Plant transcriptional regulators of abiotic stress

Abstract

The invention relates to plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties compared to a reference plant, including improved abiotic stress tolerance. Sequence information related to these polynucleotides and polypeptides can also be used in bioinformatic search methods to identify related sequences and is also disclosed.

Description

RELATIONSHIP TO COPENDING APPLICATIONS

[0001] This application claims the benefit of U.S. application Ser. No. 10/412,699, filed Apr. 10, 2003, which in turn claims the benefit of U.S. Non-provisional application Ser. No. 09/533,030, filed Mar. 22, 2000, which in turn claims the benefit of U.S. Provisional Application No. 60/125,814, filed Mar. 23, 1999, U.S. Non-provisional application Ser. No. 09/713,994, filed Nov. 16, 2000, which in turn claims the benefit of U.S. Provisional Application No. 60/166,228, filed Nov. 17, 1999, U.S. Provisional Application No. 60/197,899, filed Apr. 17, 2000, and U.S. Provisional Application No. 60/227,439, filed Aug. 22, 2000; U.S. Non-provisional application Ser. No. 10/112,887, filed Mar. 18, 2002; U.S. Non-provisional application Ser. No. 10/286,264, filed Jan. 23, 2003; U.S. Non-provisional application Ser. No. 10/225,068, filed Aug. 9, 2002; U.S. Non-provisional application Ser. No. 10/225,066, filed Aug. 9, 2002; U.S. Non-provisional application Ser. No. 10/374,780, filed Feb. 25, 2003, which claims the benefit of U.S. Non-provisional application Ser. No. 09/837,944, filed Apr. 18, 2001, U.S. Non-provisional application Ser. No. 10/171,468, filed Jun. 14, 2002, U.S. Provisional Application No. 60/310,847, filed Aug. 9, 2001, and U.S. Provisional Application No. 60/336,049, filed Nov. 19, 2001; U.S. Non-provisional application "Polynucleotides and Polypeptides in Plants", filed Sep. 18, 2003, claims the benefit of U.S. Provisional Application No. 60/434,166, filed Dec. 17, 2002, and U.S. Provisional Application No. 60/411,837, filed Sep. 18, 2002. The entire contents of all of these applications are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for modifying a plant phenotypically, said plant having altered sugar sensing and an altered response to abiotic stresses, including osmotic stresses, including germination in cold and heat, increased tolerance to drought and high salt stress.

BACKGROUND OF THE INVENTION

[0003] A plant's traits, such as its biochemical, developmental, or phenotypic characteristics, may be controlled through a number of cellular processes. One important way to manipulate that control is through transcription factors, proteins that influence the expression of a particular gene or sets of genes. Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, possess advantageous or desirable traits. Strategies for manipulating traits by altering a plant cell's transcription factor content can therefore result in plants and crops with new and/or improved commercially valuable properties.

[0004] Transcription factors can modulate gene expression, either increasing or decreasing (inducing or repressing) the rate of transcription. This modulation results in differential levels of gene expression at various developmental stages, in different tissues and cell types, and in response to different exogenous (e.g., environmental) and endogenous stimuli throughout the life cycle of the organism.

[0005] Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, plant, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. Comparisons of Arabidopsis gene sequences with those from other organisms where the structure and/or function may be known allow researchers to draw analogies and to develop model systems for testing hypotheses. These model systems are of great importance in developing and testing plant varieties with novel traits that may have an impact upon agronomy.

[0006] Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or biomolecules in plants or improvement in other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits, including traits that improve a plant's survival and yield during periods of abiotic stress, including germination in cold and hot conditions, and osmotic stress, including drought, salt stress, and other abiotic stresses, as noted below.

[0007] Problems associated with drought. A drought is a period of abnormally dry weather that persists long enough to produce a serious hydrologic imbalance (for example crop damage, water supply shortage, etc.). While much of the weather that we experience is brief and short-lived, drought is a more gradual phenomenon, slowly taking hold of an area and tightening its grip with time. In severe cases, drought can last for many years and can have devastating effects on agriculture and water supplies. With burgeoning population and chronic shortage of available fresh water, drought is not only the number one weather related problem in agriculture, it also ranks as one of the major natural disasters of all time, causing not only economic damage, but also loss of human lives. For example, losses from the US drought of 1988 exceeded $40 billion, exceeding the losses caused by Hurricane Andrew in 1992, the Mississippi River floods of 1993, and the San Francisco earthquake in 1989. In some areas of the world, the effects of drought can be far more severe. In the Horn of Africa the 1984-1985 drought led to a famine that killed 750,000 people.

[0008] Problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Drought also causes plants to become more susceptible to various diseases (Simpson (1981). "The Value of Physiological Knowledge of Water Stress in Plants", In Water Stress on Plants, (Simpson, G. M., ed.), Praeger, N.Y., pp. 235-265).

[0009] In addition to the many land regions of the world that are too arid for most if not all crop plants, overuse and over-utilization of available water is resulting in an increasing loss of agriculturally-usable land, a process which, in the extreme, results in desertification. The problem is further compounded by increasing salt accumulation in soils, as described above, which adds to the loss of available water in soils.

[0010] Problems associated with high salt levels. One in five hectares of irrigated land is damaged by salt, an important historical factor in the decline of ancient agrarian societies. This condition is only expected to worsen, further reducing the availability of arable land and crop production, since none of the top five food crops--wheat, corn, rice, potatoes, and soybean--can tolerate excessive salt.

[0011] Detrimental effects of salt on plants are a consequence of both water deficit resulting in osmotic stress (similar to drought stress) and the effects of excess sodium ions on critical biochemical processes. As with freezing and drought, high saline causes water deficit; the presence of high salt makes it difficult for plant roots to extract water from their environment (Buchanan et al. (2000) in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.). Soil salinity is thus one of the more important variables that determines where a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. To compound the problem, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture. The latter is compounded by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt level in the whole soil profile.

[0012] Problems associated with excessive heat. Germination of many crops is very sensitive to temperature. A transcription factor that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates. Seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeleton, and impairs membrane function (Buchanan et al. (2000) in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.).

[0013] Heat shock may produce a decrease in overall protein synthesis, accompanied by expression of heat shock proteins. Heat shock proteins function as chaperones and are involved in refolding proteins denatured by heat.

[0014] Heat stress often accompanies conditions of low water availability. Heat itself is seen as an interacting stress and adds to the detrimental effects caused by water deficit conditions. Evaporative demand exhibits near exponential increases with increases in daytime temperatures and can result in high transpiration rates and low plant water potentials (Hall et al. (2000) Plant Physiol. 123: 1449-1458). High-temperature damage to pollen almost always occurs in conjunction with drought stress, and rarely occurs under well-watered conditions. Thus, separating the effects of heat and drought stress on pollination is difficult. Combined stress can alter plant metabolism in novel ways; therefore understanding the interaction between different stresses may be important for the development of strategies to enhance stress tolerance by genetic manipulation.

[0015] Problems associated with excessive chilling conditions. The term "chilling sensitivity" has been used to describe many types of physiological damage produced at low, but above freezing, temperatures. Most crops of tropical origins, such as soybean, rice, maize and cotton are easily damaged by chilling. Typical chilling damage includes wilting, necrosis, chlorosis or leakage of ions from cell membranes. The underlying mechanisms of chilling sensitivity are not completely understood yet, but probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (disruption of photosynthesis due to high light intensities) often occurs under clear atmospheric conditions subsequent to cold late summer/autumn nights. For example, chilling may lead to yield losses and lower product quality through the delayed ripening of maize. Another consequence of poor growth is the rather poor ground cover of maize fields in spring, often resulting in soil erosion, increased occurrence of weeds, and reduced uptake of nutrients. A retarded uptake of mineral nitrogen could also lead to increased losses of nitrate into the ground water. By some estimates, chilling accounts for monetary losses in the United States (US) behind only to drought and flooding.

[0016] Desirability of altered sugar sensing. Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Sucrose, for example, is the major transport form of photosynthate and its flux through cells has been shown to affect gene expression and alter storage compound accumulation in seeds (source-sink relationships). Glucose-specific hexose-sensing has also been described in plants and is implicated in cell division and repression of "famine" genes (photosynthetic or glyoxylate cycles).

[0017] Water deficit is a common component of many plant stresses. Water deficit occurs in plant cells when the whole plant transpiration rate exceeds the water uptake. In addition to drought, other stresses, such as salinity and low temperature, produce cellular dehydration (McCue and Hanson (1990) Trends Biotechnol. 8: 358-362

[0018] Salt and drought stress signal transduction consist of ionic and osmotic homeostasis signaling pathways. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. The pathway regulating ion homeostasis in response to salt stress has been reviewed recently by Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139.

[0019] The osmotic component of salt stress involves complex plant reactions that overlap with drought and/or cold stress responses.

[0020] Common aspects of drought, cold and salt stress response have been reviewed recently by Xiong and Zhu (2002) supra). Those include:

[0021] (a) transient changes in the cytoplasmic calcium levels very early in the signaling event (Knight, (2000) Int. Rev. Cytol. 195: 269-324; Sanders et al. (1999) Plant Cell 11: 691-706);

[0022] (b) signal transduction via mitogen-activated and/or calcium dependent protein kinases (CDPKs;

[0023] see Xiong et al., 2002) and protein phosphatases (Merlot et al. (2001) Plant J. 25: 295-303; Thtiharju and Palva (2001) Plant J. 26: 461-470);

[0024] (c) increases in abscisic acid levels in response to stress triggering a subset of responses (Xiong et al. (2002) supra, and references therein);

[0025] (d) inositol phosphates as signal molecules (at least for a subset of the stress responsive transcriptional changes (Xiong et al. (2001) Genes Dev. 15: 1971-1984);

[0026] (e) activation of phospholipases which in turn generate a diverse array of second messenger molecules, some of which might regulate the activity of stress responsive kinases (phospholipase D functions in an ABA independent pathway, Frank et al. (2000) Plant Cell 12: 111-124);

[0027] (f) induction of late embryogenesis abundant (LEA) type genes including the CRT/DRE responsive COR/RD genes (Xiong and Zhu (2002) supra);

[0028] (g) increased levels of antioxidants and compatible osmolytes such as proline and soluble sugars (Hasegawa et al. (2000) Annu. Rev. Plant Mol. Plant Physiol. 51: 463499); and

[0029] (h) accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals (Hasegawa et al. (2000) supra).

[0030] Abscisic acid biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and -independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.

[0031] Based on the commonality of many aspects of cold, drought and salt stress responses, it can be concluded that genes that increase tolerance to cold or salt stress can also improve drought stress protection. In fact this has already been demonstrated for transcription factors (in the case of AtCBF/DREB1) and for other genes such as OsCDPK7 (Saijo et al. (2000) Plant J. 23: 319-327), or AVP1 (a vacuolar pyrophosphatase-proton-- pump, Gaxiola et al. (2001) Proc. Natl. Acad. Sci. USA 98: 11444-11449).

[0032] The present invention relates to methods and compositions for producing transgenic plants with modified traits, particularly traits that address agricultural and food needs. These traits, including altered sugar sensing and tolerance to abiotic and osmotic stress (e.g., tolerance to cold, high salt concentrations and drought), may provide significant value in that they allow the plant to thrive in hostile environments, where, for example, high or low temperature, low water availability or high salinity may limit or prevent growth of non-transgenic plants.

[0033] We have identified polynucleotides encoding transcription factors, including G482, G481, G485, G1364, G2345, G1781 and their equivalogs listed in the Sequence Listing, and structurally and functionally similar sequences, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for their tolerance to abiotic stresses, including those associated with heat, cold, or osmotic stresses such as drought and excessive salt. In so doing, we have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.

SUMMARY OF THE INVENTION

[0034] The present invention pertains to transgenic plants that comprise a recombinant polynucleotide that includes a nucleotide sequence encoding a CCAAT transcription factor with the ability to regulate abiotic stress tolerance in a plant. The nucleotide sequence is capable of hybridizing to the complement of the G482 polynucleotide sequence (SEQ ID NO:3) under stringent conditions consisting of hybridization (e.g., to filter-bound DNA, such as a hybridization procedure that includes the use of 6.times.SSC, 65.degree. C., in two wash steps of 10-30 minutes in duration. The resultant transgenic plant has increased tolerance to abiotic stress as compared to a non-transformed plant.

[0035] The invention also encompasses transgenic plant that comprise a recombinant polynucleotide that includes a nucleotide sequence encoding a CCAAT transcription factor with the ability to regulate abiotic stress tolerance in a plant; the transcription factor comprising a CCAAT-box binding conserved domain that is at least 83% identical with the conserved CCAAT-box binding or "B" domain of the G482 polypeptide (SEQ ID NO: 4). This transgenic plant has increased tolerance to abiotic stress as compared to a non-transformed plant that does not overexpress the recombinant polynucleotide.

[0036] The present invention also relates to a method of using transgenic plants transformed with the presently disclosed transcription factor sequences, their complements or their variants to grow a progeny plant by crossing the transgenic plant with either itself or another plant, selecting seed that develops as a result of the crossing; and then growing the progeny plant from the seed. The progeny plant will generally express mRNA that encodes a transcription factor: that is, a DNA-binding protein that binds to a DNA regulatory sequence and regulates gene expression, such as that of a plant trait gene. The mRNA will generally be expressed at a level greater than a non-transformed plant; and the progeny plant is characterized by a change in a plant trait compared to the non-transformed plant.

[0037] The present invention also pertains to an expression cassette. The expression cassette comprises at least two elements, including: (1) a constitutive, inducible, or tissue-specific promoter; and (2) a recombinant polynucleotide having a polynucleotide sequence, or a complementary polynucleotide sequence thereof, selected from the group consisting of

[0038] (a) the G482 polynucleotide (SEQ ID NO: 3);

[0039] (b) a polynucleotide encoding the G482 polypeptide (SEQ ID NO: 4);

[0040] (c) a nucleotide sequence that hybridizes to the polynucleotide of (a) or (b) under the stringent conditions of 6.times.SSC and 65.degree. C.; and

[0041] (d) a nucleotide sequence that is at least 83% identical with the B domain found in the G482 polypeptide (SEQ ID NO: 4).

[0042] The invention is also characterized by a host cell that contains the aforementioned expression cassette.

[0043] The present invention also pertains to methods for increasing a plant's tolerance to abiotic stress. This is accomplished through the use of a vector that comprises a polynucleotide sequence that hybridizes over its full length to the complement of the G482 polynucleotide (SEQ ID NO:3) under the stringent conditions of hybridization to filter-bound DNA in 6.times.SSC at 65.degree. C. The polynucleotide sequence encodes a CCAAT transcription factor that has the property of SEQ ID NO:4 of regulating abiotic stress tolerance in a plant. The vector also includes regulatory elements that control expression of the polynucleotide sequence in a target plant. These regulatory elements flank the polynucleotide sequence. The target plant is then transformed with the vector, which transformation process generates a plant with increased tolerance to osmotic stress.

[0044] The invention is also directed to a method for producing a plant that has increased tolerance to one or more osmotic stresses. This method is performed by selecting a polynucleotide that encodes the G482 polypeptide (SEQ ID NO: 4), inserting either this polynucleotide or its complement into an expression cassette (for example, the expression cassette described above), introducing the expression cassette into a plant or plant cell in order to overexpress the G482 polypeptide, which thereby produces a plant having increased tolerance to osmotic stress. A plant that has this increased tolerance relative to a control plant not so transformed may then be identified and selected.

[0045] The invention further pertains to an isolated nucleic acid comprising a nucleotide sequence at least 99.6% identical to G482, SEQ ID NO: 3, wherein the expression of this nucleotide sequence results in increased abiotic stress tolerance in a plant.

[0046] Also encompassed by the invention are polypeptides encoded by isolated nucleic acids that are at least 99.6% identical to G482, vectors comprising isolated nucleic acid that are at least 99.6% identical to G482, and host cells and transgenic plants transformed with these isolated nucleic acids.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0047] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0048] The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.

[0049] CD-ROM1 is a read-only memory computer-readable compact disc and contains a copy of the Sequence Listing in ASCII text format. The Sequence Listing is named "MBI0022CIP.ST25.txt" and is 163 kilobytes in size. The copies of the Sequence Listing on the CD-ROM disc are hereby incorporated by reference in their entirety.

[0050] FIG. 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Angiosperm Phylogeny Group (1998) Ann. Missouri Bot. Gard. 84: 1-49). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales. FIG. 1 was adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333.

[0051] FIG. 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including clades containing tomato and Arabidopsis; adapted from Ku et al. (2000) Proc. Natl. Acad. Sci. 97: 9121-9126; and Chase et al. (1993) Ann. Missouri Bot. Gard. 80: 528-580.

[0052] FIG. 3 is adapted from Kwong et al (2003) Plant Cell 15: 5-18, and shows crop orthologs identified through BLAST analysis of various L1L-related sequences. A phylogeny tree was then generated using ClustaIX based on whole protein sequences showing the non-LEC1-like HAP3 clade of transcription factors (large box). This clade, also contains members from other species (for example, SEQ ID NOs: 18, 20, 24, 26, 48, 50, 52, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, and other sequences appearing in Table 5) are phylogenetically distinct from the LEC 1-like proteins, some of which are also shown in FIG. 3. The smaller box delineates the G482-like subclade, containing transcription factors that are structurally most closely related to G482, and in which several members have been shown to confer improved abiotic stress tolerance and/or altered flowering time characteristics.

[0053] Similar to FIG. 3, FIG. 4 shows the phylogenic relationship of sequences within the G482-subclade (within the smaller box) and the non-LEC1-like clade (larger box).

[0054] FIG. 5 shows the domain structure of HAP3 proteins. HAP3 proteins contain an amino-terminal A domain, a central B domain, and a carboxy-terminal C domain. There may be relatively little sequence similarity between HAP3 proteins in the A and C domains. The A and C domains could thus provide a degree of specificity to each member of the HAP3 family. The B domain is the conserved region that specifies DNA binding and subunit association.

[0055] In FIGS. 6A-6F, the alignments of HAP3 polypeptides are presented, including G481, G482, G485, G1364, G2345, G1781 and related sequences from Arabidopsis aligned with soybean, rice and corn sequences, showing the B domains (indicated by the line that spans FIGS. 6B through 6D). Consensus residues within the listed sequences are indicated by boldface. The boldfaced residues in the consensus sequence that appears at the bottom of FIGS. 6A through 6C in their respective positions are uniquely found in the non-LEC1-like clade. The underlined serine residue appearing in the consensus sequence in its respective positions is uniquely found within the G482-like subclade. As discussed in greater detail below in Example IX, the residue positions indicated by the arrows in FIG. 6B are associated with an alteration of flowering time when these polypeptides are overexpressed.

[0056] FIGS. 7A-7D show the effects of water deprivation and recovery from this treatment on Arabidopsis control and 35S::G481-overexpressing lines. After eight days of drought treatment overexpressing plants had a darker green and less withered appearance (FIG. 7C) than those in the control group (FIG. 7A). The differences in appearance between the control and G481-overexpressing plants after they were rewatered was even more striking. Most (11 of 12 plants; FIG. 7B) of this set of control plants died after rewatering, indicating the inability to recover following severe water deprivation, whereas all nine of the overexpressor plants of the line shown recovered from this drought treatment (FIG. 7D). The results shown in FIGS. 7A-7D were typical of a number of control and 35 S::G481-overexpressing lines.

[0057] FIGS. 8A and 8B show the effects of salt stress on Arabidopsis seed germination. The three lines of G481-and G482 overexpressors on these two plate had longer roots and showed greater cotyledon expansion (arrows) after three days on 150 mM NaCl than the control seedlings on the right-hand sides of the plates.

[0058] In FIG. 9A, G481 null mutant seedlings (labeled K481) show reduced tolerance of osmotic stress, relative to the control seedlings in FIG. 8B, as evidenced by the reduced cotyledon expansion and root growth in the former group. Without salt stress tolerance on control media, (FIGS. 9C, G481 null mutants; and 9D, control seedlings), the knocked out and control plants appear the same.

[0059] FIGS. 10A-10D show the effects of stress-related treatments on G485 overexpressing seedlings (35S::G485 lines) in plate assays. In each treatment, including cold, high sucrose, high salt and ABA germination assays, the overexpressors fared much better than the wild-type controls exposed to the same treatments in FIGS. 10E-10H, respectively, as evidenced by the enhanced cotyledon expansion and root growth seen with the overexpressing seedlings.

[0060] FIGS. 11A-11C depict the effects of G485 knockout and overexpression on flowering time and maturation. As seen in FIG. 10A, a T-DNA insertion knockout mutation containing a SALK.sub.--062245 insertion was shown to flower several days later than wild-type control plants. The plants in FIG. 11A are shown 44 days after germination. FIG. 11C shows that G485 primary transformants flowered distinctly earlier than wild-type controls. These plants are shown 24 days after germination. These effects were observed in each of two independent T1 plantings derived from separate transformation dates. Additionally, accelerated flowering was also seen in plants that overexpressed G485 from a two component system (35S::LexA;op-LexA::G485). These studies indicated that G485 is both sufficient to act as a floral activator, and is also necessary in that role within the plant. G485 overexpressor plants also matured and set siliques much more rapidly than wild type controls, as shown in FIG. 11B with plants 39 days post-germination.

DESCRIPTION OF THE INVENTION

[0061] In an important aspect, the present invention relates to polynucleotides and polypeptides, for example, for modifying phenotypes of plants, particularly those associated with osmotic stress tolerance. Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses, for example. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of "incorporation by reference" is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.

[0062] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a plant" includes a plurality of such plants, and a reference to "a stress" is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.

[0063] Definitions

[0064] "Nucleic acid molecule" refers to a oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA).

[0065] "Polynucleotide" is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides, optionally at least about 30 consecutive nucleotides, at least about 50 consecutive nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. "Oligonucleotide" is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single stranded.

[0066] "Gene" or "gene sequence" refers to the partial or complete coding sequence of a gene, its complement, and its 5' or 3' untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as splicing and folding to obtain a functional protein or polypeptide. A gene may be isolated, partially isolated, or be found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.

[0067] Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and which may be used to determine the limits of the genetically active unit (Rieger et al. (1976) Glossary of Genetics and Cytogenetics: Classical and Molecular, 4th ed., Springer Verlag. Berlin). A gene generally includes regions preceding ("leaders"; upstream) and following ("trailers"; downstream) of the coding region. A gene may also include intervening, non-coding sequences, referred to as "introns", located between individual coding segments, referred to as "exons". Most genes have an associated promoter region, a regulatory sequence 5' of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.

[0068] A "recombinant polynucleotide" is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.

[0069] An "isolated polynucleotide" is a polynucleotide whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.

[0070] A "polypeptide" is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) a DNA-binding domain, or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.

[0071] "Protein" refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.

[0072] "Portion", as used herein, refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules which specifically bind to that portion or for the production of antibodies.

[0073] A "recombinant polypeptide" is a polypeptide produced by translation of a recombinant polynucleotide. A "synthetic polypeptide" is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An "isolated polypeptide," whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.

[0074] "Homology" refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.

[0075] "Hybridization complex" refers to a complex between two nucleic acid molecules by virtue of the formation of hydrogen bonds between purines and pyrimidines.

[0076] "Identity" or "similarity" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity" and "% identity" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[0077] The term "amino acid consensus motif" refers to the portion or subsequence of a polypeptide sequence that is substantially conserved among the polypeptide transcription factors listed in the Sequence Listing.

[0078] "Alignment" refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (ire., nucleotide bases or amino acid residues) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those of FIGS. 6A-6F may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, Calif.).

[0079] A "conserved domain" or "conserved region" as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. A CCAAT-box binding conserved domain, such as one of the domains shown in Table 1, is an example of a conserved domain.

[0080] With respect to polynucleotides encoding presently disclosed transcription factors, a conserved domain is preferably at least 10 base pairs (bp) in length.

[0081] A "conserved domain", with respect to presently disclosed polypeptides refers to a domain within a transcription factor family that exhibits a higher degree of sequence homology, such as at least 26% sequence similarity, at least 16% sequence identity, preferably at least 40% sequence identity, preferably at least 65% sequence identity including conservative substitutions, and more preferably at least 80% sequence identity, and even more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% amino acid residue sequence identity of a polypeptide of consecutive amino acid residues. A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular transcription factor class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be "outside a conserved domain" if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.

[0082] As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000) supra). Thus, by using alignment methods well known in the art, the conserved domains of the plant transcription factors for the CAAT-element binding proteins (Forsburg and Guarente (1989) Genes Dev. 3: 1166-1178) may be determined.

[0083] The CCAAT-box binding conserved domains or conserved domains for SEQ ID NO: 2, 4, 6, 8 and 10 and similar sequences are listed in Table 1. Also, the polypeptides of Table 1 have CCAAT-box binding conserved domains specifically indicated by start and stop sites. A comparison of the regions of the polypeptides in Table 1 allows one of skill in the art to identify "B" or CCAAT-box binding conserved domains, or conserved domains for any of the polypeptides listed or referred to in this disclosure.

[0084] "Complementary" refers to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T (5'->3') forms hydrogen bonds with its complements A-C-G-T (5'->3') or A-C-G-U (5'->3'). Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or "completely complementary" if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of the hybridization and amplification reactions. "Fully complementary" refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.

[0085] The terms "highly stringent" or "highly stringent condition" refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985) Nature 313:402404, and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y ("Sambrook"); and by Haymes et al., "Nucleic Acid Hybridization: A Practical Approach", IRL Press, Washington, D.C. (1985), which references are incorporated herein by reference.

[0086] In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known transcription factor sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate transcription factor sequences having similarity to transcription factor sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed transcription factor sequences, such as, for example, encoded transcription factors having 60% or greater identity with disclosed transcription factors, or 83% or greater identity with the B domain of disclosed transcription factors.

[0087] Regarding the terms "paralog" and "ortholog", homologous polynucleotide sequences and homologous polypeptide sequences may be paralogs or orthologs of the claimed polynucleotide or polypeptide sequence. Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event. Sequences that are sufficiently similar to one another will be appreciated by those of skill in the art and may be based upon percentage identity of the complete sequences, percentage identity of a conserved domain or sequence within the complete sequence, percentage similarity to the complete sequence, percentage similarity to a conserved domain or sequence within the complete sequence, and/or an arrangement of contiguous nucleotides or peptides particular to a conserved domain or complete sequence. Sequences that are sufficiently similar to one another will also bind in a similar manner to the same DNA binding sites of transcriptional regulatory elements using methods well known to those of skill in the art.

[0088] The term "equivalog" describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) world wide web (www) website, "tigr.org " under the heading "Terms associated with TIGRFAMs".

[0089] The term "variant", as used herein, may refer to polynucleotides or polypeptides, that differ from the presently disclosed polynucleotides or polypeptides, respectively, in sequence from each other, and as set forth below.

[0090] With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences o may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.

[0091] Also within the scope of the invention is a variant of a transcription factor nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide.

[0092] "Allelic variant" or "polynucleotide allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be "silent" or may encode polypeptides having altered amino acid sequence. "Allelic variant" and "polypeptide allelic variant" may also be used with respect to polypeptides, and in this case the term refer to a polypeptide encoded by an allelic variant of a gene.

[0093] "Splice variant" or "polynucleotide splice variant" as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. This, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. "Splice variant" or "polypeptide splice variant" may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.

[0094] As used herein, "polynucleotide variants" may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. "Polypeptide variants" may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.

[0095] Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent transcription factor. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. A polypeptide sequence variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the functional or biological activity of the transcription factor is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine (for more detail on conservative substitutions, see Table 3). More rarely, a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or 0-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see U.S. Pat. No. 5,840,544).

[0096] "Ligand" refers to any molecule, agent, or compound that will bind specifically to a complementary site on a nucleic acid molecule or protein. Such ligands stabilize or modulate the activity of nucleic acid molecules or proteins of the invention and may be composed of at least one of the following: inorganic and organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.

[0097] "Modulates" refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and either a nucleic acid molecule or a protein.

[0098] The term "plant" includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. (See for example, FIG. 1, adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333; FIG. 2, adapted from Ku et al. (2000) Proc. Natl. Acad. Sci. 97: 9121-9126; and see also Tudge in The Variety of Life, Oxford University Press, New York, N.Y. (2000) pp. 547-606).

[0099] A "transgenic plant" refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.

[0100] A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.

[0101] "Control plant" refers to a plant that serves as a standard of comparison for testing the results of a treatment or genetic alteration, or the degree of altered expression of a gene or gene product. Examples of control plants include plants that are untreated, or genetically unaltered (i.e., wild-type).

[0102] "Wild type", as used herein, refers to a cell, tissue or plant that has not been genetically modified to knock out or overexpress one or more of the presently disclosed transcription factors. Wild-type cells, tissue or plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants in which transcription factor expression is altered or ectopically expressed, e.g., in that it has been knocked out or overexpressed.

[0103] "Fragment", with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A polynucleotide fragment" refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the transcription factor polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes a B domain of a transcription factor, for example, amino acid residues 26-116 of G482 (SEQ ID NO: 4), as noted in Table 1.

[0104] Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length. Exemplary polypeptide fragments are the first twenty consecutive amino acids of a mammalian protein encoded by are the first twenty consecutive amino acids of the transcription factor polypeptides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a B domain of a transcription factor, for example, amino acid residues 26-116 of G482 (SEQ ID NO: 4), as noted in Table 1.

[0105] The invention also encompasses production of DNA sequences that encode transcription factors and transcription factor derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding transcription factors or any fragment thereof.

[0106] "Derivative" refers to the chemical modification of a nucleic acid molecule or amino acid sequence. Chemical modifications can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process that retains or enhances biological activity or lifespan of the molecule or sequence.

[0107] A "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as osmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.

[0108] "Trait modification" refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease in an observed trait (difference), at least a 5% difference, at least about a 10% difference, at least about a 20% difference, at least about a 30%, at least about a 50%, at least about a 70%, or at least about a 100%, or an even greater difference compared with a wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution of the trait in the plants compared with the distribution observed in wild-type plants.

[0109] The term "transcript profile" refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell knocking out or overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.

[0110] "Ectopic expression or altered expression" in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term "ectopic expression or altered expression" further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.

[0111] The term "overexpression" as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong expression signal, such as one of the promoters described herein (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may occur throughout a plant or in specific tissues of the plant, depending on the promoter used, as described below.

[0112] Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the transcription factor in the plant, cell or tissue.

[0113] The term "transcription regulating region" refers to a DNA regulatory sequence that regulates expression of one or more genes in a plant when a transcription factor having one or more specific binding domains binds to the DNA regulatory sequence. Transcription factors of the present invention possess an AP2 domain, a B3 domain, or both of these binding domains. The AP2 domain of the transcription factor binds to a transcription regulating region comprising the motif CAACA, and the B3 domain of the same transcription factor binds to a transcription regulating region comprising the motif CACCTG. The transcription factors of the invention also comprise an amino acid subsequence that forms a transcription activation domain that regulates expression of one or more abiotic stress tolerance genes in a plant when the transcription factor binds to the regulating region.

[0114] The term "phase change" refers to a plant's progression from embryo to adult, and, by some definitions, the transition wherein flowering plants gain reproductive competency. It is believed that phase change occurs either after a certain number of cell divisions in the shoot apex of a developing plant, or when the shoot apex achieves a particular distance from the roots. Thus, altering the timing of phase changes may affect a plant's size, which, in turn, may affect yield and biomass.

[0115] A "sample" with respect to a material containing nucleic acid molecules may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a forensic sample; and the like. In this context "substrate" refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores. A substrate may also refer to a reactant in a chemical or biological reaction, or a substance acted upon (e.g., by an enzyme).

[0116] "Substantially purified" refers to nucleic acid molecules or proteins that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free, from other components with which they are naturally associated.

DETAILED DESCRIPTION

[0117] Transcription Factors Modify Expression of Endogenous Genes

[0118] A transcription factor may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes. As one of ordinary skill in the art recognizes, transcription factors can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding site or DNA-binding site motif (see, for example, Riechmann et al. (2000) Science 290: 2105-2110). The plant transcription factors may belong to the CAAT-element binding protein transcription factor family (Forsburg and Guarente (1989) supra).

[0119] Generally, the transcription factors encoded by the present sequences are involved in cell differentiation and proliferation and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits related to osmotic stresses. The sequences of the invention may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement.

[0120] The sequences of the present invention may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the invention may also include fragments of the present amino acid sequences. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

[0121] In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like; as substrates for cloning e.g., including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations.

[0122] Expression of genes that encode transcription factors that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997, Genes Development 11: 3194-3205) and Peng et al. (1999, Nature, 400: 256-261). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001, Plant Cell 13: 1791-1802); Nandi et al. (2000, Curr. Biol. 10: 215-218); Coupland (1995, Nature 377: 482483); and Weigel and Nilsson (1995, Nature 377: 482-500).

[0123] In another example, Mandel et al. (1992, Cell 71-133-143) and Suzuki et al.(2001, Plant J. 28: 409-418) teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al. 1992, supra; Suzuki et al. 2001, supra).

[0124] Other examples include Miller et al. (2001, Plant J. 28: 169-179); Kim et al. (2001, Plant J. 25: 247-259); Kyozuka and Shimamoto (2002, Plant Cell Physiol. 43: 130-135); Boss and Thomas (2002, Nature, 416: 847-850); He et al. (2000, Transgenic Res. 9: 223-227); and Robson et al. (2001, Plant J. 28: 619-631).

[0125] In yet another example, Gilmour et al. (1998, Plant J. 16: 433442) teach an Arabidopsis AP2 transcription factor, CBF1 (SEQ ID NO: 96), which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al. ((2001) Plant Physiol. 127: 910-917) further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. (See Jaglo et al. supra.) Transcription factors mediate cellular responses and control traits through altered expression of genes containing cis-acting nucleotide sequences that are targets of the introduced transcription factor. It is well appreciated in the Art that the effect of a transcription factor on cellular responses or a cellular trait is determined by the particular genes whose expression is either directly or indirectly (e.g., by a cascade of transcription factor binding events and transcriptional changes) altered by transcription factor binding. In a global analysis of transcription comparing a standard condition with one in which a transcription factor is overexpressed, the resulting transcript profile associated with transcription factor overexpression is related to the trait or cellular process controlled by that transcription factor. For example, the PAP2 gene (and other genes in the MYB family) have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al. (2000) Plant Cell 12: 65-79; and Borevitz et al. (2000) Plant Cell 12: 2383-2393). Further, global transcript profiles have been used successfully as diagnostic tools for specific cellular states (e.g., cancerous vs. non-cancerous; Bhattacharjee et al. (2001) Proc. Natl. Acad. Sci. USA 98: 13790-13795; and Xu et al. (2001) Proc Natl Acad Sci, USA 98: 15089-15094). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different transcription factors would indicate similarity of transcription factor function.

[0126] CCAAT-Element Binding Protein Transcription Factor Family

[0127] The CAAT family of transcription factors, also be referred to as the "CCAAT" or "CCAAT-box" family, are characterized by their ability to bind to the CCAAT-box element located 80 to 300 bp 5' from a transcription start site (Gelinas et al. (1985) Nature 313: 323-325). The CCAAT-box is a conserved cis-acting regulatory element with the consensus sequence CCAAT that is found in the promoters of genes from all eukaryotic species. The element can act in either orientation, alone or as multimeric regions with possible cooperation with other cis regulatory elements (Tasanen et al. (1992) (J. Biol. Chem. 267: 11513-11519). It has been estimated that 25% of eukaryotic promoters harbor this element (Bucher (1988) J. Biomol. Struct. Dyn. 5: 1231-1236). CCAAT-box elements have been shown to function in the regulation of gene expression in plants (Rieping and Schoffl (1992) Mol. Gen. Genet. 231: 226-232; Kehoe et al. (1994) Plant Cell 6: 1123-1134; Ito et al. (1995) Plant Cell Physiol. 36: 1281-1289). Several reports have described the importance of the CCAAT-binding element for regulated expression; including the regulation of genes that are responsive to light (Kusnetsov et al. (1999) J. Biol. Chem. 274: 36009-36014; Carre and Kay (1995) Plant Cell 7: 2039-2051) as well as stress (Rieping and Schoffl (1992) supra). Specifically, a CCAAT-box motif was shown to be important for the light regulated expression of the CAB2 promoter in Arabidopsis, however, the proteins that bind to the site were not identified (Carre and Kay (1995) supra). To date, no specific Arabidopsis CCAAT-box binding protein has been functionally associated with its corresponding target genes. In October of 2002 at an EPSO meeting on Plant Networks, a seminar was given by Detlef Weigel (Tuebingen) on the control of the AGAMOUS (a floral organ identity gene) gene in Arabidopsis. In order to find important cis-elements that regulate AGAMOUS activity, he aligned the promoter regions from 29 different Brassicaceae species and showed that there were two highly conserved regions; one well characterized site that binds LEAFY/WUS heterodimers and another putative CCAAT-box binding motif. We have discovered several CCAAT-box genes that regulate flowering time and are candidates for binding to the AGAMOUS promoter. One of these genes, G485, is a HAP3-like protein that is closely related to G481. Gain of function and loss of function studies on G485 reveal opposing effects on flowering time, indicating that the gene is both sufficient to act as a floral activator, and is also necessary in that role within the plant.

