO: 88), and Ceres
ANNOT ID no.857222 (SEQ ID NO: 90).
[0087] In some cases, a cold tolerance-modulating polypeptide has
an amino acid sequence with at least 45% sequence identity, e.g.,
50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO: 93 Amino acid sequences of polypeptides having
greater than 45% sequence identity to the polypeptide set forth in
SEQ ID NO: 93 are provided in FIG. 4 and in the Sequence Listing.
Examples of such polypeptides include Ceres CLONE ID no.1831117
(SEQ ID NO: 95), Ceres CLONE ID no.1844076 (SEQ ID NO: 98), Ceres
CLONE ID no.473040 (SEQ ID NO: 104), Ceres CLONE ID no.922223 (SEQ
ID NO: 106), and GI ID no.125528967 (SEQ ID NO: 107).
[0088] In some cases, a cold tolerance-modulating tasiRNA acts upon
an ARF amino acid sequence with at least 45% sequence identity,
e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98%, or 99% sequence identity, to the amino acid sequence set
forth in SEQ ID NO: 116 Amino acid sequences of polypeptides having
greater than 45% sequence identity to the polypeptide set forth in
SEQ ID NO: 116 are provided in FIG. 5 and in the Sequence Listing.
Examples of such polypeptides include LOCUS ID no. AT5G62000 (SEQ
ID NO: 112), Ceres ANNOT ID no.1527370 (SEQ ID NO: 114), GI ID
no.62319853 (SEQ ID NO: 117), GI ID no.62319903 (SEQ ID NO:118), GI
ID no.47716275 (SEQ ID NO: 119), GI ID no.125534572 (SEQ ID
NO:120), GI ID no.26251300(SEQ ID NO:121), GI ID no.115441981 (SEQ
ID NO:123), GI ID no.23893346 (SEQ ID NO:124), GI ID no.115485689
(SEQ ID NO:125), GI ID no.108864435 (SEQ ID NO:126), GI ID
no.50511471 (SEQ ID NO:127), LOCUS ID no. At2g33860 (SEQ ID
NO:128), GI ID no.2245390 (SEQ ID NO:131), GI ID no.3228517 (SEQ ID
NO:132), Ceres CLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE ID
no.1598488 (SEQ ID NO: 136), GI ID no.125553314 (SEQ ID NO: 138),
and Ceres CLONE ID no.462443 (SEQ ID NO:143).
E. Other Sequences
[0089] It should be appreciated that a cold tolerance-modulating
polypeptide can include additional amino acids that are not
involved in cold tolerance modulation, and thus such a polypeptide
can be longer than would otherwise be the case. For example, a cold
tolerance-modulating polypeptide can include a purification tag, a
chloroplast transit peptide, a mitochondrial transit peptide, an
amyloplast transit peptide, a lysosome signal peptide or a leader
sequence added to the amino or carboxy terminus. In some
embodiments, a cold tolerance-modulating polypeptide includes an
amino acid sequence that functions as a reporter, e.g., a green
fluorescent protein or yellow fluorescent protein.
III. Nucleic Acids
[0090] Nucleic acids described herein include nucleic acids that
are effective to modulate cold tolerance levels when transcribed in
a plant or plant cell. Such nucleic acids include, without
limitation, those that encode a cold tolerance-modulating
polypeptide and those that can be used to inhibit expression of a
cold tolerance-modulating polypeptide via a nucleic acid based
method.
A. Cold Tolerance-Modulating Nucleic Acids
[0091] Nucleic acids encoding cold tolerance-modulating
polypeptides are described herein. Examples of such nucleic acids
include SEQ ID NOs: 3, 5, 7, 9, 11, 14, 16, 18, 19, 21, 23, 25, 27,
31, 33, 35, 37, 39, 41, 55, 57, 70, 75, 78, 80, 84, 87, 89, 91, 92,
94, 97, 99, 103, 105, 109, 113, 115, 129, 133, 135, 140, and 142,
as described in more detail below. A nucleic acid also can be a
fragment that is at least 40% (e.g., at least 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 99%) of the length of the full-length
nucleic acid set forth in SEQ ID NOs: 3, 5, 7, 9, 11, 14, 16, 18,
19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41, 55, 57, 70, 75, 78, 80,
84, 87, 89, 91, 92, 94, 97, 99, 103, 105, 109, 113, 115, 129, 133,
135, 140, and 142 or of the length of the full-length nucleic acid
set forth in the sequence listing identified as functional homologs
of the sequences of FIG. 3.
[0092] A cold tolerance-modulating nucleic acid can comprise the
nucleotide sequence set forth in SEQ ID NO: 1. Alternatively, a
cold tolerance-modulating nucleic acid can be a variant of the
nucleic acid having the nucleotide sequence set forth in SEQ ID NO:
1. For example, a cold tolerance-modulating nucleic acid can have a
nucleotide sequence with at least 80% sequence identity, e.g., 81%,
85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
nucleotide sequence set forth in SEQ ID NO: 1.
[0093] A cold tolerance-modulating nucleic acid can comprise the
nucleotide sequence set forth in SEQ ID NO: 19. Alternatively, a
cold tolerance-modulating nucleic acid can be a variant of the
nucleic acid having the nucleotide sequence set forth in SEQ ID NO:
19. For example, a cold tolerance-modulating nucleic acid can have
a nucleotide sequence with at least 80% sequence identity, e.g.,
81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
nucleotide sequence set forth in SEQ ID NO: 19.
[0094] A cold tolerance-modulating nucleic acid can comprise the
nucleotide sequence set forth in SEQ ID NO: 92. Alternatively, a
cold tolerance-modulating nucleic acid can be a variant of the
nucleic acid having the nucleotide sequence set forth in SEQ ID NO:
92. For example, a cold tolerance-modulating nucleic acid can have
a nucleotide sequence with at least 80% sequence identity, e.g.,
81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
nucleotide sequence set forth in SEQ ID NO: 92.
[0095] A cold tolerance-modulating nucleic acid can comprise the
nucleotide sequence set forth in SEQ ID NO: 97. Alternatively, a
cold tolerance-modulating nucleic acid can be a variant of the
nucleic acid having the nucleotide sequence set forth in SEQ ID NO:
97. For example, a cold tolerance-modulating nucleic acid can have
a nucleotide sequence with at least 80% sequence identity, e.g.,
81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
nucleotide sequence set forth in SEQ ID NO: 97.
[0096] A cold tolerance-modulating nucleic acid can comprise the
nucleotide sequence set forth in SEQ ID NO: 111. Alternatively, a
cold tolerance-modulating nucleic acid can be a variant of the
nucleic acid having the nucleotide sequence set forth in SEQ ID NO:
111. For example, a cold tolerance-modulating nucleic acid can have
a nucleotide sequence with at least 80% sequence identity, e.g.,
81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
nucleotide sequence set forth in SEQ ID NO: 111.
[0097] A cold tolerance-modulating sequence can be at least a
fragment of a nucleotide sequence such as Ceres ANNOT ID no.1473961
(SEQ ID NO: 116) or homologs thereof. For example, the cold
tolerance-modulating nucleotide may be a tasiRNA. Such cold
tolerance-modulating nucleotide sequences can act upon a protein
that comprises an auxin response factor motif. This motif is
present in SEQ ID NO: 112, which sets forth the amino acid sequence
of an Arabidopsis clone, identified herein as LOCUS ID no.
AT5G62000 (SEQ ID NO: 112), that is predicted to encode an
polypeptide comprising an auxin response factor motif. In certain
embodiments, the protein comprising an auxin response factor motif
is an ARF protein. The ARFs are key regulators of auxin-modulated
gene expression. There are multiple ARF proteins, some of which
activate, while others repress transcription. ARF proteins bind to
auxin-responsive cis-acting promoter elements (AuxREs) using an
N-terminal DNA-binding domain. It is thought that Aux/IAA proteins
activate transcription by modifying ARF activity through the
C-terminal protein-protein interaction domains found in both
Aux/IAA and ARF proteins.
[0098] A cold tolerance-modulating sequence can be at least a
fragment of a nucleotide sequence such as Ceres ANNOT ID no.1473961
(SEQ ID NO: 116) or homologs thereof. For example, the cold
tolerance-modulating nucleotide may be a tasiRNA. Such cold
tolerance-modulating nucleotide sequences can act upon a protein
that comprises a B3 DNA binding domain. This domain is present in
SEQ ID NO: 112, which sets forth the amino acid sequence of an
Arabidopsis clone, identified herein as LOCUS ID no. AT5G62000 (SEQ
ID NO: 112), that is predicted to encode an polypeptide comprising
an B3 DNA binding domain. In certain embodiments, the protein
comprising a B3 DNA binding domain is an ARF protein.
[0099] In some embodiments, a cold tolerance-modulating sequence is
a tasiRNA sequence or a homolog thereof, such tasiRNA sequence
being encoded by a nucleic acid sequence that comprises a domain
having 80% or greater sequence identity to nucleic acid residues
305 to about 346 of SEQ ID NO: 111, residues 21 to about 62 of SEQ
ID NO: 66, residues 20 to about 61 of SEQ ID NO: 67, residues 21 to
about 62 of SEQ ID NO: 72, residues 21 to about 62 of SEQ ID NO:
73, residues 77 to about 118 of SEQ ID NO: 144, residues 292 to
about 313 of SEQ ID NO: 145, residues 37 to about 78 of SEQ ID NO:
146, residues 56 to about 97 of SEQ ID NO: 147, residues 37 to
about 78 of SEQ ID NO: 148, residues 45 to about 86 of SEQ ID NO:
149, residues 46 to about 98 of SEQ ID NO: 150, residues 476 to
about 497 of SEQ ID NO: 151, residues 21 to about 62 of SEQ ID NO:
152, residues 21 to about 62 of SEQ ID NO: 153, residues 21 to
about 62 of SEQ ID NO: 154, residues 21 to about 62 of SEQ ID NO:
155, or residues 21 to about 62 of SEQ ID NO: 156.
[0100] In some embodiments, a cold tolerance-modulating sequence is
a nucleotide sequence or a homolog thereof, such as a tasiRNA
sequence, wherein said nucleotide is encoded by a nucleic acid
sequence that also comprises an miR390 recognition sequence having
80% or greater sequence identity to nucleic acid residues 109 to
about 129 of SEQ ID NO: 66, residues 114 to about 135 of SEQ ID NO:
67, residues 119 to about 139 of SEQ ID NO: 72, residues 108 to
about 128 of SEQ ID NO: 73, residues 234 to about 254 of SEQ ID NO:
144, residues 135 to about 176 of SEQ ID NO: 145, residues 173 to
about 189 of SEQ ID NO: 147, residues 154 to about 170 of SEQ ID
NO: 148, residues 134 to about 157 of SEQ ID NO: 149, residues 154
to about 198 of SEQ ID NO: 150, residues 319 to about 360 of SEQ ID
NO: 151, residues 121 to about 141 of SEQ ID NO: 152, residues 120
to about 140 of SEQ ID NO: 153, residues 121 to about 141 of SEQ ID
NO: 154, residues 121 to about 141 of SEQ ID NO: 155, residues 121
to about 141 of SEQ ID NO: 156, or residues 462 to about 483 of SEQ
ID NO: 111. miR390 recognition sequences may guide in-phase
processing of transcription (Allen et al. 2005).
[0101] In embodiments of the invention, a cold tolerance-modulating
nucleotide, such as Ceres ANNOT ID no.1473961 (SEQ ID NO: 116) or a
homolog threof, can act upon an polypeptide that comprises a B3 DNA
binding domain having 80% or greater sequence identity to amino
acid residues 163 to 268 of SEQ ID NO: 112, residues 157 to 262 of
SEQ ID NO: 114, residues 157 to 262 of SEQ ID NO: 116, residues 163
to 268 of SEQ ID NO: 117, residues 163 to 268 of SEQ ID NO: 118,
residues 158 to 263 of SEQ ID NO: 119, residues 148 to 253 of SEQ
ID NO: 120, residues 147 to 252 of SEQ ID NO: 121, residues 123 to
228 of SEQ ID NO: 122, residues 128 to 233 of SEQ ID NO: 123,
residues 131 to 236 of SEQ ID NO: 124, residues 147 to 252 of SEQ
ID NO: 125, residues 148 to 253 of SEQ ID NO: 126, residues 141 to
246 of SEQ ID NO: 127, residues 158 to 263 of SEQ ID NO: 128,
residues 142 to 247 of SEQ ID NO: 130, residues 158 to 263 of SEQ
ID NO: 131, residues 158 to 263 of SEQ ID NO: 132, residues 126 to
231 of SEQ ID NO: 134, residues 129 to 234 of SEQ ID NO: 136,
residues 114 to 219 of SEQ ID NO: 137, residues 141 to 246 of SEQ
ID NO: 138, residues 176 to 281 of SEQ ID NO: 139, residues 152 to
257 of SEQ ID NO: 141, or residues 121 to 225 of SEQ ID NO:
143.
[0102] In embodiments of the invention, a cold tolerance-modulating
tasiRNA sequence such as Ceres ANNOT ID no.1473961 (SEQ ID NO: 116)
can act upon an ARF polypeptide that comprises an auxin response
factor motif having 80% or greater sequence identity to amino acid
residues 290 to 372 of SEQ ID NO: 112, residues 284 to 366 of SEQ
ID NO: 114, residues 284 to 366 of SEQ ID NO: 116, residues 290 to
372 of SEQ ID NO: 117, residues 290 to 372 of SEQ ID NO: 118,
residues 285 to 367 of SEQ ID NO: 119, residues 275 to 357 of SEQ
ID NO: 120, residues 274 to 356 of SEQ ID NO: 121, residues 250 to
331 of SEQ ID NO: 122, residues 255 to 336 of SEQ ID NO: 123,
residues 258 to 340 of SEQ ID NO: 124, residues 274 to 356 of SEQ
ID NO: 125, residues 275 to 357 of SEQ ID NO: 126, residues 268 to
349 of SEQ ID NO: 127, residues 285 to 367 of SEQ ID NO: 128,
residues 269 to 351 of SEQ ID NO: 130, residues 285 to 367 of SEQ
ID NO: 131, residues 285 to 367 of SEQ ID NO: 132, residues 253 to
334 of SEQ ID NO: 134, residues 256 to 337 of SEQ ID NO: 136,
residues 241 to 322 of SEQ ID NO: 137, residues 268 to 349 of SEQ
ID NO: 138, residues 302 to 384 of SEQ ID NO: 139, residues 279 to
361 of SEQ ID NO: 141, or residues 247 to 332 of SEQ ID NO:
143.
[0103] Isolated nucleic acid molecules can be produced by standard
techniques. For example, polymerase chain reaction (PCR) techniques
can be used to obtain an isolated nucleic acid containing a
nucleotide sequence described herein. PCR can be used to amplify
specific sequences from DNA as well as RNA, including sequences
from total genomic DNA or total cellular RNA. Various PCR methods
are described, for example, in PCR Primer: A Laboratory Manual,
Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory
Press, 1995. Generally, sequence information from the ends of the
region of interest or beyond is employed to design oligonucleotide
primers that are identical or similar in sequence to opposite
strands of the template to be amplified. Various PCR strategies
also are available by which site-specific nucleotide sequence
modifications can be introduced into a template nucleic acid.
Isolated nucleic acids also can be chemically synthesized, either
as a single nucleic acid molecule (e.g., using automated DNA
synthesis in the 3' to 5' direction using phosphoramidite
technology) or as a series of oligonucleotides. For example, one or
more pairs of long oligonucleotides (e.g., >100 nucleotides) can
be synthesized that contain the desired sequence, with each pair
containing a short segment of complementarity (e.g., about 15
nucleotides) such that a duplex is formed when the oligonucleotide
pair is annealed. DNA polymerase is used to extend the
oligonucleotides, resulting in a single, double-stranded nucleic
acid molecule per oligonucleotide pair, which then can be ligated
into a vector. Isolated nucleic acids of the invention also can be
obtained by mutagenesis of, e.g., a naturally occurring DNA.
B. Use of Nucleic Acids to Modulate Expression of Polypeptides
[0104] i. Expression of a Cold Tolerance-Modulating Polypeptide
[0105] A nucleic acid encoding one of the cold tolerance-modulating
polypeptides described herein can be used to express the
polypeptide in a plant species of interest, typically by
transforming a plant cell with a nucleic acid having the coding
sequence for the polypeptide operably linked in sense orientation
to one or more regulatory regions. It will be appreciated that
because of the degeneracy of the genetic code, a number of nucleic
acids can encode a particular cold tolerance-modulating
polypeptide; i.e., for many amino acids, there is more than one
nucleotide triplet that serves as the codon for the amino acid.
Thus, codons in the coding sequence for a given cold
tolerance-modulating polypeptide can be modified such that optimal
expression in a particular plant species is obtained, using
appropriate codon bias tables for that species.
[0106] In some cases, expression of a cold tolerance-modulating
polypeptide inhibits one or more functions of an endogenous
polypeptide. For example, a nucleic acid that encodes a dominant
negative polypeptide can be used to inhibit protein function. A
dominant negative polypeptide typically is mutated or truncated
relative to an endogenous wild type polypeptide, and its presence
in a cell inhibits one or more functions of the wild type
polypeptide in that cell, i.e., the dominant negative polypeptide
is genetically dominant and confers a loss of function. The
mechanism by which a dominant negative polypeptide confers such a
phenotype can vary but often involves a protein-protein interaction
or a protein-DNA interaction. For example, a dominant negative
polypeptide can be an enzyme that is truncated relative to a native
wild type enzyme, such that the truncated polypeptide retains
domains involved in binding a first protein but lacks domains
involved in binding a second protein. The truncated polypeptide is
thus unable to properly modulate the activity of the second
protein. See, e.g., US 2007/0056058. As another example, a point
mutation that results in a non-conservative amino acid substitution
in a catalytic domain can result in a dominant negative
polypeptide. See, e.g., US 2005/032221. As another example, a
dominant negative polypeptide can be a transcription factor that is
truncated relative to a native wild type transcription factor, such
that the truncated polypeptide retains the DNA binding domain(s)
but lacks the activation domain(s). Such a truncated polypeptide
can inhibit the wild type transcription factor from binding DNA,
thereby inhibiting transcription activation.
[0107] ii. Inhibition of Expression of a Cold Tolerance-Modulating
Polypeptide
[0108] Polynucleotides and recombinant constructs described herein
can be used to inhibit expression of a cold tolerance-modulating
polypeptide in a plant species of interest. See, e.g., Matzke and
Birchler, Nature Reviews Genetics 6:24-35 (2005); Akashi et al.,
Nature Reviews Mol. Cell Biology 6:413-422 (2005); Mittal, Nature
Reviews Genetics 5:355-365 (2004); Dorsett and Tuschl, Nature
Reviews Drug Discovery 3: 318-329 (2004); and Nature Reviews RNA
interference collection, October 2005 at
nature.com/reviews/focus/mai. Typically, at least a fragment of a
nucleic acid encoding cold tolerance-modulating polypeptides and/or
its complement is expressed. A fragment is typically at least 20
nucleotides long, as needed for the methods noted below. A number
of nucleic acid based methods, including antisense RNA, ribozyme
directed RNA cleavage, post-transcriptional gene silencing (PTGS),
e.g., RNA interference (RNAi), and transcriptional gene silencing
(TGS) are known to inhibit gene expression in plants. Suitable
polynucleotides include full-length nucleic acids encoding cold
tolerance-modulating polypeptides or fragments of such full-length
nucleic acids. In some embodiments, a complement of the full-length
nucleic acid or a fragment thereof can be used. Typically, a
fragment is at least 10 nucleotides, e.g., at least 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 35, 40, 50, 80,
100, 200, 500 nucleotides or more. Generally, higher homology can
be used to compensate for the use of a shorter sequence.
[0109] Antisense technology is one well-known method. In this
method, a nucleic acid segment from a gene to be repressed is
cloned and operably linked to a regulatory region and a
transcription termination sequence so that the antisense strand of
RNA is transcribed. The recombinant construct is then transformed
into plants, as described herein, and the antisense strand of RNA
is produced. The nucleic acid segment need not be the entire
sequence of the gene to be repressed, but typically will be
substantially complementary to at least a portion of the sense
strand of the gene to be repressed. Generally, higher homology can
be used to compensate for the use of a shorter sequence. Typically,
a sequence of at least 30 nucleotides is used, e.g., at least 40,
50, 80, 100, 200, 500 nucleotides or more.
[0110] In another method, a nucleic acid can be transcribed into a
ribozyme, or catalytic RNA, that affects expression of an mRNA.
See, U.S. Pat. No. 6,423,885. Ribozymes can be designed to
specifically pair with virtually any target RNA and cleave the
phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. Heterologous nucleic
acids can encode ribozymes designed to cleave particular mRNA
transcripts, thus preventing expression of a polypeptide.
Hammerhead ribozymes are useful for destroying particular mRNAs,
although various ribozymes that cleave mRNA at site-specific
recognition sequences can be used. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target RNA contains a 5'-UG-3' nucleotide sequence. The
construction and production of hammerhead ribozymes is known in the
art. See, for example, U.S. Pat. No. 5,254,678 and WO 02/46449 and
references cited therein. Hammerhead ribozyme sequences can be
embedded in a stable RNA such as a transfer RNA (tRNA) to increase
cleavage efficiency in vivo. Perriman et al., Proc. Natl. Acad.
Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methods
in Molecular Biology, Vol. 74, Chapter 43, "Expressing Ribozymes in
Plants", Edited by Turner, P. C., Humana Press Inc., Totowa, N.J.
RNA endoribonucleases which have been described, such as the one
that occurs naturally in Tetrahymena thermophila, can be useful.
See, for example, U.S. Pat. Nos. 4,987,071 and 6,423,885.
[0111] PTGS, e.g., RNAi, can also be used to inhibit the expression
of a gene. For example, a construct can be prepared that includes a
sequence that is transcribed into an RNA that can anneal to itself,
e.g., a double stranded RNA having a stem-loop structure. In some
embodiments, one strand of the stem portion of a double stranded
RNA comprises a sequence that is similar or identical to the sense
coding sequence or a fragment thereof of a cold
tolerance-modulating polypeptide, and that is from about 10
nucleotides to about 2,500 nucleotides in length. The length of the
sequence that is similar or identical to the sense coding sequence
can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides
to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from
25 nucleotides to 100 nucleotides. The other strand of the stem
portion of a double stranded RNA comprises a sequence that is
similar or identical to the antisense strand or a fragment thereof
of the coding sequence of the cold tolerance-modulating
polypeptide, and can have a length that is shorter, the same as, or
longer than the corresponding length of the sense sequence. In some
cases, one strand of the stem portion of a double stranded RNA
comprises a sequence that is similar or identical to the 3' or 5'
untranslated region, or a fragment thereof, of an mRNA encoding a
cold tolerance-modulating polypeptide, and the other strand of the
stem portion of the double stranded RNA comprises a sequence that
is similar or identical to the sequence that is complementary to
the 3' or 5' untranslated region, respectively, or a fragment
thereof, of the mRNA encoding the cold tolerance-modulating
polypeptide. In other embodiments, one strand of the stem portion
of a double stranded RNA comprises a sequence that is similar or
identical to the sequence of an intron, or a fragment thereof, in
the pre-mRNA encoding a cold tolerance-modulating polypeptide, and
the other strand of the stem portion comprises a sequence that is
similar or identical to the sequence that is complementary to the
sequence of the intron, or a fragment thereof, in the pre-mRNA. The
loop portion of a double stranded RNA can be from 3 nucleotides to
5,000 nucleotides, e.g., from 3 nucleotides to 25 nucleotides, from
15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500
nucleotides, or from 25 nucleotides to 200 nucleotides. The loop
portion of the RNA can include an intron. A double stranded RNA can
have zero, one, two, three, four, five, six, seven, eight, nine,
ten, or more stem-loop structures. A construct including a sequence
that is operably linked to a regulatory region and a transcription
termination sequence, and that is transcribed into an RNA that can
form a double stranded RNA, is transformed into plants as described
herein. Methods for using RNAi to inhibit the expression of a gene
are known to those of skill in the art. See, e.g., U.S. Pat. Nos.
5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and
6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO
98/36083; and U.S. Patent Publications 20030175965, 20030175783,
20040214330, and 20030180945.