[0128] The first proteins identified that bind to the CCAAT-box element were identified in yeast. The CCAAT-box transcription factors bind as hetero-tetrameric complex called the HAP complex (heme activator protein complex) or the CCAAT binding factor (Forsburg and Guarente (1988) Mol. Cell Biol. 8: 647-654). The HAP complex in yeast is composed of at least four subunits, HAP2, HAP3, HAP4 and HAP5. In addition, the proteins that make up the HAP2,3,4,5 complex are represented by single genes. Their function is specific for the activation of genes involved in mitochondrial biogenesis and energy metabolism (Dang et al. (1996) Mol. Microbiol. 22:681-692). In mammals, the CCAAT binding factor is a trimeric complex consisting of NF-YA (HAP2-like), NF-YB (HAP3-like) and NF-YC (HAP5-like) subunits (Maity and de Crombrugghe (1998) Trends Biochem. Sci. 23: 174-178). In plants, analogous members of the CCAAT binding factor complex are represented by small gene families, and it is likely that these genes play a more complex role in regulating gene transcription. In Arabidopsis there are ten members of the HAP2 subfamily, ten members of the HAP3 subfamily, thirteen members of the HAP5 subfamily. Plants and mammals, however, do not appear to have a protein equivalent of HAP4 of yeast. HAP4 is not required for DNA binding in yeast although it provides the primary activation domain for the complex (McNabb et al. (1995) Genes Dev. 9: 47-58; Olesen and Guarente (1990) Genes Dev. 4, 1714-1729).

[0129] In mammals, the CCAAT-box element is found in the promoters of many genes and it is therefore been proposed that CCAAT binding factors serve as general transcriptional regulators that influence the frequency of transcriptional initiation (Maity and de Crombrugghe (1998) supra). CCAAT binding factors, however, can serve to regulate target promoters in response to environmental cues and it has been demonstrated that assembly of CCAAT binding factors on target promoters occurs in response to a variety signals (Myers et al. (1986) Myers et al. (1986) Science 232: 613-618; Maity and de Crombrugghe (1998) supra; Bezhani et al. (2001) J. Biol. Chem. 276: 23785-23789). Mammalian CP 1 and NF-Y are both heterotrimeric CCAAT binding factor complexes (Johnson and McKnight (1989) Ann. Rev. Biochem. 58: 799-839. Plant CCAAT binding factors are assumed to be trimeric, as is the case in mammals, however, they could associate with other transcription factors on target promoters as part of a larger complex. The CCAAT box is generally found in close proximity of other promoter elements and it is generally accepted that the CCAAT binding factor functions synergistically with other transcription factors in the regulation of transcription. In addition, it has recently been shown that a HAP3-like protein from rice, OsNF-YB1, interacts with a MADS-box protein OsMADS18 in vitro (Masiero et al. (2002) J. Biol. Chem. 277: 26429-26435). It was also shown that the in vitro ternary complex between these two types of transcription factors requires that both; OsNF-YB1 form a dimer with a HAP5-like protein, and that OsMADS18 form a heterodimer with another MADS-box protein. Interestingly, the OsNF-YB1/HAP5 protein dimer is incapable of interacting with HAP2-like subunits and therefore cannot bind the CCAAT element. The authors therefore speculate that there is a select set of HAP3-like proteins in plants that act on non-CCAAT promoter elements by virtue of their interaction with other non-CCAAT transcription factors (Masiero et al. (2002) supra). In support of this, HAP3/HAP5 subunit dimers have been shown to be able to interact with TFIID in the absence of HAP2 subunits (Romier et al. (2003) J. Biol. Chem. 278: 1336-1345).

[0130] The CCAAT-box motif is found in the promoters of a variety of plant genes. In addition, the expression pattern of many of the HAP-like genes in Arabidopsis shows developmental regulation. We have used RT-PCR to analyze the endogenous expression of 31 of the 34 CCAAT-box proteins. Our findings suggest that while most of the CCAAT-box gene transcripts are found ubiquitously throughout the plant, in more than half of the cases, the genes are predominantly expressed in flower, embryo and/or silique tissues. Cell-type specific localization of the CCAAT genes in Arabidopsis would be very informative and could help determine the activity of various CCAAT genes in the plant.

[0131] Genetic analysis has determined the function of one Arabidopsis CCAAT gene, LEAFY COTYLEDON (LEC1). LEC1 is a HAP3 subunit homolog that accumulates only during seed development. Arabidopsis plants carrying a mutation in the LEC1 gene display embryos that are intolerant to desiccation and that show defects in seed maturation (Lotan et al. (1998) Cell 93: 1195-1205). This phenotype can be rescued if the embryos are allowed to grow before the desiccation process occurs during normal seed maturation. This result suggests LEC1 has a role in allowing the embryo to survive desiccation during seed maturation. The mutant plants also possess trichomes, or epidermal hairs on their cotyledons, a characteristic that is normally restricted to adult tissues like leaves and stems. Such an effect suggests that LEC1 also plays a role in specifying embryonic organ identity in addition to the mutant analysis, the ectopic expression (unregulated overexpression) of the wild type LEC1 gene induces embryonic programs and embryo development in vegetative cells consistent with its role in coordinating higher plant embryo development. The ortholog of LEC1 has been identified recently in maize. The expression pattern of ZmLEC1 in maize during somatic embryo development is similar to that of LEC1 in Arabidopsis during zygotic embryo development (Zhang et al. (2002) Planta 215:191-194).

[0132] Matching the CCAAT transcription factors with target promoters and the analysis of the knockout and overexpression mutant phenotypes will help sort out whether these proteins act specifically or non-specifically in the control of plant pathways. The fact that CCAAT-box elements are not present in most plant promoters suggests that plant CCAAT binding factors most likely do not function as general components of the transcriptional machinery. In addition, the very specific role of the LEC1 protein in plant developmental processes supports the idea that CCAAT-box binding complexes play very specific roles in plant growth and development.

[0133] The Domain Structure of CCAAT-Element Binding Transcription Factors and Novel Conserved Domains in Arabidopsis and Other Species

[0134] Plant CCAAT binding factors potentially bind DNA as heterotrimers composed of HAP2-like, HAP3-like and HAP5-like subunits. All subunits contain regions that are required for DNA binding and subunit association. The subunit proteins appear to lack activation domains; therefore, that function must come from proteins with which they interact on target promoters. No proteins that provide the activation domain function for CCAAT binding factors have been identified in plants. In yeast, however, the HAP4 protein provides the primary activation domain (McNabb et al. (1995) Genes Dev. 9: 47-58; Olesen and Guarente (1990) Genes Dev. 4, 1714-1729).

[0135] HAP2-, HAP3- and HAP5-like proteins have two highly conserved sub-domains, one that functions in subunit interaction and the other that acts in a direct association with DNA. Outside these two regions, non-paralogous Arabidopsis HAP-like proteins are quite divergent in sequence and in overall length.

[0136] The general domain structure of HAP3 proteins is found in FIG. 5. HAP3 proteins contain an amino-terminal A domain, a central B domain and a carboxy-terminal C domain. There is very little sequence similarity between HAP3 proteins in the A and C domains; it is therefore reasonable to assume that the A and C domains could provide a degree of functional specificity to each member of the HAP3 subfamily. The B domain is the conserved region that specifies DNA binding and subunit association.

[0137] In FIGS. 6A-6F, HAP3 proteins from Arabidopsis, soybean, rice and corn are aligned with G48 1, with the A, B and C domains and the DNA binding and subunit interaction domains indicated. As can be seen in FIG. 6B-6C, the B domain of the non-LEC1-like clade (identified in FIGS. 3 and 4) may be distinguished by the amino acid residues:

1 Ser/Gly-Arg-Ile/Leu-Met-Lys-(Xaa).sub.2-Lys/Ile/Val-Pro-X- aa-Asn-Ala/Gly-Lys-Ile/Val- Ser/Ala/Gly-Lys-Asp/Glu-Ala/S- er-Lys-Glu/Asp/Gln-Thr/Ile-Xaa-Gln-Glu-Cys-Val/Ala-Ser/Thr-Glu- Phe-Ile-Ser-Phe-Ile/Val/His-Thr/Ser-[Pro]-Gly/Ser/Cys-Glu-Ala/Leu-Se- r/Ala-Asp/Glu/Gly-Lys/Glu- Cys-Gln/His-Arg/Lys-Glu-Lys/Asn- -Arg-Lys-Thr-Ile/Val-Asn-Gly-Asp/Glu-Asp-Leu/Ile-Xaa-Trp/Phe- Ala-Met/Ile/Leu-Xaa-Thr/Asn-Leu-Gly-Phe/Leu-Glu/Asp-Xaa-Tyr-(Xaa).sub.- 2-Pro/Gln/Ala-Leu/Val- Lys/Gly;

[0138] where Xaa can be any amino acid. The proline residue that appears in brackets is an additional residue that was found in only one sequence (not shown in FIG. 6B). The boldfaced residues that appear here and in the consensus sequences of FIGS. 6B-6C in their present positions are uniquely found in the non-LEC1-like clade, and may be used to identify members of this clade. The G482-like subclade may be delineated by the underlined serine residue in its present position here and in the consensus sequence of FIGS. 6B-6C. More generally, the non-LEC1-like clade is distinguished by a B domain comprising:

2 Asn-(Xaa).sub.4-Lys-(Xaa).sub.33-34-Asn-Gly;

[0139] and the G482 subclade is distinguished by a B-domain comprising:

3 Ser-(Xaa).sub.9-Asn-(Xaa).sub.4-Lys-(Xaa).sub.33-34-Asn-Gly.

[0140] Overexpression of these polypeptides confers increased abiotic stress tolerance in a transgenic plant, as compared to a non-transformed plant that does not overexpress the polypeptide.

[0141] Table 1 shows the polypeptides identified by SEQ ID NO; Mendel Gene ID (GID) No.; the transcription factor family to which the polypeptide belongs, and conserved B domains of the polypeptide. The first column shows the polypeptide SEQ ID NO; the second column the species and identifier (GID, GenBank accession no., or other identifier); the third column shows the conserved domain in amino acid coordinates; the fourth column shows the B domain; and the fifth column shows the percentage identity to G482. The sequences are arranged in descending order of percentage identity to G482.

4TABLE 1 Gene families and B domains CCAAT-box Species/ binding GID No., conserved % ID to CCAAT- Accession domain in box binding Polypeptide No., or Amino Acid conserved domain SEQ ID NO: Identifier Coordinates B Domain of G482 4 At/G482 26-116 REQDRFLPIANVSRIMKKALPANAKISKDAKET 100% MQECVSEFISFVTGEASDKCQKEKRKTINGDD LLWAMTTLGFEDYVEPLKVYLQRFRE 20 Gm/G3475 23-113 REQDRFLPIANVSRIMKKALPANAKISKDAKET 95% VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEDYVEPLKGYLQRFRE 86 Gm/3478 23-113 REQDRFLPIANVSRIMKKALPANAKISKDAKET 95% VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEDYVEPLKGYLQRFRE 6 At/G485 20-110 REQDRFLPIANVSRIMKKALPANAKISKDAKET 94% VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEDYVEPLKVYLQKYRE 18 Gm/G3476 26-116 REQDRFLPIANVSRIMKKALPANAKISKDAKET 94% VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEEYVEPLKIYLQRFRE 48 Zm/ 22-112 REQDRFLPIANVSRIMKKALPANAKISKDAKET 93% CLUSTER VQECVSEFISFITGEASDKCQREKRKTINGDDL 90408_1 LWAMTTLGFEDYVEPLKHYLHKFRE 48 Zm/G3435 22-112 REQDRFLPIANVSRIMKKALPANAKISKDAKET 93% VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEDYVEPLKHYLHKFRE 50 Zm/G3436 20-110 REQDRFLPIANVSRIMKKALPANAKISKDAKET 93% CLUSTER VQECVSEFISFITGEASDKCQREKRKTINGDDL 90408_2 LWAMTTLGFEDYVEPLKLYLHKFRE 92 Os/G3397 23-113 REQDRFLPIANVSRIMKKALPANAKISKDAKET 92% AC120529 VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEDYVDPLKHYLHKFRE 80 Gm/G3472 25-115 REQDRFLPIANVSRIMKKALPANAKISKEAKET 92% VQECVSEFISFITGEASDKCQKEKRKTINGDDL LWAMTTLGFEEYVEPLKVYLHKYRE 82 Gm/G3474 25-115 REQDRFLPIANVSRIMKKALPANAKISKEAKET 91% CLUSTER VQECVSEFISFITGEASDKCQKEKRKTINGDDL 33504_1 LWAMTFITLGFEDYVDPLKIYLHKYRE 76 Os/G3398 21-111 REQDRFLPIANVSRIMKRALPANAKISKDAKET 90% AP005193 VQECVSEFISFITGEASDKCQREKRKTINGDDL LWAMTTLGFEDYIDPLKLYLHKFRE 94 Zm/G3437 54-144 KEQDRFLPIANVSRIMKRSLPANAKISKEAKET 87% VQECVSEFISFVTGEASDKCQREKRKTINGDDL LWAMTTLGFEAYVAPLKSYLNRYRE 28 Os/ 38-127 VRQDRFLPIANISRIMKKAIPANGKIAKDAKET 86% CLUSTER VQECVSEFISFITSEASDKCQREKRKTINGDDLL 26105_1 WAMATLGFEDYIEPLKVYLQKYRE 78 Zm/G3434 18-108 REQDRFLPIANISRIMKKAVPANGKIAKDAKET 86% LQECVSEFISFVTSEASDKCQKEKRKTINGDDL LWAMATLGFEEYVEPLKIYLQKYKE 31 Os/ 57-147 KEQDRFLPIANVSRIMKRSLPANAKISKESKET 86% OSC30077 VQECVSEFISFVTGEASDKCQREKRKTINGDDL LWAMTTLGFEAYVGPLKSYLNRYRE 88 Os/G3394 37-127 VRQDRFLPIANISRIMKKAIPANGKIAKDAKET 86% VQECVSEFISFITSEASDKCQREKRKTINGDDLL WAMATLGFEDYIEPLKVYLQKYRE 24 Gm/G3471 26-116 REQDRYLPIANISRIMKKALPPNGKIAKDAKDT 85% MQECVSEFISFITSEASEKCQKEKRKTINGDDL LWAMATLGFEDYIEPLKVYLARYRE 26 Gm/G3470 27-117 REQDRYLPIANISRIMKKALPPNGKIAKDAKDT 85% CLUSTER MQECVSEFISFITSEASEKCQKIEKRKTINGDDL 4778_3 LWAMATLGFEDYIEPLKVYLARYRE 52 Gm/G3473 23-114 REQDRFLPIANVSRIMKKALPANAKISKDAKET 85% VQECVSEFISFHSPGGLAGECQKEKRKTINGDD LLWAMTTLGFEEYVEPLKVYLHKYRE 8 At/G1364 29-119 REQDRFLPIANISRIMKRGLPANGKIAKDAKEI 85% VQEGVSEFISFVTSEASDKGQREKRKTINGDDL LWAMATLGFEDYMEPLKVYLMRYRE 10 At/G2345 28-118 REQDRFLPIANISRIMKRGLPLNGKIAKDAKET 85% MQECVSEFISFVTSEASDKGQREKRKTINGDDL LWAMATLGFEDYIDPLKVYLMRYRE 86 Gm/G3477 27-117 REQDRYLPIANISRIMKKALPPNGKIAKDAKDT 85% MQEGVSEFISFITSEASEKCQKEKRKTINGDDL LWAMATLGFEDYIEPLKVYLARYRE 2 At/G481 20-110 REQDRYLPIANISRIMKKALPPNGKIGKDAKDT 83% VQECVSEFISFITSEASDKCQKEKRKTVNGDDL LWAMATLGFEDYLEPLKIYLARYRE 72 At/G1781 35-125 KEQDRFLPIANVGRIMKKVLPGNGKISKDAKE 83% TVQECVSEFISFVTGEASDKCQREKRKTINGDD IIWAITTLGFEDYVAPLKVYLCKYRD 74 Os/G3395 19-109 REQDRFLPIANISRIMIKKAVPANGKIAKDAKET 83% LQECVSEFISFVTSEASDKCQKEKRKTINGEDL LFAMGTLGFEEYVDPLKIYLHKYRE Os/ 19-109 REQDRFLPIANISRIMKKAVPANGKIAKDAKET 83% AP004366 LQECVSEFISFVTSEASDKCQKEKRKTINGEDL LFAMGTLGFEEYVDPLKIYLHKYRE 70 At/G1248 50-140 KEQDRLLPIANVGRIMKNILPANAKVSKEAKE 77% TMQECVSEFISFVTGEASDKCHKEKRKTVNGD DICWAMANLGFDDYAAQLKKYLHRYRV 90 Os/G3396 21-111 KEQDRFLPIANIGRIMRRAVPENGKIAKDSKES 75% VQECVSEFISFITSEASDKCLKEKRKTINGDDLI WSMGTLGFEDYVEPLKLYLRLYRE 60 At/G1821 28-118 REQDRFMPIANVIRIMRRILPAHAKISDDSKETI 69% L1L QECVSEYISFITGEANERCQREQRKTITAEDVL WAMSKLGFDDYIEPLTLYLHRYRE At/ 28-118 REQDQYMPIANVIRIMRKTLPSHAKISDDAKET 67% AAC39488 IQECVSEYISFVTGEANERCQREQRKTITAEDIL LEC1 WAMSKLGFDNYVDPLTVFINRYRE At/G486 2-92 TDEDRLLPIANVGRLMKQILPSNAKISKEAKQT 60% VQECATEFISFVTCEASEKCHRENRKTVNGDDI WWALSTLGLDNYADAVGRHLHKYRE Abbreviations: At Arabidopsis thaliana Gm Glycine max Os Oryza sativa Zm Zea mays

[0142] The transcription factors of the present invention each possess a B or conserved domain, including the orthologs of G482 found by BLAST analysis, as described below. Generally, the B domain of the transcription factors will bind to a transcription-regulating region comprising the motif CCAAT. As shown in Table 1, the B domains of G481, G485 and rice G3395 are at least 83% identical to the corresponding domains of G482, and all four of these transcription factors, which rely on the binding specificity of their B domains, have similar or identical functions in plants, conferring increased abiotic, including osmotic, stress tolerance when overexpressed.

[0143] Polypeptides and Polynucleotides of the Invention

[0144] The present invention provides, among other things, transcription factors (TFs), and transcription factor homolog polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of transcription factors derived from the specific sequences provided here. These polypeptides and polynucleotides may be employed to modify a plant's characteristics.

[0145] Exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. In addition, further exemplary polynucleotides encoding the polypeptides of the invention were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.

[0146] Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure, using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5' and 3' ends. The full-length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5' and 3' ends. Exemplary sequences are provided in the Sequence Listing.

[0147] The polynucleotides of the invention can be or were ectopically expressed in overexpressor or knockout plants and the changes in the characteristic(s) or trait(s) of the plants observed. Therefore, the polynucleotides and polypeptides can be employed to improve the characteristics of plants.

[0148] The polynucleotides of the invention can be or were ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be employed to change expression levels of a genes, polynucleotides, and/or proteins of plants.

[0149] The CCAAT Family Members Under Study

[0150] The correct sequences for G482, and trait disclosures for G481, G482 and G485, were first disclosed in U.S. Provisional Patent Application 60/166,228, filed Nov. 17, 1999.

[0151] G481, G482 and G485 (polynucleotide SEQ ID NOs: 1, 3 and 5) were chosen for study based on observations that Arabidopsis plants overexpressing these genes had resistance to abiotic stresses, such as osmotic stress, and including drought-related stress (see Example VIII, below). G481, G482 and G485 are members of the CCAAT family, proteins that act in a multi-subunit complex and are believed to bind CCAAT boxes in promoters of target genes as trimers or tetramers.

[0152] In Arabidopsis, three types of CCAAT binding proteins exist: HAP2, HAP3 and HAP5. The G481, G482 and G485 polypeptides, as well as a number of other proteins in the Arabidopsis proteome, belong to the HAP3 class. As reported in the scientific literature thus far, only two genes from the HAP3 class have been functionally analyzed to a substantial degree. These are LEAFY COTYLEDON1 (LEC1) and its most closely related subunit, LEC1-LIKE (L1L). LEC1 and L1L are expressed primarily during seed development. Both appear to be essential for embryo survival of desiccation during seed maturation (Kwong et al. (2003) Plant Cell 15: 5-18). LEC1 is a critical regulator required for normal development during the early and late phases of embryo genesis that is sufficient to induce embryonic development in vegetative cells. Kwong et al. showed that ten Arabidopsis HAP3 subunits can be divided into two classes based on sequence identity in their central, conserved B domain. LEC1 and L1L constitute LEC1-type HAP3 subunits, whereas the remaining HAP3 subunits were designated non-LEC1-type.

[0153] Phylogenetic trees based on sequential relatedness of the HAP3 genes are shown in FIG. 3 and 4. As can be seen in these figures, G1364 and G2345 are closely related to G481, and G482 and G485 are more related to G481 than either LEC1 or L1L, which are found on somewhat more distant nodes.

[0154] Producing Polypeptides

[0155] The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homolog polypeptides and sequences complementary thereto, as well as unique fragments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, e.g., DNA or RNA, e.g., mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homolog polypeptide, in isolation, in combination with additional coding sequences (e.g., a purification tag, a localization signal, as a fusion-protein, as a pre-protein, or the like), in combination with non-coding sequences (e.g., introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homolog polypeptide is an endogenous or exogenous gene.

[0156] A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152 Academic Press, Inc., San Diego, Calif. ("Berger"); Sambrook et al. Molecular Cloning--A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular Biology, Ausubel et al. eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2000) ("Ausubel").

[0157] Alternatively, polynucleotides of the invention, can be produced by a variety of in vitro amplification methods adapted to the present invention by appropriate selection of specific or degenerate primers. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qbeta-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger (supra), Sambrook (supra), and Ausubel (supra), as well as Mullis et al. (1987) PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al. U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.

[0158] Alternatively, polynucleotides and oligonucleotides of the invention can be assembled from fragments produced by solid-phase synthesis methods. Typically, fragments of up to approximately 100 bases are individually synthesized and then enzymatically or chemically ligated to produce a desired sequence, e.g., a polynucleotide encoding all or part of a transcription factor. For example, chemical synthesis using the phosphoramidite method is described, e.g., by Beaucage et al. (1981) Tetrahedron Letters 22: 1859-1869; and Matthes et al. (1984) EMBO J. 3: 801-805. According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.

[0159] Homologous Sequences

[0160] Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing, derived from Arabidopsis thaliana or from other plants of choice, are also an aspect of the invention. Homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn (maize), potato, cotton, rice, rape, oilseed rape (including canola), sunflower, alfalfa, clover, sugarcane, and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, tomatillo, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops, including fruits and vegetables, whose phenotype can be changed and which comprise homologous sequences include barley; rye; millet; sorghum; currant; avocado; citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries; nuts such as the walnut and peanut; endive; leek; roots such as arrowroot, beet, cassava, turnip, radish, yam, and sweet potato; and beans. The homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus, or mint or other labiates. In addition, homologous sequences may be derived from plants that are evolutionarily related to crop plants, but which may not have yet been used as crop plants. Examples include deadly nightshade (Atropa belladona), related to tomato; jimson weed (Datura strommium), related to peyote; and teosinte (Zea species), related to corn (maize).

[0161] Orthologs and Paralogs

[0162] Homologous sequences as described above can comprise orthologous or paralogous sequences. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. Three general methods for defining orthologs and paralogs are described; an ortholog, paralog or homolog may be identified by one or more of the methods described below.

[0163] Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.

[0164] Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same lade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126: 122-132), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16: 433-442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the lade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., page 543.)

[0165] Speciation, the production of new species from a parental species, can also give rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) supra) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.

[0166] Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75: 519-530; Lin et al. (1991) Nature 353: 569-571; Sadowski et al. (1988) Nature 335: 563-564). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions.

[0167] Orthologous genes from different organisms have highly conserved functions, and very often essentially identical functions (Lee et al. (2002) Genome Res. 12: 493-502; Remm et al. (2001) J. Mol. Biol. 314: 1041-1052). Paralogous genes, which have diverged through gene duplication, may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably with respect to certain embodiments of the instant invention (for example, transgenic expression of a coding sequence). An example of such highly related paralogs is the CBF family, with three well-defined members in Arabidopsis and at least one ortholog in Brassica napus (SEQ ID NOs: 96, 98, 100, and 102, respectively), all of which control pathways involved in both freezing and drought stress (Gilmour et al. (1998) Plant J. 16: 433-442; Jaglo et al. (1998) Plant Physiol. 127: 910-917).

[0168] The following references represent a small sampling of the many studies that demonstrate that conserved transcription factor genes from diverse species are likely to function similarly (i.e., regulate similar target sequences and control the same traits), and that transcription factors may be transformed into diverse species to confer or improve traits.

[0169] (1) The Arabidopsis NPR1 gene regulates systemic acquired resistance (SAR); over-expression of NPR1 leads to enhanced resistance in Arabidopsis. When either Arabidopsis NPR1 or the rice NPR1 ortholog was overexpressed in rice (which, as a monocot, is diverse from Arabidopsis), challenge with the rice bacterial blight pathogen Xanthomonas oryzae pv. Oryzae, the transgenic plants displayed enhanced resistance (Chern et al. (2001) Plant J. 27: 101-113). NPR1 acts through activation of expression of transcription factor genes, such as TGA2 (Fan and Dong (2002) Plant Cell 14: 1377-1389).

[0170] (2) E2F genes are involved in transcription of plant genes for proliferating cell nuclear antigen (PCNA). Plant E2Fs share a high degree of similarity in amino acid sequence between monocots and dicots, and are even similar to the conserved domains of the animal E2Fs. Such conservation indicates a functional similarity between plant and animal E2Fs. E2F transcription factors that regulate meristem development act through common cis-elements, and regulate related (PCNA) genes (Kosugi and Ohashi, (2002) Plant J. 29: 45-59).

[0171] (3) The ABI5 gene (ABA insensitive 5) encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Co-transformation experiments with ABI5 cDNA constructs in rice protoplasts resulted in specific transactivation of the ABA-inducible wheat, Arabidopsis, bean, and barley promoters. These results demonstrate that sequentially similar ABI5 transcription factors are key targets of a conserved ABA signaling pathway in diverse plants. (Gampala et al. (2001) J. Biol. Chem. 277: 1689-1694).

[0172] (4) Sequences of three Arabidopsis GAMYB-like genes were obtained on the basis of sequence similarity to GAMYB genes from barley, rice, and L. temulentum. These three Arabadopsis genes were determined to encode transcription factors (AtMYB33, AtMYB65, and AtMYB101) and could substitute for a barley GAMYB and control alpha-amylase expression (Gocal et al. (2001) Plant Physiol. 127: 1682-1693).

[0173] (5) The floral control gene LEAFY from Arabidopsis can dramatically accelerate flowering in numerous dictoyledonous plants. Constitutive expression of Arabidopsis LEAFY also caused early flowering in transgenic rice (a monocot), with a heading date that was 26-34 days earlier than that of wild-type plants. These observations indicate that floral regulatory genes from Arabidopsis are useful tools for heading date improvement in cereal crops (He et al. (2000) Transgenic Res. 9: 223-227).

[0174] (6) Bioactive gibberellins (GAs) are essential endogenous regulators of plant growth. GA signaling tends to be conserved across the plant kingdom. GA signaling is mediated via GAI, a nuclear member of the GRAS family of plant transcription factors. Arabidopsis GAI has been shown to function in rice to inhibit gibberellin response pathways (Fu et al. (2001) Plant Cell 13: 1791-1802).

[0175] (7) The Arabidopsis gene SUPERMAN (SUP), encodes a putative transcription factor that maintains the boundary between stamens and carpels. By over-expressing Arabidopsis SUP in rice, the effect of the gene's presence on whorl boundaries was shown to be conserved. This demonstrated that SUP is a conserved regulator of floral whorl boundaries and affects cell proliferation (Nandi et al. (2000) Curr. Biol. 10: 215-218).

[0176] (8) Maize, petunia and Arabidopsis myb transcription factors that regulate flavonoid biosynthesis are very genetically similar and affect the same trait in their native species, therefore sequence and function of these myb transcription factors correlate with each other in these diverse species (Borevitz et al. (2000) Plant Cell 12: 2383-2394).

[0177] (9) Wheat reduced height-1 (Rht-B1/Rht-D1) and maize dwarf-8 (d8) genes are orthologs of the Arabidopsis gibberellin insensitive (GAI) gene. Both of these genes have been used to produce dwarf grain varieties that have improved grain yield. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signaling. Transgenic rice plants containing a mutant GAI allele from Arabidopsis have been shown to produce reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologs could be used to increase yield in a wide range of crop species (Peng et al. (1999) Nature 400: 256-261).

[0178] Transcription factors that are homologous to the listed sequences will typically share at least about 70% amino acid sequence identity in B domain. More closely related transcription factors can share at least about 75% or about 80% or about 90% or about 95% or about 98% or more sequence identity with the listed sequences, or with the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site, or with the listed sequences excluding one or all conserved domains. Factors that are most closely related to the listed sequences share, e.g., at least about 85%, about 90% or about 95% or more % sequence identity to the listed sequences, or to the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site or outside one or all conserved domain. At the nucleotide level, the sequences will typically share at least about 40% nucleotide sequence identity, preferably at least about 50%, about 60%, about 70% or about 80% sequence identity, and more preferably about 85%, about 90%, about 95% or about 97% or more sequence identity to one or more of the listed sequences, or to a listed sequence but excluding or outside a known consensus sequence or consensus DNA-binding site, or outside one or all conserved domain. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein. B domains within the CCAAT-binding transcription factor family may exhibit a higher degree of sequence homology, such as at least 70% amino acid sequence identity including conservative substitutions, and preferably at least 80% sequence identity, and more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% sequence identity. Transcription factors that are homologous to the listed sequences should share at least 30%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% at least about 95% amino acid sequence identity over the entire length of the polypeptide or the homolog.

[0179] Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method. (See, for example, Higgins and Sharp (1988) Gene 73: 237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).

[0180] Other techniques for alignment are described in Methods in Enzymology, vol. 266, Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997) Methods Mol. Biol. 70: 173-187). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.

[0181] The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein (1990) Methods Enzymol. 183: 626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see U.S. patent application No. 20010010913).

[0182] Thus, the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

[0183] In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1993) J. Mol. Evol. 36: 290-300; Altschul et al. (1990) supra), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Res. 19: 6565-6572), Hidden Markov Models (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6: 361-365; Sonnhammer et al. (1997) Proteins 28: 405420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y., p 856-853).

[0184] A further method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related transcription factors. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, more preferably with greater than 70% regulated transcripts in common, most preferably with greater than 90% regulated transcripts in common) will have highly similar functions. Fowler et al. (2002, Plant Cell, 14: 1675-79) have shown that three paralogous AP2 family genes (CBF1, CBF2 and CBF3), each of which is induced upon cold treatment, and each of which can condition improved freezing tolerance, have highly similar transcript profiles. Once a transcription factor has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether putative paralogs or orthologs have the same function.

[0185] Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and B domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide which comprises a known function with a polypeptide sequence encoded by a polynucleotide sequence which has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.

[0186] Orthologs and paralogs of presently disclosed transcription factors may be cloned using compositions provided by the present invention according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present transcription factors. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present transcription factor sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Transcription factor-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed transcription factor gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, methods disclosed herein such as microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.

[0187] In addition to the Sequences listed in the Sequence Listing, the invention encompasses isolated nucleotide sequences that are sequentially and structurally similar to G481, G482, and G485, SEQ ID NO: 1, 3, and 5, and function in a plant in a manner similar to G481, G482 and G485 by regulating abiotic stress tolerance. The nucleotide sequences of G481 and G485 are 88% and 82% identical to the polynucleotide sequence of G482, respectively. Since all three polynucleotide sequences are phylogenetically related, sequentially similar, and have been shown to regulate abiotic stress tolerance, one skilled in the art would predict that other similar, phylogenetically related sequences would also regulate abiotic stress tolerance. A sequence that was 99.5% identical (861 of 865 bases) to G482 has been taught by Edwards et al., ((1998) Plant Physiol. 117: 1015-1022), but with no analysis of the function of this gene.

[0188] The present invention is also directed to polypeptide encoded by isolated nucleic acid that are similar tc G481, G482 and G485, vectors comprising isolated nucleic acid that are similar to G481, G482 and G485, and transgenic plants transformed with these isolated nucleic acids.

[0189] Identifying Polynucleotides or Nucleic Acids by Hybridization

[0190] Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited above.

[0191] Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (See, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399407; and Kimmel (1987) Methods Enzymol. 152: 507-511). In addition to the nucleotide sequences in the Sequence Listing, full-length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

[0192] With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual" (2nd ed., Cold Spring Harbor Laboratory); Berger and Kimmel, eds., (1987) "Guide to Molecular Cloning Techniques", In Methods in Enzymology: 152: 467469; and Anderson and Young (1985) "Quantitative Filter Hybridisation. " In: Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical Approach. Oxford, IRL Press, 73-111.

[0193] Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (T.sub.m) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equations:

[0194] (I) DNA-DNA:

T.sub.m(.degree. C.)=81.5+16.6(log [Na+])+0.41(% G+C)-0.62(% formamide)-500/L

[0195] (II) DNA-RNA:

T.sub.m(.degree. C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C).sup.2-0.5(% formamide)-820/L

[0196] (III) RNA-RNA:

T.sub.m(.degree. C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C).sup.2-0.35(% formamide)-820/L

[0197] where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1.degree. C. is required to reduce the melting temperature for each 1% mismatch.

[0198] Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson et al. (1985) supra). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.

[0199] Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at T.sub.m-5.degree. C. to T.sub.m-20.degree. C., moderate stringency at T.sub.m-20.degree. C. to T.sub.m-35.degree. C. and low stringency at T.sub.m-35.degree. C. to T.sub.m-50.degree. C. for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50.degree. C. below T.sub.m), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T.sub.m-25.degree. C. for DNA-DNA duplex and T.sub.m-15.degree. C. for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.

[0200] High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5.degree. C. to 20.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50.degree. C. and about 70.degree. C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50.degree. C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.

[0201] Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCI and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30.degree. C., more preferably of at least about 37.degree. C., and most preferably of at least about 42.degree. C. with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.

[0202] The washing steps that follow hybridization may also vary in stringency; the post-hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.

[0203] Thus, hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present transcription factors include, for example:

[0204] 6.times.SSC at 65.degree. C.;

[0205] 50% formamide, 4.times.SSC at 42.degree. C.; or

[0206] 0.5.times.SSC, 0.1% SDS at 65.degree. C.;

[0207] with, for example, two wash steps of 10-30 minutes each. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0208] A person of skill in the art would not expect substantial variation among polynucleotide species encompassed within the scope of the present invention because the highly stringent conditions set forth in the above formulae yield structurally similar polynucleotides.

[0209] If desired, one may employ wash steps of even greater stringency, including about 0.2.times.SSC, 0.1% SDS at 65.degree. C. and washing twice, each wash step being about 30 min, or about 0.1.times.SSC, 0.1% SDS at 65.degree. C. and washing twice for 30 min. The temperature for the wash solutions will ordinarily be at least about 25.degree. C., and for greater stringency at least about 42.degree. C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3.degree. C. to about 5.degree. C., and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6.degree. C. to about 9.degree. C. For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 50.degree. C.

[0210] An example of a low stringency wash step employs a solution and conditions of at least 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42.degree. C. in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Even higher stringency wash conditions are obtained at 65.degree. C.-68.degree. C. in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, U.S. patent application No. 20010010913).

[0211] Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10.times. higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15.times. or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2.times. or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a calorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

[0212] Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the present polynucleotide sequences, and, in particular, to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, polynucleotides that encode polypeptide SEQ ID NOs: 29-32, and fragments thereof under various conditions of stringency. (See, e.g., Wahl and Berger (1987) Methods Enzymol. 152: 399-407; Kimmel (1987) Methods Enzymol. 152: 507-511.) Estimates of homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.