[0112] microRNA (miRNA) and tasiRNA, which are non-protein coding
RNAs, can also be used to inhibit the expression of a gene. The
gene targeted for inhibition may be an endogenous plant gene, a
viral gene, a bacterial gene, a fungal gene, or an insect gene.
miRNAs and tasiRNAs are regulatory agents consisting of about 19 to
25 ribonucleotides. miRNA are highly efficient at inhibiting the
expression of endogenous genes and/or can guide in-phase processing
of tasiRNA primary transcripts. tasiRNAs similarly inhibit gene
expression by interacting with target mRNAs and guide cleavage by
the same mechanism as do plant miRNAs, but differ from miRNAs in
that they arise from double-stranded RNA, which may require
RNA-dependent RNA polymerases.
[0113] For example, a tasiRNA can act upon an auxin responsive
protein (ARF) (e.g., ARF3 or ARF4). In particular, inhibition of
the expression of an ARF encoding gene (e.g., ARF3 or ARF4) may be
obtained by interference by expression of a nucleic acid sequence
encoding a tasiRNA. Transcription of a tasiRNA encoding nucleic
acid sequence can be under the control of a promoter, such as, but
not limited to, those promoters and regulatory regions described
herein, or under promotional control of a tasiRNA coding sequence's
own promoter. For such interference, the expression cassette is
designed to express an RNA molecule that is modeled on an
endogenous tasiRNA encoding sequence. The tasiRNA encoding sequence
encodes an RNA that forms a hairpin structure containing a
nucleotide sequence that is complementary to another endogenous
gene (target sequence). For suppression of ARF protein expression,
the nucleotide sequence is selected from an ARF transcript sequence
and contains about 19 to 25 nucleotides of said ARF protein
sequence in sense orientation and about 19 to 25 nucleotides of a
corresponding antisense sequence that is complementary to the sense
sequence. tasiRNA molecules are highly efficient at inhibiting the
expression of endogenous genes. In Arabidopsis, a nuclear DCL
enzyme is believed to be required for mature miRNA formation (Xie
et al. (2004) PLoS Biol., 2:642-652, which is incorporated by
reference herein) Inhibition of gene expression by miRNAs and
tasiRNAs and methods for inhibition are known to those of skill in
the art. See, for example, Javier, et al., (2003) Nature
425:257-263; Bartel (2004) Cell, 116:281-297; Kim (2005) Nature
Rev. Mol. Cell Biol., 6:376-385; and Allen et al. (2005) Cell,
121:207-221, all of which are incorporated by reference herein.
[0114] Constructs containing regulatory regions operably linked to
nucleic acid molecules in sense orientation can also be used to
inhibit the expression of a gene. The transcription product can be
similar or identical to the sense coding sequence, or a fragment
thereof, of a cold tolerance-modulating polypeptide. The
transcription product also can be unpolyadenylated, lack a 5' cap
structure, or contain an unspliceable intron. Methods of inhibiting
gene expression using a full-length cDNA as well as a partial cDNA
sequence are known in the art. See, e.g., U.S. Pat. No.
5,231,020.
[0115] In some embodiments, a construct containing a nucleic acid
having at least one strand that is a template for both sense and
antisense sequences that are complementary to each other is used to
inhibit the expression of a gene. The sense and antisense sequences
can be part of a larger nucleic acid molecule or can be part of
separate nucleic acid molecules having sequences that are not
complementary. The sense or antisense sequence can be a sequence
that is identical or complementary to the sequence of an mRNA, the
3' or 5' untranslated region of an mRNA, or an intron in a pre-mRNA
encoding a cold tolerance-modulating polypeptide, or a fragment of
such sequences. In some embodiments, the sense or antisense
sequence is identical or complementary to a sequence of the
regulatory region that drives transcription of the gene encoding a
cold tolerance-modulating polypeptide. In each case, the sense
sequence is the sequence that is complementary to the antisense
sequence.
[0116] The sense and antisense sequences can be any length greater
than about 10 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides).
For example, an antisense sequence can be 21 or 22 nucleotides in
length. Typically, the sense and antisense sequences range in
length from about 15 nucleotides to about 30 nucleotides, e.g.,
from about 18 nucleotides to about 28 nucleotides, or from about 21
nucleotides to about 25 nucleotides.
[0117] In some embodiments, an antisense sequence is a sequence
complementary to an mRNA sequence, or a fragment thereof, encoding
a cold tolerance-modulating polypeptide described herein. The sense
sequence complementary to the antisense sequence can be a sequence
present within the mRNA of the cold tolerance-modulating
polypeptide. Typically, sense and antisense sequences are designed
to correspond to a 15-30 nucleotide sequence of a target mRNA such
that the level of that target mRNA is reduced.
[0118] In some embodiments, a construct containing a nucleic acid
having at least one strand that is a template for more than one
sense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense
sequences) can be used to inhibit the expression of a gene.
Likewise, a construct containing a nucleic acid having at least one
strand that is a template for more than one antisense sequence
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antisense sequences) can
be used to inhibit the expression of a gene. For example, a
construct can contain a nucleic acid having at least one strand
that is a template for two sense sequences and two antisense
sequences. The multiple sense sequences can be identical or
different, and the multiple antisense sequences can be identical or
different. For example, a construct can have a nucleic acid having
one strand that is a template for two identical sense sequences and
two identical antisense sequences that are complementary to the two
identical sense sequences. Alternatively, an isolated nucleic acid
can have one strand that is a template for (1) two identical sense
sequences 20 nucleotides in length, (2) one antisense sequence that
is complementary to the two identical sense sequences 20
nucleotides in length, (3) a sense sequence 30 nucleotides in
length, and (4) three identical antisense sequences that are
complementary to the sense sequence 30 nucleotides in length. The
constructs provided herein can be designed to have any arrangement
of sense and antisense sequences. For example, two identical sense
sequences can be followed by two identical antisense sequences or
can be positioned between two identical antisense sequences.
[0119] A nucleic acid having at least one strand that is a template
for one or more sense and/or antisense sequences can be operably
linked to a regulatory region to drive transcription of an RNA
molecule containing the sense and/or antisense sequence(s). In
addition, such a nucleic acid can be operably linked to a
transcription terminator sequence, such as the terminator of the
nopaline synthase (nos) gene. In some cases, two regulatory regions
can direct transcription of two transcripts: one from the top
strand, and one from the bottom strand. See, for example, Yan et
al., Plant Physiol., 141:1508-1518 (2006). The two regulatory
regions can be the same or different. The two transcripts can form
double-stranded RNA molecules that induce degradation of the target
RNA. In some cases, a nucleic acid can be positioned within a T-DNA
or plant-derived transfer DNA (P-DNA) such that the left and right
T-DNA border sequences, or the left and right border-like sequences
of the P-DNA, flank or are on either side of the nucleic acid. See,
US 2006/0265788. The nucleic acid sequence between the two
regulatory regions can be from about 15 to about 300 nucleotides in
length. In some embodiments, the nucleic acid sequence between the
two regulatory regions is from about 15 to about 200 nucleotides in
length, from about 15 to about 100 nucleotides in length, from
about 15 to about 50 nucleotides in length, from about 18 to about
50 nucleotides in length, from about 18 to about 40 nucleotides in
length, from about 18 to about 30 nucleotides in length, or from
about 18 to about 25 nucleotides in length.
[0120] In some nucleic-acid based methods for inhibition of gene
expression in plants, a suitable nucleic acid can be a nucleic acid
analog. Nucleic acid analogs can be modified at the base moiety,
sugar moiety, or phosphate backbone to improve, for example,
stability, hybridization, or solubility of the nucleic acid.
Modifications at the base moiety include deoxyuridine for
deoxythymidine, and 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the
sugar moiety include modification of the 2' hydroxyl of the ribose
sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose
phosphate backbone can be modified to produce morpholino nucleic
acids, in which each base moiety is linked to a six-membered
morpholino ring, or peptide nucleic acids, in which the
deoxyphosphate backbone is replaced by a pseudopeptide backbone and
the four bases are retained. See, for example, Summerton and
Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et
al., Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the
deoxyphosphate backbone can be replaced with, for example, a
phosphorothioate or phosphorodithioate backbone, a
phosphoroamidite, or an alkyl phosphotriester backbone.
C. Constructs/Vectors
[0121] Recombinant constructs provided herein can be used to
transform plants or plant cells in order to modulate cold tolerance
levels. A recombinant nucleic acid construct can comprise a nucleic
acid encoding a cold tolerance-modulating polypeptide as described
herein, operably linked to a regulatory region suitable for
expressing the cold tolerance-modulating polypeptide in the plant
or cell. Thus, a nucleic acid can comprise a coding sequence that
encodes any of the cold tolerance-modulating polypeptides as set
forth in SEQ ID NOs: 2, 20, 93, 74, or a homologs thereof. Examples
of nucleic acids encoding cold tolerance-modulating polypeptides
are set forth in SEQ ID NOs: 1, 19, 92, 97, or 111, and in FIGS.
1-5 and in the Sequence Listing. The cold tolerance-modulating
polypeptide encoded by a recombinant nucleic acid can be a native
cold tolerance-modulating polypeptide, or can be heterologous to
the cell. In some cases, the recombinant construct contains a
nucleic acid that inhibits expression of a cold
tolerance-modulating polypeptide, operably linked to a regulatory
region. Examples of suitable regulatory regions are described in
the section entitled "Regulatory Regions."
[0122] Vectors containing recombinant nucleic acid constructs such
as those described herein also are provided. Suitable vector
backbones include, for example, those routinely used in the art
such as plasmids, viruses, artificial chromosomes, BACs, YACs, or
PACs. Suitable expression vectors include, without limitation,
plasmids and viral vectors derived from, for example,
bacteriophage, baculoviruses, and retroviruses. Numerous vectors
and expression systems are commercially available from such
corporations as Novagen (Madison, Wis.), Clontech (Palo Alto,
Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life
Technologies (Carlsbad, Calif.).
[0123] The vectors provided herein also can include, for example,
origins of replication, scaffold attachment regions (SARs), and/or
markers. A marker gene can confer a selectable phenotype on a plant
cell. For example, a marker can confer biocide resistance, such as
resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or
hygromycin), or an herbicide (e.g., glyphosate, chlorsulfuron or
phosphinothricin). In addition, an expression vector can include a
tag sequence designed to facilitate manipulation or detection
(e.g., purification or localization) of the expressed polypeptide.
Tag sequences, such as luciferase, .beta.-glucuronidase (GUS),
green fluorescent protein (GFP), glutathione S-transferase (GST),
polyhistidine, c-myc, hemagglutinin, or Flag.TM. tag (Kodak, New
Haven, Conn.) sequences typically are expressed as a fusion with
the encoded polypeptide. Such tags can be inserted anywhere within
the polypeptide, including at either the carboxyl or amino
terminus.
D. Regulatory Regions
[0124] The choice of regulatory regions to be included in a
recombinant construct depends upon several factors, including, but
not limited to, efficiency, selectability, inducibility, desired
expression level, and cell- or tissue-preferential expression. It
is a routine matter for one of skill in the art to modulate the
expression of a coding sequence by appropriately selecting and
positioning regulatory regions relative to the coding sequence.
Transcription of a nucleic acid can be modulated in a similar
manner
[0125] Some suitable regulatory regions initiate transcription
only, or predominantly, in certain cell types. Methods for
identifying and characterizing regulatory regions in plant genomic
DNA are known, including, for example, those described in the
following references: Jordano et al., Plant Cell, 1:855-866 (1989);
Bustos et al., Plant Cell, 1:839-854 (1989); Green et al., EMBO J.,
7:4035-4044 (1988); Meier et al., Plant Cell, 3:309-316 (1991); and
Zhang et al., Plant Physiology, 110:1069-1079 (1996).
[0126] Examples of various classes of regulatory regions are
described below. Some of the regulatory regions indicated below as
well as additional regulatory regions are described in more detail
in U.S. Patent Application Ser. Nos. 60/505,689; 60/518,075;
60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140;
60/757,544; 60/776,307; 10/957,569; 11/058,689; 11/172,703;
11/208,308; 11/274,890; 60/583,609; 60/612,891; 11/097,589;
11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;
PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343;
and PCT/US06/038236; PCT/US06/040572; and PCT/US07/62762.