[0213] Identifying Polynucleotides or Nucleic Acids with Expression Libraries

[0214] In addition to hybridization methods, transcription factor homolog polypeptides can be obtained by screening an expression library using antibodies specific for one or more transcription factors. With the provision herein of the disclosed transcription factor, and transcription factor homolog nucleic acid sequences, the encoded polypeptide(s) can be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for the polypeptide(s) in question. Antibodies can also be raised against synthetic peptides derived from transcription factor, or transcription factor homolog, amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologs, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.

[0215] Sequence Variations

[0216] It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (i.e., peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the Sequence Listing due to degeneracy in the genetic code, are also within the scope of the invention.

[0217] Altered polynucleotide sequences encoding polypeptides include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding a polypeptide with at least one functional characteristic of the instant polypeptides. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding the instant polypeptides, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding the instant polypeptides.

[0218] Allelic variant refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. Splice variant refers to alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.

[0219] Those skilled in the art would recognize that, for example, G482, SEQ ID NO: 4, represents a single transcription factor; allelic variation and alternative splicing may be expected to occur. Allelic variants of SEQ ID NO: 3 can be cloned by probing cDNA or genomic libraries from different individual organisms according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 3, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 4. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the transcription factor are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individual organisms or tissues according to standard procedures known in the art (see U.S. Pat. No. 6,388,064).

[0220] Thus, in addition to the sequences set forth in the Sequence Listing, the invention also encompasses related nucleic acid molecules that include allelic or splice variants SEQ ID NO: 1, 3, 5, 7, 9, 11-21, 27-52, 55, 57, 59, 61, 63, 65, 67, 69, 71, 75, 77, and 79, and include sequences which are complementary to these nucleotide sequences. Related nucleic acid molecules also include nucleotide sequences encoding a polypeptide comprising or consisting essentially of a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide as set forth in any of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 29, 30, 31, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. Such related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues.

[0221] For example, Table 2 illustrates, e.g., that the codons AGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine. Accordingly, at each position in the sequence where there is a codon encoding serine, any of the above trinucleotide sequences can be used without altering the encoded polypeptide.

5TABLE 2 Amino acid Possible Codons Alanine Ala A GCA GCC GCG GCT Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAA GAG Phenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGT Histidine His H CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTT Methionine Met M ATG Asparagine Asn N AAC AAT Proline Pro P CCA CCC CCG CCT Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGT Serine Ser S AGC AGT TCA TCC TCG TCT Threonine Thr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

[0222] Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed "silent" variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.

[0223] In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention.

[0224] For example, substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing, are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (ed.) Methods Enzymol. (1993) vol. 217, Academic Press) or the other methods noted below. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from I to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.

[0225] Conservative substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 3 when it is desired to maintain the activity of the protein. Table 3 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.

6 TABLE 3 Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0226] Similar substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 4 when it is desired to maintain the activity of the protein. Table 4 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as structural and functional substitutions. For example, a residue in column 1 of Table 4 may be substituted with a residue in column 2; in addition, a residue in column 2 of Table 4 may be substituted with the residue of column 1.

7 TABLE 4 Residue Similar Substitutions Ala Ser; Thr; Gly; Val; Leu; Ile Arg Lys; His; Gly Asn Gln; His; Gly; Ser; Thr Asp Glu, Ser; Thr Gln Asn; Ala Cys Ser; Gly Glu Asp Gly Pro; Arg His Asn; Gln; Tyr; Phe; Lys; Arg Ile Ala; Leu; Val; Gly; Met Leu Ala; Ile; Val; Gly; Met Lys Arg; His; Gln; Gly; Pro Met Leu; Ile; Phe Phe Met; Leu; Tyr; Trp; His; Val; Ala Ser Thr; Gly; Asp; Ala; Val; Ile; His Thr Ser; Val; Ala; Gly Trp Tyr; Phe; His Tyr Trp; Phe; His Val Ala; Ile; Leu; Gly; Thr; Ser; Glu

[0227] Substitutions that are less conservative than those in Table 3 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

[0228] Further Modifying Sequences of the Invention--Mutation/Forced Evolution

[0229] In addition to generating silent or conservative substitutions as noted, above, the present invention optionally includes methods of modifying the sequences of the Sequence Listing. In the methods, nucleic acid or protein modification methods are used to alter the given sequences to produce new sequences and/or to chemically or enzymatically modify given sequences to change the properties of the nucleic acids or proteins.

[0230] Thus, in one embodiment, given nucleic acid sequences are modified, e.g., according to standard mutagenesis or artificial evolution methods to produce modified sequences. The modified sequences may be created using purified natural polynucleotides isolated from any organism or may be synthesized from purified compositions and chemicals using chemical means well know to those of skill in the art. For example, Ausubel, supra, provides additional details on mutagenesis methods. Artificial forced evolution methods are described, for example, by Stemmer (1994) Nature 370: 389-391, Stemmer (1994) Proc. Natl. Acad. Sci. 91: 10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and 6,242,568. Methods for engineering synthetic transcription factors and other polypeptides are described, for example, by Zhang et al. (2000) J. Biol. Chem. 275: 33850-33860, Liu et al. (2001) J. Biol. Chem. 276: 11323-11334, and Isalan et al. (2001) Nature Biotechnol. 19: 656-660. Many other mutation and evolution methods are also available and expected to be within the skill of the practitioner.

[0231] Similarly, chemical or enzymatic alteration of expressed nucleic acids and polypeptides can be performed by standard methods. For example, sequence can be modified by addition of lipids, sugars, peptides, organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like. For example, protein modification techniques are illustrated in Ausubel, supra. Further details on chemical and enzymatic modifications can be found herein. These modification methods can be used to modify any given sequence, or to modify any sequence produced by the various mutation and artificial evolution modification methods noted herein.

[0232] Accordingly, the invention provides for modification of any given nucleic acid by mutation, evolution, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, e.g., using the sequences herein as a starting substrate for the various modification approaches.

[0233] For example, optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be used e.g., to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced using a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for Saccharomyces cerevisiae and mammals are TAA and TGA, respectively. The preferred stop codon for monocotyledonous plants is TGA, whereas insects and E. coli prefer to use TAA as the stop codon.

[0234] The polynucleotide sequences of the present invention can also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the sequence to facilitate cloning, processing and/or expression of the gene product. For example, alterations are optionally introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.

[0235] Furthermore, a fragment or domain derived from any of the polypeptides of the invention can be combined with domains derived from other transcription factors or synthetic domains to modify the biological activity of a transcription factor. For instance, a DNA-binding domain derived from a transcription factor of the invention can be combined with the activation domain of another transcription factor or with a synthetic activation domain. A transcription activation domain assists in initiating transcription from a DNA-binding site. Examples include the transcription activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl. Acad. Sci. 95: 376-381; Aoyama et al. (1995) Plant Cell 7: 1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51: 113-119) and synthetic peptides (Giniger and Ptashne (1987) Nature 330: 670-672).

[0236] Expression and Modification of Polypeptides

[0237] Typically, polynucleotide sequences of the invention are incorporated into recombinant DNA (or RNA) molecules that direct expression of polypeptides of the invention in appropriate host cells, transgenic plants, in vitro translation systems, or the like. Due to the inherent degeneracy of the genetic code, nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can be substituted for any listed sequence to provide for cloning and expressing the relevant homolog.

[0238] The transgenic plants of the present invention comprising recombinant polynucleotide sequences are generally derived from parental plants, which may themselves be non-transformed (or non-transgenic) plants. These transgenic plants may either have a transcription factor gene "knocked out" (for example, with a genomic insertion by homologous recombination, an antisense or ribozyme construct) or expressed to a normal or wild-type extent. However, overexpressing transgenic "progeny" plants will exhibit greater mRNA levels, wherein the mRNA encodes a transcription factor, that is, a DNA-binding protein that is capable of binding to a DNA regulatory sequence and inducing transcription, and preferably, expression of a plant trait gene. Preferably, the mRNA expression level will be at least three-fold greater than that of the parental plant, or more preferably at least ten-fold greater mRNA levels compared to said parental plant, and most preferably at least fifty-fold greater compared to said parental plant.

[0239] Vectors, Promoters, and Expression Systems

[0240] The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

[0241] General texts that describe molecular biological techniques useful herein, including the use and production of vectors, promoters and many other relevant topics, include Berger, Sambrook, supra and Ausubel, supra. Any of the identified sequences can be incorporated into a cassette or vector, e.g., for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvin et al. (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed by Herrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) Nucleic Acids Res. 12: 8711-8721, Klee (1985) Bio/Technology 3: 637-642, for dicotyledonous plants.

[0242] Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993) Plant Physiol. 102: 1077-1084; Vasil (1993) Bio/Technology 10: 667-674; Wan and Lemeaux (1994) Plant Physiol. 104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996) Nature Biotechnol. 14: 745-750).

[0243] Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.

[0244] A potential utility for the transcription factor polynucleotides disclosed herein is the isolation of promoter elements from these genes that can be used to program expression in plants of any genes. Each transcription factor gene disclosed herein is expressed in a unique fashion, as determined by promoter elements located upstream of the start of translation, and additionally within an intron of the transcription factor gene or downstream of the termination codon of the gene. As is well known in the art, for a significant portion of genes, the promoter sequences are located entirely in the region directly upstream of the start of translation. In such cases, typically the promoter sequences are located within 2.0 kb of the start of translation, or within 1.5 kb of the start of translation, frequently within 1.0 kb of the start of translation, and sometimes within 0.5 kb of the start of translation.

[0245] The promoter sequences can be isolated according to methods known to one skilled in the art.

[0246] Examples of constitutive plant promoters which can be useful for expressing the TF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odell et al. (1985) Nature 313: 810-812); the nopaline synthase promoter (An et al. (1988) Plant Physiol. 88: 547-552); and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1: 977-984).

[0247] The transcription factors of the invention may be operably linked with a specific promoter that causes the transcription factor to be expressed in response to environmental, tissue-specific or temporal signals. A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a TF sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like. Numerous known promoters have been characterized and can favorably be employed to promote expression of a polynucleotide of the invention in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol. Biol. 11: 651-662), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active in vascular tissue (Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988), flower-specific (Kaiser et al. (1995) Plant Mol. Biol. 28: 231-243), pollen (Baerson et al. (1994) Plant Mol. Biol. 26: 1947-1959), carpels (Ohl et al. (1990) Plant Cell 2: 837-848), pollen and ovules (Baerson et al. (1993) Plant Mol. Biol. 22: 255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al., (1999) Plant Cell 11: 323-334), cytokinin-inducible promoter (Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promoters responsive to gibberellin (Shi et al. (1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825) and the like. Additional promoters are those that elicit expression in response to heat (Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1: 471-478, and the maize rbcS promoter, Schafffner and Sheen (1991) Plant Cell 3: 997-1012); wounding (e.g., wunl, Siebertz et al. (1989) Plant Cell 1: 961-968); pathogens (such as the PR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40: 387-396, and the PDF1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38: 1071-1080), and chemicals such as methyl jasmonate or salicylic acid (Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (Odell et al. (1994) Plant Physiol. 106: 447-458).

[0248] Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3'-untranslated region of plant genes, e.g., a 3' terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3' terminator regions.

[0249] Additional Expression Elements

[0250] Specific initiation signals can aid in efficient translation of coding sequences. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.

[0251] Expression Hosts

[0252] The present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (i.e., nucleic acids are introduced, e.g., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector comprising the relevant nucleic acids herein. The vector is optionally a plasmid, a viral particle, a phage, a naked nucleic acid, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the relevant gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook, supra and Ausubel, supra.

[0253] The host cell can be a eukaryotic cell, such as a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Plant protoplasts are also suitable for some applications. For example, the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (Fromm et al. (1985) Proc. Natl. Acad. Sci. 82: 5824-5828, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors Academic Press, New York, N.Y., pp. 549-560; U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al. (1987) Nature 327: 70-73), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233: 496-498; Fraley et al. (1983) Proc. Natl. Acad. Sci. 80: 4803-4807).

[0254] The cell can include a nucleic acid of the invention that encodes a polypeptide, wherein the cell expresses a polypeptide of the invention. The cell can also include vector sequences, or the like. Furthermore, cells and transgenic plants that include any polypeptide or nucleic acid above or throughout this specification, e.g., produced by transduction of a vector of the invention, are an additional feature of the invention.

[0255] For long-term, high-yield production of recombinant proteins, stable expression can be used. Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides encoding mature proteins of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.

[0256] Modified Amino Acid Residues

[0257] Polypeptides of the invention may contain one or more modified amino acid residues. The presence of modified amino acids may be advantageous in, for example, increasing polypeptide half-life, reducing polypeptide antigenicity or toxicity, increasing polypeptide storage stability, or the like.

[0258] Amino acid residue(s) are modified, for example, co-translationally or post-translationally during recombinant production or modified by synthetic or chemical means.

[0259] Non-limiting examples of a modified amino acid residue include incorporation or other use of acetylated amino acids, glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, PEG modified (e.g., "PEGylated") amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References adequate to guide one of skill in the modification of amino acid residues are replete throughout the literature.

[0260] The modified amino acid residues may prevent or increase affinity of the polypeptide for another molecule, including, but not limited to, polynucleotide, proteins, carbohydrates, lipids and lipid derivatives, and other organic or synthetic compounds.

[0261] Identification of Additional Factors

[0262] A transcription factor provided by the present invention can also be used to identify additional endogenous or exogenous molecules that can affect a phentoype or trait of interest. On the one hand, such molecules include organic (small or large molecules) and/or inorganic compounds that affect expression of (i.e., regulate) a particular transcription factor. Alternatively, such molecules include endogenous molecules that are acted upon either at a transcriptional level by a transcription factor of the invention to modify a phenotype as desired. For example, the transcription factors can be employed to identify one or more downstream genes that are subject to a regulatory effect of the transcription factor. In one approach, a transcription factor or transcription factor homolog of the invention is expressed in a host cell, e.g., a transgenic plant cell, tissue or explant, and expression products, either RNA or protein, of likely or random targets are monitored, e.g., by hybridization to a microarray of nucleic acid probes corresponding to genes expressed in a tissue or cell type of interest, by two-dimensional gel electrophoresis of protein products, or by any other method known in the art for assessing expression of gene products at the level of RNA or protein. Alternatively, a transcription factor of the invention can be used to identify promoter sequences (such as binding sites on DNA sequences) involved in the regulation of a downstream target. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence can be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA-binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences can be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the transcription factors with their promoters (Bulyk et al. (1999) Nature Biotechnol. 17: 573-577).

[0263] The identified transcription factors are also useful to identify proteins that modify the activity of the transcription factor. Such modification can occur by covalent modification, such as by phosphorylation, or by protein-protein (homo or-heteropolymer) interactions. Any method suitable for detecting protein-protein interactions can be employed. Among the methods that can be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns, and the two-hybrid yeast system.

[0264] The two-hybrid system detects protein interactions in vivo and is described in Chien et al. ((1991) Proc. Natl. Acad. Sci. 88: 9578-9582) and is commercially available from Clontech (Palo Alto, Calif.). In such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the TF polypeptide and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

[0265] Then, the library plasmids responsible for reporter gene expression are isolated and sequenced to identify the proteins encoded by the library plasmids. After identifying proteins that interact with the transcription factors, assays for compounds that interfere with the TF protein-protein interactions can be preformed.

[0266] Identification of Modulators

[0267] In addition to the intracellular molecules described above, extracellular molecules that alter activity or expression of a transcription factor, either directly or indirectly, can be identified. For example, the methods can entail first placing a candidate molecule in contact with a plant or plant cell. The molecule can be introduced by topical administration, such as spraying or soaking of a plant, or incubating a plant in a solution containing the molecule, and then the molecule's effect on the expression or activity of the TF polypeptide or the expression of the polynucleotide monitored. Changes in the expression of the TF polypeptide can be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis or the like. Changes in the expression of the corresponding polynucleotide sequence can be detected by use of microarrays, Northerns, quantitative PCR, or any other technique for monitoring changes in mRNA expression. These techniques are exemplified in Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons (1998, and supplements through 2001). Changes in the activity of the transcription factor can be monitored, directly or indirectly, by assaying the function of the transcription factor, for example, by measuring the expression of promoters known to be controlled by the transcription factor (using promoter-reporter constructs), measuring the levels of transcripts using microarrays, Northern blots, quantitative PCR, etc. Such changes in the expression levels can be correlated with modified plant traits and thus identified molecules can be useful for soaking or spraying on fruit, vegetable and grain crops to modify traits in plants.

[0268] Essentially any available composition can be tested for modulatory activity of expression or activity of any nucleic acid or polypeptide herein. Thus, available libraries of compounds such as chemicals, polypeptides, nucleic acids and the like can be tested for modulatory activity. Often, potential modulator compounds can be dissolved in aqueous or organic (e.g., DMSO-based) solutions for easy delivery to the cell or plant of interest in which the activity of the modulator is to be tested. Optionally, the assays are designed to screen large modulator composition libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on micrometer plates in robotic assays).

[0269] In one embodiment, high throughput screening methods involve providing a combinatorial library containing a large number of potential compounds (potential modulator compounds). Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as target compounds.

[0270] A combinatorial chemical library can be, e.g., a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis. For example, a combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (e.g., in one example, amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound of a set length). Exemplary libraries include peptide libraries, nucleic acid libraries, antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnol. 14: 309-314 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. Science (1996) 274: 1520-1522 and U.S. Pat. No. 5,593,853), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), and small organic molecule libraries (see, e.g., benzodiazepines, in Baum Chem. & Engineering News Jan. 18, 1993, page 33; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337) and the like.

[0271] Preparation and screening of combinatorial or other libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, (1991) Int. J. Pept. Prot. Res. 37: 487493; and Houghton et al. (1991) Nature 354: 84-88). Other chemistries for generating chemical diversity libraries can also be used.

[0272] In addition, as noted, compound screening equipment for high-throughput screening is generally available, e.g., using any of a number of well known robotic systems that have also been developed for solution phase chemistries useful in assay systems. These systems include automated workstations including an automated synthesis apparatus and robotic systems utilizing robotic arms. Any of the above devices are suitable for use with the present invention, e.g., for high-throughput screening of potential modulators. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.

[0273] Indeed, entire high-throughput screening systems are commercially available. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. Similarly, microfluidic implementations of screening are also commercially available.

[0274] The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. The integrated systems herein, in addition to providing for sequence alignment and, optionally, synthesis of relevant nucleic acids, can include such screening apparatus to identify modulators that have an effect on one or more polynucleotides or polypeptides according to the present invention.

[0275] In some assays it is desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. That is, known transcriptional activators or inhibitors can be incubated with cells or plants, for example, in one sample of the assay, and the resulting increase/decrease in transcription can be detected by measuring the resulting increase in RNA levels and/or protein expression, for example, according to the methods herein. It will be appreciated that modulators can also be combined with transcriptional activators or inhibitors to find modulators that inhibit transcriptional activation or transcriptional repression. Either expression of the nucleic acids and proteins herein or any additional nucleic acids or proteins activated by the nucleic acids or proteins herein, or both, can be monitored.

[0276] In an embodiment, the invention provides a method for identifying compositions that modulate the activity or expression of a polynucleotide or polypeptide of the invention. For example, a test compound, whether a small or large molecule, is placed in contact with a cell, plant (or plant tissue or explant), or composition comprising the polynucleotide or polypeptide of interest and a resulting effect on the cell, plant, (or tissue or explant) or composition is evaluated by monitoring, either directly or indirectly, one or more of: expression level of the polynucleotide or polypeptide, activity (or modulation of the activity) of the polynucleotide or polypeptide. In some cases, an alteration in a plant phenotype can be detected following contact of a plant (or plant cell, or tissue or explant) with the putative modulator, e.g., by modulation of expression or activity of a polynucleotide or polypeptide of the invention. Modulation of expression or activity of a polynucleotide or polypeptide of the invention may also be caused by molecular elements in a signal transduction second messenger pathway and such modulation can affect similar elements in the same or another signal transduction second messenger pathway.

[0277] Subsequences

[0278] Also contemplated are uses of polynucleotides, also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least 20, 30, or 50 bases, which hybridize under at least highly stringent (or ultra-high stringent or ultra-ultra-high stringent conditions) conditions to a polynucleotide sequence described above. The polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted supra.

[0279] Subsequences of the polynucleotides of the invention, including polynucleotide fragments and oligonucleotides are useful as nucleic acid probes and primers. An oligonucleotide suitable for use as a probe or primer is at least about 15 nucleotides in length, more often at least about 18 nucleotides, often at least about 21 nucleotides, frequently at least about 30 nucleotides, or about 40 nucleotides, or more in length. A nucleic acid probe is useful in hybridization protocols, e.g., to identify additional polypeptide homologs of the invention, including protocols for microarray experiments. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods. See Sambrook, supra, and Ausubel, supra.

[0280] In addition, the invention includes an isolated or recombinant polypeptide including a subsequence of at least about 15 contiguous amino acids encoded by the recombinant or isolated polynucleotides of the invention. For example, such polypeptides, or domains or fragments thereof, can be used as immunogens, e.g., to produce antibodies specific for the polypeptide sequence, or as probes for detecting a sequence of interest. A subsequence can range in size from about 15 amino acids in length up to and including the full length of the polypeptide.

[0281] To be encompassed by the present invention, an expressed polypeptide which comprises such a polypeptide subsequence performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA binding domain that activates transcription, e.g., by binding to a specific DNA promoter region an activation domain, or a domain for protein-protein interactions.

[0282] Production of Transgenic Plants

[0283] Modification of Traits

[0284] The polynucleotides of the invention are favorably employed to produce transgenic plants with various traits, or characteristics, that have been modified in a desirable manner, e.g., to improve the seed characteristics of a plant. For example, alteration of expression levels or patterns (e.g., spatial or temporal expression patterns) of one or more of the transcription factors (or transcription factor homologs) of the invention, as compared with the levels of the same protein found in a wild-type plant, can be used to modify a plant's traits. An illustrative example of trait modification, improved characteristics, by altering expression levels of a particular transcription factor is described further in the Examples and the Sequence Listing.

[0285] Arabidopsis as a Model System

[0286] Arabidopsis thaliana is the object of rapidly growing attention as a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz et al., eds., Methods in Arabidopsis Research (1992) World Scientific, New Jersey, N.J., in "Preface"). Because of its small size, short life cycle, obligate autogamy and high fertility, Arabidopsis is also a choice organism for the isolation of mutants and studies in morphogenetic and development pathways, and control of these pathways by transcription factors (Koncz supra, p. 72). A number of studies introducing transcription factors into A. thaliana have demonstrated the utility of this plant for understanding the mechanisms of gene regulation and trait alteration in plants. (See, for example, Koncz supra, and U.S. Pat. No. 6,417,428).

[0287] Arabidopsis Genes in Transgenic Plants.

[0288] Expression of genes which encode transcription factors modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997 Genes and Development 11: 3194-3205) and Peng et al. (1999 Nature 400: 256-261). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001 Plant Cell 13: 1791-1802); Nandi et al. (2000 Curr. Biol. 10: 215-218); Coupland (1995 Nature 377: 482-483); and Weigel and Nilsson (1995, Nature 377: 482-500).

[0289] Homologous Genes Introduced Into Transgenic Plants.

[0290] Homologous genes that may be derived from any plant, or from any source whether natural, synthetic, semi-synthetic or recombinant, and that share significant sequence identity or similarity to those provided by the present invention, may be introduced into plants, for example, crop plants, to confer desirable or improved traits. Consequently, transgenic plants may be produced that comprise a recombinant expression vector or cassette with a promoter operably linked to one or more sequences homologous to presently disclosed sequences. The promoter may be, for example, a plant or viral promoter.

[0291] The invention thus provides for methods for preparing transgenic plants, and for modifying plant traits. These methods include introducing into a plant a recombinant expression vector or cassette comprising a functional promoter operably linked to one or more sequences homologous to presently disclosed sequences. Plants and kits for producing these plants that result from the application of these methods are also encompassed by the present invention.

[0292] Transcription Factors of Interest for the Modification of Plant Traits

[0293] Currently, the existence of a series of maturity groups for different latitudes represents a major barrier to the introduction of new valuable traits. Any trait (e.g. disease resistance) has to be bred into each of the different maturity groups separately, a laborious and costly exercise. The availability of single strain, which could be grown at any latitude, would therefore greatly increase the potential for introducing new traits to crop species such as soybean and cotton.

[0294] For the specific effects, traits and utilities conferred to plants, one or more transcription factor genes of the present invention may be used to increase or decrease, or improve or prove deleterious to a given trait. For example, knocking out a transcription factor gene that naturally occurs in a plant, or suppressing the gene (with, for example, antisense suppression), may cause decreased tolerance to an osmotic stress relative to non-transformed or wild-type plants. By overexpressing this gene, the plant may experience increased tolerance to the same stress. More than one transcription factor gene may be introduced into a plant, either by transforming the plant with one or more vectors comprising two or more transcription factors, or by selective breeding of plants to yield hybrid crosses that comprise more than one introduced transcription factor.

[0295] Genes, Traits and Utilities that Affect Plant Characteristics

[0296] Plant transcription factors can modulate gene expression, and, in turn, be modulated by the environmental experience of a plant. Significant alterations in a plant's environment invariably result in a change in the plant's transcription factor gene expression pattern. Altered transcription factor expression patterns generally result in phenotypic changes in the plant. Transcription factor gene product(s) in transgenic plants then differ(s) in amounts or proportions from that found in wild-type or non-transformed plants, and those transcription factors likely represent polypeptides that are used to alter the response to the environmental change. By way of example, it is well accepted in the art that analytical methods based on altered expression patterns may be used to screen for phenotypic changes in a plant far more effectively than can be achieved using traditional methods.

[0297] Sugar Sensing.

[0298] In addition to their important role as an energy source and structural component of the plant cell, sugars are central regulatory molecules that control several aspects of plant physiology, metabolism and development (Hsieh et al. (1998) Proc. Natl. Acad. Sci. 95: 13965-13970). It is thought that this control is achieved by regulating gene expression and, in higher plants, sugars have been shown to repress or activate plant genes involved in many essential processes such as photosynthesis, glyoxylate metabolism, respiration, starch and sucrose synthesis and degradation, pathogen response, wounding response, cell cycle regulation, pigmentation, flowering and senescence. The mechanisms by which sugars control gene expression are not understood.

[0299] Several sugar sensing mutants have turned out to be allelic to abscisic acid (ABA) and ethylene mutants. ABA is found in all photosynthetic organisms and acts as a key regulator of transpiration, stress responses, embryogenesis, and seed germination. Most ABA effects are related to the compound acting as a signal of decreased water availability, whereby it triggers a reduction in water loss, slows growth, and mediates adaptive responses. However, ABA also influences plant growth and development via interactions with other phytohormones. Physiological and molecular studies indicate that maize and Arabidopsis have almost identical pathways with regard to ABA biosynthesis and signal transduction. For further review, see Finkelstein and Rock ((2002) Abscisic acid biosynthesis and response (In The Arabidopsis Book, Editors: Somerville and Meyerowitz (American Society of Plant Biologists, Rockville, Md.).

[0300] This potentially implicates G481 and G482 in hormone signaling based on the sucrose sugar sensing phenotype of 35S::G481 and 35S::G482 transgenic lines. On the other hand, under the laboratory conditions we use at Mendel, the sucrose treatment (9.5% w/v) could also be an osmotic stress. Therefore, one could interpret this data to indicate that the 35S::G481 transgenic lines are more tolerant to osmotic stress. Interestingly, the Mendel RT-PCR expression profiling studies have shown that more than half of the CCAAT transcription factors are up-regulated in tissues with developing seeds. One example is the well-characterized HAP3-like protein, LEC1, which is required for desiccation tolerance during seed maturation. LEC1 is also ABA and drought inducible. This information, combined with the fact that CCAAT genes are disproportionately responsive to osmotic stress suggests that this family of transcription factors could control pathways involved in both ABA responses and desiccation tolerance.

[0301] Because sugars are important signaling molecules, the ability to control either the concentration of a signaling sugar or how the plant perceives or responds to a signaling sugar could be used to control plant development, physiology or metabolism. For example, the flux of sucrose (a disaccharide sugar used for systemically transporting carbon and energy in most plants) has been shown to affect gene expression and alter storage compound accumulation in seeds. Manipulation of the sucrose-signaling pathway in seeds may therefore cause seeds to have more protein, oil or carbohydrate, depending on the type of manipulation. Similarly, in tubers, sucrose is converted to starch which is used as an energy store. It is thought that sugar-signaling pathways may partially determine the levels of starch synthesized in the tubers. The manipulation of sugar signaling in tubers could lead to tubers with a higher starch content.

[0302] Thus, the presently disclosed transcription factor genes that manipulate the sugar signal transduction pathway, including, for example, G481, along with its equivalogs, may lead to altered gene expression to produce plants with desirable traits. In particular, manipulation of sugar signal transduction pathways could be used to alter source-sink relationships in seeds, tubers, roots and other storage organs leading to increase in yield.

[0303] Osmotic stress. Modification of the expression of a number of presently disclosed transcription factor genes may be used to increase germination rate or growth under adverse osmotic conditions, which could impact survival and yield of seeds and plants. Osmotic stresses may be regulated by specific molecular control mechanisms that include genes controlling water and ion movements, functional and structural stress-induced proteins, signal perception and transduction, and free radical scavenging, and many others (Wang et al. (2001) Acta Hort. (ISHS) 560: 285-292). Instigators of osmotic stress include freezing, drought and high salinity, each of which is discussed in more detail below.

[0304] In many ways, freezing, high salt and drought have similar effects on plants, not the least of which is the induction of common polypeptides that respond to these different stresses. For example, freezing is similar to water deficit in that freezing reduces the amount of water available to a plant. Exposure to freezing temperatures may lead to cellular dehydration as water leaves cells and forms ice crystals in intercellular spaces (Buchanan, supra). As with high salt concentration and freezing, the problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Thus, the incorporation of transcription factors that modify a plant's response to osmotic stress into, for example, a crop or ornamental plant, may be useful in reducing damage or loss. Specific effects caused by freezing, high salt and drought are addressed below.

[0305] Salt and Drought Tolerance

[0306] Plants are subject to a range of environmental challenges. Several of these, including salt stress, general osmotic stress, drought stress and freezing stress, have the ability to impact whole plant and cellular water availability. Not surprisingly, then, plant responses to this collection of stresses are related. In a recent review, Zhu notes that "most studies on water stress signaling have focused on salt stress primarily because plant responses to salt and drought are closely related and the mechanisms overlap" (Zhu (2002) Ann. Rev. Plant Biol. 53: 247-273). Many examples of similar responses and pathways to this set of stresses have been documented. For example, the CBF transcription factors have been shown to condition resistance to salt, freezing and drought (Kasuga et al. (1999) Nature Biotech. 17: 287-291). The Arabidopsis rd29B gene is induced in response to both salt and dehydration stress, a process that is mediated largely through an ABA signal transduction process (Uno et al. (2000) Proc. Natl. Acad. Sci. USA 97: 11632-11637), resulting in altered activity of transcription factors that bind to an upstream element within the rd29B promoter. In Mesembryanthemum crystallinum (ice plant), Patharker and Cushman have shown that a calcium-dependent protein kinase (McCDPK1) is induced by exposure to both drought and salt stresses (Patharker and Cushman (2000) Plant J. 24: 679-691). The stress-induced kinase was also shown to phosphorylate a transcription factor, presumably altering its activity, although transcript levels of the target transcription factor are not altered in response to salt or drought stress. Similarly, Saijo et al. demonstrated that a rice salt/drought-induced calmodulin-dependent protein kinase (OsCDPK7) conferred increased salt and drought tolerance to rice when overexpressed (Saijo et al. (2000) Plant J. 23: 319-327).

[0307] Exposure to dehydration invokes similar survival strategies in plants as does freezing stress (see, for example, Yelenosky (1989) Plant Physiol 89: 444-451) and drought stress induces freezing tolerance (see, for example, Siminovitch et al. (1982) Plant Physiol 69: 250-255; and Guy et al. (1992) Planta 188: 265-270). In addition to the induction of cold-acclimation proteins, strategies that allow plants to survive in low water conditions may include, for example, reduced surface area, or surface oil or wax production.

[0308] Consequently, one skilled in the art would expect that some pathways involved in resistance to one of these stresses, and hence regulated by an individual transcription factor, will also be involved in resistance to another of these stresses, regulated by the same or homologous transcription factors. Of course, the overall resistance pathways are related, not identical, and therefore not all transcription factors controlling resistance to one stress will control resistance to the other stresses. Nonetheless, if a transcription factor conditions resistance to one of these stresses, it would be apparent to one skilled in the art to test for resistance to these related stresses.

[0309] Thus, modifying the expression of a number of presently disclosed transcription factor genes, such as G481 or G482, may be used to increase a plant's tolerance to low water conditions and provide the benefits of improved survival, increased yield and an extended geographic and temporal planting range.

[0310] Salt. The genes of the sequence listing, including, for example, G482, that provide tolerance to salt may be used to engineer salt tolerant crops and trees that can flourish in soils with high saline content or under drought conditions. In particular, increased salt tolerance during the germination stage of a plant enhances survival and yield. Presently disclosed transcription factor genes that provide increased salt tolerance during germination, the seedling stage, and throughout a plant's life cycle, would find particular value for imparting survival and yield in areas where a particular crop would not normally prosper.

[0311] Increased anthocyanin level in plant organs and tissues. Presently disclosed transcription factor genes (i.e., G481 and its equivalogs) can be used to alter anthocyanin levels in one or more tissues, depending on the organ in which these genes are expressed. The potential utilities of these genes include alterations in pigment production for horticultural purposes, and possibly increasing stress resistance, including osmotic stress resistance. In addition, plants with increased anthocyanin may provide health-promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of the oxidation of lipids.

[0312] Summary of altered plant characteristics. A clade of structurally and functionally related sequences that derive from a wide range of plants, including polynucleotide SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, polynucleotides that encode polypeptide SEQ ID NOs: 29-32, fragments thereof, paralogs, orthologs, equivalogs, and fragments thereof, is provided. These sequences have been shown in laboratory and field experiments to confer altered size and abiotic stress tolerance phenotypes in plants. The invention also provides polypeptides comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 30, 31, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, and fragments thereof, conserved domains thereof, paralogs, orthologs, equivalogs, and fragments thereof. Plants that overexpress these sequences have been observed to be more tolerant to a wide variety of abiotic stresses, including, germination in heat and cold, and osmotic stresses such as drought and high salt levels. Many of the orthologs of these sequences are listed in the Sequence Listing, and due to the high degree of structural similarity to the sequences of the invention, it is expected that these sequences may also function to increase plant biomass and/or abiotic stress tolerance. The invention also encompasses the complements of the polynucleotides. The polynucleotides are useful for screening libraries of molecules or compounds for specific binding and for creating transgenic plants having increased biomass and/or abiotic stress tolerance.

[0313] Antisense and Co-Suppression

[0314] In addition to expression of the nucleic acids of the invention as gene replacement or plant phenotype modification nucleic acids, the nucleic acids are also useful for sense and anti-sense suppression of expression, e.g. to down-regulate expression of a nucleic acid of the invention, e.g. as a further mechanism for modulating plant phenotype. That is, the nucleic acids of the invention, or subsequences or anti-sense sequences thereof, can be used to block expression of naturally occurring homologous nucleic acids. A variety of sense and anti-sense technologies are known in the art, e.g. as set forth in Lichtenstein and Nellen (1997) Antisense Technology: A Practical Approach IRL Press at Oxford University Press, Oxford, U.K. Antisense regulation is also described in Crowley et al. (1985) Cell 43: 633-641; Rosenberg et al. (1985) Nature 313: 703-706; Preiss et al. (1985) Nature 313: 27-32; Melton (1985) Proc. Natl. Acad. Sci. 82: 144-148; Izant and Weintraub (1985) Science 229: 345-352; and Kim and Wold (1985) Cell 42: 129-138. Additional methods for antisense regulation are known in the art. Antisense regulation has been used to reduce or inhibit expression of plant genes in, for example in European Patent Publication No. 271988. Antisense RNA may be used to reduce gene expression to produce a visible or biochemical phenotypic change in a plant (Smith et al. (1988) Nature, 334: 724-726; Smith et al. (1990) Plant Mol. Biol. 14: 369-379). In general, sense or anti-sense sequences are introduced into a cell, where they are optionally amplified, e.g. by transcription. Such sequences include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.