[0127] For example, the sequences of regulatory regions p326,
YP0144, YP0190, p13879, YP0050, p32449, 21876, YP0158, YP0214,
YP0380, PT0848, PT0633, YP0128, YP0275, PT0660, PT0683, PT0758,
PT0613, PT0672, PT0688, PT0837, YP0092, PT0676, PT0708, YP0396,
YP0007, YP0111, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115,
YP0119, YP0120, YP0374, YP0101, YP0102, YP0110, YP0117, YP0137,
YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156, PT0650,
PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886,
PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286,
YP0377, PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188,
YP0263, PT0743 and YP0096 are set forth in the sequence listing of
PCT/US06/040572; the sequence of regulatory region PT0625 is set
forth in the sequence listing of PCT/US05/034343; the sequences of
regulatory regions PT0623, YP0388, YP0087, YP0093, YP0108, YP0022
and YP0080 are set forth in the sequence listing of U.S. patent
application Ser. No. 11/172,703; the sequence of regulatory region
PR0924 is set forth in the sequence listing of PCT/US07/62762; and
the sequences of regulatory regions p530c10, pOsFIE2-2, pOsMEA,
pOsYp102, and pOsYp285 are set forth in the sequence listing of
PCT/US06/038236.
[0128] It will be appreciated that a regulatory region may meet
criteria for one classification based on its activity in one plant
species, and yet meet criteria for a different classification based
on its activity in another plant species.
[0129] ii Broadly Expressing Promoters
[0130] A promoter can be said to be "broadly expressing" when it
promotes transcription in many, but not necessarily all, plant
tissues. For example, a broadly expressing promoter can promote
transcription of an operably linked sequence in one or more of the
shoot, shoot tip (apex), and leaves, but weakly or not at all in
tissues such as roots or stems. As another example, a broadly
expressing promoter can promote transcription of an operably linked
sequence in one or more of the stem, shoot, shoot tip (apex), and
leaves, but can promote transcription weakly or not at all in
tissues such as reproductive tissues of flowers and developing
seeds. Non-limiting examples of broadly expressing promoters that
can be included in the nucleic acid constructs provided herein
include the p326, YP0144, YP0190, p13879, YP0050, p32449, 21876,
YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additional
examples include the cauliflower mosaic virus (CaMV) 35S promoter,
the mannopine synthase (MAS) promoter, the 1' or 2' promoters
derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic
virus 34S promoter, actin promoters such as the rice actin
promoter, and ubiquitin promoters such as the maize ubiquitin-1
promoter. In some cases, the CaMV 35S promoter is excluded from the
category of broadly expressing promoters.
[0131] ii. Root Promoters
[0132] Root-active promoters confer transcription in root tissue,
e.g., root endodermis, root epidermis, or root vascular tissues. In
some embodiments, root-active promoters are root-preferential
promoters, i.e., confer transcription only or predominantly in root
tissue. Root-preferential promoters include the YP0128, YP0275,
PT0625, PT0660, PT0683, and PT0758 promoters. Other
root-preferential promoters include the PT0613, PT0672 , PT0688,
and PT0837 promoters, which drive transcription primarily in root
tissue and to a lesser extent in ovules and/or seeds. Other
examples of root-preferential promoters include the root-specific
subdomains of the CaMV 35S promoter (Lam et al., Proc. Natl. Acad.
Sci. USA, 86:7890-7894 (1989)), root cell specific promoters
reported by Conkling et al., Plant Physiol., 93:1203-1211 (1990),
and the tobacco RD2 promoter.
[0133] iii. Maturing Endosperm Promoters
[0134] In some embodiments, promoters that drive transcription in
maturing endosperm can be useful. Transcription from a maturing
endosperm promoter typically begins after fertilization and occurs
primarily in endosperm tissue during seed development and is
typically highest during the cellularization phase. Most suitable
are promoters that are active predominantly in maturing endosperm,
although promoters that are also active in other tissues can
sometimes be used. Non-limiting examples of maturing endosperm
promoters that can be included in the nucleic acid constructs
provided herein include the napin promoter, the Arcelin-5 promoter,
the phaseolin promoter (Bustos et al., Plant Cell, 1(9):839-853
(1989)), the soybean trypsin inhibitor promoter (Riggs et al.,
Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,
Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP
desaturase promoter (Slocombe et al., Plant Physiol.,
104(4):167-176 (1994)), the soybean .alpha.' subunit of
.beta.-conglycinin promoter (Chen et al., Proc. Natl. Acad. Sci.
USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al., Plant
Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the
15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter,
22 kD zein promoter and 27 kD zein promoter. Also suitable are the
Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al., Mol.
Cell Biol., 13:5829-5842 (1993)), the beta-amylase promoter, and
the barley hordein promoter. Other maturing endosperm promoters
include the YP0092, PT0676, and PT0708 promoters.
[0135] iv. Ovary Tissue Promoters
[0136] Promoters that are active in ovary tissues such as the ovule
wall and mesocarp can also be useful, e.g., a polygalacturonidase
promoter, the banana TRX promoter, the melon actin promoter,
YP0396, and PT0623. Examples of promoters that are active primarily
in ovules include YP0007, YP0111, YP0092, YP0103, YP0028, YP0121,
YP0008, YP0039, YP0115, YP0119, YP0120, and YP0374.
[0137] v. Embryo Sac/Early Endosperm Promoters
[0138] To achieve expression in embryo sac/early endosperm,
regulatory regions can be used that are active in polar nuclei
and/or the central cell, or in precursors to polar nuclei, but not
in egg cells or precursors to egg cells. Most suitable are
promoters that drive expression only or predominantly in polar
nuclei or precursors thereto and/or the central cell. A pattern of
transcription that extends from polar nuclei into early endosperm
development can also be found with embryo sac/early
endosperm-preferential promoters, although transcription typically
decreases significantly in later endosperm development during and
after the cellularization phase. Expression in the zygote or
developing embryo typically is not present with embryo sac/early
endosperm promoters.
[0139] Promoters that may be suitable include those derived from
the following genes: Arabidopsis viviparous-1 (see, GenBank No.
U93215); Arabidopsis atmycl (see, Urao (1996) Plant Mol. Biol.,
32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE
(GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank
No. AF096096); and FIE 1.1 (U.S. Pat. No. 6,906,244). Other
promoters that may be suitable include those derived from the
following genes: maize MAC1 (see, Sheridan (1996) Genetics,
142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993)
Plant Mol. Biol., 22:10131-1038). Other promoters include the
following Arabidopsis promoters: YP0039, YP0101, YP0102, YP0110,
YP0117, YP0119, YP0137, DME, YP0285, and YP0212. Other promoters
that may be useful include the following rice promoters: p530c10,
pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285.
[0140] vi. Embryo Promoters
[0141] Regulatory regions that preferentially drive transcription
in zygotic cells following fertilization can provide
embryo-preferential expression. Most suitable are promoters that
preferentially drive transcription in early stage embryos prior to
the heart stage, but expression in late stage and maturing embryos
is also suitable. Embryo-preferential promoters include the barley
lipid transfer protein (Ltp1) promoter (Plant Cell Rep (2001)
20:647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650,
PT0695, PT0723, PT0838, PT0879, and PT0740.
[0142] vii. Photosynthetic Tissue Promoters
[0143] Promoters active in photosynthetic tissue confer
transcription in green tissues such as leaves and stems. Most
suitable are promoters that drive expression only or predominantly
in such tissues. Examples of such promoters include the
ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the
RbcS promoter from eastern larch (Larix laricina), the pine cab6
promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778 (1994)),
the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,
15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et
al., Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from
rice (Luan et al., Plant Cell, 4:971-981 (1992)), the pyruvate
orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al.,
Proc. Natl. Acad. Sci. USA, 90:9586-9590 (1993)), the tobacco
Lhcb1*2 promoter (Cerdan et al., Plant Mol. Biol., 33:245-255
(1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter
promoter (Truernit et al., Planta, 196:564-570 (1995)), and
thylakoid membrane protein promoters from spinach (psaD, psaF,
psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissue
promoters include PT0535, PT0668, PT0886, YP0144, YP0380 and
PT0585.
[0144] viii. Vascular Tissue Promoters
[0145] Examples of promoters that have high or preferential
activity in vascular bundles include YP0087, YP0093, YP0108,
YP0022, and YP0080. Other vascular tissue-preferential promoters
include the glycine-rich cell wall protein GRP 1.8 promoter (Keller
and Baumgartner, Plant Cell, 3(10):1051-1061 (1991)), the Commelina
yellow mottle virus (CoYMV) promoter (Medberry et al., Plant Cell,
4(2):185-192 (1992)), and the rice tungro bacilliform virus (RTBV)
promoter (Dai et al., Proc. Natl. Acad. Sci. USA, 101(2):687-692
(2004)).
[0146] ix. Inducible Promoters
[0147] Inducible promoters confer transcription in response to
external stimuli such as chemical agents or environmental stimuli.
For example, inducible promoters can confer transcription in
response to hormones such as giberellic acid or ethylene, or in
response to light or drought. Examples of drought-inducible
promoters include YP0380, PT0848, YP0381, YP0337, PT0633, YP0374,
PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286,
YP0377, PD1367, and PD0901. Examples of nitrogen-inducible
promoters include PT0863, PT0829, PT0665, and PT0886. Examples of
shade-inducible promoters include PR0924 and PT0678. An example of
a promoter induced by salt is rd29A (Kasuga et al. (1999) Nature
Biotech 17: 287-291).
[0148] x. Basal Promoters
[0149] A basal promoter is the minimal sequence necessary for
assembly of a transcription complex required for transcription
initiation. Basal promoters frequently include a "TATA box" element
that may be located between about 15 and about 35 nucleotides
upstream from the site of transcription initiation. Basal promoters
also may include a "CCAAT box" element (typically the sequence
CCAAT) and/or a GGGCG sequence, which can be located between about
40 and about 200 nucleotides, typically about 60 to about 120
nucleotides, upstream from the transcription start site.
[0150] xi. Stem Promoters
[0151] A stem promoter may be specific to one or more stem tissues
or specific to stem and other plant parts. Stem promoters may have
high or preferential activity in, for example, epidermis and
cortex, vascular cambium, procambium, or xylem. Examples of stem
promoters include YP0018 which is disclosed in US20060015970 and
CryIA(b) and CryIA(c) (Braga et al. 2003, Journal of New Seeds
5:209-221).
[0152] xii. Other Promoters
[0153] Other classes of promoters include, but are not limited to,
shoot-preferential, callus-preferential, trichome
cell-preferential, guard cell-preferential such as PT0678,
tuber-preferential, parenchyma cell-preferential, and
senescence-preferential promoters. Promoters designated YP0086,
YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, and YP0096, as
described in the above-referenced patent applications, may also be
useful.
[0154] xiii. Other Regulatory Regions
[0155] A 5' untranslated region (UTR) can be included in nucleic
acid constructs described herein. A 5' UTR is transcribed, but is
not translated, and lies between the start site of the transcript
and the translation initiation codon and may include the +1
nucleotide. A 3' UTR can be positioned between the translation
termination codon and the end of the transcript. UTRs can have
particular functions such as increasing mRNA stability or
attenuating translation. Examples of 3' UTRs include, but are not
limited to, polyadenylation signals and transcription termination
sequences, e.g., a nopaline synthase termination sequence.
[0156] It will be understood that more than one regulatory region
may be present in a recombinant polynucleotide, e.g., introns,
enhancers, upstream activation regions, transcription terminators,
and inducible elements. Thus, for example, more than one regulatory
region can be operably linked to the sequence of a polynucleotide
encoding a cold tolerance-modulating polypeptide.
[0157] Regulatory regions, such as promoters for endogenous genes,
can be obtained by chemical synthesis or by subcloning from a
genomic DNA that includes such a regulatory region. A nucleic acid
comprising such a regulatory region can also include flanking
sequences that contain restriction enzyme sites that facilitate
subsequent manipulation.
IV. Transgenic Plants and Plant Cells
A. Transformation
[0158] The invention also features transgenic plant cells and
plants comprising at least one recombinant nucleic acid construct
described herein. A plant or plant cell can be transformed by
having a construct integrated into its genome, i.e., can be stably
transformed. Stably transformed cells typically retain the
introduced nucleic acid with each cell division. A plant or plant
cell can also be transiently transformed such that the construct is
not integrated into its genome. Transiently transformed cells
typically lose all or some portion of the introduced nucleic acid
construct with each cell division such that the introduced nucleic
acid cannot be detected in daughter cells after a sufficient number
of cell divisions. Both transiently transformed and stably
transformed transgenic plants and plant cells can be useful in the
methods described herein.
[0159] Transgenic plant cells used in methods described herein can
constitute part or all of a whole plant. Such plants can be grown
in a manner suitable for the species under consideration, either in
a growth chamber, a greenhouse, or in a field. Transgenic plants
can be bred as desired for a particular purpose, e.g., to introduce
a recombinant nucleic acid into other lines, to transfer a
recombinant nucleic acid to other species, or for further selection
of other desirable traits. Alternatively, transgenic plants can be
propagated vegetatively for those species amenable to such
techniques. As used herein, a transgenic plant also refers to
progeny of an initial transgenic plant provided, as long as the
progeny inherits the transgene. Seeds produced by a transgenic
plant can be grown and then selfed (or outcrossed and selfed) to
obtain seeds homozygous for the nucleic acid construct.
[0160] Transgenic plants can be grown in suspension culture, or
tissue or organ culture. For the purposes of this invention, solid
and/or liquid tissue culture techniques can be used. When using
solid medium, transgenic plant cells can be placed directly onto
the medium or can be placed onto a filter that is then placed in
contact with the medium. When using liquid medium, transgenic plant
cells can be placed onto a flotation device, e.g., a porous
membrane that contacts the liquid medium. A solid medium can be,
for example, Murashige and Skoog (MS) medium containing agar and a
suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic
acid (2,4-D), and a suitable concentration of a cytokinin, e.g.,
kinetin.
[0161] When transiently transformed plant cells are used, a
reporter sequence encoding a reporter polypeptide having a reporter
activity can be included in the transformation procedure and an
assay for reporter activity or expression can be performed at a
suitable time after transformation. A suitable time for conducting
the assay typically is about 1-21 days after transformation, e.g.,
about 1-14 days, about 1-7 days, or about 1-3 days. The use of
transient assays is particularly convenient for rapid analysis in
different species, or to confirm expression of a heterologous cold
tolerance-modulating polypeptide whose expression has not
previously been confirmed in particular recipient cells.
[0162] Techniques for introducing nucleic acids into
monocotyledonous and dicotyledonous plants are known in the art,
and include, without limitation, Agrobacterium-mediated
transformation, viral vector-mediated transformation,
electroporation and particle gun transformation, e.g., U.S. Pat.
Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or
cultured tissue is used as the recipient tissue for transformation,
plants can be regenerated from transformed cultures if desired, by
techniques known to those skilled in the art.
B. Screening/Selection
[0163] A population of transgenic plants can be screened and/or
selected for those members of the population that have a trait or
phenotype conferred by expression of the transgene. For example, a
population of progeny of a single transformation event can be
screened for those plants having a desired level of expression of a
cold tolerance-modulating polypeptide or nucleic acid. Physical and
biochemical methods can be used to identify expression levels.
These include Southern analysis or PCR amplification for detection
of a polynucleotide; Northern blots, 51 RNAse protection,
primer-extension, or RT-PCR amplification for detecting RNA
transcripts; enzymatic assays for detecting enzyme or ribozyme
activity of polypeptides and polynucleotides; and protein gel
electrophoresis, Western blots, immunoprecipitation, and
enzyme-linked immunoassays to detect polypeptides. Other techniques
such as in situ hybridization, enzyme staining, and immunostaining
also can be used to detect the presence or expression of
polypeptides and/or polynucleotides. Methods for performing all of
the referenced techniques are known. As an alternative, a
population of plants comprising independent transformation events
can be screened for those plants having a desired trait, such as a
modulated level of cold tolerance. Selection and/or screening can
be carried out over one or more generations, and/or in more than
one geographic location. In some cases, transgenic plants can be
grown and selected under conditions which induce a desired
phenotype or are otherwise necessary to produce a desired phenotype
in a transgenic plant. In addition, selection and/or screening can
be applied during a particular developmental stage in which the
phenotype is expected to be exhibited by the plant. Selection
and/or screening can be carried out to choose those transgenic
plants having a statistically significant difference in a cold
tolerance level relative to a control plant that lacks the
transgene. Selected or screened transgenic plants have an altered
phenotype as compared to a corresponding control plant, as
described in the "Transgenic Plant Phenotypes" section herein.
C. Plant Species
[0164] The polynucleotides and vectors described herein can be used
to transform a number of monocotyledonous and dicotyledonous plants
and plant cell systems, including species from one of the following
families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,
Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,
Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,
Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,
Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae,
Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae,
Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae,
Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae,
Sapindaceae, Solanaceae, Taxaceae, Theaceae, or Vitaceae.
[0165] Suitable species may include members of the genus
Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas,
Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis,
Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis,
Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum,
Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita,
Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra,
Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus,
Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus,
Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon,
Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana,
Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris,
Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa,
Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,
Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,
Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.
[0166] Suitable species include Panicum spp. or hybrids thereof,
Sorghum spp. or hybrids thereof, sudangrass, Miscanthus spp. or
hybrids thereof, Saccharum spp. or hybrids thereof, Erianthus spp.,
Populus spp., Andropogon gerardii (big bluestem), Pennisetum
purpureum (elephant grass) or hybrids thereof (e.g., Pennisetum
purpureum.times.Pennisetum typhoidum), Phalaris arundinacea (reed
canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea
(tall fescue), Spartina pectinata (prairie cord-grass), Medicago
sativa (alfalfa), Arundo donax (giant reed) or hybrids thereof,
Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.
(eucalyptus), Triticosecale (Triticum--wheat X rye), Tripsicum
dactyloides (Eastern gammagrass), Leymus cinereus (basin wildrye),
Leymus condensatus (giant wildrye), and bamboo.
[0167] In some embodiments, a suitable species can be a wild,
weedy, or cultivated sorghum species such as, but not limited to,
Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum
arundinaceum, Sorghum bicolor (such as bicolor, guinea, caudatum,
kafir, and durra), Sorghum brachypodum, Sorghum bulbosum, Sorghum
burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum
ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense,
Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum
leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum
miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum,
Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum,
Sorghum sudanensese, Sorghum timorense, Sorghum trichocladum,
Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, or hybrids
such as Sorghum.times.almum, Sorghum.times.sudangrass or
Sorghum.times.drummondii.
[0168] Suitable species also include Helianthus annuus (sunflower),
Carthamus tinctorius (safflower), Jatropha curcas (jatropha),
Ricinus communis (castor), Elaeis guineensis (palm), Linum
usitatissimum (flax), and Brassica juncea.
[0169] Suitable species also include Beta vulgaris (sugarbeet), and
Manihot esculenta (cassava).
[0170] Suitable species also include Lycopersicon esculentum
(tomato), Lactuca sativa (lettuce), Musa paradisiaca (banana),
Solanum tuberosum (potato), Brassica oleracea (broccoli,
cauliflower, brusselsprouts), Camellia sinensis (tea), Fragaria
ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica
(coffee), Vitis vinifera (grape), Ananas comosus (pineapple),
Capsicum annum (hot & sweet pepper), Allium cepa (onion),
Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima
(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),
Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), and
Solanum melongena (eggplant).
[0171] Suitable species also include Papaver somniferum (opium
poppy), Papaver orientale, Taxus baccata, Taxus brevifolia,
Artemisia annua, Cannabis sativa, Camptotheca acuminate,
Catharanthus roseus, Vinca rosea, Cinchona officinalis, Colchicum
autumnale, Veratrum californica, Digitalis lanata, Digitalis
purpurea, Dioscorea spp., Andrographis paniculata, Atropa
belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp.,
Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus
wornorii, Scopolia spp., Lycopodium serratum (=Huperzia serrata),
Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria
canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum
parthenium, Coleus forskohlii, and Tanacetum parthenium.
[0172] Suitable species also include Parthenium argentatum
(guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha
piperita (mint), Bixa orellana, and Alstroemeria spp.
[0173] Suitable species also include Rosa spp. (rose), Dianthus
caryophyllus (carnation), Petunia spp. (petunia) and Poinsettia
pulcherrima (poinsettia).
[0174] Suitable species also include Nicotiana tabacum (tobacco),
Lupinus albus (lupin), Uniola paniculata (oats), bentgrass
(Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine),
Abies spp. (fir), Acer spp. (maple, Hordeum vulgare (barley), Poa
pratensis (bluegrass), Lolium spp. (ryegrass) and Phleum pratense
(timothy).
[0175] Thus, the methods and compositions can be used over a broad
range of plant species, including species from the dicot genera
Brassica, Carthamus, Glycine, Gossypium, Helianthus, Jatropha,
Parthenium, Populus, and Ricinus; and the monocot genera Elaeis,
Festuca, Hordeum, Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa,
Saccharum, Secale, Sorghum, Triticosecale, Triticum, and Zea. In
some embodiments, a plant is a member of the species Panicum
virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass),
Miscanthus giganteus (miscanthus), Saccharum sp. (energycane),
Populus balsamifera (poplar), Zea mays (corn), Glycine max
(soybean), Brassica napus (canola), Triticum aestivum (wheat),
Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus
(sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet),
or Pennisetum glaucum (pearl millet).
[0176] In certain embodiments, the polynucleotides and vectors
described herein can be used to transform a number of
monocotyledonous and dicotyledonous plants and plant cell systems,
wherein such plants are hybrids of different species or varieties
of a specific species (e.g., Saccharum sp..times.Miscanthus sp.,
Panicum virgatum.times.Panicum amarum, Panicum
virgatum.times.Panicum amarulum, and Pennisetum
purpureum.times.Pennisetum typhoidum).
D. Transgenic Plant Phenotypes
[0177] In some embodiments, a plant in which expression of a cold
tolerance-modulating polypeptide is modulated can have increased
levels of cold tolerance and/or biomass in vegetative tissues. Cold
tolerance can be measured by means well know to those of skill in
the art, including, but not limited to, seedling survival,
decreased photosynthesis and membrane damage (measured by
electrolyte leakage), seedling area, yield, and or biomass. For
example, a cold tolerance-modulating polypeptide or nucleic acid
described herein can be expressed in a transgenic plant, resulting
in increased levels of cold tolerance and/or biomass. The cold
tolerance level can be increased by at least 2 percent, e.g., 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 or more than 100
percent, as compared to the cold tolerance level in a corresponding
control plant that does not express the transgene. In some
embodiments, a plant in which expression of a cold
tolerance-modulating polypeptide or polynucleotide is modulated can
have increased levels of biomass. The biomass level can be
increased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100 percent, as
compared to the biomass level in a corresponding control plant that
does not express the transgene. In some embodiments, differences
can be measured for a plant in which expression of a cold
tolerance-modulating polypeptide is modulated can be exposed to
cold for one or more periods of time that may vary depending on
climatic conditions. For example, for periods of about 1/2 hour, 1
hour, 3 hours, 6 hours, 12 hours, 1 day, 3 days, 5 days, 10 days, 1
month, 3 months, 6 months, 12 months, or the entire lifespan of
such a plant.
[0178] Increases in cold tolerance in such plants can provide
improved nutritional quantity and content in geographic locales
where cold affects plants. Increases in cold tolerance in such
plants can be useful in situations where plant parts such as, but
not limited to, seeds, tubers, stems, leaves or roots are harvested
for human or animal consumption.
[0179] Decrease in cold tolerance in such plants can be useful for
species or varieties of plants that benefit from cold exposure. For
example, cold sensitive plants might be able to undergo
vernalization more easily. Decreases in cold tolerance in such
plants can be useful in situations where plant parts such as, but
not limited to, seeds, tubers, stems, leaves or roots are harvested
for human or animal consumption.
[0180] Typically, a difference in the level of cold tolerance in a
transgenic plant or cell relative to a control plant or cell is
considered statistically significant at p.ltoreq.0.05 with an
appropriate parametric or non-parametric statistic, e.g.,
Chi-square test, Student's t-test, Mann-Whitney test, or F-test. In
some embodiments, a difference in the level of cold tolerance is
statistically significant at p<0.01, p<0.005, or p<0.001.
A statistically significant difference in, for example, the level
of cold tolerance in a transgenic plant compared to the amount in
cells of a control plant indicates that the recombinant nucleic
acid present in the transgenic plant results in altered cold
tolerance levels.