[0315] For example, a reduction or elimination of expression (i.e., a "knock-out") of a transcription factor or transcription factor homolog polypeptide in a transgenic plant, e.g., to modify a plant trait, can be obtained by introducing an antisense construct corresponding to the polypeptide of interest as a cDNA. For antisense suppression, the transcription factor or homolog cDNA is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. The introduced sequence need not be the full-length cDNA or gene, and need not be identical to the cDNA or gene found in the plant type to be transformed. Typically, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. Preferably, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous transcription factor gene in the plant cell.

[0316] Suppression of endogenous transcription factor gene expression can also be achieved using RNA interference (RNAi) or microRNA-based methods (Llave et al. (2002) Science 297: 2053-2056; Tang et al. (2003) Genes Dev. 17: 49-63). RNAi is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to incite degradation of messenger RNA (mRNA) containing the same sequence as the dsRNA (Constans, (2002) The Scientist 16: 36). Small interfering RNAs, or siRNAs are produced in at least two steps: an endogenous ribonuclease cleaves longer dsRNA into shorter, 21-23 nucleotide-long RNAs (Plasterk (2002) Science 296: 1263-1265). The siRNA segments then mediate the degradation of the target mRNA (Zamore, (2001) Nature Struct. Biol., 8:746-50). RNAi has been used for gene function determination in a manner similar to antisense oligonucleotides (Constans, (2002) The Scientist 16:36). Expression vectors that continually express siRNAs in transiently and stably transfected have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (Brummelkamp et al., (2002) Science 296:550-553, and Paddison, et al. (2002) Genes & Dev. 16:948-958). Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hammond et al. (2001) Nature Rev Gen 2: 110-119, Fire et al. (1998) Nature 391: 806-811 and Timmons and Fire (1998) Nature 395: 854. Vectors in which RNA encoded by a transcription factor or transcription factor homolog cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Pat. No. 5,231,020 to Jorgensen. Such co-suppression (also termed sense suppression) does not require that the entire transcription factor cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous transcription factor gene of interest. However, as with antisense suppression, the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous transcription factor gene is increased.

[0317] Vectors expressing an untranslatable form of the transcription factor mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous transcription factor, thereby reducing or eliminating its activity and modifying one or more traits. Methods for producing such constructs are described in U.S. Pat. No. 5,583,021. Preferably, such constructs are made by introducing a premature stop codon into the transcription factor gene. Alternatively, a plant trait can be modified by gene silencing using double-strand RNA (Sharp (1999) Genes and Development 13: 139-141). Another method for abolishing the expression of a gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a transcription factor or transcription factor homolog gene. Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation. Such methods are well known to those of skill in the art (See for example Koncz et al. (1992) Methods in Arabidopsis Research, World Scientific Publishing Co. Pte. Ltd., River Edge, N.J.).

[0318] Alternatively, a plant phenotype can be altered by eliminating an endogenous gene, such as a transcription factor or transcription factor homolog, e.g., by homologous recombination (Kempin et al. (1997) Nature 389: 802-803).

[0319] A plant trait can also be modified by using the Cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.

[0320] The polynucleotides and polypeptides of this invention can also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means, such as, for example, by ectopically expressing a gene by T-DNA activation tagging (Ichikawa et al. (1997) Nature 390 698-701; Kakimoto et al. (1996) Science 274: 982-985). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated. In another example, the transcriptional machinery in a plant can be modified so as to increase transcription levels of a polynucleotide of the invention (See, e.g., PCT Publications WO 96/06166 and WO 98/53057 which describe the modification of the DNA-binding specificity of zinc finger proteins by changing particular amino acids in the DNA-binding motif).

[0321] The transgenic plant can also include the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, for example, by altering the phosphorylation state of the polypeptide to maintain it in an activated state.

[0322] Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well established techniques as described above. Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homolog, of the invention, standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.

[0323] The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al., eds., (1984) Handbook of Plant Cell Culture--Crop Species, Macmillan Publ. Co., New York, N.Y.; Shimamoto et al. (1989) Nature 338: 274-276; Fromm et al. (1990) Bio/Technol. 8: 833-839; and Vasil et al. (1990) Bio/Technol. 8: 429-434.

[0324] Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells are now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.

[0325] Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

[0326] Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.

[0327] After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

[0328] Integrated Systems--Sequence Identity

[0329] Additionally, the present invention may be an integrated system, computer or computer readable medium that comprises an instruction set for determining the identity of one or more sequences in a database. In addition, the instruction set can be used to generate or identify sequences that meet any specified criteria. Furthermore, the instruction set may be used to associate or link certain functional benefits, such improved characteristics, with one or more identified sequence.

[0330] For example, the instruction set can include, e.g., a sequence comparison or other alignment program, e.g., an available program such as, for example, the Wisconsin Package Version 10.0, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.). Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PHYTOSEQ sequence database (Incyte Genomics, Palo Alto, Calif.) can be searched.

[0331] Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482489, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448, by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Ausubel et al. supra.

[0332] A variety of methods for determining sequence relationships can be used, including manual alignment and computer assisted sequence alignment and analysis. This later approach is a preferred approach in the present invention, due to the increased throughput afforded by computer assisted methods. As noted above, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.

[0333] One example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available, e.g., through the National Library of Medicine's National Center for Biotechnology Information (ncbi.nlm.nih; see at world wide web (www) National Institutes of Health US government (gov) website). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. 89: 10915-10919). Unless otherwise indicated, "sequence identity" here refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter "off" (see, for example, NIH NLM NCBI website at ncbi.nlm.nih, supra).

[0334] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. Karlin and Altschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001. An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.

[0335] The integrated system, or computer typically includes a user input interface allowing a user to selectively view one or more sequence records corresponding to the one or more character strings, as well as an instruction set which aligns the one or more character strings with each other or with an additional character string to identify one or more region of sequence similarity. The system may include a link of one or more character strings with a particular phenotype or gene function. Typically, the system includes a user readable output element that displays an alignment produced by the alignment instruction set.

[0336] The methods of this invention can be implemented in a localized or distributed computing environment. In a distributed environment, the methods may implemented on a single computer comprising multiple processors or on a multiplicity of computers. The computers can be linked, e.g. through a common bus, but more preferably the computer(s) are nodes on a network. The network can be a generalized or a dedicated local or wide-area network and, in certain preferred embodiments, the computers may be components of an intra-net or an internet.

[0337] Thus, the invention provides methods for identifying a sequence similar or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an inter or intra net) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

[0338] Any sequence herein can be entered into the database, before or after querying the database. This provides for both expansion of the database and, if done before the querying step, for insertion of control sequences into the database. The control sequences can be detected by the query to ensure the general integrity of both the database and the query. As noted, the query can be performed using a web browser based interface. For example, the database can be a centralized public database such as those noted herein, and the querying can be done from a remote terminal or computer across an internet or intranet.

[0339] Any sequence herein can be used to identify a similar, homologous, paralogous, or orthologous sequence in another plant. This provides means for identifying endogenous sequences in other plants that may be useful to alter a trait of progeny plants, which results from crossing two plants of different strain. For example, sequences that encode an ortholog of any of the sequences herein that naturally occur in a plant with a desired trait can be identified using the sequences disclosed herein. The plant is then crossed with a second plant of the same species but which does not have the desired trait to produce progeny which can then be used in further crossing experiments to produce the desired trait in the second plant. Therefore the resulting progeny plant contains no transgenes; expression of the endogenous sequence may also be regulated by treatment with a particular chemical or other means, such as EMR. Some examples of such compounds well known in the art include: ethylene; cytokinins; phenolic compounds, which stimulate the transcription of the genes needed for infection; specific monosaccharides and acidic environments which potentiate vir gene induction; acidic polysaccharides which induce one or more chromosomal genes; and opines; other mechanisms include light or dark treatment (for a review of examples of such treatments, see, Winans (1992) Microbiol. Rev. 56: 12-31; Eyal et al. (1992) Plant Mol. Biol. 19: 589-599; Chrispeels et al. (2000) Plant Mol. Biol. 42: 279-290; Piazza et al. (2002) Plant Physiol. 128: 1077-1086).

[0340] Table 5 lists sequences discovered to be orthologous to a number of representative transcription factors of the present invention. The column headings include the transcription factors listed by (a) the SEQ ID NO: of the ortholog or nucleotide encoding the ortholog; (b) the GID sequence identifier; (c) the Sequence Identifier or GenBank Accession Number;(d) the species from which the orthologs to the transcription factors are derived; (e) the smallest sum probability relationship to G482 determined by BLAST analysis; and (f) the percent identity of the B domain of the sequence to the same domain in G482.

8TABLE 5 Paralogs and Orthologs and Other Related Genes of Representative Arabidopsis Transcription Factor Genes identified using BLAST SEQ ID NO: of Ortholog or Nucleotide Smallest Sum Percent Identity Encoding Sequence Identifier or Species from Which Probability to of B domain to B Ortholog GID No. Accession Number Ortholog is Derived G482 domain of G482 1 G481 Arabidopsis thaliana 83% 3 G482 Arabidopsis thaliana 0.0 100% 5 G485 Arabidopsis thaliana 94% 7 G1364 Arabidopsis thaliana 85% 9 G2345 Arabidopsis thaliana 85% 11 GLYMA-28NOV01- Glycine max 5E-29 84% CLUSTER24839_1 13 GLYMA-28NOV01- Glycine max 2E-31 85% CLUSTER31103_1 15 GLYMA-28NOV01- Glycine max 1E-41 91% CLUSTER33504_1 17 G3476 GLYMA-28NOV01- Glycine max 3E-58 94% CLUSTER33504_3 19 G3475 GLYMA-28NOV01- Glycine max 6E-58 95% CLUSTER33504_5 21 GLYMA-28NOV01- Glycine max 6E-45 92% CLUSTER33504_6 23 G3471 GLYMA-28NOV01- Glycine max 9E-57 92% CLUSTER4778_1 81 G3472 Glycine max 9E-57 92% 25 G3470 GLYMA-28NOV01- Glycine max 8E-9 85% CLUSTER4778_3 87 G3394 ORYSA-22JAN02- Oryza sativa 3E-18 86% CLUSTER26105_1 73 G3395 Oryza sativa 1E-44 83% 29 OSC12630.C1.p5.fg Oryza sativa 2E-55 90% 30 OSC1404.C1.p3.fg Oryza sativa 4E-39 75% 31 OSC30077.C1.p6.fg Oryza sativa 3E-50 86% 32 OSC5489.C1.p2.fg Oryza sativa 8E-44 83% 60 G3398 Oryza sativa 2E-57 90% 33 L1B3732-044-Q6-K6- Zea mays 2E-23 87% C4 35 ZEAMA-08NOV01- Zea mays 7E-19 86% CLUSTER719_1 37 ZEAMA-08NOV01- Zea mays 7E-11 86% CLUSTER719_10 39 ZEAMA-08NOV01- Zea mays 6E-19 86% CLUSTER719_2 41 ZEAMA-08NOV01- Zea mays 6E-7 80% CLUSTER719_3 43 ZEAMA-08NOV01- Zea mays 8E-17 86% CLUSTER719_4 45 ZEAMA-08NOV01- Zea mays 4E-17 86% CLUSTER719_5 47 ZEAMA-08NOV01- Zea mays 5E-23 93% CLUSTER90408_1 49 G3436 ZEAMA-08NOV01- Zea mays 7E-55 93% CLUSTER90408_2 77 G3434 Zea mays 2E-44 86% 79 G3435 Zea mays 1E-58 93% 51 G3473 GLYMA-28NOV01- Glycine max 7E-17 83% CLUSTER33504_4 83 G3474 Glycine max 6E-57 91% 85 G3477 Glycine max 5E-47 85% 87 G3478 Glycine max 4E-58 95% 53 ORYSA-22JAN02- Oryza sativa 9E-21 83% CLUSTER119015_1 55 Zm_S11418173 Zea mays 3E-17 86% 57 Zm_S11434692 Zea mays 1E-19 85% 59 Ta_S45374 Triticum aestivum 2E-24 85% 61 Ta_S50443 Triticum aestivum 9E-24 90% 63 SGN-UNIGENE-46859 Lycopersicon esculentum 2E-6 87% 65 SGN-UNIGENE-47447 Lycopersicon esculentum 3E-11 91% BU238020 Descurainia sophia 1.00E-70 BG440251 Gossypium arboreum 3.00E-56 CB290513 Citrus sinensis 3.00E-55 BF071234 Glycine max 1.00E-54 BQ799965 Vitis vinifera 3.00E-54 AX584261 Eucalyptus grandis 5.00E-54 AX584259 Momordica charantia 7.00E-54 CD848631 Helianthus annuus 6.00E-53 BQ488908 Beta vulgaris 6.00E-53 CD573484 Zea mays 8.00E-53 gi115840 Zea mays 2.40E-51 86% gi30409461 Oryza sativa (japonica 3.50E-50 86% cultivar-group) AP004366 Oryza sativa 3E-50 AC120529 Oryza sativa (japonica 7E-46 cultivar-group) gi15408794 Oryza sativa 8.70E-38 75% AC108500 Oryza sativa 2E-20 CD574709 Poncirus trifoliata 8.00E-60 BQ505706 Solanum tuberosum 9.00E-59 AC122165 Medicago truncatula 9.00E-57 AC120529 Oryza sativa (japonica 6E-56 cultivar-group) BQ104671 Rosa hybrid cultivar 3.00E-55 AX584271 Glycine max 6.00E-55 AX584265 Zea mays 1.00E-54 AAAA01003638 Oryza sativa (indica 2.00E-54 cultivar-group) AP005193 Oryza sativa (japonica 2.00E-54 cultivar-group) BU880488 Populus balsamifera 2.00E-53 subsp. trichocarpa BJ248969 Triticum aestivum 3.00E-53 gi115840 Zea mays 1.80E-46 86% gi30409461 Oryza sativa (japonica 8.80E-45 86% cultivar-group) AP004366 Oryza sativa 4E-44 gi15408794 Oryza sativa 1.80E-37 75% AP005193 Oryza sativa (japonica 9E-21 cultivar-group) AC108500 Oryza sativa 5E-15 CD574709 Poncirus trifoliata 9.00E-62 BQ505706 Solanum tuberosum 4.00E-60 BQ996905 Lactuca sativa 2.00E-58 AAAA01003638 Oryza sativa (indica 3.00E-57 cultivar-group) AP005193 Oryza sativa (japonica 3.00E-57 cultivar-group) BQ592365 Beta vulgaris 9.00E-57 CD438068 Zea mays 9.00E-57 AX288144 Physcomitrella patens 3.00E-56 BU880488 Populus balsamifera 1.00E-55 subsp. trichocarpa AX584277 Glycine max 6.00E-55 gi30409461 Oryza sativa (japonica 4.60E-48 86% cultivar-group) gi30349365 Oryza sativa (indica 1.10E-39 cultivar-group) gi15408794 Oryza sativa 1.60E-38 75% CD823119 Brassica napus 1.00E-64 BG642751 Lycopersicon esculentum 2.00E-60 BQ629472 Glycine max 6.00E-60 BQ405785 Gossypium arboreum 6.00E-60 BQ488908 Beta vulgaris 1.00E-59 AX584261 Eucalyptus grandis 3.00E-59 BQ799965 Vitis vinifera 6.00E-59 CB290513 Citrus sinensis 3.00E-58 CD848631 Helianthus annuus 3.00E-58 CF069249 Medicago truncatula 2.00E-57 gi115840 Zea mays 2.10E-50 86% gi30409461 Oryza sativa (japonica 9.50E-48 82% cultivar-group) CD823119 Brassica napus 2.00E-75 BG445358 Gossypium arboreum 1.00E-64 BG642751 Lycopersicon esculentum 2.00E-64 BQ629472 Glycine max 5.00E-63 BQ488908 Beta vulgaris 6.00E-63 AX584261 Eucalyptus grandis 7.00E-62 BQ799965 Vitis vinifera 1.00E-61 CD848631 Helianthus annuus 2.00E-61 CF069249 Medicago truncatula 6.00E-61 BG599785 Solanum tuberosum 7.00E-61 82% gi115840 Zea mays 6.80E-54 86% gi30409459 Oryza sativa (japonica 1.00E-50 83% cultivar-group)

[0341] Molecular Modeling

[0342] Another means that may be used to confirm the utility and function of transcription factor sequences that are orthologous or paralogous to presently disclosed transcription factors is through the use of molecular modeling software. Molecular modeling is routinely used to predict polypeptide structure, and a variety of protein structure modeling programs, such as "Insight II" (Accelrys, Inc.) are commercially available for this purpose. Modeling can thus be used to predict which residues of a polypeptide can be changed without altering function (Crameri et al. (2003) U.S. Pat. No. 6,521,453). Thus, polypeptides that are sequentially similar can be shown to have a high likelihood of similar function by their structural similarity, which may, for example, be established by comparison of regions of superstructure. The relative tendencies of amino acids to form regions of superstructure (for example, helixes and _-sheets) are well established. For example, ONeil et al. ((1990) Science 250: 646-651) have discussed in detail the helix forming tendencies of amino acids. Tables of relative structure forming activity for amino acids can be used as substitution tables to predict which residues can be functionally substituted in a given region, for example, in DNA-binding domains of known transcription factors and equivalogs. Homologs that are likely to be functionally similar can then be identified.

[0343] Of particular interest is the structure of a transcription factor in the region of its conserved domains, such as those identified in Table 1. Structural analyses may be performed by comparing the structure of the known transcription factor around its conserved domain with those of orthologs and paralogs. Analysis of a number of polypeptides within a transcription factor group or lade, including the functionally or sequentially similar polypeptides provided in the Sequence Listing, may also provide an understanding of structural elements required to regulate transcription within a given family.

EXAMPLES

[0344] The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. It will be recognized by one of skill in the art that a transcription factor that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.

[0345] The complete descriptions of the traits associated with each polynucleotide of the invention are fully disclosed in Example VIII. The complete description of the transcription factor gene family and identified B domains of the polypeptide encoded by the polynucleotide is fully disclosed in Table 1.

Example I

Full Length Gene Identification and Cloning

[0346] Putative transcription factor sequences (genomic or ESTs) related to known transcription factors were identified in the Arabidopsis thaliana GenBank database using the tblastn sequence analysis program using default parameters and a P-value cutoff threshold of -4 or -5 or lower, depending on the length of the query sequence. Putative transcription factor sequence hits were then screened to identify those containing particular sequence strings. If the sequence hits contained such sequence strings, the sequences were confirmed as transcription factors.

[0347] Alternatively, Arabidopsis thaliana cDNA libraries derived from different tissues or treatments, or genomic libraries were screened to identify novel members of a transcription family using a low stringency hybridization approach. Probes were synthesized using gene specific primers in a standard PCR reaction (annealing temperature 60.degree. C.) and labeled with .sup.32P dCTP using the High Prime DNA Labeling Kit (Boehringer Mannheim Corp. (now Roche Diagnostics Corp., Indianapolis, Ind.). Purified radiolabelled probes were added to filters immersed in Church hybridization medium (0.5 M NaPO.sub.4 pH 7.0, 7% SDS, 1% w/v bovine serum albumin) and hybridized overnight at 60.degree. C. with shaking. Filters were washed two times for 45 to 60 minutes with 1.times.SCC, 1% SDS at 60.degree. C.

[0348] To identify additional sequence 5' or 3' of a partial cDNA sequence in a cDNA library, 5' and 3' rapid amplification of cDNA ends (RACE) was performed using the MARATHON cDNA amplification kit (Clontech, Palo Alto, Calif.). Generally, the method entailed first isolating poly(A) mRNA, performing first and second strand cDNA synthesis to generate double stranded cDNA, blunting cDNA ends, followed by ligation of the MARATHON Adaptor to the cDNA to form a library of adaptor-ligated ds cDNA.

[0349] Gene-specific primers were designed to be used along with adaptor specific primers for both 5' and 3' RACE reactions. Nested primers, rather than single primers, were used to increase PCR specificity. Using 5' and 3' RACE reactions, 5' and 3' RACE fragments were obtained, sequenced and cloned. The process can be repeated until 5' and 3' ends of the full-length gene were identified. Then the full-length cDNA was generated by PCR using primers specific to 5' and 3' ends of the gene by end-to-end PCR.

Example II

Construction of Expression Vectors

[0350] The sequence was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. The expression vector was pMEN20 or pMEN65, which are both derived from pMON316 (Sanders et al. (1987) Nucleic Acids Res. 15:1543-1558) and contain the CAMV 35S promoter to express transgenes. To clone the sequence into the vector, both pMEN20 and the amplified DNA fragment were digested separately with SalI and NotI restriction enzymes at 37.degree. C. for 2 hours. The digestion products were subject to electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. The DNA fragments containing the sequence and the linearized plasmid were excised and purified by using a QIAQUICK gel extraction kit (Qiagen, Valencia Calif.). The fragments of interest were ligated at a ratio of 3:1 (vector to insert). Ligation reactions using T4 DNA ligase (New England Biolabs, Beverly Mass.) were carried out at 16.degree. C. for 16 hours. The ligated DNAs were transformed into competent cells of the E. coli strain DH5alpha by using the heat shock method. The transformations were plated on LB plates containing 50 mg/l kanamycin (Sigma Chemical Co. St. Louis Mo.). Individual colonies were grown overnight in five milliliters of LB broth containing 50 mg/l kanamycin at 37.degree. C. Plasmid DNA was purified by using Qiaquick Mini Prep kits (Qiagen).

Example III

Transformation of Agrobacterium with the Expression Vector

[0351] After the plasmid vector containing the gene was constructed, the vector was used to transform Agrobacterium tumefaciens cells expressing the gene products. The stock of Agrobacterium tumefaciens cells for transformation was made as described by Nagel et al. (1990) FEMS Microbiol Letts. 67: 325-328. Agrobacterium strain ABI was grown in 250 ml LB medium (Sigma) overnight at 28.degree. C. with shaking until an absorbance over 1 cm at 600 nm (A.sub.600) of 0.5-1.0 was reached. Cells were harvested by centrifugation at 4,000.times.g for 15 min at 4.degree. C. Cells were then resuspended in 250 .mu.l chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 .mu.l chilled buffer. Cells were then centrifuged and resuspended two more times in the same HEPES buffer as described above at a volume of 100 .mu.l and 750 .mu.l respectively. Resuspended cells were then distributed into 40 l.sup.1l aliquots, quickly frozen in liquid nitrogen, and stored at -80.degree. C.

[0352] Agrobacterium cells were transformed with plasmids prepared as described above following the protocol described by Nagel et al. (supra). For each DNA construct to be transformed, 50-100 ng DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was mixed with 40 .mu.l of Agrobacterium cells. The DNA/cell mixture was then transferred to a chilled cuvette with a 2 mm electrode gap and subject to a 2.5 kV charge dissipated at 25 .mu.F and 200 .mu.F using a Gene Pulser II apparatus (Bio-Rad, Hercules, Calif.). After electroporation, cells were immediately resuspended in 1.0 ml LB and allowed to recover without antibiotic selection for 2-4 hours at 28.degree. C. in a shaking incubator. After recovery, cells were plated onto selective medium of LB broth containing 100 .mu.g/ml spectinomycin (Sigma) and incubated for 2448 hours at 28.degree. C. Single colonies were then picked and inoculated in fresh medium. The presence of the plasmid construct was verified by PCR amplification and sequence analysis.

Example IV

Transformation of Arabidopsis Plants with Agrobacteriumn tumefaciens with Expression Vector

[0353] After transformation of Agrobacterium tumefaciens with plasmid vectors containing the gene, single Agrobacterium colonies were identified, propagated, and used to transform Arabidopsis plants. Briefly, 500 ml cultures of LB medium containing 50 mg/l kanamycin were inoculated with the colonies and grown at 28.degree. C. with shaking for 2 days until an optical absorbance at 600 nm wavelength over 1 cm (A.sub.600) of >2.0 is reached. Cells were then harvested by centrifugation at 4,000.times.g for 10 min, and resuspended in infiltration medium (1/2.times. Murashige and Skoog salts (Sigma), 1.times. Gamborg's B-5 vitamins (Sigma), 5.0% (w/v) sucrose (Sigma), 0.044 JIM benzylamino purine (Sigma), 200 .mu.l/l Silwet L-77 (Lehle Seeds) until an A.sub.600 of 0.8 was reached.

[0354] Prior to transformation, Arabidopsis thaliana seeds (ecotype Columbia) were sown at a density of .about.10 plants per 4" pot onto Pro-Mix BX potting medium (Hummert International) covered with fiberglass mesh (18 mm.times.16 mm). Plants were grown under continuous illumination (50-75 .mu.E/m.sup.2/sec) at 22-23.degree. C. with 65-70% relative humidity. After about 4 weeks, primary inflorescence stems (bolts) are cut off to encourage growth of multiple secondary bolts. After flowering of the mature secondary bolts, plants were prepared for transformation by removal of all siliques and opened flowers.

[0355] The pots were then immersed upside down in the mixture of Agrobacterium infiltration medium as described above for 30 sec, and placed on their sides to allow draining into a 1'.times.2' flat surface covered with plastic wrap. After 24 h, the plastic wrap was removed and pots are turned upright. The immersion procedure was repeated one week later, for a total of two immersions per pot. Seeds were then collected from each transformation pot and analyzed following the protocol described below.

Example V

Identification of Arabidopsis Primary Transformants

[0356] Seeds collected from the transformation pots were sterilized essentially as follows. Seeds were dispersed into in a solution containing 0.1% (v/v) Triton X-100 (Sigma) and sterile water and washed by shaking the suspension for 20 min. The wash solution was then drained and replaced with fresh wash solution to wash the seeds for 20 min with shaking. After removal of the ethanol/detergent solution, a solution containing 0.1% (v/v) Triton X-100 and 30% (v/v) bleach (CLOROX; Clorox Corp. Oakland Calif.) was added to the seeds, and the suspension was shaken for 10 min. After removal of the bleach/detergent solution, seeds were then washed five times in sterile distilled water. The seeds were stored in the last wash water at 4.degree. C. for 2 days in the dark before being plated onto antibiotic selection medium (1.times. Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH), 1.times. Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin). Seeds were germinated under continuous illumination (50-75 .mu.E/m.sup.2/sec) at 22-230 C. After 7-10 days of growth under these conditions, kanamycin resistant primary transformants (T1 generation) were visible and obtained. These seedlings were transferred first to fresh selection plates where the seedlings continued to grow for 3-5 more days, and then to soil (Pro-Mix BX potting medium).

[0357] Primary transformants were crossed and progeny seeds (T.sub.2) collected; kanamycin resistant seedlings were selected and analyzed. The expression levels of the recombinant polynucleotides in the transformants vary from about a 5% expression level increase to a least a 100% expression level increase. Similar observations are made with respect to polypeptide level expression.

Example VI

Identification of Arabidopsis Plants with Transcription Factor Gene Knockouts

[0358] The screening of insertion mutagenized Arabidopsis collections for null mutants in a known target gene was essentially as described in Krysan et al. (1999) Plant Cell 11: 2283-2290. Briefly, gene-specific primers, nested by 5-250 base pairs to each other, were designed from the 5' and 3' regions of a known target gene. Similarly, nested sets of primers were also created specific to each of the T-DNA or transposon ends (the "right" and "left" borders). All possible combinations of gene specific and T-DNA/transposon primers were used to detect by PCR an insertion event within or close to the target gene. The amplified DNA fragments were then sequenced which allows the precise determination of the T-DNA/transposon insertion point relative to the target gene. Insertion events within the coding or intervening sequence of the genes were deconvoluted from a pool comprising a plurality of insertion events to a single unique mutant plant for functional characterization. The method is described in more detail in Yu and Adam, U.S. application Ser. No. 09/177,733 filed Oct. 23, 1998.

Example VII

Identification of Modified Phenotypes in Overexpression or Gene Knockout Plants

[0359] Experiments were performed to identify those transformants or knockouts that exhibited modified biochemical characteristics.

[0360] Calibration of NIRS response was performed using data obtained by wet chemical analysis of a population of Arabidopsis ecotypes that were expected to represent diversity of oil and protein levels.

[0361] Experiments were performed to identify those transformants or knockouts that exhibited modified sugar-sensing. For such studies, seeds from transformants were germinated on media containing 5% glucose or 9.4% sucrose which normally partially restrict hypocotyl elongation. Plants with altered sugar sensing may have either longer or shorter hypocotyls than normal plants when grown on this media. Additionally, other plant traits may be varied such as root mass.

[0362] In some instances, expression patterns of the stress-induced genes may be monitored by microarray experiments. In these experiments, cDNAs are generated by PCR and resuspended at a final concentration of .about.100 ng/ul in 3.times.SSC or 150 mM Na-phosphate (Eisen and Brown (1999) Methods Enzymol. 303: 179-205). The cDNAs are spotted on microscope glass slides coated with polylysine. The prepared cDNAs are aliquoted into 384 well plates and spotted on the slides using, for example, an x-y-z gantry (OmniGrid) which may be purchased from GeneMachines (Menlo Park, Calif.) outfitted with quill type pins which may be purchased from Telechem International (Sunnyvale, Calif.). After spotting, the arrays are cured for a minimum of one week at room temperature, rehydrated and blocked following the protocol recommended by Eisen and Brown (1999; supra).

[0363] Sample total RNA (10 .mu.g) samples are labeled using fluorescent Cy3 and Cy5 dyes. Labeled samples are resuspended in 4.times.SSC/0.03% SDS/4 .mu.g salmon sperm DNA/2 .mu.g tRNA/ 50 mM Na-pyrophosphate, heated for 95.degree. C. for 2.5 minutes, spun down and placed on the array. The array is then covered with a glass coverslip and placed in a sealed chamber. The chamber is then kept in a water bath at 62.degree. C. overnight. The arrays are washed as described in Eisen and Brown (1999, supra) and scanned on a General Scanning 3000 laser scanner. The resulting files are subsequently quantified using IMAGENE, software (BioDiscovery, Los Angeles Calif.).

[0364] RT-PCR experiments may be performed to identify those genes induced after exposure to osmotic stress. Generally, the gene expression patterns from ground plant leaf tissue is examined. Reverse transcriptase PCR was conducted using gene specific primers within the coding region for each sequence identified. The primers were designed near the 3' region of each DNA binding sequence initially identified.

[0365] Total RNA from these ground leaf tissues was isolated using the CTAB extraction protocol. Once extracted total RNA was normalized in concentration across all the tissue types to ensure that the PCR reaction for each tissue received the same amount of cDNA template using the 28S band as reference. Poly(A+) RNA was purified using a modified protocol from the Qiagen OLIGOTEX purification kit batch protocol. cDNA was synthesized using standard protocols. After the first strand cDNA synthesis, primers for Actin 2 were used to normalize the concentration of cDNA across the tissue types. Actin 2 is found to be constitutively expressed in fairly equal levels across the tissue types we are investigating.

[0366] For RT PCR, cDNA template was mixed with corresponding primers and Taq DNA polymerase. Each reaction consisted of 0.2 .mu.l cDNA template, 2 .mu.l 10.times. Tricine buffer, 2 .mu.l 10.times. Tricine buffer and 16.8 .mu.l water, 0.05 .mu.l Primer 1, 0.05 .mu.l, Primer 2, 0.3 .mu.l Taq DNA polymerase and 8.6 .mu.l water.

[0367] The 96 well plate is covered with microfilm and set in the thermocycler to start the reaction cycle. By way of illustration, the reaction cycle may comprise the following steps:

[0368] Step 1: 93.degree. C. for 3 min;

[0369] Step 2: 93.degree. C. for 30 sec;

[0370] Step 3: 650 C for 1 min;

[0371] Step 4: 72.degree. C. for 2 min;

[0372] Steps 2, 3 and 4 are repeated for 28 cycles;

[0373] Step 5: 72.degree. C. for 5 min; and

[0374] STEP 6 4.degree. C.

[0375] To amplify more products, for example, to identify genes that have very low expression, additional steps may be performed: The following method illustrates a method that may be used in this regard. The PCR plate is placed back in the thermocycler for 8 more cycles of steps 2-4.

[0376] Step 2 93.degree. C. for 30 sec;

[0377] Step 3 65.degree. C. for 1 min;

[0378] Step 4 72.degree. C. for 2 min, repeated for 8 cycles; and

[0379] Step 5 4.degree. C.

[0380] Eight microliters of PCR product and 1.5 .mu.l of loading dye are loaded on a 1.2% agarose gel for analysis after 28 cycles and 36 cycles. Expression levels of specific transcripts are considered low if they were only detectable after 36 cycles of PCR. Expression levels are considered medium or high depending on the levels of transcript compared with observed transcript levels for an internal control such as actin2. Transcript levels are determined in repeat experiments and compared to transcript levels in control (e.g., non-transformed) plants.

[0381] Experiments were performed to identify those transformants or knockouts that exhibited an improved environmental stress tolerance. For such studies, the transformants were exposed to a variety of environmental stresses.

[0382] Germination assays all followed modifications of the same basic protocol. Sterile seeds were sown on the following conditional media. Plates were incubated at 22.degree. C. under 24-hour light (120-130 .mu.Ein/m.sup.2/s) in a growth chamber. Evaluation of germination and seedling vigor was conducted 3 to 15 days after planting. The basal media was 80% Murashige-Skoog medium (MS)+vitamins.

[0383] For salt and osmotic stress experiments, the medium was supplemented with 150 mM NaCl or 300 mM mannitol.

[0384] Carbon/nitrogen sensing experiments were conducted in basal media minus nitrogen plus 3% sucrose (--N) or in--basal media minus nitrogen plus 3% sucrose and 1 mM glutamine (N/+Gln).

[0385] Growth regulator sensitivity assays were performed in MS media, vitamins, and either 0.3 .mu.M ABA, 9.4% sucrose 9.4%, or 5% glucose.

[0386] Temperature stress cold germination experiments were carried out at 8.degree. C. Heat stress germination experiments were conducted at 32.degree. C. to 37.degree. C. for 6 hours of exposure.

[0387] For stress experiments conducted with more mature plants, seeds were germinated and grown for seven days on MS+vitamins+1% sucrose at 22.degree. C. and then transferred to chilling and heat stress conditions. The plants were either exposed to chilling stress (6 hour exposure to 4-8.degree. C. ), or heat stress (32.degree. C. was applied for five days, after which the plants were transferred back 22.degree. C. for recovery and evaluated after 5 days relative to controls not exposed to the depressed or elevated temperature).

[0388] high salt stress (6 hour exposure to 200 mM NaCl), drought stress (168 hours after removing water from trays), osmotic stress (6 hour exposure to 3 M mannitol), or nutrient limitation (nitrogen, phosphate, and potassium) (nitrogen: all components of MS medium remained constant except N was reduced to 20 mg/l of NH.sub.4NO.sub.3; phosphate: all components of MS medium except KH2PO.sub.4, which was replaced by K.sub.2SO.sub.4; potassium: all components of MS medium except removal of KNO3 and KH.sub.2PO.sub.4, which were replaced by NaH.sub.4PO.sub.4).

[0389] Modified phenotypes observed for particular overexpressor or knockout plants are provided. For a particular overexpressor that shows a less beneficial characteristic, it may be more useful to select a plant with a decreased expression of the particular transcription factor. For a particular knockout that shows a less beneficial characteristic, it may be more useful to select a plant with an increased expression of the particular transcription factor.

[0390] The sequences of the Sequence Listing, can be used to prepare transgenic plants and plants with altered osmotic stress tolerance. The specific transgenic plants listed below are produced from the sequences of the Sequence Listing, as noted.

Example VIII

Genes that Confer Significant Improvements to Plants

[0391] Examples of genes and homologs that confer significant improvements to knockout or overexpressing plants are noted below. Experimental observations made by us with regard to specific genes whose expression has been modified in overexpressing or knock-out plants, and potential applications based on these observations, are also presented.

[0392] This example provides experimental evidence for increased biomass and abiotic stress tolerance controlled by the transcription factor polypeptides and polypeptides of the invention.

[0393] Salt stress assays are intended to find genes that confer better germination, seedling vigor or growth in high salt. Evaporation from the soil surface causes upward water movement and salt accumulation in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt concentration of in the whole soil profile.

[0394] Plants differ in their tolerance to NaCl depending on their stage of development, therefore seed germination, seedling vigor, and plant growth responses are evaluated.

[0395] Osmotic stress assays (including NaCl and mannitol assays) are intended to determine if an osmotic stress phenotype is NaCl-specific or if it is a general osmotic stress related phenotype. Plants tolerant to osmotic stress could also have more tolerance to drought and/or freezing.

[0396] Drought assays are intended to find genes that mediate better plant survival after short-term, severe water deprivation. Ion leakage will be measured if needed. Osmotic stress tolerance would also support a drought tolerant phenotype.

[0397] Temperature stress assays are intended to find genes that confer better germination, seedling vigor or plant growth under temperature stress (cold, freezing and heat).