[0181] The phenotype of a transgenic plant is evaluated relative to
a control plant. A plant is said "not to express" a polypeptide
when the plant exhibits less than 10%, e.g., less than 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount
of polypeptide or mRNA encoding the polypeptide exhibited by the
plant of interest. Expression can be evaluated using methods
including, for example, RT-PCR, Northern blots, S1 RNAse
protection, primer extensions, Western blots, protein gel
electrophoresis, immunoprecipitation, enzyme-linked immunoassays,
chip assays, and mass spectrometry. It should be noted that if a
polypeptide is expressed under the control of a tissue-preferential
or broadly expressing promoter, expression can be evaluated in the
entire plant or in a selected tissue. Similarly, if a polypeptide
is expressed at a particular time, e.g., at a particular time in
development or upon induction, expression can be evaluated
selectively at a desired time period.
V. Plant Breeding
[0182] Genetic polymorphisms are discrete allelic sequence
differences in a population. Typically, an allele that is present
at 1% or greater is considered to be a genetic polymorphism. The
discovery that polypeptides disclosed herein can modulate cold
tolerance content is useful in plant breeding, because genetic
polymorphisms exhibiting a degree of linkage with loci for such
polypeptides are more likely to be correlated with variation in a
cold tolerance trait. For example, genetic polymorphisms linked to
the loci for such polypeptides are more likely to be useful in
marker-assisted breeding programs to create lines having a desired
modulation in the cold tolerance trait.
[0183] Thus, one aspect of the invention includes methods of
identifying whether one or more genetic polymorphisms are
associated with variation in a cold tolerance trait. Such methods
involve determining whether genetic polymorphisms in a given
population exhibit linkage with the locus for one of the
polypeptides depicted in FIGS. 1 to 5 and/or functional homologs
thereof, such as, but not limited to those identified in the
Sequence Listing of this application. The correlation is measured
between variation in the cold tolerance trait in plants of the
population and the presence of the genetic polymorphism(s) in
plants of the population, thereby identifying whether or not the
genetic polymorphism(s) are associated with variation for the
trait. If the presence of a particular allele is statistically
significantly correlated with a desired modulation in the cold
tolerance trait, the allele is associated with variation for the
trait and is useful as a marker for the trait. If, on the other
hand, the presence of a particular allele is not significantly
correlated with the desired modulation, the allele is not
associated with variation for the trait and is not useful as a
marker.
[0184] Such methods are applicable to populations containing the
naturally occurring endogenous polypeptide rather than an exogenous
nucleic acid encoding the polypeptide, i.e., populations that are
not transgenic for the exogenous nucleic acid. It will be
appreciated, however, that populations suitable for use in the
methods may contain a transgene for another, different trait, e.g.,
herbicide resistance.
[0185] Genetic polymorphisms that are useful in such methods
include simple sequence repeats (SSRs, or microsatellites), rapid
amplification of polymorphic DNA (RAPDs), single nucleotide
polymorphisms (SNPs), amplified fragment length polymorphisms
(AFLPs) and restriction fragment length polymorphisms (RFLPs). SSR
polymorphisms can be identified, for example, by making sequence
specific probes and amplifying template DNA from individuals in the
population of interest by PCR. If the probes flank an SSR in the
population, PCR products of different sizes will be produced. See,
e.g., U.S. Pat. No. 5,766,847. Alternatively, SSR polymorphisms can
be identified by using PCR product(s) as a probe against Southern
blots from different individuals in the population. See, U. H.
Refseth et al., (1997) Electrophoresis 18: 1519. The identification
of RFLPs is discussed, for example, in Alonso-Blanco et al.
(Methods in Molecular Biology, vol. 82, "Arabidopsis Protocols",
pp. 137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998
by Humana Press, Totowa, N.J.); Burr ("Mapping Genes with
Recombinant Inbreds", pp. 249-254, in Freeling, M. and V. Walbot
(Ed.), The Maize Handbook, c. 1994 by Springer-Verlag New York,
Inc.: New York, N.Y., USA; Berlin Germany; Burr et al. Genetics
(1998) 118: 519; and Gardiner, J. et al., (1993) Genetics 134:
917). The identification of AFLPs is discussed, for example, in EP
0 534 858 and U.S. Pat. No. 5,878,215.
[0186] In some embodiments, the methods are directed to breeding a
plant line. Such methods use genetic polymorphisms identified as
described above in a marker assisted breeding program to facilitate
the development of lines that have a desired alteration in the cold
tolerance trait. Once a suitable genetic polymorphism is identified
as being associated with variation for the trait, one or more
individual plants are identified that possess the polymorphic
allele correlated with the desired variation. Those plants are then
used in a breeding program to combine the polymorphic allele with a
plurality of other alleles at other loci that are correlated with
the desired variation. Techniques suitable for use in a plant
breeding program are known in the art and include, without
limitation, backcrossing, mass selection, pedigree breeding, bulk
selection, crossing to another population and recurrent selection.
These techniques can be used alone or in combination with one or
more other techniques in a breeding program. Thus, each identified
plants is selfed or crossed a different plant to produce seed which
is then germinated to form progeny plants. At least one such
progeny plant is then selfed or crossed with a different plant to
form a subsequent progeny generation. The breeding program can
repeat the steps of selfing or outcrossing for an additional 0 to 5
generations as appropriate in order to achieve the desired
uniformity and stability in the resulting plant line, which retains
the polymorphic allele. In most breeding programs, analysis for the
particular polymorphic allele will be carried out in each
generation, although analysis can be carried out in alternate
generations if desired.
[0187] In some cases, selection for other useful traits is also
carried out, e.g., selection for fungal resistance or bacterial
resistance. Selection for such other traits can be carried out
before, during or after identification of individual plants that
possess the desired polymorphic allele.
VI. Articles of Manufacture
[0188] Transgenic plants provided herein have various uses in the
agricultural and energy production industries. For example,
transgenic plants described herein can be used to make animal feed
and food products. Such plants, however, are often particularly
useful as a feedstock for energy production.
[0189] Transgenic plants described herein often produce higher
yields of grain and/or biomass per hectare, relative to control
plants that lack the exogenous nucleic acid. In some embodiments,
such transgenic plants provide equivalent or even increased yields
of grain and/or biomass per hectare relative to control plants when
grown under conditions of reduced inputs such as fertilizer and/or
water. Thus, such transgenic plants can be used to provide yield
stability at a lower input cost and/or under environmentally
stressful conditions such as drought. In some embodiments, plants
described herein have a composition that permits more efficient
processing into free sugars, and subsequently ethanol, for energy
production. In some embodiments, such plants provide higher yields
of ethanol, butanol, other biofuel molecules, and/or sugar-derived
co-products per kilogram of plant material, relative to control
plants. Such processing efficiencies are believed to be derived
from the chemical composition of the plant material. By providing
higher yields at an equivalent or even decreased cost of production
relative to control plants that do not have increased levels of
cold tolerance, the transgenic plants described herein improve
profitability for farmers and processors as well as decrease costs
to consumers.
[0190] Seeds from transgenic plants described herein can be
conditioned and bagged in packaging material by means known in the
art to form an article of manufacture. Packaging material such as
paper and cloth are well known in the art. A package of seed can
have a label, e.g., a tag or label secured to the packaging
material, a label printed on the packaging material, or a label
inserted within the package, that describes the nature of the seeds
therein.
[0191] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
VII. Examples
Example 1
Transgenic Arabidopsis Plants
[0192] The following symbols are used in the Examples with respect
to Arabidopsis transformation: T1: first generation transformant;
T2: second generation, progeny of self-pollinated T1 plants; T3:
third generation, progeny of self-pollinated T2 plants; T4: fourth
generation, progeny of self-pollinated T3 plants. Independent
transformations are referred to as events.
[0193] The following is a list of nucleic acids that were isolated
from Arabidopsis thaliana plants, Clone 2273, Clone 924103, and
Clone 13209. The nucleic acids designated Clone 6639 and Clone
924103 were isolated from the species Triticum aestivum.
[0194] Each isolated nucleic acid described above was cloned into a
Ti plasmid vector containing a phosphinothricin acetyltransferase
gene which confers Finale.TM. resistance to transformed plants. A
Ti plasmid vector useful for these constructs is CRS 338. Unless
otherwise indicated, each Ceres Clone and/or Seedline derived from
a Clone is in the sense orientation relative to either the 35S
promoter in a Ti plasmid. Wild-type Arabidopsis thaliana ecotype
Wassilewskija (Ws) plants were transformed separately with each
construct. The transformations were performed essentially as
described in Bechtold et al., C.R. Acad. Sci. Paris, 316:1194-1199
(1993).
[0195] Wild-type Arabidopsis Wassilewskija (Ws) plants were
transformed with a Ti plasmid containing Clone 924103 in the sense
orientation relative to the 326F promoter. The Ti plasmid vector
used for this construct, CRS814, contains the Ceres-constructed,
plant selectable marker gene phosphinothricin acetyltransferase
(PAT) which confers herbicide resistance to transformed plants.
[0196] Wild-type Arabidopsis Wassilewskija (Ws) plants were
transformed with a Ti plasmid containing clone 2273 in the sense
orientation relative to the 32449 promoter.
[0197] Wild-type Arabidopsis Wassilewskija (Ws) plants were
transformed with a Ti plasmid containing clone 13209 in the sense
orientation relative to the 32449 promoter. The Ti plasmid vector
used for this construct, CRS311, contains the Ceres-constructed,
plant selectable marker gene phosphinothricin acetyltransferase
(PAT) which confers herbicide resistance to transformed plants
[0198] Transgenic Arabidopsis lines containing Clone 2273, Clone
6639, Clone 924103, or Clone 13209 were designated ME00327,
ME04315, ME17294, or ME00572 respectively. The presence of each
vector containing a nucleic acid described above in the respective
transgenic Arabidopsis line transformed with the vector was
confirmed by Finale.TM. resistance, PCR amplification from green
leaf tissue extract, and/or sequencing of PCR products. As
controls, wild-type Arabidopsis ecotype Ws plants were transformed
with the empty vector SR0059.
Example 2
Screening for Cold Tolerance in Transgenic Plants
[0199] How plants respond to stress in the environment dictates
their ability to survive and reproduce. There are probably many
mechanisms by which plants regulate the temperatures under which
they will germinate (Lu and Hills, 2003). A number of
polynucleotides that result in stress tolerance when over-expressed
have been identified in model species such as Arabidopsis.
[0200] Over-expression of these polynucleotides could be useful for
increasing low temperature, chilling or cold tolerance in crops.
Assays described here focus on low temperature, chilling or cold
tolerance in seedlings. The ability to germinate and grow under low
temperature, chilling or cold, and wet conditions would allow a
longer growing season and mitigate damage caused by unexpected low
temperature, chilling or cold periods. If this trait is
recapitulated in crops overexpressing these polynucleotides, the
result could be very valuable in agriculture in many crops and
environments and make a significant contribution to sustainable
farming. Furthermore, low temperature, chilling or cold tolerance
may be modulated by expressing these polynucleotides under the
control of a low temperature, chilling or cold inducible
promoter.
1. Cold Growth Superpool Screen
[0201] Plates of solidified agar MS medium are prepared for the
screen as follows. One liter of medium is prepared by mixing 2.15 g
of MS basal salt mixture (from Phytotech M524) and 7 g of agar
(from EM Science, 1.01614.1000) in water, and adjusting the pH to
5.7 with a 10N KOH solution. After autoclaving, 45 ml of media are
transferred under sterile conditions per 100 mm square.times.15 mm
deep plate.
[0202] Individual superpool and control seeds are sterilized in a
30% bleach solution for 5 minutes. Seeds are then rinsed repeatedly
with sterile water to eliminate all bleach solution. Seeds are
plated using a COPAS.TM. robot (Union Biometrica, Holliston, Mass.)
at a density of 72 seeds per plate. The plates are wrapped with
vent tape and transferred to a dark 4.degree. C. refrigerator for 3
days to promote uniform germination. The plates are then placed
horizontally in a Conviron growth chamber set at 22.degree. C.,
16:8 hour light:dark cycle, 70% humidity with fluorescent lamps
emitting a light intensity of .about.100 .mu.Einsteins. Normal
growth is allowed to occur for 3-5 days. At end of 3-5 days of
growth, images of the plates are scanned using an Epson perfection
4870 scanner. Then, cold-growth treatment is applied for 1-3 weeks.
Accordingly, plates are transferred in a horizontal position to an
8.degree. C. Conviron chamber under constant light at .about.100
.mu.Einsteins. After a defined number of days of cold-growth
treatment, for example 7 or 14, the plates are scanned again. The
WinRhizo software program (Regent Instruments Inc., Canada) is used
to determine the area for each seedling from the scanned
images.
[0203] Individual seedlings that perform better in the cold growth
screen are identified by visual inspection for those showing
obvious morphological differences and by statistical analysis of
the seedling area data. DNA from these candidate seedlings is
extracted and the transgene amplified using PCR. The PCR product is
sequenced to determine the identity of the transgene and
consequently the ME line from which the candidate is derived.
2. Cold Growth Assay
[0204] The cold growth assay is used to validate candidate
misexpression (ME) lines obtained from screens for enhanced growth
under cold conditions. This procedure allows a high-throughput
methodology for assessing transgenic Arabidopsis candidates that
have germinated at normal temperature (22.degree. C.) and light
(.about.100 to 200 .mu.Einsteins) in a walk-in growth chamber on
agar solidified MS medium before transfer to cold temperature. It
relies on the ability to discriminate between seedlings that have
become significantly larger during cold growth than controls by
imaging the seedlings when they are transferred to the cold and
then periodically thereafter under cold growth conditions.
[0205] Plate preparation for the cold growth assay and the growth
conditions are the same as those described for the cold growth
screen as described above. Seeds from independent transformation
events for each ME line are bleach sterilized and then plated at a
density of 40 seeds per plate (30 seeds from the event and 10
wild-type control seeds). After cold-growth treatment, the
seedlings are then FINALE.RTM.-treated to identify the plants
carrying the ME vector.
[0206] Cold growth is characterized by statistical analysis as
follows. The control population is the internal non-transgenic
segregants for that particular event. When there are not enough
internal non-transgenic segregants for an event, a pool of all
non-transgenic segregants from all events associated with that ME
line is used (i.e. when non-transgenics are less than five for the
event or the event appears to be homozygous). Pooling is only done
for events associated with the same ME line and within an
experiment (an experiment is the set of plates with a common sow
date). Thus in the final analysis, the pooled control population
may be different for generations T.sub.2 and T.sub.3.
[0207] The WinRhizo software program (Regent Instruments Inc.,
Canada) is used to determine the area for each seedling. The change
in area is calculated for a defined number of days of treatment. A
one-tailed t-test is used to compare change in area and the mean
size of the transgenic seedlings within an event to the internal
non-transgenic segregants. Significance is assessed at an
.alpha.-value of 0.05.
3. Cold Flux Assay
[0208] The cold flux growth assay is used to validate candidate
misexpression (ME) lines obtained from screens for enhanced growth
under fluctuating cold conditions. This procedure allows a
high-throughput methodology for assessing transgenic Arabidopsis
candidates that have germinated at normal temperature (22.degree.
C.) and light (.about.100 to 200 .mu.Einsteins) in a walk-in growth
chamber on agar solidified MS medium before transfer to cold
temperature. It relies on the ability to discriminate between
seedlings that have become significantly larger during growth under
fluctuating cold conditions than controls by imaging the seedlings
when they are transferred to the cold and then periodically
thereafter under cold growth conditions.
[0209] Plate preparation for the cold growth assay and the growth
conditions are the same as those described for the cold growth
screen as described above. Seeds from independent transformation
events for each ME line are bleach sterilized and then plated at a
density of 61 seeds per plate (including both seeds from the event
and wild-type control seeds). After cold flux-growth treatment, the
seedlings are then FINALE.RTM.-treated to identify the plants
carrying the ME vector.
[0210] Normal growth is allowed to occur for 3-5 days. At end of
3-5 days of growth, images of the plates are scanned using an Epson
perfection 4870 scanner. After 3-5 days growth in normal
conditions, the plates are transferred in a horizontal position to
an 8.degree. C. Conviron under constant light at .about.100
.mu.Einsteins. All transfers take place in the morning. Growth is
allowed at 8.degree. C. for 3-4 days. After 3-4 days growth at
8.degree. C., plates are transferred to 1.degree. C. Percival under
constant light at .about.70 .mu.Einsteins. Growth is allowed at
1.degree. C. for 3-4 days. 8.degree. C./1.degree. C. cycling is
repeated for a total of 14 days. The plates are imaged using CF
imager and Winrhizo scanner. Individual seedlings are selected
which are significantly larger and/or exhibit increased
photosynthetic efficiency (Fv/Fm). Plates are visually observed as
well. DNA from these candidate seedlings is extracted and the
transgene amplified using PCR. The PCR product is sequenced to
determine the identity of the transgene and consequently the ME
line from which the candidate is derived. These seedlings are then
grown for progeny seed.
[0211] Cold flux growth is characterized by statistical analysis as
follows. The control population is the internal non-transgenic
segregants for that particular event. When there are not enough
internal non-transgenic segregants for an event, a pool of all
non-transgenic segregants from all events associated with that ME
line is used (i.e. when non-transgenics are less than five for the
event or the event appears to be homozygous). Pooling is only done
for events associated with the same ME line and within an
experiment (an experiment is the set of plates with a common sow
date). Thus in the final analysis, the pooled control population
may be different for generations T2 and T3.
[0212] The WinRhizo software program (Regent Instruments Inc.,
Canada) is used to determine the area for each seedling. The change
in area is calculated for a defined number of days of treatment. A
one-tailed t-test is used to compare change in area and the mean
size of the transgenic seedlings within an event to the internal
non-transgenic segregants. Significance is assessed at an
.alpha.-value of 0.05.
Example 3
Results for ME00327 Events (SEQ ID NO:2)
[0213] Ectopic expression of clone 2273 (from Arabidopsis thaliana)
under the control of the 32449 promoter in the ME00327 plants
results in larger seedlings after 14 days fluctuation between
8.degree. C. and 1.degree. C.
[0214] The seedling area of transgenic plants within a seed line
was compared to that of non-transgenic segregants within the same
seed line after 14 days of growth at fluctuating temperatures of
8.degree. C. and 1.degree. C. Six events of ME00327 were analyzed
as described in the cold flux assay (Example 2). Events-04 and -06
were significant in at least two generations at p<0.05 using a
one-tailed t-test assuming unequal variance (Table 1). The
transgenic plants were visibly larger than the controls.
[0215] Event-04 segregated 3:1 (R:S) for Finale.TM. resistance in
the T.sub.2 generation. Event-06 appears to segregate for two
inserts (15:1) in the T.sub.2 generation.
TABLE-US-00001 TABLE 1 t-test comparison of seedling area between
transgenic seedlings and control non-transgenic segregants after 14
days fluctuation between 8.degree. C. and 1.degree. C. Control
Event- Transgenic Non-Transgenics.sup.a t-test Events Gen Avg SE N
Avg SE N p-value ME00327-04 -04-T2 0.0383 0.0014 16 0.0297 0.0011
14 2.58E-05 ME00327-04- -04-T3 0.0490 0.0023 27 0.0410 0.0019 32
4.48E-03 02 ME00327-04- -04-T3 0.0522 0.0018 32 0.0378 0.0020 26
9.46E-07 03 ME00327-04- -04-T3 0.0497 0.0020 35 0.0364 0.0022 25
1.38E-05 04 ME00327-06.sup.c -06-T2 0.0314 0.0011 29 0.0272 0.0009
39 1.98E-03 ME00327-06.sup.bc -06-T2 0.0392 0.0012 54 0.0346 0.0008
194 8.17E-04 ME00327-06- -06-T3 0.0296 0.0015 41 0.0232 0.0016 17
2.47E-03 01 ME00327-06- -06-T3 0.0356 0.0015 42 0.0290 0.0019 15
4.44E-03 03 .sup.aTransgenic seedlings were compared to internal
non-transgenic segregants within an event unless otherwise
indicated. .sup.bThese events were sown twice. The first time was
to identify ME00327 as a Hit. They were repeated the second time
with the next generation to identify ME00327 as a Lead. .sup.cThese
events did not segregate non-transgenic seedlings and were compared
to pooled non-transgenics for the line.
[0216] Plants from Events-04 and -06 which are hemizygous or
homozygous for clone 2273 do not show any negative phenotypes under
standard conditions. Events-04 and -06 of ME00327 were tested for
negative phenotypes compared to the empty vector control SR00559.
The results showed no detectable reduction in germination rate, the
plants appeared wild-type in all instances, and no statistical
differences in days to flowering, rosette area 7 days post-bolting,
or fertility (silique number and seed fill).
Example 4
Results for ME04315 Events (SEQ ID NO: 20)
[0217] Candidate ME04315 was identified by superpool screen
described above in Example 2. Ectopic expression of Clone 6639
under the control of the 35S promoter in ME04315 plants results in
early germination at 8.degree. C. resulting in larger seedlings
after 10 days at 8.degree. C.
[0218] The seedling area of transgenic plants within a seed line
was compared to that of non-transgenic segregants within the same
seed line after 10 days of growth at 8.degree. C. Nine events of
ME04315 were analyzed as described in the Cold Growth Assay
described in Example 2 and showed significant tolerance under cold
conditions in two generations. Three Events, -02, -03 and -06, were
significant in both generations at p<0.05 using a one-tailed
t-test assuming unequal variance (Table 2). `-99` signifies that
seeds were pooled from several plants. Events-02 and -06 were from
the T3 generation because T2 seed was not available. Subsequently,
next generation seeds for three of the events (T3 or T4 as needed)
were evaluated under cold germination conditions.
[0219] The transgenic plants were visibly larger and lighter in
color than the controls. Under cold conditions, seedlings typically
become darker, presumably due to the accumulation of anthocyanin
The lighter color exhibited by ME04315 seedlings suggests a
decrease in this stress response. ME04315 plants grown under
standard conditions in soil did not appear different in color than
controls.
[0220] Event-03 segregated 3:1 (R:S) for Finale.TM. resistance in
the T.sub.2 generation. Seed collected from individual, hemizygous
plants was not available for Events-02 and -06. However, the
T.sub.3 generation seeds that were pooled from several T.sub.2
plants segregated approximately 2:1 in a manner consistent with a
single insert for Event-06 (only transgenic plants were pooled).
Pooled T.sub.3 generation seeds for Event-02 segregated 1:3.
TABLE-US-00002 TABLE 2 T-test comparison of seedling area between
transgenic seedlings and control non-transgenic segregants after 10
days at 8.degree. C. Control Non- Event- Transgenic
Transgenics.sup.a t-test Events Gen Avg SE N Avg SE N p-value
ME04315-02-99.sup.b 02-T3 0.0071 0.0013 10 0.0049 0.0007 24
7.50E-02 ME04315-02-99 02-T3 0.0046 0.0007 16 0.0036 0.0002 50
7.92E-02 ME04315-02-99-02.sup.a 02-T4 0.0070 0.0002 61 0.0047
0.0002 213 6.66E-16 ME04315-02-99-03 02-T4 0.0072 0.0002 45 0.0057
0.0007 3 2.08E-02 ME04315-03.sup.b 03-T2 0.0026 0.0002 18 0.0017
0.0003 7 0.0070 ME04315-03 03-T2 0.0017 0.0001 35 0.0016 0.0001 19
0.2221 ME04315-03-02 03-T3 0.0078 0.0005 12 0.0045 0.0011 7
6.69E-03 ME04315-03-03 03-T3 0.0063 0.0003 45 0.0053 0.0004 24
0.0153 ME04315-06-99.sup.b 06-T3 0.0034 0.0006 16 0.0023 0.0003 14
0.0499 ME04315-06-99 06-T3 0.0029 0.0002 42 0.0021 0.0001 28
9.52E-04 ME04315-06-99-02.sup.a 06-T4 0.0072 0.0002 61 0.0047
0.0002 213 3.33E-16 .sup.aTransgenic seedlings were compared to
non-transgenic segregants within a seed line except for the T.sub.4
generation of Events -02 and -06. Since these seed lines were
homozygous, they were compared to pooled non-transgenic segregants
from another T.sub.4 generation event that was grown in the same
flat as the T.sub.4 generation of Events -02 and -06. .sup.bThese
events were sown twice. The first time was to identify ME04315 as a
hit. They were repeated the second time with two generations to
identify ME04315 as a Lead.
[0221] Plants from Events-02, -03 and -06 which are hemizygous or
homozygous for Clone 6639 do not show any negative phenotypes under
long-day conditions. The physical appearance of eight of the nine
T.sub.1 plants was identical to the controls. Event-06 was smaller
and had fewer rosette leaves.