[0398] Sugar sensing assays are intended to find genes involved in sugar sensing by germinating seeds on high concentrations of sucrose and glucose and looking for degrees of hypocotyl elongation. The germination assay on mannitol controls for responses related to osmotic stress. Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Sucrose is the major transport form of photosynthate and its flux through cells has been shown to affect gene expression and alter storage compound accumulation in seeds (source-sink relationships). Glucose-specific hexose-sensing has also been described in plants and is implicated in cell division and repression of "famine" genes (photosynthetic or glyoxylate cycles).

[0399] Germination assays followed modifications of the same basic protocol. Sterile seeds were sown on the conditional media listed below. Plates were incubated at 22.degree. C. under 24-hour light (120-130 .mu.Ein/m.sup.2/s) in a growth chamber. Evaluation of germination and seedling vigor was conducted 3 to 15 days after planting. The basal media was 80% Murashige-Skoog medium (MS)+vitamins.

[0400] For salt and osmotic stress germination experiments, the medium was supplemented with 150 mM NaCl or 300 mM mannitol. Growth regulator sensitivity assays were performed in MS media, vitamins, and either 0.3 .mu.M ABA, 9.4% sucrose, or 5% glucose.

[0401] Temperature stress cold germination experiments were carried out at 8.degree. C. Heat stress germination experiments were conducted at 32.degree. C. to 37.degree. C. for 6 hours of exposure.

[0402] For stress experiments conducted with more mature plants, seeds were germinated and grown for seven days on MS +vitamins +1% sucrose at 22.degree. C. and then transferred to chilling and heat stress conditions. The plants were either exposed to chilling stress (6 hour exposure to 4-8.degree. C. ), or heat stress (32.degree. C. was applied for five days, after which the plants were transferred back 22.degree. C. for recovery and evaluated after 5 days relative to controls not exposed to the depressed or elevated temperature).

[0403] Results:

[0404] The overexpression of A. thaliana genes G481, G482, G485 and rice ortholog G3395 has been shown to increase osmotic stress tolerance. As noted below, changes in the activity of the G481 clade also produce alterations in flowering time.

[0405] G481 (Polynucleotide SEQ ID NO: 1)

[0406] Published Information

[0407] G481 is equivalent to AtHAP3a which was identified by Edwards et al., ((1998) Plant Physiol. 117: 1015-1022) as an EST with extensive sequence homology to the yeast HAP3. Northern blot data from five different tissue samples indicates that G481 is primarily expressed in flower and/or silique, and root tissue. No other functional data is available for G481 in Arabidopsis.

[0408] Closely Related Genes from Other Species

[0409] There are several genes in the database from higher plants that show significant homology to G481 including, X59714 from corn, and two ESTs from tomato, AJ486503 and A1782351.

[0410] Experimental Observations

[0411] The function of G481 was analyzed through its ectopic overexpression in plants. Except for darker color in one line (noted below), plants overexpressing G481 had a wild-type morphology. G481 overexpressors were found to be more tolerant to high sucrose and high salt (the latter is seen in FIG. 8A), having better germination, longer radicles, and more cotyledon expansion. There was a consistent difference in the hypocotyl and root elongation in the overexpressor compared to wild-type controls. These results indicated that G481 is involved in sucrose-specific sugar sensing. Sucrose-sensing has been implicated in the regulation of source-sink relationships in plants.

[0412] In the T2 generation, one overexpressing line was darker green than wild-type plants, which may indicate a higher photosynthetic rate that would be consistent with the role of G481 in sugar sensing.

[0413] 35S::G481 plants were also significantly larger and greener in a soil-based drought assay than wild-type controls plants After eight days of drought treatment overexpressing lines had a darker green and less withered appearance (FIG. 7C) than those in the control group (FIG. 7A). The differences in appearance between the control and G481-overexpressing plants after they were rewatered was even more striking. Eleven of twelve plants of this set of control plants died after rewatering (FIG. 7B), indicating the inability to recover following severe water deprivation, whereas all nine of the overexpressor plants of the line shown recovered from this drought treatment (FIG. 7D). The results shown in FIGS. 7A-7D were typical of a number of control and 35S::G481-overexpressing lines.

[0414] One line of plants in which G481 was overexpressed under the control of the ARSK1 root-specific promoter was found to germinate better under cold conditions than wild-type plants.

[0415] Interestingly, in one Arabidopsis line in which G481 was knocked out, the plants were found to be more sensitive to high salt in a plate-based assay than wild-type plants, which indicates the importance of the role played by G481 in regulating osmotic stress tolerance, and demonstrates that the gene is both necessary and sufficient to fulfill that function.

[0416] A number of the 35S::G481 plants evaluated had a late flowering phenotype.

[0417] Utilities

[0418] The potential utility of G481 includes altering photosynthetic rate, which could also impact yield in vegetative tissues as well as seed. Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Sucrose is the major transport form of photosynthate and its flux through cells has been shown to affect gene expression and alter storage compound accumulation in seeds (source-sink relationships).

[0419] Since G481 overexpressing plants performed better than controls in drought experiments, this gene or its equivalogs may be used to improve seedling vigor, plant survival, as well as yield, quality, and range.

[0420] G482 (Polynucleotide SEQ ID NO: 3)

[0421] Published Information

[0422] G482 is equivalent to AtHAP3b which was identified by Edwards et al. (1998) Plant Physiol. 117: 1015-1022) as an EST with homology to the yeast gene HAP3b. Their northern blot data suggests that AtHAP3b is expressed primarily in roots. No other functional information regarding G482 is publicly available.

[0423] Closely Related Genes from Other Species

[0424] The closest homology in the non-Arabidopsis plant database is within the B domain of G482, and therefore no potentially orthologous genes are available in the public domain.

[0425] Experimental Observations

[0426] RT-PCR analysis of endogenous levels of G482 transcripts indicated that this gene is expressed constitutively in all tissues tested. A cDNA array experiment supports the RT-PCR derived tissue distribution data. G482 is not induced above basal levels in response to any environmental stress treatments tested.

[0427] A T-DNA insertion mutant for G482 was analyzed and was found to flower slightly later than control plants.

[0428] The function of G482 was also analyzed through its ectopic overexpression in plants. Plants overexpressing G482 had a wild-type morphology. Germination assays to measure salt tolerance demonstrated increased seedling growth when germinated on the high salt medium (FIG. 8B).

[0429] 35S::G482 transgenic plants also displayed an osmotic stress response phenotype similar to 35S::G481 transgenic lines. Five of ten overexpressing lines had increased seedling growth on medium containing 80% MS plus vitamins with 300 mM mannitol.

[0430] Three of ten 35S::G482 lines also demonstrated enhanced germination relative to controls after 6 h exposure to 32.degree. C.

[0431] The majority of these 35S::G482 lines also demonstrated a slightly early flowering phenotype.

[0432] Utilities

[0433] The potential utilities of this gene include the ability to confer osmotic stress tolerance, as measured by salt, heat tolerance and improved germination in mannitol-containing media, during the germination stage of a crop plant. This would most likely impact survivability and yield. Evaporation of water from the soil surface causes upward water movement and salt accumulation in the upper soil layer, where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt concentration in the whole soil profile.

[0434] Improved osmotic stress tolerance is also likely to result in enhanced seedling vigor, plant survival, improved yield, quality, and range. Osmotic stress assays, including subjecting plants to aqueous dissolved sugars, are often used as surrogate assays for improved water-stress (e.g., drought) response. Thus, G482 may also be used to improve plant performance under conditions of water deprivation, including increased seedling vigor, plant survival, yield, quality, and range.

[0435] G485 (Polynucleotide SEQ ID NO: 5)

[0436] Published Information

[0437] G485 is a member of the HAP3-like subfamily of CCAAT-box binding transcription factors. G485 corresponds to gene At4g14540, annotated by the Arabidopsis Genome Initiative. The gene corresponds to sequence 1042 from patent application W00216655 (Harper et al. (2002)) on stress-regulated genes, transgenic plants and methods of use. In this application, G485 was reported to be cold responsive in their microarray analysis. No information is available about the function(s) of G485.

[0438] Experimental Observations

[0439] RT-PCR analyses of the endogenous levels of G485 indicated that this gene is expressed in all tissues and under all conditions tested.

[0440] A T-DNA insertion mutant for G485 was analyzed and was found to flower several days later than control plants (FIG. 11A).

[0441] The effects of G485 overexpression were also studied. Interestingly, the gain of function and loss of function studies on G485 reveal opposing effects on flowering time. Under conditions of continuous light, approximately half of the 35S::G485 primary transformants flowered distinctly earlier than wild-type controls (up to a week sooner in 24-hour light) (FIG. 11C). These effects were observed in each of two independent T1 plantings derived from separate transformation dates. Additionally, accelerated flowering was also seen in plants that overexpressed G485 from a two component system (35S::LexA;op-LexA::G485). These studies indicated that G485 is both sufficient to act as a floral activator, and is also necessary in that role within the plant. It should be noted that overexpression of G1820 (SEQ ID NO: 68), a member of the HAP5-like subfamily of CCAAT-box binding transcription factors had a similar effect on flowering time as G485. It is possible that G1820 interacts with G485 as part of a complex that binds and regulates the promoters of target genes involved in the regulation of flowering.

[0442] G485 overexpressor plants also matured and set siliques much more rapidly than wild type controls (FIG. 11B ).

[0443] G485 overexpressing plants were shown to have enhanced response to stress-related treatments in plate-based germination assays. As seen in FIGS. 10A-10D and Table 6, 35S::G485 lines showed enhanced cotyledon expansion and root growth seen in the overexpressing seedlings in cold, high sucrose, high salt and ABA treatments, as compared to wild-type controls with the same treatments seen in FIGS. 10E-10H.

[0444] Utilities

[0445] Based on the loss of function and gain of function phenotypes, G485 could be used to modify flowering time.

[0446] The delayed flowering displayed by G485 knockouts suggests that the gene might be used to manipulate the flowering time of commercial species. In particular, an extension of vegetative growth can significantly increase biomass and result in substantial yield increases. In some species (for example sugar beet), where the vegetative parts of the plant constitute the crop, it would be advantageous to delay or suppress flowering in order to prevent resources being diverted into reproductive development. Additionally, delaying flowering beyond the normal time of harvest could alleviate the risk of transgenic pollen escape from such crops.

[0447] The early flowering effects see in the G485 overexpressors could be applied to accelerate flowering, or eliminate any requirement for vernalization. In some instances, a faster cycling time might allow additional harvests of a crop to be made within a given growing season. Shortening generation times could also help speed-up breeding programs, particularly in species such as trees, which typically grow for many years before flowering.

[0448] Table 6 provides a summary of the data collected from one series of experiments conducted with plants overexpressing G482 or a paralog of G482. In each case the promoter used for regulating the introduced transcription factor was the cauliflower mosaic virus 35 S transcription initiation region. The column headings include the transcription factors used to transform the Arabidopsis plants listed by Gene ID (GID) numbers, the corresponding polypeptide SEQ ID NO; the project type indicating the nature of the promoter-gene interaction, and the ratio of lines determine to have one of the enhanced abiotic stress phenotypes listed over the number of lines tested

[0449] G3395 (Polynucleotide SEQ ID NO: 73)

[0450] Published Information

[0451] G3395, an ortholog of G482, is a member of the HAP3-like subfamily of CCAAT-box binding transcription factors. G3395 corresponds to polypeptide BAC76331 ("NF-YB subunit of rice").

[0452] Closely Related Genes from Other Species

[0453] The most closely related gene sequence found in GenBank appears to be the nearly identical AB095438 ("OsNF-YB2 mRNA for NF-YB").

[0454] Experimental Observations

[0455] The function of G3395 was analyzed through its ectopic overexpression in plants. One of the lines of G3395 overexpressors tested was found to be more tolerant to high salt levels, producing larger and greener seedlings in a high salt germination assay.

[0456] Utilities

[0457] The potential utilities of this gene include the ability to confer osmotic stress tolerance, particularly during the germination stage of a crop plant.

9TABLE 6 Summary of Results of Physiological Assays. One or two Overexpressor lines showing phenotype Component Improved Improved Improved Polypeptide Transformation Heat Drought germ. in germ. in ABA germ. in GID SEQ ID NO Promoter Type tolerance tolerance high NaCl high sugar sens. cold G482 2 CaMV 35S 2-components- + +* supTfn CaMV 35S Direct + promoter-fusion G481 4 CaMV 35S Direct + ++ promoter-fusion ARSK1 2-components- ++ supTfn CaMV 35S Superactivation + CaMV 35S RNAi (GS) ++ + +** G485 6 CaMV 35S 2-components- + +** + + supTfn G3395 74 CaMV 35S Direct + promoter-fusion *Mannitol **Sucrose Abbreviations: Sens. Sensitivity Germ. Germination + Moderate trait manifestation in one or more lines tested ++ Strong trait manifestation in one or more lines tested

EXAMPLE IX

CCAAT Family Transcription Factors and Flowering Time

[0458] We have also found that overexpressed CCAAT genes also have a highly noticeable effect on the timing of onset of flowering. G482 (SEQ ID NO: 3), G485 (SEQ ID NO: 5), G1248 (SEQ ID NO: 69), G1781 (SEQ ID NO: 71) and related crop orthologs G3398 (SEQ ID NO: 75), G3435 (SEQ ID NO: 47), and G3436 (SEQ ID NO: 49), accelerate onset of flowering when overexpressed in Arabidopsis.

[0459] Conversely, overexpression of G481, G1364 and related crop orthologs G3471 (SEQ ID NO:

[0460] 23), G3434 (SEQ ID NO: 77), and G3395 (SEQ ID NO: 73), produce a slight but reproducible delay in flowering in Arabidopsis. Results of knockout and RNAi studies confirm these findings. Knocked-out G485 and G482 plants exhibit a delay in flowering, and RNAi lines (using a construct designed to knock-out any member of the clade) are late flowering.

[0461] Thus, it appears that genes in the node of the tree clustered around G481 act to repress flowering, whereas those clustered around G482 and G485 act to promote flowering.

[0462] Interestingly, the addition of an activation domain appears to convert a floral repressor to a floral activator. Overexpression of a fusion protein comprising G481 fused at its carboxyl end with a GAL4 activation domain causes early flowering that is comparable to the effects caused by G482 or G482 overexpression.

[0463] An alignment of some of these HAP3 genes, seen in FIGS. 6A-6F, shows the high degree of conservation within the B domain, particularly in the B domain extending from FIG. 6B through FIG. 6C. These proteins are almost identical within the B domain, but the composition of two residue positions within the B domain correlates with effects of expression on flowering. These positions are indicated by arrows in FIG. 6B. The residue position indicated by the downward-pointing arrow in FIG. 6B is a serine residue in G1364, G2345 and G481 and a glycine residue in G482 and G485. The composition at this position correlate with flowering time when the polypeptide is overexpressed. The former group with a serine residue at this position induces late flowering when overexpressed, whereas the latter group with the glycine residue is distinguished by very early flowering upon overexpression. This study was expanded to include other polypeptides of the HAP3 family that compared the effects on flowering time and the relationship to the serine/glycine residue, including orthologous soy, corn and rice polypeptides. In each case, a glycine present at this position was associated with early flowering, and a serine residue was associated with a delay in flowering (G486 was found to possess a cysteine residue at this position, and one overexpressing T2 line appeared to have a late flowering phenotype). Similar observations were made with respect the other residue position, as indicated by the upward-pointing arrow in FIG. 6B) where orthologous polypeptides that cause late flowering, including soy, corn and rice polypeptides, possess a glycine or alanine residue at this position, and orthologs derived these species that produce an early flowering phenotype have a serine residue at the position. These results suggest that these residue positions are essential for determining whether these polypeptides are able to interact effectively with their partners in the multi-subunit complex and bind effectively to a promoter CCAAT box.

Example X

Identification of Homologous Sequences

[0464] This example describes identification of genes that are orthologous to Arabidopsis thaliana transcription factors from a computer homology search.

[0465] Homologous sequences, including those of paralogs and orthologs from Arabidopsis and other plant species, were identified using database sequence search tools, such as the Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215: 403-410; and Altschul et al. (1997) Nucleic Acid Res. 25: 3389-3402). The tblastx sequence analysis programs were employed using the BLOSUM-62 scoring matrix (Henikoff and Henikoff(1992) Proc. Natl. Acad. Sci. 89: 10915-10919). The entire NCBI GenBank database was filtered for sequences from all plants except Arabidopsis thaliana by selecting all entries in the NCBI GenBank database associated with NCBI taxonomic ID 33090 (Viridiplantae; all plants) and excluding entries associated with taxonomic ID 3701 (Arabidopsis thaliana).

[0466] These sequences are compared to sequences SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or polynucleotides that encode polypeptide SEQ ID NOs: 29-32, using the Washington University TBLASTX algorithm (version 2.0a19MP) at the default settings using gapped alignments with the filter "off". For each these genes, individual comparisons were ordered by probability score (P-value), where the score reflects the probability that a particular alignment occurred by chance. For example, a score of 3.6e40 is 3.6.times.10-40. In addition to P-values, comparisons were also scored by percentage identity. Percentage identity reflects the degree to which two segments of DNA or protein are identical over a particular length. Examples of sequences so identified are presented in Table 5. The percent sequence identity among these sequences can be as low as 49%, or even lower sequence identity.

[0467] Candidate paralogous sequences were identified among Arabidopsis transcription factors through alignment, identity, and phylogenic relationships. Paralogs of G481 so determined include G482, G485, G1364, and G2345. Candidate orthologous sequences were identified from proprietary unigene sets of plant gene sequences in Zea mays, Glycine max and Oryza sativa based on significant homology to Arabidopsis transcription factors. These candidates were reciprocally compared to the set of Arabidopsis transcription factors. If the candidate showed maximal similarity in the protein domain to the eliciting transcription factor or to a paralog of the eliciting transcription factor, then it was considered to be an ortholog. Identified non-Arabidopsis sequences that were shown in this manner to be orthologous to the Arabidopsis sequences are provided in Table 5.

Example XI

Screen of Plant cDNA Library for Sequence Encoding a Transcription Factor DNA Binding Domain that Binds to a Transcription Factor Binding Promoter Element and Demonstration of Protein Transcription Regulation Activity

[0468] The "one-hybrid" strategy (Li and Herskowitz (1993) Science 262: 1870-1874) is used to screen for plant cDNA clones encoding a polypeptide comprising a transcription factor DNA binding domain, a conserved domain. In brief, yeast strains are constructed that contain a lacZ reporter gene with either wild-type or mutant transcription factor binding promoter element sequences in place of the normal UAS (upstream activator sequence) of the GALL promoter. Yeast reporter strains are constructed that carry transcription factor binding promoter element sequences as UAS elements are operably linked upstream (5') of a lacZ reporter gene with a minimal GAL1 promoter. The strains are transformed with a plant expression library that contains random cDNA inserts fused to the GAL4 activation domain (GAL4-ACT) and screened for blue colony formation on X-gal-treated filters (X-gal: 5-bromo-4-chloro-3-indolyl-.beta.-D-galacto- side; Invitrogen Corporation, Carlsbad Calif.). Alternatively, the strains are transformed with a cDNA polynucleotide encoding a known transcription factor DNA binding domain polypeptide sequence.

[0469] Yeast strains carrying these reporter constructs produce low levels of beta-galactosidase and form white colonies on filters containing X-gal. The reporter strains carrying wild-type transcription factor binding promoter element sequences are transformed with a polynucleotide that encodes a polypeptide comprising a plant transcription factor DNA binding domain operably linked to the acidic activator domain of the yeast GAL4 transcription factor, "GAL4-ACT". The clones that contain a polynucleotide encoding a transcription factor DNA binding domain operably linked to GLA4-ACT can bind upstream of the lacZ reporter genes carrying the wild-type transcription factor binding promoter element sequence, activate transcription of the lacZ gene and result in yeast forming blue colonies on X-gal-treated filters.

[0470] Upon screening about 2.times.10.sup.6 yeast transformants, positive cDNA clones are isolated; i.e., clones that cause yeast strains carrying lacZ reporters operably linked to wild-type transcription factor binding promoter elements to form blue colonies on X-gal-treated filters. The cDNA clones do not cause a yeast strain carrying a mutant type transcription factor binding promoter elements fused to LacZ to turn blue. Thus, a polynucleotide encoding transcription factor DNA binding domain, a conserved domain, is shown to activate transcription of a gene.

Example XII

Gel Shift Assays

[0471] The presence of a transcription factor comprising a DNA binding domain which binds to a DNA transcription factor binding element is evaluated using the following gel shift assay. The transcription factor is recombinantly expressed and isolated from E. coli or isolated from plant material. Total soluble protein, including transcription factor, (40 ng) is incubated at room temperature in 10 .mu.l of 1.times. binding buffer (15 mM HEPES (pH 7.9), 1 mM EDTA, 30 mM KCl, 5% glycerol, 5% bovine serum albumin, 1 mM DTT) plus 50 ng poly(dl-dC):poly(dl-dC) (Pharmacia, Piscataway N.J.) with or without 100 ng competitor DNA. After 10 minutes incubation, probe DNA comprising a DNA transcription factor binding element (1 ng) that has been .sup.32P-labeled by end-filling (Sambrook et al. (1989) supra) is added and the mixture incubated for an additional 10 minutes. Samples are loaded onto polyacrylamide gels (4% w/v) and fractionated by electrophoresis at 150V for 2 h (Sambrook et al. supra). The degree of transcription factor-probe DNA binding is visualized using autoradiography. Probes and competitor DNAs are prepared from oligonucleotide inserts ligated into the BamHI site of pUC 118 (Vieira et al. (1987) Methods Enzymol. 153: 3-1 1). Orientation and concatenation number of the inserts are determined by dideoxy DNA sequence analysis (Sambrook et al. supra). Inserts are recovered after restriction digestion with EcoRI and HindIII and fractionation on polyacrylamide gels (12% w/v) (Sambrook et al. supra).

Example XIII

Introduction of Polynucleotides Into Dicotyledonous Plants

[0472] SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, polynucleotides that encode polypeptide SEQ ID NOs: 29-32, paralogous, and orthologous sequences recombined into pMEN20 or pMEN65 expression vectors are transformed into a plant for the purpose of modifying plant traits. The cloning vector may be introduced into a variety of cereal plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. It is now routine to produce transgenic plants using most dicot plants (see Weissbach and Weissbach, (1989) supra; Gelvin et al. (1990) supra; Herrera-Estrella et al. (1983) supra; Bevan (1984) supra; and Klee (1985) supra). Methods for analysis of traits are routine in the art and examples are disclosed above.

Example XIV

Transformation of Cereal Plants with an Expression Vector

[0473] Cereal plants such as, but not limited to, corn, wheat, rice, sorghum, or barley, may also be transformed with the present polynucleotide sequences in pMEN20 or pMEN65 expression vectors for the purpose of modifying plant traits. For example, pMEN020 may be modified to replace the NptII coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin. The KpnI and BgIII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.

[0474] The cloning vector may be introduced into a variety of cereal plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. It is now routine to produce transgenic plants of most cereal crops (Vasil (1994) Plant Mol. Biol. 25: 925-937) such as corn, wheat, rice, sorghum (Cassas et al. (1993) Proc. Natl. Acad. Sci. 90: 11212-11216, and barley (Wan and Lemeaux (1994) Plant Physiol. 104:37-48. DNA transfer methods such as the microprojectile can be used for corn (Fromm et al. (1990) Bio/Technol. 8: 833-839); Gordon-Kamm et al. (1990) Plant Cell 2: 603-618; Ishida (1990) Nature Biotechnol. 14:745-750), wheat (Vasil et al. (1992) Bio/Technol. 10:667-674; Vasil et al. (1993) Bio/Technol. 11:1553-1558; Weeks et al. (1993) Plant Physiol. 102:1077-1084), rice (Christou (1991) Bio/Technol. 9:957-962; Hiei et al. (1994) Plant J. 6:271-282; Aldemita and Hodges (1996) Planta 199:612-617; and Hiei et al. (1997) Plant Mol. Biol. 35:205-218). For most cereal plants, embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al. (1997) Plant Mol. Biol. 35:205-218; Vasil (1994) Plant Mol. Biol. 25: 925-937).

[0475] Vectors according to the present invention may be transformed into corn embryogenic cells derived from immature scutellar tissue by using microprojectile bombardment, with the A288XB73 genotype as the preferred genotype (Fromm et al. (1990) Bio/Technol. 8: 833-839; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al. (1990) Plant Cell 2: 603-618). Transgenic plants are regenerated by standard corn regeneration techniques (Fromm et al. (1990) Bio/Technol. 8: 833-839; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618).

[0476] The plasmids prepared as described above can also be used to produce transgenic wheat and rice plants (Christou (1991) Bio/Technol. 9:957-962; Hiei et al. (1994) Plant J. 6:271-282; Aldemita and Hodges (1996) Planta 199:612-617; and Hiei et al. (1997) Plant Mol. Biol. 35:205-218) that coordinately express genes of interest by following standard transformation protocols known to those skilled in the art for rice and wheat (Vasil et al. (1992) Bio/Technol. 10:667-674; Vasil et al. (1993) Bio/Technol. 11:1553-1558; and Weeks et al. (1993) Plant Physiol. 102:1077-1084), where the bar gene is used as the selectable marker.

Example XV

Genes that Confer Significant Improvements to Non-Arabidopsis Species

[0477] The function of orthologs of G481 and G482 may be analyzed through their ectopic overexpression in plants using the CaMV 35S or other appropriate promoter, as identified above. These genes encode members of the HAP3 subfamily of CCAAT-box binding transcription factors and include those found in Table 5, FIGS. 3 and 4, and, for example, polynucleotide sequences from Arabidopsis thaliana (SEQ ID NO: 1, 3, 5, 7, 9, 69, and 71), Glycine max (SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 51, 79, 81, 83, and 85), Solanum tuberosum (BQ505706), Medicago truncatula (AC122165), Lycopersicon esculentum (SEQ ID NO: 63, SEQ ID NO: 65, and BG642751), Rosa hybrid (BQ104671), Poncirus trifoliata (CD574709), Populus balsamifera subsp. trichocarpa (BU880488), Zea mays (SEQ ID NO: 33, 35, 37, 39,41, 43, 45, 47, 49, 55, 57, 77, 93, CC429501; and AX584265), Oryza sativa (SEQ ID NO: 27, 53, 73, 75, 87, 89, AAAA01003638, AP005193, AC108500, AP004366, AP003266, AP004179, AC104284, and AP120529), and Triticum aestivum (SEQ ID NO: 59, 61, and BJ248969). The function of specific HAP3 subfamily of CCAAT-box binding transcription factor genes that may be analyzed through ectopic overexpression in plants also includes rice nucleic acid sequences that encode polypeptides SEQ ID NO: 29-32, corn sequence gi115840, and wheat sequence gi16902058. These polynucleotide and polypeptide sequences derived from monocots may be used to transform both monocot and dicot plants, and those derived from dicots may also be used to transform either group, although some of these sequences will function best if the gene is transformed into the a plant from the same group as that from which the sequence is derived.

[0478] Seeds of these transgenic plants are subjected to germination assays to measure sucrose sensing. Sterile monocot seeds, including, but not limited to, corn, rice, wheat, rye and sorghum, as well as dicots including, but not limited to soybean and alfalfa, are sown on 80% MS medium plus vitamins with 9.4% sucrose; control media lack sucrose. All assay plates are then incubated at 22.degree. C. under 24-hour light, 120-130 .mu.Ein/m.sup.2/s, in a growth chamber. Evaluation of germination and seedling vigor is then conducted three days after planting. Overexpressors of these genes may be found to be more tolerant to high sucrose by having better germination, longer radicles, and more cotyledon expansion. These results would indicate that overexpressors of G482 orthologs are involved in sucrose-specific sugar sensing.

[0479] Plants overexpressing G482 orthologs may also be subjected to soil-based drought assays to identify those lines that are more tolerant to water deprivation than wild-type control plants. Generally, 35S: G482 ortholog overexpressing plants will appear significantly larger and greener, with less wilting or desiccation, than wild-type controls plants, particularly after a period of water deprivation is followed by rewatering and a subsequent incubation period.

Example XVI

Identification of Orthologous and Paralogous Sequences

[0480] Orthologs to Arabidopsis genes may identified by several methods, including hybridization, amplification, or bioinformatically. This example describes how one may identify homologs to the Arabidopsis AP2 family transcription factor CBF1 (polynucleotide SEQ ID NO: 95, encoded polypeptide SEQ ID NO: 96), which confers tolerance to abiotic stresses (Thomashow et al. (2002) U.S. Pat. No. 6,417,428), and an example to confirm the function of homologous sequences. In this example, orthologs to CBF1 were found in canola (Brassica napus) using polymerase chain reaction (PCR).

[0481] Degenerate primers were designed for regions of AP2 binding domain and outside of the AP2 (carboxyl terminal domain):

10 Mol 368 5'- CAY CCN ATH TAY MGN (SEQ ID NO: 103) (reverse) GGN GT -3' Mol 378 5'- GGN ARN ARC ATN CCY (SEQ ID NO: 104) (forward) TCN GCC -3' (Y: C/T, N: A/C/G/T, H: A/C/T, M: A/C, R: A/G)

[0482] Primer Mol 368 is in the AP2 binding domain of CBF1 (amino acid sequence: His-Pro-Ile-Tyr-Arg-Gly-Val) while primer Mol 378 is outside the AP2 domain (carboxyl terminal domain) (amino acid sequence: Met-Ala-Glu-Gly-Met-Leu-Leu-Pro).

[0483] The genomic DNA isolated from B. napus was PCR-amplified by using these primers following these conditions: an initial denaturation step of 2 min at 93.degree. C.; 35 cycles of 93.degree. C. for 1 min, 55.degree. C. for 1 min, and 72.degree. C. for 1 min; and a final incubation of 7 min at 72.degree. C. at the end of cycling

[0484] The PCR products were separated by electrophoresis on a 1.2% agarose gel and transferred to nylon membrane and hybridized with the AT CBF1 probe prepared from Arabidopsis genomic DNA by PCR amplification. The hybridized products were visualized by colorimetric detection system (Boehringer Mannheim) and the corresponding bands from a similar agarose gel were isolated using the Qiagen Extraction Kit (Qiagen). The DNA fragments were ligated into the TA clone vector from TOPO TA Cloning Kit (Invitrogen) and transformed into E. coli strain TOP 10 (Invitrogen).

[0485] Seven colonies were picked and the inserts were sequenced on an ABI 377 machine from both strands of sense and antisense after plasmid DNA isolation. The DNA sequence was edited by sequencer and aligned with the AtCBF1 by GCG software and NCBI blast searching.

[0486] The nucleic acid sequence and amino acid sequence of one canola ortholog found in this manner (bnCBF1; polynucleotide SEQ ID NO: 101 and polypeptide SEQ ID NO: 102) identified by this process is shown in the Sequence Listing.

[0487] The aligned amino acid sequences show that the bnCBF1 gene has 88% identity with the Arabidopsis sequence in the AP2 domain region and 85% identity with the Arabidopsis sequence outside the AP2 domain when aligned for two insertion sequences that are outside the AP2 domain.

[0488] Similarly, paralogous sequences to Arabidopsis genes, such as CBF1, may also be identified.

[0489] Two paralogs of CBF1 from Arabidopsis thaliana: CBF2 and CBF3. CBF2 and CBF3 have been cloned and sequenced as described below. The sequences of the DNA SEQ ID NO: 97 and 99 and encoded proteins SEQ ID NO: 98 and 100 are set forth in the Sequence Listing.

[0490] A lambda cDNA library prepared from RNA isolated from Arabidopsis thaliana ecotype Columbia (Lin and Thomashow (1992) Plant Physiol. 99: 519-525) was screened for recombinant clones that carried inserts related to the CBF1 gene (Stockinger et al. (1997) Proc. Natl. Acad. Sci. 94:1035-1040). CBF1 was .sup.32P-radiolabeled by random priming (Sambrook et al. supra) and used to screen the library by the plaque-lift technique using standard stringent hybridization and wash conditions (Hajela et al. (1990) Plant Physiol. 93:1246-1252; Sambrook et al. supra) 6.times.SSPE buffer, 60.degree. C. for hybridization and 0.1.times.SSPE buffer and 60.degree. C. for washes). Twelve positively hybridizing clones were obtained and the DNA sequences of the cDNA inserts were determined. The results indicated that the clones fell into three classes. One class carried inserts corresponding to CBF1. The two other classes carried sequences corresponding to two different homologs of CBF1, designated CBF2 and CBF3. The nucleic acid sequences and predicted protein coding sequences for Arabidopsis CBF1, CBF2 and CBF3 are listed in the Sequence Listing (SEQ ID NOs:95, 97, 99 and SEQ ID NOs: 96, 98, and 100, respectively). The nucleic acid sequences and predicted protein coding sequence for Brassica napus CBF ortholog is listed in the Sequence Listing (SEQ ID NOs: 101 and 102, respectively).

[0491] A comparison of the nucleic acid sequences of Arabidopsis CBF1, CBF2 and CBF3 indicate that they are 83 to 85% identical as shown in Table 7.

11 TABLE 7 Percent identity.sup.a DNA.sup.b Polypeptide cbf1/cbf2 85 86 cbf1/cbf3 83 84 cbf2/cbf3 84 85 .sup.aPercent identity was determined using the Clustal algorithm from the Megalign program (DNASTAR, Inc.). .sup.bComparisons of the nucleic acid sequences of the open reading frames are shown.

[0492] Similarly, the amino acid sequences of the three CBF polypeptides range from 84 to 86% identity. An alignment of the three amino acidic sequences reveals that most of the differences in amino acid sequence occur in the acidic C-terminal half of the polypeptide. This region of CBF 1 serves as an activation domain in both yeast and Arabidopsis (not shown).

[0493] Residues 47 to 106 of CBF1 correspond to the AP2 domain of the protein, a DNA binding motif that to date, has only been found in plant proteins. A comparison of the AP2 domains of CBF1, CBF2 and CBF3 indicates that there are a few differences in amino acid sequence. These differences in amino acid sequence might have an effect on DNA binding specificity.

Example XVII

Transformation of Canola with a Plasmid Containing CBF1, CBF2, or CBF3

[0494] After identifying homologous genes to CBF 1, canola was transformed with a plasmid containing the Arabidopsis CBF1, CBF2, or CBF3 genes cloned into the vector pGA643 (An (1987) Methods Enzymol. 253: 292). In these constructs the CBF genes were expressed constitutively under the CaMV 35S promoter. In addition, the CBF1 gene was cloned under the control of the Arabidopsis COR15 promoter in the same vector pGA643. Each construct was transformed into Agrobacterium strain GV3101. Transformed Agrobacteria were grown for 2 days in minimal AB medium containing appropriate antibiotics.

[0495] Spring canola (B. napus cv. Westar) was transformed using the protocol of Moloney et al. ((1989) Plant Cell Reports 8: 238) with some modifications as described. Briefly, seeds were sterilized and plated on half strength MS medium, containing 1% sucrose. Plates were incubated at 24.degree. C. under 60-80 .mu.E/m.sup.2s light using a16 hour light/8 hour dark photoperiod. Cotyledons from 4-5 day old seedlings were collected, the petioles cut and dipped into the Agrobacterium solution. The dipped cotyledons were placed on co-cultivation medium at a density of 20 cotyledons/plate and incubated as described above for 3 days. Explants were transferred to the same media, but containing 300 mg/l timentin (SmithKline Beecham, Pa.) and thinned to 10 cotyledons/plate. After 7 days explants were transferred to Selection/Regeneration medium. Transfers were continued every 2-3 weeks (2 or 3 times) until shoots had developed. Shoots were transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots were transferred to rooting medium. Once good roots had developed, the plants were placed into moist potting soil.

[0496] The transformed plants were analyzed for the presence of the NPTII gene/ kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, Colo.). Approximately 70% of the screened plants were NPTII positive; these plants were further analyzed.

[0497] From Northern blot analysis of the plants that were transformed with the constitutively expressing constructs, showed expression of the CBF genes and all CBF genes were capable of inducing the Brassica napus cold-regulated gene BN115 (homolog of the Arabidopsis COR15 gene). Most of the transgenic plants appear to exhibit a normal growth phenotype. As expected, the transgenic plants are more freezing tolerant than the wild-type plants. Using the electrolyte leakage of leaves test, the control showed a 50% leakage at -2 to -3.degree. C. Spring canola transformed with either CBF1 or CBF2 showed a 50% leakage at -6 to -7.degree. C. Spring canola transformed with CBF3 shows a 50% leakage at about -10 to -15.degree. C. Winter canola transformed with CBF3 may show a 50% leakage at about -16 to -20.degree. C. Furthermore, if the spring or winter canola are cold acclimated the transformed plants may exhibit a further increase in freezing tolerance of at least -2.degree. C.