[0222] Events-02, -03 and -06 of ME04315 exhibited no statistically
significant negative phenotypes compared to empty vector control
SR00559. There was no detectable reduction in germination rate, the
plants appeared wild-type in all instances, and there was no
observable or statistical differences between experimentals and
controls for days to flowering, rosette area 7 days post-bolting or
fertility (silique number and seed fill).
Example 5
Results for ME17294 Events (SEO ID NO:93) 5' Truncated
[0223] Nine events of ME17294 (Clone 924103 from Triticum aestivum)
were analyzed as described in the cold germination assay (Example
2). In this study, the seedling area (a measure of germination
timing and cotyledon expansion) of transgenic plants within a seed
line was compared to that of non-transgenic segregants within the
same seed line, except for the T3 generation of both events. These
seed lines were homozygous for the transgene. For these seed lines,
we used pooled non-transgenic segregants from another T3 generation
event of ME17294 that were collected from plants grown in the same
flat as the T3 generation of Events-08 and -09.
[0224] The two events, -08 and -09, were significant in two
generations at p<0.05 using a one-tailed t-test assuming unequal
variance (Table 3). The transgenic plants are visibly larger.
[0225] Events-08 and -09 segregated 3:1 (R:S) for Finale.TM.
resistance in the T.sub.2 generation. No T.sub.1 phenotypes were
reported for this line.
TABLE-US-00003 TABLE 3 t-test comparison of seedling area between
transgenic seedlings and control non-transgenic segregants after 10
days at 8.degree. C. Control Non- Event- Transgenic
Transgenics.sup.a t-test Events Gen Avg SE N Avg SE N p-value
ME17294-08.sup.b 08-T2 0.0032 0.0003 24 0.0022 0.0002 9 2.57E-03
ME17294-08 08-T2 0.0021 0.0001 41 0.0018 0.0001 10 4.52E-03
ME17294-08-02.sup.a 08-T3 0.0083 0.0003 48 0.0064 0.0002 234
3.05E-08 ME17294-08-04 08-T3 0.0075 0.0003 54 0.0064 0.0002 234
9.19E-04 ME17294-09.sup.b 09-T2 0.0058 0.0003 22 0.0035 0.0002 9
1.02E-06 ME17294-09 09-T2 0.0039 0.0002 41 0.0029 0.0002 16
4.59E-04 ME17294-09-01.sup.a 09-T3 0.0073 0.0003 46 0.0064 0.0002
234 5.53E-03 ME17294-09-04.sup.a 09-T3 0.0087 0.0003 63 0.0064
0.0002 234 -9.10E-11 .sup.aTransgenic seedlings were compared to
internal non-transgenic segregants within a seed line except for
the T.sub.3 generation of Events-08 and -09. Since these seed lines
were homozygous, they were compared to pooled non-transgenic
segregants from another T.sub.3 generation event that was grown in
the same flat as the T.sub.3 generation of Events -08 and -09.
.sup.bThese events were sown twice. The first time was to identify
ME17294 as a Hit. They were repeated the second time with two
generations to identify ME17294 as a Lead.
[0226] Plants from Events-08 and -09 which are hemizygous or
homozygous for clone 924103 do not show any negative phenotypes
under standard conditions. Events-08 and -09 of ME17294 exhibited
no statistically significant negative phenotypes compared to empty
vector control SR00559. There was no detectable reduction in
germination rate, the plants appeared wild-type in all instances,
and there were no statistical differences between experimentals and
controls for days to flowering, rosette area 7 days post-bolting,
or fertility (silique number and seed fill).
Example 6
Results for ME00572 Events (SEO ID NO:111) tasiRNA
[0227] Clone 13209, in ME00572 plants, is a trans-acting small
interfering RNA (tasiRNA) that interacts with ARFs (Auxin Response
Factors). A megapool containing superpools 9-12 was screened for
seedlings that grew more vigorously than controls after transfer to
fluctuating cold conditions according to Example 2. Seven
candidates were chosen from this megapool. ME00572 was represented
two times in this set.
[0228] Four events of ME00572 showed significant tolerance under
cold fluctuating conditions in at least two generations. The
seedling area of transgenic plants within a seed line was compared
to that of non-transgenic segregants within the same seed line
after 14 days of growth at fluctuating temperatures of 8.degree. C.
and 1.degree. C. Five events of ME00572 were analyzed as described
in the Cold Flux Assay described in Example 2. Events-01, -03, -04
and -05 were significant in at least two generations at p<0.05
using a one-tailed t-test assuming unequal variance (Table 4). The
transgenic plants were visibly larger than the controls.
[0229] Events-01 and -05 segregated 3:1 (R:S) for Finale.TM.
resistance in the T2 generation. Event-04 segregated 3:1 in the T3
generation. Event-03 segregated 1:1 in the T2 generation.
TABLE-US-00004 TABLE 4 t-test comparison of seedling area between
transgenic seedlings and control non-transgenic segregants after 14
days fluctuation between 8.degree. C. and 1.degree. C. Control
Event- Transgenic Non-Transgenics.sup.a t-test Events Gen Avg SE N
Avg SE N p-value ME00572-01 -01-T2 0.0338 0.0293 26 0.0233 0.0010 4
0.155617 ME00572-01.sup.b -01-T2 0.0541 0.0021 45 0.0442 0.0030 13
4.92E-03 ME00572-01- -01-T3 0.0347 0.0017 40 0.0246 0.0014 14
1.53E-05 01 ME00572-01- -01-T3 0.0331 0.0013 59 0.0267 0.0005 294
2.35E-06 02.sup.c ME00572-01- -01-T3 0.0360 0.0010 59 0.0267 0.0005
294 -9.39E-11 03.sup.c ME00572-01- -01-T3 0.0329 0.0013 46 0.0233
0.0018 14 3.19E-05 04 ME00572-03 -03-T2 0.0312 0.0016 11 0.0223
0.0013 19 9.71E-05 ME00572-03.sup.b -03-T2 0.0383 0.0024 24 0.0341
0.0023 25 1.04E-01 ME00572-03- -03-T3 0.0328 0.0014 30 0.0236
0.0008 28 1.42E-07 01 ME00572-03- -03-T3 0.0311 0.0010 26 0.0261
0.0015 33 3.81E-03 02 ME00572-03- -03-T3 0.0352 0.0011 28 0.0252
0.0009 32 1.39E-09 03 ME00572-03- -03-T3 0.0331 0.0019 23 0.0260
0.0014 35 1.97E-03 04 ME00572-04- -04-T3 0.0235 0.0009 23 0.0158
0.0020 7 6.98E-04 99 ME00572-04- -04-T3 0.0336 0.0012 31 0.0255
0.0010 29 2.01E-06 99.sup.b ME00572-04- -04-T4 0.0388 0.0014 57
0.0267 0.0005 294 -9.39E-11 99-01.sup.c ME00572-04- -04-T4 0.0345
0.0011 41 0.0253 0.0018 19 3.93E-05 99-02 ME00572-04- -04-T4 0.0433
0.0012 60 0.0267 0.0005 294 -3.33E-16 99-03.sup.c ME00572-04-
-04-T4 0.0315 0.0010 44 0.0239 0.0012 16 3.22E-06 99-04 ME00572-05
-05-T2 0.0362 0.0020 19 0.0219 0.0020 10 1.13E-05 ME00572-05-
-05-T3 0.0337 0.0011 57 0.0267 0.0005 294 6.21E-09 01.sup.c
ME00572-05- -05-T3 0.0320 0.0014 39 0.0232 0.0020 17 3.59E-04 02
ME00572-05- -05-T3 0.0379 0.0011 59 0.0267 0.0005 294 -9.39E-11
03.sup.c ME00572-05- -05-T3 0.0344 0.0020 37 0.0268 0.0022 19
7.26E-03 04 .sup.aTransgenic seedlings were compared to internal
non-transgenic segregants within an event unless otherwise
indicated. .sup.bThese events were sown twice. The first time was
to identify ME00572 as a Hit. They were repeated the second time
with the next generation to identify ME00572 as a Lead. .sup.cThese
events did not segregate non-transgenic seedlings and were compared
to pooled non- transgenics for the line.
[0230] Plants from Events-01, -03, -04 and -05 which are hemizygous
or homozygous for clone 13209 do not show any negative phenotypes
under standard conditions. Events-01, -03, -04 and -05 of ME00572
were tested for negative phenotypes compared to the empty vector
control SR00559. There was no detectable reduction in germination
rate, the plants appeared wild-type in all instances, and there was
no statistical differences between experimentals and controls for
days to flowering, rosette area 7 days post-bolting, and fertility
(silique number and seed fill).
Example 7
Determination of Functional Homologs by Reciprocal BLAST
[0231] A candidate sequence was considered a functional homolog of
a reference sequence if the candidate and reference sequences
encoded proteins having a similar function and/or activity. A
process known as Reciprocal BLAST (Rivera et al., Proc. Natl. Acad.
Sci. USA, 95:6239-6244 (1998)) was used to identify potential
functional homolog sequences from databases consisting of all
available public and proprietary peptide sequences, including NR
from NCBI and peptide translations from Ceres clones.
[0232] Before starting a Reciprocal BLAST process, a specific
reference polypeptide was searched against all peptides from its
source species using BLAST in order to identify polypeptides having
BLAST sequence identity of 80% or greater to the reference
polypeptide and an alignment length of 85% or greater along the
shorter sequence in the alignment. The reference polypeptide and
any of the aforementioned identified polypeptides were designated
as a cluster.
[0233] The BLASTP version 2.0 program from Washington University at
Saint Louis, Mo., USA was used to determine BLAST sequence identity
and E-value. The BLASTP version 2.0 program includes the following
parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5;
and 3) the -postsw option. The BLAST sequence identity was
calculated based on the alignment of the first BLAST HSP
(High-scoring Segment Pairs) of the identified potential functional
homolog sequence with a specific reference polypeptide. The number
of identically matched residues in the BLAST HSP alignment was
divided by the HSP length, and then multiplied by 100 to get the
BLAST sequence identity. The HSP length typically included gaps in
the alignment, but in some cases gaps were excluded.
[0234] The main Reciprocal BLAST process consists of two rounds of
BLAST searches; forward search and reverse search. In the forward
search step, a reference polypeptide sequence, "polypeptide A,"
from source species SA was BLASTed against all protein sequences
from a species of interest. Top hits were determined using an
E-value cutoff of 10.sup.-5 and a sequence identity cutoff of 35%.
Among the top hits, the sequence having the lowest E-value was
designated as the best hit, and considered a potential functional
homolog or ortholog. Any other top hit that had a sequence identity
of 80% or greater to the best hit or to the original reference
polypeptide was considered a potential functional homolog or
ortholog as well. This process was repeated for all species of
interest.
[0235] In the reverse search round, the top hits identified in the
forward search from all species were BLASTed against all protein
sequences from the source species SA. A top hit from the forward
search that returned a polypeptide from the aforementioned cluster
as its best hit was also considered as a potential functional
homolog.
[0236] Functional homologs were identified by manual inspection of
potential functional homolog sequences. Representative functional
homologs for SEQ ID NO: 2, 20, 74, 93, and 116, are shown in FIGS.
1-5, respectively. Additional exemplary homologs are correlated to
certain Figures in the Sequence Listing.
Example 8
Determination of Functional Homologs by Hidden Markov Models
[0237] Hidden Markov Models (HMMs) were generated by the program
HMMER 2.3.2. To generate each HMM, the default HMMER 2.3.2 program
parameters, configured for glocal alignments, were used.
[0238] An HMM was generated using the sequences shown in FIG. 1 as
input. These sequences were fitted to the model and a
representative HMM bit score for each sequence is shown in the
Sequence Listing. Additional sequences were fitted to the model,
and representative HMM bit scores for any such additional sequences
are shown in the Sequence Listing. The results indicate that these
additional sequences are functional homologs of SEQ ID NO: 2.
[0239] The procedure above was repeated and an HMM was generated
for each group of sequences shown in FIGS. 2, 3, 4, and 5 using the
sequences shown in each Figure as input for that HMM. A
representative bit score for each sequence is shown in the Sequence
Listing. Additional sequences were fitted to certain HMMs, and
representative HMM bit scores for such additional sequences are
shown in the Sequence Listing. The results indicate that these
additional sequences are functional homologs of the sequences used
to generate that HMM.
Other Embodiments
[0240] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110061122A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110061122A1).
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