[0498] To test salinity tolerance of the transformed plants, plants were watered with 150 mM NaCl. Plants overexpressing CBF1, CBF2 or CBF3 grew better compared with plants that had not been transformed with CBF1, CBF2 or CBF3.

[0499] These results demonstrate that homologs of Arabidopsis transcription factors can be identified and shown to confer similar functions in non-Arabidopsis plant species.

Example XVIII

Cloning of Transcription Factor Promoters

[0500] Promoters are isolated from transcription factor genes that have gene expression patterns useful for a range of applications, as determined by methods well known in the art (including transcript profile analysis with cDNA or oligonucleotide microarrays, Northern blot analysis, semi-quantitative or quantitative RT-PCR). Interesting gene expression profiles are revealed by determining transcript abundance for a selected transcription factor gene after exposure of plants to a range of different experimental conditions, and in a range of different tissue or organ types, or developmental stages. Experimental conditions to which plants are exposed for this purpose includes cold, heat, drought, osmotic challenge, varied hormone concentrations (ABA, GA, auxin, cytokinin, salicylic acid, brassinosteroid), pathogen and pest challenge. The tissue types and developmental stages include stem, root, flower, rosette leaves, cauline leaves, siliques, germinating seed, and meristematic tissue. The set of expression levels provides a pattern that is determined by the regulatory elements of the gene promoter.

[0501] Transcription factor promoters for the genes disclosed herein are obtained by cloning 1.5 kb to 2.0 kb of genomic sequence immediately upstream of the translation start codon for the coding sequence of the encoded transcription factor protein. This region includes the 5'-UTR of the transcription factor gene, which can comprise regulatory elements. The 1.5 kb to 2.0 kb region is cloned through PCR methods, using primers that include one in the 3' direction located at the translation start codon (including appropriate adaptor sequence), and one in the 5' direction located from 1.5 kb to 2.0 kb upstream of the translation start codon (including appropriate adaptor sequence). The desired fragments are PCR-amplified from Arabidopsis Col-0 genomic DNA using high-fidelity Taq DNA polymerase to minimize the incorporation of point mutation(s). The cloning primers incorporate two rare restriction sites, such as Not1 and Sfi1, found at low frequency throughout the Arabidopsis genome. Additional restriction sites are used in the instances where a Not1 or Sfi1 restriction site is present within the promoter.

[0502] The 1.5-2.0 kb fragment upstream from the translation start codon, including the 5'-untranslated region of the transcription factor, is cloned in a binary transformation vector immediately upstream of a suitable reporter gene, or a transactivator gene that is capable of programming expression of a reporter gene in a second gene construct. Reporter genes used include green fluorescent protein (and related fluorescent protein color variants), beta-glucuronidase, and luciferase. Suitable transactiivator genes include LexA-GAL4, along with a transactivatable reporter in a second binary plasmid (as disclosed in U.S. patent application Ser. No. 09/958,131, incorporated herein by reference). The binary plasmid(s) is transferred into Agrobacterium and the structure of the plasmid confirmed by PCR. These strains are introduced into Arabidopsis plants as described in other examples, and gene expression patterns determined according to standard methods know to one skilled in the art for monitoring GFP fluorescence, beta-glucuronidase activity, or luminescence.

[0503] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0504] The present invention is not limited by the specific embodiments described herein. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. Modifications that become apparent from the foregoing description and accompanying figures fall within the scope of the claims.

Sequence CWU 1

1

104 1 832 DNA Arabidopsis thaliana G481 1 gagcgtttcg tagaaaaatt cgatttctct aaagccctaa aactaaaacg actatcccca 60 attccaagtt ctagggtttc catcttcccc aatctagtat aaatggcgga tacgccttcg 120 agcccagctg gagatggcgg agaaagcggc ggttccgtta gggagcagga tcgatacctt 180 cctatagcta atatcagcag gatcatgaag aaagcgttgc ctcctaatgg taagattgga 240 aaagatgcta aggatacagt tcaggaatgc gtctctgagt tcatcagctt catcactagc 300 gaggccagtg ataagtgtca aaaagagaaa aggaaaactg tgaatggtga tgatttgttg 360 tgggcaatgg caacattagg atttgaggat tacctggaac ctctaaagat atacctagcg 420 aggtacaggg agttggaggg tgataataag ggatcaggaa agagtggaga tggatcaaat 480 agagatgctg gtggcggtgt ttctggtgaa gaaatgccga gctggtaaaa gaagttgcaa 540 gtagtgatta agaacaatcg ccaaatgatc aagggaaatt agagatcagt gagttgttta 600 tagttgagct gatcgacaac tatttcgggt ttactctcaa tttcggttat gttagtttga 660 acgtttggtt tattgtttcc ggtttagttg gttgtattta aagatttctc tgttagatgt 720 tgagaacact tgaatgaagg aaaaatttgt ccacatcctg ttgttatttt cgattcactt 780 tcggaatttc atagctaatt tattctcatt taataccaaa tccttaaatt aa 832 2 141 PRT Arabidopsis thaliana G481 polypeptide 2 Met Ala Asp Thr Pro Ser Ser Pro Ala Gly Asp Gly Gly Glu Ser Gly 1 5 10 15 Gly Ser Val Arg Glu Gln Asp Arg Tyr Leu Pro Ile Ala Asn Ile Ser 20 25 30 Arg Ile Met Lys Lys Ala Leu Pro Pro Asn Gly Lys Ile Gly Lys Asp 35 40 45 Ala Lys Asp Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55 60 Thr Ser Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Val 65 70 75 80 Asn Gly Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Asp 85 90 95 Tyr Leu Glu Pro Leu Lys Ile Tyr Leu Ala Arg Tyr Arg Glu Leu Glu 100 105 110 Gly Asp Asn Lys Gly Ser Gly Lys Ser Gly Asp Gly Ser Asn Arg Asp 115 120 125 Ala Gly Gly Gly Val Ser Gly Glu Glu Met Pro Ser Trp 130 135 140 3 1065 DNA Arabidopsis thaliana G482 3 tcgacccacg cgtccggaca cttaacaatt cacaccttct ctttttactc ttcctaaaac 60 cctaaatttc ctcgcttcag tcttcccact caagtcaacc accaattgaa ttcgatttcg 120 aatcattgat ggaaatgatt tgaaaaaaga gtaaagttta tttttttatt ccttgtaatt 180 ttcagaaatg ggggattccg acagggattc cggtggaggg caaaacggga acaaccagaa 240 cggacagtcc tccttgtctc caagagagca agacaggttc ttgccgatcg ctaacgtcag 300 ccggatcatg aagaaggcct tgcccgccaa cgccaagatc tctaaagatg ccaaagagac 360 gatgcaggag tgtgtctccg agttcatcag cttcgtcacc ggagaagcat ctgataagtg 420 tcagaaggag aagaggaaga cgatcaacgg agacgatttg ctctgggcta tgactactct 480 aggttttgag gattatgttg agccattgaa agtttacttg cagaggttta gggagatcga 540 aggggagagg actggactag ggaggccaca gactggtggt gaggtcggag agcatcagag 600 agatgctgtc ggagatggcg gtgggttcta cggtggtggt ggtgggatgc agtatcacca 660 acatcatcag tttcttcacc agcagaacca tatgtatgga gccacaggtg gcggtagcga 720 cagtggaggt ggagctgcct ccggtaggac aaggacttaa caaagattgg tgaagtggat 780 ctctctctgt atatagatac ataaatacat gtatacacat gcctattttt acgacccata 840 taaggtatct atcatgtgat agaacgaaca ttggtgttgg tgatgtaaaa tcagatgtgc 900 attaagggtt tagattttga ggctgtgtaa aagaagatca agtgtgcttt gttggacaat 960 aggattcact aacgaatctg cttcattgga tcttgtatgt aactaaagcc attgtattga 1020 atgcaaatgt tttcatttgg gatgctttaa aaaaaaaaaa aaaaa 1065 4 190 PRT Arabidopsis thaliana G482 polypeptide 4 Met Gly Asp Ser Asp Arg Asp Ser Gly Gly Gly Gln Asn Gly Asn Asn 1 5 10 15 Gln Asn Gly Gln Ser Ser Leu Ser Pro Arg Glu Gln Asp Arg Phe Leu 20 25 30 Pro Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn 35 40 45 Ala Lys Ile Ser Lys Asp Ala Lys Glu Thr Met Gln Glu Cys Val Ser 50 55 60 Glu Phe Ile Ser Phe Val Thr Gly Glu Ala Ser Asp Lys Cys Gln Lys 65 70 75 80 Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr 85 90 95 Thr Leu Gly Phe Glu Asp Tyr Val Glu Pro Leu Lys Val Tyr Leu Gln 100 105 110 Arg Phe Arg Glu Ile Glu Gly Glu Arg Thr Gly Leu Gly Arg Pro Gln 115 120 125 Thr Gly Gly Glu Val Gly Glu His Gln Arg Asp Ala Val Gly Asp Gly 130 135 140 Gly Gly Phe Tyr Gly Gly Gly Gly Gly Met Gln Tyr His Gln His His 145 150 155 160 Gln Phe Leu His Gln Gln Asn His Met Tyr Gly Ala Thr Gly Gly Gly 165 170 175 Ser Asp Ser Gly Gly Gly Ala Ala Ser Gly Arg Thr Arg Thr 180 185 190 5 486 DNA Arabidopsis thaliana G485 5 atggcggatt cggacaacga ttcaggagga cacaaagacg gtggaaatgc ttcgacacgt 60 gagcaagata ggtttctacc gatcgctaac gttagcagga tcatgaagaa agcacttcct 120 gcgaacgcaa aaatctctaa ggatgctaaa gaaacggttc aagagtgtgt atcggaattc 180 ataagtttca tcaccggtga ggcttctgac aagtgtcaga gagagaagag gaagacaatc 240 aacggtgacg atcttctttg ggcgatgact acgctagggt ttgaggacta cgtggagcct 300 ctcaaggttt atctgcaaaa gtatagggag gtggaaggag agaagactac tacggcaggg 360 agacaaggcg ataaggaagg tggaggagga ggcggtggag ctggaagtgg aagtggagga 420 gctccgatgt acggtggtgg catggtgact acgatgggac atcaattttc ccatcatttt 480 tcttaa 486 6 161 PRT Arabidopsis thaliana G485 polypeptide 6 Met Ala Asp Ser Asp Asn Asp Ser Gly Gly His Lys Asp Gly Gly Asn 1 5 10 15 Ala Ser Thr Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Ser 20 25 30 Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp 35 40 45 Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55 60 Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile 65 70 75 80 Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp 85 90 95 Tyr Val Glu Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Val Glu 100 105 110 Gly Glu Lys Thr Thr Thr Ala Gly Arg Gln Gly Asp Lys Glu Gly Gly 115 120 125 Gly Gly Gly Gly Gly Ala Gly Ser Gly Ser Gly Gly Ala Pro Met Tyr 130 135 140 Gly Gly Gly Met Val Thr Thr Met Gly His Gln Phe Ser His His Phe 145 150 155 160 Ser 7 537 DNA Arabidopsis thaliana G1364 7 atggcggagt cgcaggccaa gagtcccgga ggctgtggaa gccatgagag tggtggagat 60 caaagtccca ggtcgttaca tgttcgtgag caagataggt ttcttccgat tgctaacata 120 agccgtatca tgaaaagagg tcttcctgct aatgggaaaa tcgctaaaga tgctaaggag 180 attgtgcagg aatgtgtctc tgaattcatc agtttcgtca ccagcgaagc gagtgataaa 240 tgtcaaagag agaaaaggaa gactattaat ggagatgatt tgctttgggc aatggctact 300 ttaggatttg aagactacat ggaacctctc aaggtttacc tgatgagata tagagagggt 360 gacacaaagg gatcagcaaa aggtggggat ccaaatgcaa agaaagatgg gcaatcaagc 420 caaaatggcc agttctcgca gcttgctcac caaggtcctt atgggaactc tcaagtaact 480 tttcctctct tctcttcaca ctcaagcaat acgcatcatt ctcttctaat ttgttaa 537 8 178 PRT Arabidopsis thaliana G1364 polypeptide 8 Met Ala Glu Ser Gln Ala Lys Ser Pro Gly Gly Cys Gly Ser His Glu 1 5 10 15 Ser Gly Gly Asp Gln Ser Pro Arg Ser Leu His Val Arg Glu Gln Asp 20 25 30 Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Arg Gly Leu 35 40 45 Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Ile Val Gln Glu 50 55 60 Cys Val Ser Glu Phe Ile Ser Phe Val Thr Ser Glu Ala Ser Asp Lys 65 70 75 80 Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp 85 90 95 Ala Met Ala Thr Leu Gly Phe Glu Asp Tyr Met Glu Pro Leu Lys Val 100 105 110 Tyr Leu Met Arg Tyr Arg Glu Gly Asp Thr Lys Gly Ser Ala Lys Gly 115 120 125 Gly Asp Pro Asn Ala Lys Lys Asp Gly Gln Ser Ser Gln Asn Gly Gln 130 135 140 Phe Ser Gln Leu Ala His Gln Gly Pro Tyr Gly Asn Ser Gln Val Thr 145 150 155 160 Phe Pro Leu Phe Ser Ser His Ser Ser Asn Thr His His Ser Leu Leu 165 170 175 Ile Cys 9 687 DNA Arabidopsis thaliana G2345 9 atggccgaat cgcaaaccgg tggtggtggt ggtggaagcc atgagagtgg cggtgatcag 60 agcccgaggt ctttgaatgt tcgtgagcag gacaggtttc ttccgattgc taacataagc 120 cgtatcatga agagaggttt acctctaaat ggcaaaatcg ctaaagatgc taaagagact 180 atgcaggaat gtgtctctga attcatcagc ttcgtcacca gcgaggctag tgataagtgc 240 caaagagaga aaaggaagac catcaatgga gatgatttgc tttgggctat ggccacttta 300 ggattcgaag attacatcga tcccctcaag gtttacctga tgcgatatag agagatggag 360 ggtgacacta aaggatcagg aaaaggcggg gaatcgagtg caaagagaga tggtcaacca 420 agccaagtgt ctcagttctc gcaggttcct caacaaggct cattctcaca gggtccttat 480 ggaaactctc aatctctgag gttcggcaat agcatcgagc atcttgaagt gttaatgagt 540 agtactagga cactattcat cacaatcttc cgagactcga ctatgcctgt tgtgtctgag 600 aatctgagtg atccactttc catagatatg gattgtgaag ctatttatca ccacttcatt 660 ggcctgttga ttctttcatg caagtga 687 10 228 PRT Arabidopsis thaliana G2345 polypeptide 10 Met Ala Glu Ser Gln Thr Gly Gly Gly Gly Gly Gly Ser His Glu Ser 1 5 10 15 Gly Gly Asp Gln Ser Pro Arg Ser Leu Asn Val Arg Glu Gln Asp Arg 20 25 30 Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Arg Gly Leu Pro 35 40 45 Leu Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Thr Met Gln Glu Cys 50 55 60 Val Ser Glu Phe Ile Ser Phe Val Thr Ser Glu Ala Ser Asp Lys Cys 65 70 75 80 Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala 85 90 95 Met Ala Thr Leu Gly Phe Glu Asp Tyr Ile Asp Pro Leu Lys Val Tyr 100 105 110 Leu Met Arg Tyr Arg Glu Met Glu Gly Asp Thr Lys Gly Ser Gly Lys 115 120 125 Gly Gly Glu Ser Ser Ala Lys Arg Asp Gly Gln Pro Ser Gln Val Ser 130 135 140 Gln Phe Ser Gln Val Pro Gln Gln Gly Ser Phe Ser Gln Gly Pro Tyr 145 150 155 160 Gly Asn Ser Gln Ser Leu Arg Phe Gly Asn Ser Ile Glu His Leu Glu 165 170 175 Val Leu Met Ser Ser Thr Arg Thr Leu Phe Ile Thr Ile Phe Arg Asp 180 185 190 Ser Thr Met Pro Val Val Ser Glu Asn Leu Ser Asp Pro Leu Ser Ile 195 200 205 Asp Met Asp Cys Glu Ala Ile Tyr His His Phe Ile Gly Leu Leu Ile 210 215 220 Leu Ser Cys Lys 225 11 1131 DNA Glycine max GLYMA-28NOV01-CLUSTER24839_1 11 attcggctcg agataatcca gagagaggag agagaagtaa aaaggtggag gaagaagcga 60 aaagcgagtg agggcagtgt tgcttaataa aagaaaacga acggtggtga taggcttcag 120 tctagatctc aatcgtctcc accttgcttt cttctccagc gtccgattct ctcaccgatc 180 tcgcgccaaa tacaaattcg tgtcaaccca acccagggtt ccggcgagca tggccgacgg 240 tccggctagc ccaggcggcg gcagccacga gagcggcgac cacagccctc gctctaacgt 300 gcgcgagcag gacaggtacc tccctatcgc taacataagc cgcatcatga agaaggcact 360 tcctgccaac ggtaaaatcg caaaggacgc caaagagacc gttcaggaat gcgtctccga 420 gttcatcagc ttcatcacca gcgaggcctc tgataagtgt cagagagaaa agagaaagac 480 tattaacggc gatgatttgc tctgggcgat ggccactctc ggtttcgagg attatatgga 540 tcctcttaaa atttacctca ctagataccg agagatggag ggtgatacga agggctctgc 600 caagggtgga gactcatctg ctaagagaga tgttcagcca agtcctaatg ctcagcttgc 660 tcatcaaggt tctttctcac aaaatgttac ttacccgaat tctcagggtc gacatatgat 720 ggttccaatg caaggcccgg agtaggtatc aagtttatta ttgaccctct tgttgtaacg 780 tatgttttct acgccagtta ccaagtgctc acggcatatt gaatgtcttt ttatgttatg 840 tgaatactga caggagatgt tggttcttgt gtccgttttt ttttttaaat taaggtttgt 900 atattatctt tggattcgaa ttattatttg aaagttatta ttatattgta aatcctagag 960 ccctgttgtc tgaatccatc aggcggcttg gtaaagaccg agattttagg actgattgta 1020 agcataaatc cgaatattct tttcctaatt tctttgcgca ataatgtatg aaaaaggctc 1080 gagcttcttt ttaaaaaaaa aaaaaaagga acaaaaaaaa aaaagggggg g 1131 12 171 PRT Glycine max GLYMA-28NOV01-CLUSTER24839_1 polypeptide 12 Met Ala Asp Gly Pro Ala Ser Pro Gly Gly Gly Ser His Glu Ser Gly 1 5 10 15 Asp His Ser Pro Arg Ser Asn Val Arg Glu Gln Asp Arg Tyr Leu Pro 20 25 30 Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Gly 35 40 45 Lys Ile Ala Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu 50 55 60 Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Asp Lys Cys Gln Arg Glu 65 70 75 80 Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Ala Thr 85 90 95 Leu Gly Phe Glu Asp Tyr Met Asp Pro Leu Lys Ile Tyr Leu Thr Arg 100 105 110 Tyr Arg Glu Met Glu Gly Asp Thr Lys Gly Ser Ala Lys Gly Gly Asp 115 120 125 Ser Ser Ala Lys Arg Asp Val Gln Pro Ser Pro Asn Ala Gln Leu Ala 130 135 140 His Gln Gly Ser Phe Ser Gln Asn Val Thr Tyr Pro Asn Ser Gln Gly 145 150 155 160 Arg His Met Met Val Pro Met Gln Gly Pro Glu 165 170 13 993 DNA Glycine max GLYMA-28NOV01-CLUSTER31103_1 13 attcggctcg ggaggaacgt gaaagtaaaa cggacggtgg cgatagaagc gtctctcatc 60 tccatcgtct cctcactcct ctcttctcca gcgttcattt tttctcgcgc ccaaatacaa 120 aatcacatca caacagggtt ccggcgacca tgtccgatgc tccggcgagt ccatgcggcg 180 gcggcggcgg aggcagccac gagagcggcg agcacagtcc ccgctccaat ttccgcgagc 240 aggaccgctt cctccccatc gccaacatca gccgcatcat gaagaaagcg cttcctccca 300 acgggaaaat cgccaaggac gccaaggaaa ccgtgcagga atgcgtctcc gagttcatca 360 gcttcgtcac cagcgaagcg agcgataagt gtcagagaga gaagaggaag accatcaacg 420 gcgacgattt gctttgggct atgaccactt taggtttcga ggagtatatt gatccgctca 480 aggtttacct cgccgcttac agagagattg agggtgattc aaagggttcg gccaagggtg 540 gagatgcatc tgctaagaga gatgtttatc agagtcctaa tggccaggtt gctcatcaag 600 gttctttctc acaaggtgtt aattatacga attcttagcc ccaggctcaa catatgatag 660 ttccgatgca aggccaagag tagatattga tcctctcctt cagtgtttga catgtgtgat 720 ctaaatgcca gtggaacttt tatgtcaata tgtgcccttg gtatattgaa tgcattttat 780 gttatgtaaa cactacatgc ggggatgttg gttcttgtga ccagatatta tttattaaga 840 cttacattta tctttggaaa agaatcatta ttcataagtt atattgtaaa ttctggaaca 900 atgcttgtct gattccatca atcgtcctgg taacgatttt atgtacctga ttggaagcat 960 aaattggtat attctttccc ttcgttgtct gtt 993 14 162 PRT Glycine max GLYMA-28NOV01-CLUSTER31103_1 polypeptide 14 Met Ser Asp Ala Pro Ala Ser Pro Cys Gly Gly Gly Gly Gly Gly Ser 1 5 10 15 His Glu Ser Gly Glu His Ser Pro Arg Ser Asn Phe Arg Glu Gln Asp 20 25 30 Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Leu 35 40 45 Pro Pro Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Thr Val Gln Glu 50 55 60 Cys Val Ser Glu Phe Ile Ser Phe Val Thr Ser Glu Ala Ser Asp Lys 65 70 75 80 Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp 85 90 95 Ala Met Thr Thr Leu Gly Phe Glu Glu Tyr Ile Asp Pro Leu Lys Val 100 105 110 Tyr Leu Ala Ala Tyr Arg Glu Ile Glu Gly Asp Ser Lys Gly Ser Ala 115 120 125 Lys Gly Gly Asp Ala Ser Ala Lys Arg Asp Val Tyr Gln Ser Pro Asn 130 135 140 Gly Gln Val Ala His Gln Gly Ser Phe Ser Gln Gly Val Asn Tyr Thr 145 150 155 160 Asn Ser 15 1000 DNA Glycine max GLYMA-28NOV01-CLUSTER33504_- 1 15 taataaggtt gtatatggtt tggtgggatg gctcgagagt ctttagaaaa gatatccatg 60 gctgagtccg acaacgagtc aggaggtcac acggggaacg cgagcgggag caacgagttg 120 tccggttgca gggagcaaga caggttcctc ccaatagcaa acgtgagcag gatcatgaag 180 aaggcgttgc cggcgaacgc gaagatatcg aaggaggcga aggagacggt gcaggagtgc 240 gtgtcggagt tcatcagctt cataacagga gaggcttccg ataagtgcca gaaggagaag 300 aggaagacga tcaacggcga cgatcttctc tgggccatga ctaccctggg cttcgaggac 360 tacgtggatc ctctcaagat ttacctgcac aagtataggg agatggaggg ggagaaaacc 420 gctatgatgg gaaggccaca tgagagggat gagggttatg gccatggcca tggtcatgca 480 actcctatga tgacgatgat gatggggcat cagccccagc accagcacca gcaccagcac 540 cagcaccagc accagggaca cgtgtatgga tctggatcag catcttctgc aagaactaga 600 taacatgtgt catctgttta agcttaattg attttattat gaggatgata tgatataaga 660 tttatattcg tatatgtttg gttttagaaa tacaccagct ccagcttgta attgcttgaa 720 acttccttgt tgagagaata tagacattat tgtggatggt gatgtggcat atgtggcata 780 cacagaattt ttgtattctt ctttctctct atggattttt gtgtaagggc aggactatgg 840 ctttgtttgc tgatcgtata gctagtatgg tgctatctag gttcggattt ttttcttttt 900 catgtataat gaaaaattaa cggaggaaat tactcttacg ttactttgaa attaattaac

960 taaatcccgc ttctgccttt ttttttttct cctttctgag 1000 16 181 PRT Glycine max GLYMA-28NOV01-CLUSTER33504_1 polypeptide 16 Met Ala Glu Ser Asp Asn Glu Ser Gly Gly His Thr Gly Asn Ala Ser 1 5 10 15 Gly Ser Asn Glu Leu Ser Gly Cys Arg Glu Gln Asp Arg Phe Leu Pro 20 25 30 Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala 35 40 45 Lys Ile Ser Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu 50 55 60 Phe Ile Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Lys Glu 65 70 75 80 Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr 85 90 95 Leu Gly Phe Glu Asp Tyr Val Asp Pro Leu Lys Ile Tyr Leu His Lys 100 105 110 Tyr Arg Glu Met Glu Gly Glu Lys Thr Ala Met Met Gly Arg Pro His 115 120 125 Glu Arg Asp Glu Gly Tyr Gly His Gly His Gly His Ala Thr Pro Met 130 135 140 Met Thr Met Met Met Gly His Gln Pro Gln His Gln His Gln His Gln 145 150 155 160 His Gln His Gln His Gln Gly His Val Tyr Gly Ser Gly Ser Ala Ser 165 170 175 Ser Ala Arg Thr Arg 180 17 620 DNA Glycine max misc_feature (560)..(560) n is a, c, g, or t 17 tgaagaagat tcctggattg attgtgaaga tggctgagtc ggacaacgac tcgggagggg 60 cgcagaacgc gggaaacagt ggaaacttga gcgagttgtc gcctcgggaa caggaccggt 120 ttctccccat agcgaacgtg agcaggatca tgaagaaggc cttgccggcg aacgcgaaga 180 tctcgaagga cgcgaaggag acggtgcagg aatgcgtgtc ggagttcatc agcttcataa 240 cgggtgaggc gtcggacaag tgccagaggg agaagcgcaa gaccatcaac ggcgacgatc 300 ttctctgggc catgacaacc ctgggattcg aagagtacgt ggagcctctg aagatttacc 360 tccagcgctt ccgcgagatg gagggagaga agaccgtggc cgcccgcgac tcttctaagg 420 actcggcctc cgcctcctcc tatcatcagg gacacgtgta cggctcccct gcctaccatc 480 atcaagtgcc tgggcccact tatcctgccc ctggtagacc cagatgacgt gctcctctat 540 tcgccactcc ctagactttn tatattatat tatttaatta aactctcttc tccactcaac 600 ctttgcaaga tcactgggtt 620 18 165 PRT Glycine max G3476 GLYMA-28NOV01-CLUSTER33504_3 polypeptide 18 Met Ala Glu Ser Asp Asn Asp Ser Gly Gly Ala Gln Asn Ala Gly Asn 1 5 10 15 Ser Gly Asn Leu Ser Glu Leu Ser Pro Arg Glu Gln Asp Arg Phe Leu 20 25 30 Pro Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn 35 40 45 Ala Lys Ile Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser 50 55 60 Glu Phe Ile Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg 65 70 75 80 Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr 85 90 95 Thr Leu Gly Phe Glu Glu Tyr Val Glu Pro Leu Lys Ile Tyr Leu Gln 100 105 110 Arg Phe Arg Glu Met Glu Gly Glu Lys Thr Val Ala Ala Arg Asp Ser 115 120 125 Ser Lys Asp Ser Ala Ser Ala Ser Ser Tyr His Gln Gly His Val Tyr 130 135 140 Gly Ser Pro Ala Tyr His His Gln Val Pro Gly Pro Thr Tyr Pro Ala 145 150 155 160 Pro Gly Arg Pro Arg 165 19 1872 DNA Glycine max G3475 GLYMA-28NOV01-CLUSTER33504_5 19 aacataaata ataaaatatt tctttgaaca tttcttaaaa agtatgaaca taaatttaaa 60 ttattatttt atatttaatg tatttacatt aatttatttg tcttacatac acttgtaatg 120 ttctccttat atttattaaa ctataatata gtatatataa agaaaagatt ttgagaattt 180 gaataaaata agagtgtcca agtcagaggc gagcacgtgc cagataccaa agcaacggtc 240 cagatcatgg agcactcacc aaatccaagg gctcctattt gtccgtgcaa actcacactt 300 atcgcccaac aacggtccac aaagcgccac gtgttctcaa gataaagcgt tattaaccct 360 tctgatccaa cggatcctgc tcattacctc ccaaacaagc ccttccgttc cgtttcacct 420 ttcctcttcc cgccggagcc gccgtcaccc gccgccggca atcgtatcag accctcccaa 480 tacaccgtct ccgacttcca cgcagaattg cacgattcat tgatttcaat tttcaagtct 540 tgaggatttc gtttcaacag cgcttcaatt tgacgcagaa aaactgagtc aaaccaattc 600 tcccagagtt cgtgacttgg attctcaatt tatcgttcat tccgaataga atttgaaact 660 ccgaagaaaa ctgcaccgaa cactgaatct cagttaccga ggagcttctt ctacgaaccg 720 tgcttaattc cacacagaaa caccgagtca aactggttcg tgctgtgttc gtggttcaga 780 ttctcaatcg aaatttgaaa ttcagaagaa aaccgcaccg aacacagaat ttcagaatct 840 gaacaagttt cttccgttaa cagcacttca acttcacgtg gaacaagaat caaaccgttt 900 cgtggttcgg attctcaatt cctcgtccat tcgcaatcga ttttcaaatt ccgaagaaaa 960 ccgctccgaa cactgaattt cagactctga acagcgaaca gtacttcaag ttcacgtgga 1020 acgagtcaaa gcgattccaa tcaatttcgc gaactcctcc acggtgaact ccgatatttt 1080 cctgcactga cttagtgatt cgtttcatat ttctcagctt cgattatccg tttgtcgatg 1140 gcggactcgg acaacgactc cggcggcgcg cacaacgccg ggaaggggag cgagatgtcg 1200 ccgcgggagc aggaccggtt cctgccgatc gcgaacgtga gccgcatcat gaagaaggcg 1260 ctgccggcga acgcgaagat ctcgaaggac gcgaaggaga cggtgcagga gtgcgtgtcg 1320 gagttcatca gcttcatcac cggcgaggcc tccgacaagt gccagcggga gaagcgcaag 1380 acgatcaacg gcgacgacct gctctgggcg atgaccactc tcggcttcga ggactacgtc 1440 gagcctctca agggctacct ccagcgcttc cgagaaatgg aaggagagaa gaccgtggcg 1500 gcgcgtgaca aggacgcgcc tcctcctacc aatgctacca acagtgccta cgagagtcct 1560 agttatgctg ctgctcctgg tggaatcatg atgcatcagg gacacgtgta cggttctgcc 1620 ggcttccatc aagtggctgg tggtgctata aagggtgggc ctgtttatcc cgggcctgga 1680 tccaatgccg gtaggcccag gtagatgggc ctatgttatt attattatta ttattcttat 1740 tcgtaagtta aaagaaatgt gagattcaaa gtggtgatta agtgaattag taacaaaaaa 1800 gtgcgactca gttgattaaa aatatatata aattattata agtcttttaa tatgtttttg 1860 attctcacac at 1872 20 188 PRT Glycine max G3475 GLYMA-28NOV01-CLUSTER33504_5 polypeptide 20 Met Ala Asp Ser Asp Asn Asp Ser Gly Gly Ala His Asn Ala Gly Lys 1 5 10 15 Gly Ser Glu Met Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala 20 25 30 Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile 35 40 45 Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile 50 55 60 Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg 65 70 75 80 Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly 85 90 95 Phe Glu Asp Tyr Val Glu Pro Leu Lys Gly Tyr Leu Gln Arg Phe Arg 100 105 110 Glu Met Glu Gly Glu Lys Thr Val Ala Ala Arg Asp Lys Asp Ala Pro 115 120 125 Pro Pro Thr Asn Ala Thr Asn Ser Ala Tyr Glu Ser Pro Ser Tyr Ala 130 135 140 Ala Ala Pro Gly Gly Ile Met Met His Gln Gly His Val Tyr Gly Ser 145 150 155 160 Ala Gly Phe His Gln Val Ala Gly Gly Ala Ile Lys Gly Gly Pro Val 165 170 175 Tyr Pro Gly Pro Gly Ser Asn Ala Gly Arg Pro Arg 180 185 21 521 DNA Glycine max GLYMA-28NOV01-CLUSTER33504_6 21 agactttagc tttacacaac atattattgt aaggctagct agctagccat ggctgagtcg 60 gacaacgagt ccggaggtca cacggggaac gcaagcggaa gcaacgaatt ctccggttgc 120 agggagcaag acaggttcct tccgatagcg aacgtgagca ggatcatgaa gaaggcgttg 180 ccggcgaacg cgaagatctc gaaggaggcg aaggagacgg tgcaggagtg cgtgtcggag 240 ttcatcagct tcataacagg agaagcgtcc gataagtgcc agaaggagaa gaggaagacg 300 atcaacggcg atgatctgct gtgggccatg accacgctgg ggttcgagga gtacgtggag 360 cctctcaagg tttatctgca taagtatagg gagctggaag gggagaaaac tgctatgatg 420 ggaaggccac atgagaggga tgagggttat ggtcatgcaa ctcctatgat gatcatgatg 480 gggcatcagc agcagcagca tcagggacac gtgtatggat c 521 22 158 PRT Glycine max misc_feature (158)..(158) Xaa can be any naturally occurring amino acid 22 Met Ala Glu Ser Asp Asn Glu Ser Gly Gly His Thr Gly Asn Ala Ser 1 5 10 15 Gly Ser Asn Glu Phe Ser Gly Cys Arg Glu Gln Asp Arg Phe Leu Pro 20 25 30 Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala 35 40 45 Lys Ile Ser Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu 50 55 60 Phe Ile Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Lys Glu 65 70 75 80 Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr 85 90 95 Leu Gly Phe Glu Glu Tyr Val Glu Pro Leu Lys Val Tyr Leu His Lys 100 105 110 Tyr Arg Glu Leu Glu Gly Glu Lys Thr Ala Met Met Gly Arg Pro His 115 120 125 Glu Arg Asp Glu Gly Tyr Gly His Ala Thr Pro Met Met Ile Met Met 130 135 140 Gly His Gln Gln Gln Gln His Gln Gly His Val Tyr Gly Xaa 145 150 155 23 556 DNA Glycine max G3471 GLYMA-28NOV01-CLUSTER4778- _1 23 gtagggtttg tgagatgtcg gatgcgccac cgagcccgac tcatgagagt gggggcgagc 60 agagcccgcg cggttcgtcg tccggcgcga gggagcagga ccggtacctc ccgattgcca 120 acatcagccg cattatgaag aaggctctgc ctcccaacgg caagattgca aaggatgcca 180 aagacaccat gcaggaatgc gtttctgagt tcatcagctt cattaccagc gaggcgagtg 240 agaaatgcca gaaggagaag agaaagacaa tcaatggaga cgatttgcta tgggccatgg 300 ccactttagg atttgaagac tacatagagc cgcttaaggt gtacctggct aggtacagag 360 aggcggaggg tgacactaaa ggatctgcta gaagtggtga tggatctgct acaccagatc 420 aagttggcct tgcaggtcaa aattctcagc ttgttcatca gggttcgctg aactatattg 480 gtttgcaggt gcaaccacaa catctggtta tgccttcaat gcaaagccat gaatagttta 540 gatgcttcta cgcatc 556 24 173 PRT Glycine max G3471 GLYMA-28NOV01-CLUSTER4778_1 polypeptide 24 Met Ser Asp Ala Pro Pro Ser Pro Thr His Glu Ser Gly Gly Glu Gln 1 5 10 15 Ser Pro Arg Gly Ser Ser Ser Gly Ala Arg Glu Gln Asp Arg Tyr Leu 20 25 30 Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Leu Pro Pro Asn 35 40 45 Gly Lys Ile Ala Lys Asp Ala Lys Asp Thr Met Gln Glu Cys Val Ser 50 55 60 Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Glu Lys Cys Gln Lys 65 70 75 80 Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Ala 85 90 95 Thr Leu Gly Phe Glu Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu Ala 100 105 110 Arg Tyr Arg Glu Ala Glu Gly Asp Thr Lys Gly Ser Ala Arg Ser Gly 115 120 125 Asp Gly Ser Ala Thr Pro Asp Gln Val Gly Leu Ala Gly Gln Asn Ser 130 135 140 Gln Leu Val His Gln Gly Ser Leu Asn Tyr Ile Gly Leu Gln Val Gln 145 150 155 160 Pro Gln His Leu Val Met Pro Ser Met Gln Ser His Glu 165 170 25 939 DNA Glycine max misc_feature (596)..(596) n is a, c, g, or t 25 taatatagtg cggctcgagc tctgctttct gtgttattgt ctggcttttg gagccgatcc 60 aaccaatcat cgctggcgcc aaatacaaaa tctcatccct tcccctttct cttactgact 120 ctctttgtca ccgggtttgt gagatgtcgg atgcaccggc gagtccgagt cacgagagtg 180 gtggcgagca gagccctcgc ggctcgttgt ccggcgcggc tagagagcag gaccggtacc 240 ttcccattgc caacatcagc cgcatcatga agaaggctct gcctcccaat ggcaagattg 300 cgaaggatgc aaaagacaca atgcaagaat gcgtttctga attcatcagc ttcattacca 360 gcgaggcgag tgagaaatgc cagaaggaga agagaaagac aatcaatgga gacgatttac 420 tatgggccat ggcaacttta gggtttgaag actacattga gccgcttaag gtgtacctgg 480 ctaggtacag agaggcggag ggtgacacta aaggatctgc tagaagtggt gatggatctg 540 ctagaccaga tcaagttggc cttgcaggtc aaaatgctca ggtgcaacca caacantctg 600 gttatgcctt caatgcaagg ccatgaatag tttagatgct tctacgcatc ttatttattt 660 cccttgaatg cttgtacgca tggcatgggt ggaaccaatt gtctggtaaa aaaatggggg 720 ggctctcgtc cccccgggtg ggggggtttt gtttcggtac tngtgtngnt ttttnttaaa 780 acacgncttg tagcgggtgt ttctcttctc aagggagaga tgtgtttagg gttatgctag 840 tgattcgaaa tgtagcttgt cagggtgaga agcacttgct tttagagttt tctttagatt 900 attatataag agagaatatt tgcagacaaa aagacttac 939 26 160 PRT Glycine max misc_feature (151)..(151) Xaa can be any naturally occurring amino acid 26 Met Ser Asp Ala Pro Ala Ser Pro Ser His Glu Ser Gly Gly Glu Gln 1 5 10 15 Ser Pro Arg Gly Ser Leu Ser Gly Ala Ala Arg Glu Gln Asp Arg Tyr 20 25 30 Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Leu Pro Pro 35 40 45 Asn Gly Lys Ile Ala Lys Asp Ala Lys Asp Thr Met Gln Glu Cys Val 50 55 60 Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Glu Lys Cys Gln 65 70 75 80 Lys Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met 85 90 95 Ala Thr Leu Gly Phe Glu Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu 100 105 110 Ala Arg Tyr Arg Glu Ala Glu Gly Asp Thr Lys Gly Ser Ala Arg Ser 115 120 125 Gly Asp Gly Ser Ala Arg Pro Asp Gln Val Gly Leu Ala Gly Gln Asn 130 135 140 Ala Gln Val Gln Pro Gln Xaa Ser Gly Tyr Ala Phe Asn Ala Arg Pro 145 150 155 160 27 1231 DNA Oryza sativa ORYSA-22JAN02-CLUSTER26105_1 27 tttttttttt tttttttttt ttttttttgg gaatcagagt aaaattgcta ccattacaga 60 gagcatctat ttctcaatag tacaacacgc tagttcagga cgaaatacaa catgaatatt 120 tatgcctccc aatacagatt atatggaacc gaatgacagc caaagaccaa ttgttaaatt 180 atcctgaata tacatacaac aacagagcta gaccacgaac cgaaactatc ctcacgggga 240 gtaatttaca tctgagaagc agccttggct cgacgcttcc aagcagcatc agttcctggt 300 tgacaaacat gggacctaga ggtggtagca agttacttcc ttcactgctc accatgaggt 360 gtaggtatat atattagcta acgactgcag caccaaataa aatccaccta gcaacagttg 420 catgaaaagg tcctatcttc agtttgagac atccccatta tggtactgag gttgcatata 480 acccattcct tgattgtatg ctgcttgttg gcccatccct tgggcacttg aactgcttcc 540 tccatgagaa ccaagtacat cctttttcac agagccatca ccagcctttg cagttaattt 600 actatcaccc tccatctctc tgtacttctg caggtagacc ttgaggggct cgatgtagtc 660 ctcgaagccc agcgtggcca tcgcccacag caagtcgtcg ccgttgatgg tcttgcgctt 720 ctccctctgg catttatcgc tcgcctcgct ggtgatgaag gagatgaact cggagacgca 780 ctcctgcacg gtctccttgg cgtccttggc gatcttcccg ttggccggga tggccttctt 840 catgatgcgg ctgatgttgg cgatggggag gaacctgtcc tgccggacga gtgggccccc 900 cccaccccca cctccccctc ctccccctcc ccccctcggg ctcccgctct cgtggctccc 960 ccctcctccc cccgggctcc ccggcccatc cgccatccca cctcccccct ccttatatag 1020 aagcgcgggc gcgcgcggag agggcgcgac gtggagagga gagagagggg ggttgggcgc 1080 gaggtggtga agcgaggagg agagagagag agagagagag agagagaggg ggggggagag 1140 gagagagaga ggaagcgggg gtgggaagcg gagcggaggt gaggcggaga ggcgagaggg 1200 ggagatcgga cgctggagaa gagaagcggc c 1231 28 185 PRT Oryza sativa ORYSA-22JAN02-CLUSTER26105_1 polypeptide 28 Met Ala Asp Gly Pro Gly Ser Pro Gly Gly Gly Gly Gly Ser His Glu 1 5 10 15 Ser Gly Ser Pro Arg Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Pro Leu Val Arg Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile 35 40 45 Ser Arg Ile Met Lys Lys Ala Ile Pro Ala Asn Gly Lys Ile Ala Lys 50 55 60 Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 65 70 75 80 Ile Thr Ser Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr 85 90 95 Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu 100 105 110 Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Met 115 120 125 Glu Gly Asp Ser Lys Leu Thr Ala Lys Ala Gly Asp Gly Ser Val Lys 130 135 140 Lys Asp Val Leu Gly Ser His Gly Gly Ser Ser Ser Ser Ala Gln Gly 145 150 155 160 Met Gly Gln Gln Ala Ala Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro 165 170 175 Gln Tyr His Asn Gly Asp Val Ser Asn 180 185 29 229 PRT Oryza sativa OSC12630.C1.p5.fg polypeptide 29 Met Pro Asp Ser Asp Asn Glu Ser Gly Gly Pro Ser Asn Ala Gly Glu 1 5 10 15 Tyr Ala Ser Ala Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val 20 25 30 Ser Arg Ile Met Lys Arg Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys 35 40 45 Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 50 55 60 Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr 65 70 75 80 Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu 85 90 95 Asp Tyr Ile Asp Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu Leu 100 105 110 Glu Gly Glu Lys Ala Ile Gly Ala Ala Gly Ser Gly Gly Gly Gly Ala

115 120 125 Ala Ser Ser Gly Gly Ser Gly Ser Gly Ser Gly Ser His His His Gln 130 135 140 Asp Ala Ser Arg Asn Asn Gly Gly Tyr Gly Met Tyr Gly Gly Gly Gly 145 150 155 160 Gly Met Ile Met Met Met Gly Gln Pro Met Tyr Gly Ser Pro Pro Ala 165 170 175 Ser Ser Ala Gly Tyr Ala Gln Pro Gln Pro Pro His His His His His 180 185 190 Gln Met Val Met Gly Gly Lys Gly Lys Val Glu Glu Val Gln Ser Lys 195 200 205 Gly Lys Ile Arg Asp Phe Leu Gln Leu Gln Ala Ser Met Leu Glu Leu 210 215 220 Ile Gln Gly Glu Asn 225 30 241 PRT Oryza sativa OSC1404.C1.p3.fg polypeptide 30 Met Ser Glu Gly Phe Asp Gly Thr Glu Asn Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Val Gly Lys Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile 20 25 30 Gly Arg Ile Met Arg Arg Ala Val Pro Glu Asn Gly Lys Ile Ala Lys 35 40 45 Asp Ser Lys Glu Ser Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 50 55 60 Ile Thr Ser Glu Ala Ser Asp Lys Cys Leu Lys Glu Lys Arg Lys Thr 65 70 75 80 Ile Asn Gly Asp Asp Leu Ile Trp Ser Met Gly Thr Leu Gly Phe Glu 85 90 95 Asp Tyr Val Glu Pro Leu Lys Leu Tyr Leu Arg Leu Tyr Arg Glu Gly 100 105 110 Asp Thr Lys Gly Ser Arg Ala Ser Glu Leu Pro Val Lys Lys Asp Val 115 120 125 Val Leu Asn Gly Asp Pro Gly Ser Ser Leu Val Asn Tyr Gly Ala Gln 130 135 140 Arg Ala Asp Ala Asn Ala Asn His Leu Asp Leu Phe Phe Leu Leu Arg 145 150 155 160 Lys Asn Pro Glu Ser Thr Thr Ala Asn Cys Met Arg Glu Asp Glu Ala 165 170 175 Lys Pro Val Thr Val Lys Ile Ile Glu Thr Val Tyr Val Glu Ala Asp 180 185 190 Thr Ala Asp Asp Phe Lys Ser Val Val Gln Arg Leu Thr Gly Lys Asp 195 200 205 Ala Val Ala Gly Asp Ala Pro Glu Leu Asn Ser Ala Gln Arg Phe Gly 210 215 220 Ser Gly Arg Glu Ala Ser Arg His Gly Asp His Lys Val Arg Ile Tyr 225 230 235 240 Glu 31 297 PRT Oryza sativa OSC30077.C1.p6.fg polypeptide 31 Met Lys Ser Arg Lys Ser Tyr Gly His Leu Leu Ser Pro Val Gly Ser 1 5 10 15 Pro Pro Leu Asp Asn Glu Ser Gly Glu Ala Ala Ala Ala Ala Ala Ala 20 25 30 Gly Gly Gly Gly Cys Gly Ser Ser Ala Gly Tyr Val Val Tyr Gly Gly 35 40 45 Gly Gly Gly Gly Asp Ser Pro Ala Lys Glu Gln Asp Arg Phe Leu Pro 50 55 60 Ile Ala Asn Val Ser Arg Ile Met Lys Arg Ser Leu Pro Ala Asn Ala 65 70 75 80 Lys Ile Ser Lys Glu Ser Lys Glu Thr Val Gln Glu Cys Val Ser Glu 85 90 95 Phe Ile Ser Phe Val Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu 100 105 110 Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr 115 120 125 Leu Gly Phe Glu Ala Tyr Val Gly Pro Leu Lys Ser Tyr Leu Asn Arg 130 135 140 Tyr Arg Glu Ala Glu Gly Glu Lys Ala Asp Val Leu Gly Gly Ala Gly 145 150 155 160 Gly Ala Ala Ala Ala Arg His Gly Glu Gly Gly Cys Cys Gly Gly Gly 165 170 175 Gly Gly Gly Ala Asp Gly Val Val Ile Asp Gly His Tyr Pro Leu Ala 180 185 190 Gly Gly Leu Ser His Ser His His Gly His Gln Gln Gln Asp Gly Gly 195 200 205 Gly Asp Val Gly Leu Met Met Gly Gly Gly Asp Ala Gly Val Gly Tyr 210 215 220 Asn Ala Gly Ala Gly Ser Thr Thr Thr Ala Phe Tyr Ala Pro Ala Ala 225 230 235 240 Thr Ala Ala Ser Gly Asn Lys Ala Tyr Cys Gly Gly Asp Gly Ser Arg 245 250 255 Val Met Glu Phe Glu Gly Ile Gly Gly Glu Glu Glu Ser Gly Gly Gly 260 265 270 Gly Gly Gly Gly Glu Arg Gly Phe Ala Gly His Leu His Gly Val Gln 275 280 285 Trp Phe Arg Leu Lys Arg Asn Thr Asn 290 295 32 285 PRT Oryza sativa OSC5489.C1.p2.fg polypeptide 32 Met Ala Asp Ala Gly His Asp Glu Ser Gly Ser Pro Pro Arg Ser Gly 1 5 10 15 Gly Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg 20 25 30 Ile Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala 35 40 45 Lys Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr 50 55 60 Ser Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn 65 70 75 80 Gly Glu Asp Leu Leu Phe Ala Met Gly Thr Leu Gly Phe Glu Glu Tyr 85 90 95 Val Asp Pro Leu Lys Ile Tyr Leu His Lys Tyr Arg Glu Met Glu Gly 100 105 110 Asp Ser Lys Leu Ser Ser Lys Ala Gly Asp Gly Ser Val Lys Lys Asp 115 120 125 Thr Ile Gly Pro His Ser Gly Ala Ser Ser Ser Ser Ala Gln Gly Met 130 135 140 Val Gly Ala Tyr Thr Gln Gly Met Gly Tyr Met Gln Pro Gln Ser Asn 145 150 155 160 Phe His Ile Leu Val Val Leu Gln Ser Phe Ala Phe Pro Tyr Met Tyr 165 170 175 Gln Val Ala Gln Ile Tyr Cys Asn Lys Tyr Glu Val Ser Arg Glu Gln 180 185 190 Ile Trp Asp Thr Pro Gln Ile Met Glu Leu Ser Pro Trp Ile Pro Tyr 195 200 205 Thr Ile Asn Arg Ile Trp Lys Glu Thr His Gly Ser Gln Asp Ile Arg 210 215 220 Ile Gln Gly Arg Pro Arg Glu Ala Ala Asn Ser Ala Leu Asp Trp Gln 225 230 235 240 Trp Pro Ser Lys His Ser Ser Leu Ala Ser Asn Phe Tyr Gly Thr Arg 245 250 255 Val Val Gly Gly His His Glu Tyr Gln Arg Ser Thr Lys Lys Asp Thr 260 265 270 Thr His Val Asn Phe Ala Ser Gly Leu Gly Asp Leu Gly 275 280 285 33 523 DNA Zea mays LIB3732-044-Q6-K6-C4 33 cccagcgtcc gaggaaggct acgggcacca gggccacctg ttgagccccg tgggcagccc 60 gctgtcggac aacgagtccg gcgccgcggc agcggccggc ggcggcgggt gcgggagcag 120 cgtggggtac tgcggcggcg gcggcggtga gtcgccggcc aaggagcaag accggttcct 180 gccgatcgcc aacgtgtcgc gcatcatgaa gcgctccctg ccggcgaacg ccaagatctc 240 caaggaggcc aaggagacgg tgcaggagtg cgtgtccgag ttcatcagct tcgtcacggg 300 ggaggcctcc gacaagtgcc agcgcgagaa gcgcaagacc atcaacggcg acgacctgct 360 ctgggccatg accacgctcg gcttcgaggc ctacgtcgcc ccactcaagt cctacctcaa 420 ccgctaccgc gaggccgagg gcgagaaggc cgccgtgcta ggcggcggcg cgcgccacgg 480 cgacggcggc ggcgcggcgg acgacgccgg cccactcgcc ggg 523 34 174 PRT Zea mays LIB3732-044-Q6-K6-C4 polypeptide 34 Pro Ala Ser Glu Glu Gly Tyr Gly His Gln Gly His Leu Leu Ser Pro 1 5 10 15 Val Gly Ser Pro Leu Ser Asp Asn Glu Ser Gly Ala Ala Ala Ala Ala 20 25 30 Gly Gly Gly Gly Cys Gly Ser Ser Val Gly Tyr Cys Gly Gly Gly Gly 35 40 45 Gly Glu Ser Pro Ala Lys Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn 50 55 60 Val Ser Arg Ile Met Lys Arg Ser Leu Pro Ala Asn Ala Lys Ile Ser 65 70 75 80 Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser 85 90 95 Phe Val Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys 100 105 110 Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe 115 120 125 Glu Ala Tyr Val Ala Pro Leu Lys Ser Tyr Leu Asn Arg Tyr Arg Glu 130 135 140 Ala Glu Gly Glu Lys Ala Ala Val Leu Gly Gly Gly Ala Arg His Gly 145 150 155 160 Asp Gly Gly Gly Ala Ala Asp Asp Ala Gly Pro Leu Ala Gly 165 170 35 1199 DNA Zea mays ZEAMA-08NOV01-CLUSTER719_1 35 cccacccgga gcgcctcctc ttctccagcg tccgatcccc attccccacc tctcctccct 60 ccgccgccag ctcccgcccc cttctctccc ctcctcgcct ccccgcgcgc gcgtttttat 120 aagggtttcg gcggaggcgc ccggtcgctg gcgatggccg acgacggcgg gagccacgag 180 ggcagcggcg gcggcggagg cgtccgggag caggaccggt tcctgcccat cgccaacatc 240 agccggatca tgaagaaggc cgtcccggcc aacggcaaga tcgccaagga cgctaaggag 300 accctgcagg agtgcgtctc cgagttcata tcattcgtga ccagcgaggc cagcgacaaa 360 tgccagaagg agaaacgaaa gacaatcaac ggggacgatt tgctctgggc gatggccact 420 ttaggattcg aggagtacgt cgagcctctc aagatttacc tacaaaagta caaagagatg 480 gagggtgata gcaagctgtc tacaaaggct ggcgagggct ctgtaaagaa ggatgcaatt 540 agtccccatg gtggcaccag tagctcaagt aatcagttgg ttcagcatgg agtctacaac 600 caagggatgg gctatatgca gccacaggta atctatcgta ctgtcatttg ttagtaaaac 660 aatactgcag ctattttccg tctcactaaa catggcagaa aatttcgatc attacattat 720 gccactaata attttctctc tgtacgcact cagtaccaca atggggaaac ctaataaagg 780 gctaatacag cagcaattta tggtaatatt attgctccct gaattttgtt aactaaagat 840 tctgtatcat gctatatgta tgtttccttt tttcttcttc tttgttttga caattgcttc 900 tttctctacg gtgtttatcc atcagctagg gaagtctctg cattgcttac catgtgtatt 960 ggcagaaaac aggaggcact tacaaagggt gttaatctct gcgatggctg cctctcaggt 1020 gtaaattggc ttcggtttag cgctgctttt gtccgtatat ttaggatgat ttgactgttg 1080 ctacttttgg caacctttta catttacaga tatgtattat tcagcataaa tataatatag 1140 tagtcctagg cctaaataat ggtgattaac ataccaaaaa aaaaaaaaaa aaaaaaaag 1199 36 166 PRT Zea mays ZEAMA-08NOV01-CLUSTER719_1 polypeptide 36 Met Ala Asp Asp Gly Gly Ser His Glu Gly Ser Gly Gly Gly Gly Gly 1 5 10 15 Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile 20 25 30 Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys 35 40 45 Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr Ser 50 55 60 Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn Gly 65 70 75 80 Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Glu Tyr Val 85 90 95 Glu Pro Leu Lys Ile Tyr Leu Gln Lys Tyr Lys Glu Met Glu Gly Asp 100 105 110 Ser Lys Leu Ser Thr Lys Ala Gly Glu Gly Ser Val Lys Lys Asp Ala 115 120 125 Ile Ser Pro His Gly Gly Thr Ser Ser Ser Ser Asn Gln Leu Val Gln 130 135 140 His Gly Val Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro Gln Val Ile 145 150 155 160 Tyr Arg Thr Val Ile Cys 165 37 564 DNA Zea mays ZEAMA-08NOV01-CLUSTER719_10 37 gccttctctt ctccagcgtc cgatctctcc cactcgcctt cctcaccgca gctctcccgg 60 ctcggtcgct tcgccacctc cgtcctcccc ccgcgctcgg tcgctcgcca cctgctctcc 120 cctccctcca cgttgctcgc gcccgcgctt atataagtgc acgaggagga gctcatggcg 180 gacgctccgg cgagccctgg gggcggaggc gggagcccca cgcagagcgg gagcccccag 240 ggccggcgga ggtggaggcg gtggcagccg tcagggagca ggacaggttc ctgcccatcg 300 ccaacatcag tcgcatcatg aagaaggcca tcccggctaa cgggaagatc gccaaggacg 360 ctaaggagac cgtgcaggag tgcgtctcgg agttcatctc cttcatcact agcgaggcga 420 gtgacaagtg ccagagggag aagcggaaga ccatcaatgg cgacgacctg ctgtgggcca 480 tggccacgct ggggtttgag gactatattg aacccctcaa ggtgtacctg cagaagtaca 540 gagagatgga gggtgatagt aagt 564 38 188 PRT Zea mays misc_feature (188)..(188) Xaa can be any naturally occurring amino acid 38 Leu Leu Phe Ser Ser Val Arg Ser Leu Pro Leu Ala Phe Leu Thr Ala 1 5 10 15 Ala Leu Pro Ala Arg Ser Leu Arg His Leu Arg Pro Pro Pro Ala Leu 20 25 30 Gly Arg Ser Pro Pro Ala Leu Pro Ser Leu His Val Ala Arg Ala Arg 35 40 45 Ala Tyr Ile Ser Ala Arg Gly Gly Ala His Gly Gly Arg Ser Gly Glu 50 55 60 Pro Trp Gly Arg Arg Arg Glu Pro His Ala Glu Arg Glu Pro Pro Gly 65 70 75 80 Pro Ala Glu Val Glu Ala Val Ala Ala Val Arg Glu Gln Asp Arg Phe 85 90 95 Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Ile Pro Ala 100 105 110 Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val 115 120 125 Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Asp Lys Cys Gln 130 135 140 Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met 145 150 155 160 Ala Thr Leu Gly Phe Glu Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu 165 170 175 Gln Lys Tyr Arg Glu Met Glu Gly Asp Ser Lys Xaa 180 185 39 1053 DNA Zea mays ZEAMA-08NOV01-CLUSTER719_2 39 cattgggtac ctcgaggccg gccggcatcg cacccacccg gagcgcctcc tcttctccag 60 cgtccgatcc ccattcccca cctctcctcc ctccgccgcc agctcccgcc cccttctctc 120 ccctcctcgc ctccccgcgc gcgcgttttt ataagggttt cggcggaggc gcccggtcgc 180 tggcgatggc cgacgacggc gggagccacg agggcagcgg cggcggcgga ggcgtccggg 240 agcaggaccg gttcctgccc atcgccaaca tcagccggat catgaagaag gccgtcccgg 300 ccaacggcaa gatcgccaag gacgctaagg agaccctgca ggagtgcgtc tccgagttca 360 tatcattcgt gaccagcgag gccagcgaca aatgccagaa ggagaaacga aagacaatca 420 acggggacga tttgctctgg gcgatggcca ctttaggatt cgaggagtac gtcgagcctc 480 tcaagattta cctacaaaag tacaaagaga tggagggtga tagcaagctg tctacaaagg 540 ctggcgaggg ctctgtaaag aaggatgcaa ttagtcccca tggtggcacc agtagctcaa 600 gtaatcagtt ggttcagcat ggagtctaca accaagggat gggctatatg cagccacagt 660 accacaatgg ggaaacctaa taaagggcta atacagcagc aatttatgct agggaagtct 720 ctgcattgct taccatgtgt attggcagaa aacaggaggc acttacaaag ggtgttaatc 780 tctgcgatgg ctgcctctca ggtgtaaatt ggcttcggtt tagcgctgct tttgtccgta 840 tatttaggat gatttgactg ttgctacttt tggcaacctt ttacatttac agatatgtat 900 tattcagcat aaatataata tagtagtcct aggcctaaat aatggtgatt aacataccaa 960 gtcttttatc aggctactcg ttttctggaa caggattcat gcttagcttt ccctcctgtc 1020 tgaatgtgat ggttgcctga atcctaattt gcc 1053 40 164 PRT Zea mays ZEAMA-08NOV01-CLUSTER719_2 polypeptide 40 Met Ala Asp Asp Gly Gly Ser His Glu Gly Ser Gly Gly Gly Gly Gly 1 5 10 15 Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile 20 25 30 Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys 35 40 45 Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr Ser 50 55 60 Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn Gly 65 70 75 80 Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Glu Tyr Val 85 90 95 Glu Pro Leu Lys Ile Tyr Leu Gln Lys Tyr Lys Glu Met Glu Gly Asp 100 105 110 Ser Lys Leu Ser Thr Lys Ala Gly Glu Gly Ser Val Lys Lys Asp Ala 115 120 125 Ile Ser Pro His Gly Gly Thr Ser Ser Ser Ser Asn Gln Leu Val Gln 130 135 140 His Gly Val Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr His 145 150 155 160 Asn Gly Glu Thr 41 1178 DNA Zea mays ZEAMA-08NOV01-CLUSTER719_3 41 gtcgacccac gcgtccgcgc accctcccgg gccgccttct cttctccagc gtccgatctc 60 ccactcccct ccctcaccgc agctctccca cctccgccct ccccccgcac gcgctcgcca 120 cctcgccctc ccctccacgt tgctcgcacc cgcgcttata taagtgcagg aggagctcat 180 ggcggaagct ccggcgagcc ctggcggcgg cggcgggagc cacgagagcg ggagccccag 240 gggaggcgga ggcggtggca gcgtcaggga gcaggacagg ttcctgccca tcgccaacat 300 cagtcgcatc atgaagaagg ccatcccggc taacgggaag accatcccgg ctaacgggaa 360 gatcgccaag gacgctaagg agaccgtgca ggagtgcgtc tccgagttca tctccttcat 420 cactagcgag gcgagtgaca agtgccagag ggagaagcgg aagaccatca atggcgacga 480 cctgctgtgg gccatggcca cgctggggtt tgaggactat attgaacccc tcaaggtgta 540 cctgcagaag tacagagaga tggagggtga tagtaagtta acttcaaaat ccagcgatgg 600 ctccattaaa aaggatgccc ttggtcatgt gggagcaagt agctcagctg tacaagggat 660 gggtcaacaa ggaacataca accaaggaat gggttatatg caaccccagt accataacgg 720 agatatctcg aactaatgaa gacatggacc ttttctgcga cagctgctct tccctgaggc 780 gattttttgg tctcagttat ttactaagta agacaccttg cggtgaccat taaagagtaa 840 ccaatcaccc tcggtaggtc cgtttttatc tgcaagaact gatgaggccg cttggtagga 900 gtaaatcgct tttcctggga acgattgttg gttagcgccg ctactgtatg tatattgaga 960 taccttaacg attggtcttt tggctgccat ttggttacat gtatttgtat cgggaggcat 1020 aaatattgtg taatttgtgt taaagactgg tgtaattgaa ctatgggaag agctgctttg 1080 gttgtaacca tattttgatg cccgtatatt aggcaaaaat agaaggctgt gggcgtgcac 1140

aacaaaaaaa aaaaggagga aaaaaaaagg gcggccgc 1178 42 185 PRT Zea mays ZEAMA-08NOV01-CLUSTER719_3 polypeptide 42 Met Ala Glu Ala Pro Ala Ser Pro Gly Gly Gly Gly Gly Ser His Glu 1 5 10 15 Ser Gly Ser Pro Arg Gly Gly Gly Gly Gly Gly Ser Val Arg Glu Gln 20 25 30 Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala 35 40 45 Ile Pro Ala Asn Gly Lys Thr Ile Pro Ala Asn Gly Lys Ile Ala Lys 50 55 60 Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 65 70 75 80 Ile Thr Ser Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr 85 90 95 Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu 100 105 110 Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Met 115 120 125 Glu Gly Asp Ser Lys Leu Thr Ser Lys Ser Ser Asp Gly Ser Ile Lys 130 135 140 Lys Asp Ala Leu Gly His Val Gly Ala Ser Ser Ser Ala Val Gln Gly 145 150 155 160 Met Gly Gln Gln Gly Thr Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro 165 170 175 Gln Tyr His Asn Gly Asp Ile Ser Asn 180 185 43 2109 DNA Zea mays ZEAMA-08NOV01-CLUSTER719_4 43 ccacgcgtcc gcccacgcgt ccgggcttgc tgagctggag ctgatggatc tagggtttgg 60 gttgcggtga tggtcctgca gcgcaggagg agctcatggc ggaagctccg gcgagccctg 120 gcggcggcgg cgggagccac gagagcggga gccccagggg aggcggaggc ggtggcagcg 180 tcagggagca ggacaggttc ctgcccatcg ccaacatcag tcgcatcatg aagaaggcca 240 tcccggctaa cgggaagatc gccaaggacg ctaaggagac cgtgcaggag tgcgtctccg 300 agttcatctc cttcatcact agcgaagcga gtgacaagtg ccagagggag aagcggaaga 360 ccatcaatgg cgacgatctg ctgtgggcca tggccacgct ggggtttgaa gactacattg 420 aacccctcaa ggtgtaccta cagaagtaca gagaggtgcg tacggtgttt gggaatttgg 480 gggtcaggtc gtgcaatcgc caatctgtca cctggccgat cgtacctctg attgaactta 540 aataatcctg ttgggcatca gcacgctaat aagtgataag tgagctatcc acttcccttc 600 caatgcttcg gccacatatt tatacttctt tagttgagga cataaagaga ccccctgttc 660 ctgtgtacta ctccagtaaa tacagctagt aaacacatta tttttataag gtgaaccaat 720 tcgaaagcac ttttatccat ttaatactga acagtgatcg aaacctctat ttgatgttct 780 tacatgggat tgagttagca ctcgtgcttg gtaagatatt ataactactc acagccctat 840 gtggctgtgt ctgttctatg atgaaaagta gatgtaatgc aaatggataa gagcggaaag 900 agctcctaca gtagtgtaat tagagcatgt tgtagtgcaa gcttttggtt gtttacacaa 960 aagatacatg aaaccattcc actgtaggtc atatacaatc ttgcttaggg tcctgaacat 1020 atctggtgca catggttcac atattaaatt atcaccatcc attctagatc taacgtcttt 1080 agttgtccat tctagatcta acatctttag ttgctctgta taattgtata ttttgcaaag 1140 aaccccttcc accactactc tctaccactt ctaccctgct ccgagggtgt tctctgcaaa 1200 aatatataga acacccatag atgttagata taggatgaat gatggtgatc taatatgtac 1260 accatgtgca ccaaactcag tgcaccagat atattctcga gtttatttag ctgtattttt 1320 ctatacgctc ttcgtattgg ttgaataatc tgtctaatga ggtttctctt ttggtcttat 1380 gtctggtgga tgacatcacg gattgcagat ggagggtgat agcaagttaa ctgctaaatc 1440 tagcgatggc tcgattaaaa aggatgctct tggtcatgtg ggagcaagta gctcagctgc 1500 agaagggatg ggccaacagg gagcatacaa ccaaggaatg ggttatatgc aacctcagta 1560 ccataacggg gatatctcaa actaatgaag gtatggacct tttctgcgac agctgctctt 1620 acctgaggcg attttttttg tcttagttat ttactaagac accttgcggt gaccattaaa 1680 gagtaaccaa tcgccctcaa taggtccgtt tttatctgcc agaactgatg aggtcgctca 1740 ctaggagtaa gtcgcttccc tgggaacggt tgtcggctag caccgctctt gtatgtatat 1800 taagagtaac ttaatgattg gtcttttggc tgcgatttga ttatatgtat ttgtatcggg 1860 aggcataaat attgtgtaat ttgtgttaaa gactagtgta attgaactat gggaagagct 1920 gctttggttg taaccatatt ttgatgcccg tatattaggc aaaaacagaa ggctgtgggc 1980 gtgcacaaca tatttactgt tcaccgaaat acttgtattg atgtatttcc gcatcaatta 2040 tagtcatcgt cagcttgtaa ctacggcaat gaataaataa aaattcactg agtaaaaaaa 2100 aaaaaaaag 2109 44 149 PRT Zea mays ZEAMA-08NOV01-CLUSTER719_4 polypeptide 44 Met Ala Glu Ala Pro Ala Ser Pro Gly Gly Gly Gly Gly Ser His Glu 1 5 10 15 Ser Gly Ser Pro Arg Gly Gly Gly Gly Gly Gly Ser Val Arg Glu Gln 20 25 30 Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala 35 40 45 Ile Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Thr Val Gln 50 55 60 Glu Cys Val Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Asp 65 70 75 80 Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu 85 90 95 Trp Ala Met Ala Thr Leu Gly Phe Glu Asp Tyr Ile Glu Pro Leu Lys 100 105 110 Val Tyr Leu Gln Lys Tyr Arg Glu Val Arg Thr Val Phe Gly Asn Leu 115 120 125 Gly Val Arg Ser Cys Asn Arg Gln Ser Val Thr Trp Pro Ile Val Pro 130 135 140 Leu Ile Glu Leu Lys 145 45 1255 DNA Zea mays ZEAMA-08NOV01-CLUSTER719_5 45 aattcggcac gaggcaccct cccgggccgc ccgcgcttct ccagcgtccg atctcccact 60 cccctccctc accgcagctc tcccacctcc gccctccccc cgcacgcgct cgccacctcg 120 ccctcccctc cacgttgctc gcacccgcgc ttatataagt gcaggaggag ctcatggcgg 180 aagctccggc gagccctggc ggcggcggcg ggagccacga gagcgggagc cccaggggag 240 gcggaggcgg tggcagcgtc agggagcagg acaggttcct gcccatcgcc aacatcagtc 300 gcatcatgaa gaaggccatc ccggctaacg ggaagatcgc caaggacgct aaggagaccg 360 tgcaggagtg cgtctccgag ttcatctcct tcatcactag cgaagcgagt gacaagtgcc 420 agagggagaa gcggaagacc atcaatggcg acgatctgct gtgggccatg gccacgctgg 480 ggtttgaaga ctacattgaa cccctcaagg tgtacctgca gaagtacaga gagatggagg 540 gtgatagcaa gttaactgca aaatctagcg atggctcaat taaaaaggat gcccttggtc 600 atgtgggagc aagtagctca gctgcacaag ggatgggcca acagggagca tacaaccaag 660 gaatgggtta tatgcaaccc cagtaccata acggggatat ctcaaactaa tgaaggcatg 720 gaccttttct gcgacagctg ctcttccccg aggcgggttt ttgtgtcgca gttatttact 780 aagtaagaca ccttgcggtg accattaaag agtaaccaat caccctgggt aggtcaattt 840 ttatctgcaa gaactgatga ggccgcttgg taggagtaaa tcgcttttcc tgggaacgat 900 tgttggttag cgccgctact gtatgtatat tgagatacct taacgattgg tcttttggct 960 gccatttggt tacatgtatt tgtatttgga ggcataagta tcgtgtaatt tgtgttatga 1020 ctagtgtatt gactattgaa ttatcagaag agctgcttta gttgtaagat cacacaaaac 1080 agcctggaaa gtataacaag attaaaactg aaccaaaaat gggcaataaa taaattatca 1140 catttacagt gaataaaaaa atccctgaac atggggcgtt cattctaaag aatccaaatt 1200 tacttgcact gcctggcaac ttttttactc ttttatgctg aaccctaagt ttaat 1255 46 178 PRT Zea mays ZEAMA-08NOV01-CLUSTER719_5 polypeptide 46 Met Ala Glu Ala Pro Ala Ser Pro Gly Gly Gly Gly Gly Ser His Glu 1 5 10 15 Ser Gly Ser Pro Arg Gly Gly Gly Gly Gly Gly Ser Val Arg Glu Gln 20 25 30 Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala 35 40 45 Ile Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu Thr Val Gln 50 55 60 Glu Cys Val Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Asp 65 70 75 80 Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu 85 90 95 Trp Ala Met Ala Thr Leu Gly Phe Glu Asp Tyr Ile Glu Pro Leu Lys 100 105 110 Val Tyr Leu Gln Lys Tyr Arg Glu Met Glu Gly Asp Ser Lys Leu Thr 115 120 125 Ala Lys Ser Ser Asp Gly Ser Ile Lys Lys Asp Ala Leu Gly His Val 130 135 140 Gly Ala Ser Ser Ser Ala Ala Gln Gly Met Gly Gln Gln Gly Ala Tyr 145 150 155 160 Asn Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr His Asn Gly Asp Ile 165 170 175 Ser Asn 47 1173 DNA Zea mays G3435 ZEAMA-08NOV01-CLUSTER90408_1 47 cattgggtac ctcgaggccg gccgggattc cccatctccc ttcattgctc gctcgctcgc 60 tcgttgctct tctccagcag cagctccttc aaatgcaaat ctctttgctg ccgacgcaga 120 gactcgccaa atttccctcc ctcctcctag ccttctcgtc gctcctgttc ttctcgcatc 180 cccagcccag gtggtgtccc ctgtcgcgtt gatgcatgct ccctcggcgg tggccttgag 240 ctgaggcggc ggagcgatgc cggactccga caacgactcc ggcgggccga gcaacgccgg 300 gggcgagctg tcgtcgccgc gggagcagga ccggttcctg cccatcgcca acgtgagccg 360 gatcatgaag aaggcgctcc cggccaacgc caagatcagc aaggacgcca aggagacggt 420 gcaggagtgc gtgtccgagt tcatctcctt catcaccggc gaggcctccg acaagtgcca 480 gcgcgagaag cgcaagacca tcaacggcga cgacctgctg tgggccatga ccacgctcgg 540 cttcgaggac tacgtcgagc cgctcaagca ctacctgcac aagttccgcg agatcgaggg 600 cgagagggcc gccgcgtccg ccggcgcctc gggctcgcag cagcagcagc agcagggcga 660 gctgcccaga ggcgccgcca atgccgccgg gtacgccggg tacggcgcgc ctggctccgg 720 cggcatgatg atgatgatga tggggcagcc catgtacggc ggctcgcagc cgcagcaaca 780 gccgccgcag cctcagccgc cacagcagca gcagcagcaa catcaacagc atcacatggc 840 aatgggaggc agaggaggat tcggccaaca aggcggcggc ggtggctcct cgtcgtcgtc 900 agggcttggc cggcaagaca gggcgtgagt tgcgacgata cgttcagaat cagaatcgct 960 gatactccta cgtagaatta tacctaccta attgatgaca ccgcaccgca cctcgttgtg 1020 ctgcctgtcc ttgtacgttt actaattatt gctgcctgta tgtaaatcaa aatctgaggc 1080 tcccatttcg aaaaaaaaaa aaaaaaaagc ggccggtgaa ctactcttcc cgtttcgttt 1140 catacgagaa tcgaactcgt tttcaattaa aaa 1173 48 223 PRT Zea mays G3435 ZEAMA-08NOV01-CLUSTER90408_1 polypeptide 48 Met Pro Asp Ser Asp Asn Asp Ser Gly Gly Pro Ser Asn Ala Gly Gly 1 5 10 15 Glu Leu Ser Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn 20 25 30 Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser 35 40 45 Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser 50 55 60 Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys 65 70 75 80 Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe 85 90 95 Glu Asp Tyr Val Glu Pro Leu Lys His Tyr Leu His Lys Phe Arg Glu 100 105 110 Ile Glu Gly Glu Arg Ala Ala Ala Ser Ala Gly Ala Ser Gly Ser Gln 115 120 125 Gln Gln Gln Gln Gln Gly Glu Leu Pro Arg Gly Ala Ala Asn Ala Ala 130 135 140 Gly Tyr Ala Gly Tyr Gly Ala Pro Gly Ser Gly Gly Met Met Met Met 145 150 155 160 Met Met Gly Gln Pro Met Tyr Gly Gly Ser Gln Pro Gln Gln Gln Pro 165 170 175 Pro Gln Pro Gln Pro Pro Gln Gln Gln Gln Gln Gln His Gln Gln His 180 185 190 His Met Ala Met Gly Gly Arg Gly Gly Phe Gly Gln Gln Gly Gly Gly 195 200 205 Gly Gly Ser Ser Ser Ser Ser Gly Leu Gly Arg Gln Asp Arg Ala 210 215 220 49 1064 DNA Zea mays G3436 ZEAMA-08NOV01-CLUSTER90408_2 49 ctcagtctca aactccccgc tctcccccgc ccgtccagct cgtgctccgc ctccgctgct 60 ctgtcctctt ccctcctctg cgtttctcct cagagctgtt tgacttgacc ggacagtgct 120 gttcggtggc tcggccgcga tgccggactc cgacaacgag tccggcgggc cgagcaacgc 180 ggagttctcg tcgccgcggg agcaggaccg gttcctgccg atcgcgaacg tgagccggat 240 catgaagaag gcgctcccgg ccaacgccaa gatctccaag gacgccaagg agacggtgca 300 ggagtgcgtg tccgagttca tctccttcat caccggcgag gcctccgaca agtgccagcg 360 cgagaagcgc aagaccatca acggcgacga cctgctctgg gccatgacca cgctcggctt 420 cgaggactac gtcgagccgc tcaagctcta cctgcacaag ttccgcgagc tcgagggcga 480 gaaggcggcc acgacgagcg cctcctccgg cccgcagccg ccgctgcaca gggagacgac 540 gccgtcgtcg tcaacgcaca atggcgcggg cgggcccgtc gggggatacg gcatgtacgg 600 cggcgcgggc gggggaagcg gtatgatcat gatgatgggg cagcccatgt acggcggctc 660 cccgccggcc gcgtcgtccg ggtcgtaccc gcaccaccag atggccatgg gcggaaaagg 720 tggcgcctat ggctacggcg gaggctcgtc gtcgtcgccg tcagggctcg gcaggtagga 780 caggttgtga ccgtcgccgt ccatgcttgc atggccatgg ccatggctcg gctcccgccg 840 ccggcttctt gcttggtgtc ggtaattagc gctggtggcc tgcgctggtt aagttaacct 900 tcggtttttc ccccttttct tttcgtggta agtaatgttg tgctgaatgg agacagtgat 960 atggttaaga tagctccata acctctcggt aattaatcct gtgatttgta ctcccaagct 1020 gctgctaaac tgagctatga cacaatacaa atgctgccat taac 1064 50 212 PRT Zea mays G3436 ZEAMA-08NOV01-CLUSTER90408_2 polypeptide 50 Met Pro Asp Ser Asp Asn Glu Ser Gly Gly Pro Ser Asn Ala Glu Phe 1 5 10 15 Ser Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Ser 20 25 30 Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp 35 40 45 Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55 60 Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile 65 70 75 80 Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp 85 90 95 Tyr Val Glu Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu Leu Glu 100 105 110 Gly Glu Lys Ala Ala Thr Thr Ser Ala Ser Ser Gly Pro Gln Pro Pro 115 120 125 Leu His Arg Glu Thr Thr Pro Ser Ser Ser Thr His Asn Gly Ala Gly 130 135 140 Gly Pro Val Gly Gly Tyr Gly Met Tyr Gly Gly Ala Gly Gly Gly Ser 145 150 155 160 Gly Met Ile Met Met Met Gly Gln Pro Met Tyr Gly Gly Ser Pro Pro 165 170 175 Ala Ala Ser Ser Gly Ser Tyr Pro His His Gln Met Ala Met Gly Gly 180 185 190 Lys Gly Gly Ala Tyr Gly Tyr Gly Gly Gly Ser Ser Ser Ser Pro Ser 195 200 205 Gly Leu Gly Arg 210 51 1818 DNA Glycine max G3473 GLYMA-28NOV01-CLUSTER33504_4 51 tttttaaata ataaaatgtt tctttggaaa tttcttaaaa agtatgaaca taaatttaaa 60 ttattatttt atattaaatg cacttatgtt aatttatttg tcttgcatac acatttaatg 120 ttatccttct ttatatctat attaaactat atatataaag aaaagatttt gaaatttgaa 180 taagataaga gtgtccaggt cagaggcgag cacgtgccag ataccaaagc aacggtccag 240 atcatggagc actcaccaaa tccaagggct ccaattcgtc cgtggacact cacacttatc 300 gactaacaac ggtccacaaa tcgccacgtg tcctcaagat aaagcgttat taacccttct 360 gatccaacgg atcctgctca ttatctccca aacaaacccc tccgttccgt ttcacctttc 420 cccttcccgc cggagccgcc gtcaccggtc gctggccacc gtatccgacc ctcccaatac 480 accctttccg agtcccacac aaaattgcac gattctgtga tttcaatttt caggtctcga 540 ggatttcgtt tcagaagcgc ttccatttga cgcagaacca ccgactcaaa ccgattcgcg 600 ccgagttcgt gactcgaatt ttcaacttct cattcatatt ccaaatcgaa tttgaaactc 660 cgaagaaaaa ttcaccgaac actgaatctc agtttccaag gagcttcttc tacgaagagc 720 gcttcaattc cacgcagaac caccaagtca agccggttcg tgactcggat tctcaattcc 780 tcgttcattc ccgaacgaat tttaaattcc gaagaaaacc gcaccgaaca ctgaatttca 840 gattctgaac aagtttcttc cgcgaaacag cacagcactt caatttcacg tggaacagag 900 acaaagggat tcgtggttcg aattctcaat cgattttcaa attccgaaca gcgaacagta 960 cttcaatttc acgtcgaact agtcaaagcg attcaaatcg atttcgcgaa ctcgtccgat 1020 attttccctg cactgactta gtgattcgtt tcatctttct cagcgcgtct tcgatttttc 1080 cgttagtcga tggcggactc cgacaacgac tccggcggcg cgcacaacgg cggcaagggg 1140 agcgagatgt cgccgcggga gcaggaccgg tttctcccga tcgcgaacgt gagccgcatc 1200 atgaagaagg cgctgccggc gaacgcgaag atctcgaagg acgcgaagga gacggtgcag 1260 gagtgcgtgt cggagttcat cagcttccat tcaccggggg gcctcgccgg tgagtgccag 1320 aaggagaaga ggaagacgat caacggcgat gatctgctgt gggccatgac cacgctggga 1380 ttcgaggagt acgtggagcc tctcaaggtt tatctgcata agtataggga gctggaaggg 1440 gagaaaactg ctatgatggg aaggccacat gagagggatg agggttatgg tcatgcaact 1500 cctatgatga tcatgatggg gcatcagcag cagcagcatc agggacacgt gtatggatct 1560 ggaactacta ctggatcagc atcttctgca agaactagat aacaggttta tgcatgtgtt 1620 atctcatctg tttaagctta ttaagggtgg tctttttgga tggtgatttt gtttgatttt 1680 agaaacaccc cagctccagc ttgtaattgt tgcttgaaac ttcgttgttg agagaatata 1740 gccattattg tggatggtga tgtgacatgc acagaatttt tgtattcttc tttcttccaa 1800 tggatttatc tcgggccc 1818 52 170 PRT Glycine max G3473 GLYMA-28NOV01-CLUSTER33504_4 polypeptide 52 Met Ala Asp Ser Asp Asn Asp Ser Gly Gly Ala His Asn Gly Gly Lys 1 5 10 15 Gly Ser Glu Met Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala 20 25 30 Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile 35 40 45 Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile 50 55 60 Ser Phe His Ser Pro Gly Gly Leu Ala Gly Glu Cys Gln Lys Glu Lys 65 70 75 80 Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu 85 90 95 Gly Phe Glu Glu Tyr Val Glu Pro Leu Lys Val Tyr Leu His Lys Tyr 100 105 110 Arg Glu Leu Glu Gly Glu Lys Thr Ala Met Met Gly Arg Pro His Glu 115 120 125 Arg Asp Glu Gly Tyr Gly His Ala Thr Pro Met Met Ile Met Met Gly 130 135 140 His Gln Gln Gln Gln His Gln Gly His Val Tyr Gly Ser Gly Thr Thr 145 150 155

160 Thr Gly Ser Ala Ser Ser Ala Arg Thr Arg 165 170 53 943 DNA Oryza sativa ORYSA-22JAN02-CLUSTER119015_1 53 ctccgccccc ccccgcgcct tcccccctct ctctcctctc ctctccgcga ctccctccac 60 ccccgcgcgc gcgcgttttt ttttttgcgt aagggttttt ggagggcggc gcggggatgg 120 cggacgcggg gcacgacgag agcgggagcc cgccgaggag cggcggggtg agggagcagg 180 acaggttcct gcccatcgcc aacatcagcc gcatcatgaa gaaggccgtc ccggcgaacg 240 gcaagatcgc caaggacgcc aaggagaccc tgcaggagtg cgtctcggag ttcatctcct 300 tcgtcaccag cgaggcgagc gacaaatgtc agaaggagaa gcgcaagacc atcaacgggg 360 aagatctcct ctttgcgatg ggtacgcttg gctttgagga gtacgttgat ccgttgaaga 420 tctatttaca caagtacaga gagatggagg gtgatagtaa gctgtcctca aaggctggtg 480 atggttcagt aaagaaggat acaattggtc cgcacagtgg cgctagtagc tcaagtgcgc 540 aagggatggt tggggcttac acccaaggga tgggttatat gcaacctcag tatcataatg 600 gggacaccta aagatgagga cagtgaaaat tttcagtaac tggtgtcctc tgtgagttat 660 tatccatctg ttaaggaaga acccacatta gggccatatt tattagtaga agactaaagc 720 acttgaaggg tgttggttta gaaagggtgt taacagttgg ctgtggcgat tgcttcacag 780 atgtaaattg cttcataagt ggtttaatgc ttgtttttgc ctgtatattc agagcaattt 840 tcacatattg gtagttctgc aatcttttgc attcccatac atgtatcagg tggcacaaat 900 ctattgcaag taccctagca ttgaataatg ctggttaaca tat 943 54 164 PRT Oryza sativa ORYSA-22JAN02-CLUSTER119015_1 polypeptide 54 Met Ala Asp Ala Gly His Asp Glu Ser Gly Ser Pro Pro Arg Ser Gly 1 5 10 15 Gly Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg 20 25 30 Ile Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala 35 40 45 Lys Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr 50 55 60 Ser Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn 65 70 75 80 Gly Glu Asp Leu Leu Phe Ala Met Gly Thr Leu Gly Phe Glu Glu Tyr 85 90 95 Val Asp Pro Leu Lys Ile Tyr Leu His Lys Tyr Arg Glu Met Glu Gly 100 105 110 Asp Ser Lys Leu Ser Ser Lys Ala Gly Asp Gly Ser Val Lys Lys Asp 115 120 125 Thr Ile Gly Pro His Ser Gly Ala Ser Ser Ser Ser Ala Gln Gly Met 130 135 140 Val Gly Ala Tyr Thr Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr His 145 150 155 160 Asn Gly Asp Thr 55 870 DNA Zea mays Zm_S11418173 55 gaattccgga taagcgcagg aggagctcat ggcggaagct ccggcgagcc ctggcggcgg 60 cggcgggagc cacgagagcg ggagccccag gggaggcgga ggcggtggca gcgtcaggga 120 gcaggacagg ttcctgccca tcgccaacat cagtcgcatc atgaagaagg ccatcccggc 180 taacgggaag atcgccaagg acgctaagga gaccgtgcag gagtgcgtct ccgagttcat 240 ctccttcatc actagcgaag cgagtgacaa gtgccagagg gagaagcgga agaccatcaa 300 tggcgacgat ctgctgtggg ccatggccac gctggggttt gaagactaca ttgaacccct 360 caaggtgtac ctacagaagt acagagagat ggagggtgat agcaagttaa ctgctaaatc 420 tagcgatggc tcgattaaaa aggatgctct tggtcatgtg ggagcaagta gctcagctgc 480 agaagggatg ggccaacagg gagcatacaa ccaaggaatg ggttatatgc aacctcagta 540 ccataacggg gatatctcaa actaatgaag gtatggacct tttctgcgac agctgctctt 600 acctgaggcg attttttttg tcttagttat ttactaagac accttgcggt gaccattaaa 660 gagtaaccaa tcgccctcaa taggtccgtt tttatctgcc agaactgatg aggtcgctca 720 ctaggagtaa gtcgcttccc tgggaacggt tgtcggctag caccgctctt gtatgtatat 780 taagagtaac ttaatgattg gtcttttggc tgcgatttga tttgattata tgtatttgta 840 tcgggaggca taaatattgt gtaattgtgt 870 56 183 PRT Zea mays Zm_S11418173 polypeptide 56 Ala Gln Glu Glu Leu Met Ala Glu Ala Pro Ala Ser Pro Gly Gly Gly 1 5 10 15 Gly Gly Ser His Glu Ser Gly Ser Pro Arg Gly Gly Gly Gly Gly Gly 20 25 30 Ser Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg 35 40 45 Ile Met Lys Lys Ala Ile Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala 50 55 60 Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile Thr 65 70 75 80 Ser Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn 85 90 95 Gly Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Asp Tyr 100 105 110 Ile Glu Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Met Glu Gly 115 120 125 Asp Ser Lys Leu Thr Ala Lys Ser Ser Asp Gly Ser Ile Lys Lys Asp 130 135 140 Ala Leu Gly His Val Gly Ala Ser Ser Ser Ala Ala Glu Gly Met Gly 145 150 155 160 Gln Gln Gly Ala Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr 165 170 175 His Asn Gly Asp Ile Ser Asn 180 57 734 DNA Zea mays misc_feature (712)..(712) n is a, c, g, or t 57 tttttttttt ttttttaatc accattattt aggcctagga ctactatatt atatttatgc 60 tgaataatac atatctgtaa atgtaaaagg ttgccaaaag tagcaacagt caaatcatcc 120 taaatatacg gacaaaagca gcgctaaacc gaagccaatt tacacctgag aggcagccat 180 cgcagagatt aacacccttt gtaagtgcct cctgttttct gccaatacac atggtaagca 240 atgcagagac ttccctagca taaattgctg ctgtattagc cctttattag gtttccccat 300 tgtggtactg tggctgcata tagcccatcc cttggttgta gactccatgc tgaaccaact 360 gattacttga gctactggtg ccaccatggg gactaattgc atccttcttt acagagccct 420 cgccagcctt tgtagacagc ttgctatcac cctccatctc tttgtacttt tgtaggtaaa 480 tcttgagagg ctcgacgtac tcctcgaatc ctaaagtggc catcgcccag agcaaatcgt 540 ccccgttgat tgtctttcgt ttctccttct ggcatttgtc gctggcctcg ctggtcacga 600 atgatatgaa ctcggagacg cactcctgca gggtctcctt agcgtccttg gcgatcttgc 660 cgttggccgg gacggccttc ttcatgatcc ggctgatgtt ggcgatgggc angaaccggt 720 cctgctcccg gacg 734 58 148 PRT Zea mays misc_feature (8)..(8) Xaa can be any naturally occurring amino acid 58 Val Arg Glu Gln Asp Arg Phe Xaa Pro Ile Ala Asn Ile Ser Arg Ile 1 5 10 15 Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys 20 25 30 Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr Ser 35 40 45 Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn Gly 50 55 60 Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Glu Tyr Val 65 70 75 80 Glu Pro Leu Lys Ile Tyr Leu Gln Lys Tyr Lys Glu Met Glu Gly Asp 85 90 95 Ser Lys Leu Ser Thr Lys Ala Gly Glu Gly Ser Val Lys Lys Asp Ala 100 105 110 Ile Ser Pro His Gly Gly Thr Ser Ser Ser Ser Asn Gln Leu Val Gln 115 120 125 His Gly Val Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr His 130 135 140 Asn Gly Glu Thr 145 59 720 DNA Triticum aestivum misc_feature (2)..(2) n is a, c, g, or t 59 cnccccccan aannagtttg attacccctg ctcgaaatta accctcacta aagggaacaa 60 aagctggagc tccaccgcgg tggcggccgc tctagaacta gtggatcccc cgggctgcag 120 gaattcggca ccagccacca ccttccctcc ctccacgcgc ccgtctatat aaggaggagg 180 gccggatgtc ggacgcgccg gcgagccccc cgggcggcgg cggcggcgga ggaggcggcg 240 gcagcgacga cggcggcggc ggcggcggct tcggcggcgt cagggagcag gacaggttcc 300 tgcccatcgc caacatcagc cgcatcatga agaaggccat cccggccaac ggcaagatcg 360 ccaaggacgc caaggagacc gtgcaggagt gcgtctccga gttcatctcc ttcatcacca 420 gcgaggcgag cgacaagtgc cagagggaga agcgcaagac catcaacggc gacgacctgc 480 tctgggcgat ggcgacgctg ggcttcgagg agtacatcga gcccctcaag gtttatctgc 540 agaagtacag agagacggag ggtgatagta agctagctgg aaagtctggt gaagtctctg 600 ttaaaaagga tgcacttggt cctcatggag gagcaagtgg cacaagtgcg caagggatgg 660 gccaacaagt acatacaatc caagaatggn ttatatgcaa cctcagtacc ataatggggg 720 60 179 PRT Triticum aestivum misc_feature (169)..(169) Xaa can be any naturally occurring amino acid 60 Met Ser Asp Ala Pro Ala Ser Pro Pro Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Ser Asp Asp Gly Gly Gly Gly Gly Gly Phe Gly Gly Val 20 25 30 Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met 35 40 45 Lys Lys Ala Ile Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu 50 55 60 Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu 65 70 75 80 Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp 85 90 95 Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Glu Tyr Ile Glu 100 105 110 Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Thr Glu Gly Asp Ser 115 120 125 Lys Leu Ala Gly Lys Ser Gly Glu Val Ser Val Lys Lys Asp Ala Leu 130 135 140 Gly Pro His Gly Gly Ala Ser Gly Thr Ser Ala Gln Gly Met Gly Gln 145 150 155 160 Gln Val His Thr Ile Gln Glu Trp Xaa Ile Cys Asn Leu Ser Thr Ile 165 170 175 Met Gly Xaa 61 924 DNA Triticum aestivum misc_feature (285)..(285) n is a, c, g, or t 61 ggcacgagca cagatcgagg aggaggagga gccatgccgg agtcggacaa cgactccggc 60 gggccgagca acaccggcgg ggagggggag ctgtcatcgc cgcgggagca ggaccgcttc 120 ctgcccatcg ccaatgtcag ccggatcatg aagaaggcgc tcccggccaa cgccaagatc 180 agcaaggacg ccaaggagac ggtgcaggag tgcgtctccg agttcatctc cttcatcacc 240 ggcgaggcct ccgacaagtg ccagcgcgag aagcgcaaga ccatnaacgg cgacgacctg 300 ctctgggcca tgaccaccct cggcttcgag gactatgtcg acccgctcaa gcactacctn 360 cacaagttcc gcgagatcga gggcnagagg gccgccgcca catcaacatc aaccacgccc 420 gacatgccaa gaaacaacaa caacaatgcc cgccggttac cccgacgccc cgggaggcat 480 gatgatgatg gggcagccca tgtaccggtt ngccggccgc accacaagga gcangnaccc 540 aacatnaaaa ttgcaatggg gaggggagaa gcgggctttt nctattttgg aggcgggggn 600 gggtcntcgt natcctnnng ggttttgacc gaaaaaanng ganacctttt cctttttctt 660 ttcttttctt tttggannct gaccnnaagg ggaggggntt ttcaaacttn tgttncttct 720 ttttgggtga aaaccctnct tgtnanctta aaattctttt cnnccccagg ggnggggaan 780 atnttntttt ttccccncgt tgnttgaaaa cctttttttt ttaaantttt ncgnttnttc 840 ccctgcnaaa aaaanttttt ttttttttna aaaaaaaaaa aaaaaaaaat tngaggnttt 900 tttaagnggg gcggggcccn annt 924 62 268 PRT Triticum aestivum misc_feature (95)..(95) Xaa can be any naturally occurring amino acid 62 Gly Thr Ser Thr Asp Arg Gly Gly Gly Gly Ala Met Pro Glu Ser Asp 1 5 10 15 Asn Asp Ser Gly Gly Pro Ser Asn Thr Gly Gly Glu Gly Glu Leu Ser 20 25 30 Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Ser Arg 35 40 45 Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp Ala 50 55 60 Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile Thr 65 70 75 80 Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Xaa Asn 85 90 95 Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp Tyr 100 105 110 Val Asp Pro Leu Lys His Tyr Xaa His Lys Phe Arg Glu Ile Glu Gly 115 120 125 Xaa Arg Ala Ala Ala Thr Ser Thr Ser Thr Thr Pro Asp Met Pro Arg 130 135 140 Asn Asn Asn Asn Asn Ala Arg Arg Leu Pro Arg Arg Pro Gly Arg His 145 150 155 160 Asp Asp Asp Gly Ala Ala His Val Pro Val Xaa Arg Pro His His Lys 165 170 175 Glu Xaa Xaa Pro Asn Xaa Lys Ile Ala Met Gly Arg Gly Glu Ala Gly 180 185 190 Phe Xaa Tyr Phe Gly Gly Gly Xaa Gly Xaa Ser Xaa Ser Xaa Xaa Val 195 200 205 Leu Thr Glu Lys Xaa Gly Xaa Leu Phe Leu Phe Leu Phe Phe Ser Phe 210 215 220 Trp Xaa Leu Thr Xaa Arg Gly Gly Xaa Phe Gln Thr Xaa Val Xaa Ser 225 230 235 240 Phe Trp Val Lys Thr Xaa Leu Xaa Xaa Leu Lys Phe Phe Xaa Xaa Pro 245 250 255 Gly Xaa Gly Xaa Xaa Xaa Phe Phe Pro Xaa Val Xaa 260 265 63 935 DNA Lycopersicon esculentum SGN-UNIGENE-46859 63 agagaaaaga gattcttttt atatatagtt ataaaaaaat ttcagagttt tctttgtaaa 60 acgaacggtg ttgatagggc aaaatccaaa actctgcctc atctcagtcg tctccctttc 120 tcccttcttc tccaacgtcc gatcttccag ttccctccat ccccagtatg gcggatggtc 180 aaggttcgtc taggtcaccg gcgagtccaa acggaggtgg tagtcatgag agtggtgggg 240 accagagtcc gaggtctaat gtacgtgaac aggacaggtt tttaccaata gctaatatta 300 gtagaatcat gaagaaggca cttcctgcta atggaaaaat tgcgaaggat gctaaggaga 360 ctgttcagga atgtgtttct gagttcatca gcttcattac tagcgaggca agtgacaagt 420 gccagagaga gaaaaggaag actattaatg gtgacgattt gctatgggca atggcaactc 480 ttgggtttga agattatatt gaaccactca aggtgtatct tgctcgatac agagagatgg 540 agggaacgtc aaaggctgct gatggctcta ctaaaagaga tgggatgcaa cctggtccta 600 attcacagct tgcacatcag ggttcatact cacaaggaat gaattatggg aattctcagg 660 gtcagcatat gatggtcccg atgcaaggaa ctgagtaaaa atccgatctt cgtcctgttt 720 gagaagacgg gtggagttga aaacatatta tatatataga tggttcttct gctgtaacct 780 ctgtaacatg gtttattaat tctagtgctc tctagtgagt gccatgtcat atttaaagtt 840 tgtaaattga ggagatgttt taagaaatat tatagacatg attgtttgta gtaataatga 900 aaaccattac ctagtaaaaa aaaaaaaaaa aaaaa 935 64 176 PRT Lycopersicon esculentum SGN-UNIGENE-46859 polypeptide 64 Met Ala Asp Gly Gln Gly Ser Ser Arg Ser Pro Ala Ser Pro Asn Gly 1 5 10 15 Gly Gly Ser His Glu Ser Gly Gly Asp Gln Ser Pro Arg Ser Asn Val 20 25 30 Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile Met 35 40 45 Lys Lys Ala Leu Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys Glu 50 55 60 Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu 65 70 75 80 Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly Asp 85 90 95 Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Asp Tyr Ile Glu 100 105 110 Pro Leu Lys Val Tyr Leu Ala Arg Tyr Arg Glu Met Glu Gly Thr Ser 115 120 125 Lys Ala Ala Asp Gly Ser Thr Lys Arg Asp Gly Met Gln Pro Gly Pro 130 135 140 Asn Ser Gln Leu Ala His Gln Gly Ser Tyr Ser Gln Gly Met Asn Tyr 145 150 155 160 Gly Asn Ser Gln Gly Gln His Met Met Val Pro Met Gln Gly Thr Glu 165 170 175 65 1004 DNA Lycopersicon esculentum SGN-UNIGENE-47447 65 agtgcttcaa catttttctc cctgacatat tgtttattat tagtttcata aaaaaaatta 60 taaaaatttt ctccattttc ttgttcttaa agcttgtgta ctatcatagg caaatacaag 120 actgcgtata catcaatgtc ttcggacttt actcgtagaa ttacatttac gacagaataa 180 agttgtgcat atcgtaccac ttgtgagatt actccgggta atttctcttt tgtattgatc 240 ggaacagaat ttaggcgatt tcgatggcgg attcggataa tgaatcagga ggacatagag 300 ataacagtaa cattgagagt tccctaagag aacaagacag gttccttccc atagcaaatg 360 taagcagaat catgaagaaa gctttaccag ctaacgcgaa aatctcaaaa gatgctaagg 420 aggtagttca agaatgtgtt tctgaattca taagtttcat cacaggggaa gcatcagata 480 agtgtcaaag agaaaagaga aagacaatca atggtgatga tctgttgtgg gcaatgacaa 540 ctcttggttt tgaagaatac attgagccac tcaagattta tttgcagagg tttagggatt 600 tggaagggca aaaaagtggt gtctctggag agaaggatca tagtggatca gtgggttatg 660 ttgaggacta ccatggcatg atgatgatgg ggagtcaaca tcatcaagga cgcgggtatg 720 gcaccggtgt atacaatcat catacggggg agaatgctgc aggggttggt acaggagggt 780 cgcggtttcc tgacgttggg aggcaaaggt gaagctgtga catccgcgga ctacaaagat 840 gtatcaggac gcgatgtatc aactgtcaac aggttgaagt atggacactg aaagacagga 900 acagactttg tagtttgtat tctacagaga tgtaaattgg taaacatgtg tgtacattac 960 aatgtgggtg taaacatgga ttgtaattgt ctattaaaaa aaaa 1004 66 182 PRT Lycopersicon esculentum SGN-UNIGENE-47447 polypeptide 66 Met Ala Asp Ser Asp Asn Glu Ser Gly Gly His Arg Asp Asn Ser Asn 1 5 10 15 Ile Glu Ser Ser Leu Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn 20 25 30 Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser 35 40 45 Lys Asp Ala Lys Glu Val Val Gln Glu Cys Val Ser Glu Phe Ile Ser 50 55 60 Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys 65 70 75 80 Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe 85 90 95 Glu Glu Tyr Ile Glu Pro Leu Lys Ile Tyr Leu Gln Arg Phe Arg Asp 100 105 110 Leu Glu Gly Gln Lys Ser Gly Val Ser Gly Glu Lys Asp His Ser Gly 115 120 125 Ser Val Gly Tyr Val Glu Asp Tyr His Gly Met Met Met Met Gly Ser 130 135 140 Gln His His Gln Gly Arg Gly Tyr Gly Thr Gly Val Tyr Asn His His 145 150 155

160 Thr Gly Glu Asn Ala Ala Gly Val Gly Thr Gly Gly Ser Arg Phe Pro 165 170 175 Asp Val Gly Arg Gln Arg 180 67 609 DNA Arabidopsis thaliana G1820 67 atggctgaga acaacaacaa caacggcgac aacatgaaca acgacaacca ccagcaacca 60 ccgtcgtact cgcagctgcc gccgatggca tcatccaacc ctcagttacg taattactgg 120 attgagcaga tggaaaccgt ctcggatttc aaaaaccgtc agcttccatt ggctcgaatt 180 aagaagatca tgaaggctga tccagatgtg cacatggtct ccgcagaggc tccgatcatc 240 ttcgcaaagg cttgcgaaat gttcatcgtt gatctcacga tgcggtcgtg gctcaaagcc 300 gaggagaaca aacgccacac gcttcagaaa tcggatatct ccaacgcagt ggctagctct 360 ttcacctacg atttccttct tgatgttgtc cctaaggacg agtctatcgc caccgctgat 420 cctggctttg tggctatgcc acatcctgac ggtggaggag taccgcaata ttattatcca 480 ccgggagtgg tgatgggaac tcctatggtt ggtagtggaa tgtacgcgcc atcgcaggcg 540 tggccagcag cggctggtga cggggaggat gatgctgagg ataatggagg aaacggcggc 600 ggaaattga 609 68 202 PRT Arabidopsis thaliana G1820 polypeptide 68 Met Ala Glu Asn Asn Asn Asn Asn Gly Asp Asn Met Asn Asn Asp Asn 1 5 10 15 His Gln Gln Pro Pro Ser Tyr Ser Gln Leu Pro Pro Met Ala Ser Ser 20 25 30 Asn Pro Gln Leu Arg Asn Tyr Trp Ile Glu Gln Met Glu Thr Val Ser 35 40 45 Asp Phe Lys Asn Arg Gln Leu Pro Leu Ala Arg Ile Lys Lys Ile Met 50 55 60 Lys Ala Asp Pro Asp Val His Met Val Ser Ala Glu Ala Pro Ile Ile 65 70 75 80 Phe Ala Lys Ala Cys Glu Met Phe Ile Val Asp Leu Thr Met Arg Ser 85 90 95 Trp Leu Lys Ala Glu Glu Asn Lys Arg His Thr Leu Gln Lys Ser Asp 100 105 110 Ile Ser Asn Ala Val Ala Ser Ser Phe Thr Tyr Asp Phe Leu Leu Asp 115 120 125 Val Val Pro Lys Asp Glu Ser Ile Ala Thr Ala Asp Pro Gly Phe Val 130 135 140 Ala Met Pro His Pro Asp Gly Gly Gly Val Pro Gln Tyr Tyr Tyr Pro 145 150 155 160 Pro Gly Val Val Met Gly Thr Pro Met Val Gly Ser Gly Met Tyr Ala 165 170 175 Pro Ser Gln Ala Trp Pro Ala Ala Ala Gly Asp Gly Glu Asp Asp Ala 180 185 190 Glu Asp Asn Gly Gly Asn Gly Gly Gly Asn 195 200 69 483 DNA Arabidopsis thaliana G1248 69 atggcgggga attatcattc gttccaaaat ccaatccctc gataccagaa ttacaacttc 60 gggagcagct catctaatca tcaacatgaa catgatgggt tagtggtggt ggtggaggat 120 caacagcaag aagaaagcat gatggtaaaa gaacaagaca ggctacttcc gatagcaaac 180 gtaggaagga tcatgaagaa catcctccca gcaaacgcaa aggtctctaa agaagccaaa 240 gagactatgc aagaatgtgt gtccgagttc attagcttcg tcacgggaga agcatccgat 300 aaatgccaca aggagaagcg aaagaccgtt aatggagacg atatctgttg ggctatggct 360 aatctagggt ttgatgatta cgccgcccag ctcaagaagt acttacatcg ttaccgagtt 420 ctcgaaggtg agaaacctaa tcatcacggc aaaggaggac ctaaatcctc gccagataat 480 taa 483 70 160 PRT Arabidopsis thaliana G1248 polypeptide 70 Met Ala Gly Asn Tyr His Ser Phe Gln Asn Pro Ile Pro Arg Tyr Gln 1 5 10 15 Asn Tyr Asn Phe Gly Ser Ser Ser Ser Asn His Gln His Glu His Asp 20 25 30 Gly Leu Val Val Val Val Glu Asp Gln Gln Gln Glu Glu Ser Met Met 35 40 45 Val Lys Glu Gln Asp Arg Leu Leu Pro Ile Ala Asn Val Gly Arg Ile 50 55 60 Met Lys Asn Ile Leu Pro Ala Asn Ala Lys Val Ser Lys Glu Ala Lys 65 70 75 80 Glu Thr Met Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr Gly 85 90 95 Glu Ala Ser Asp Lys Cys His Lys Glu Lys Arg Lys Thr Val Asn Gly 100 105 110 Asp Asp Ile Cys Trp Ala Met Ala Asn Leu Gly Phe Asp Asp Tyr Ala 115 120 125 Ala Gln Leu Lys Lys Tyr Leu His Arg Tyr Arg Val Leu Glu Gly Glu 130 135 140 Lys Pro Asn His His Gly Lys Gly Gly Pro Lys Ser Ser Pro Asp Asn 145 150 155 160 71 757 DNA Arabidopsis thaliana G1781 71 cgtcgaccag attgatcaca tgtggttaac atcaatcaaa aaaaaaaaca aagagataga 60 gatatgactg aggagagccc agaagaagat catgggtctc ctggagtagc tgaaacaaat 120 ccaggaagcc cttcttcaaa gaccaacaac aacaacaaca acaacaaaga acaagaccgg 180 tttcttccca ttgcgaatgt cggaaggatc atgaaaaaag ttcttcccgg taacggtaag 240 atctcaaaag acgctaaaga aaccgttcaa gaatgtgtct cggagttcat tagtttcgtc 300 actggtgaag cttctgacaa gtgtcaaaga gaaaagagga agaccatcaa tggagatgat 360 atcatttggg ctatcacaac tctcggtttc gaagactacg tggctccatt aaaggtctac 420 ctctgcaaat atagagacac cgaaggagag aaagttaaca gcccaaaaca acaacaacaa 480 agacaacaac aacagcagat tcaacaacag aatcatcata attatcagtt tcaagaacaa 540 gaccaaaaca ataacaacat gtcatgtact agttacatct ctcatcatca tccttctcca 600 ttcctaccag tggatcatca accttttccc aatattgctt tctctcctaa atcattgcag 660 aaacagttcc cgcagcagca tgataataac attgattcaa ttcactggtg agagagacat 720 ttgcttgcgg gccgctctag gcgggaaaag cccgaat 757 72 215 PRT Arabidopsis thaliana G1781 polypeptide 72 Met Thr Glu Glu Ser Pro Glu Glu Asp His Gly Ser Pro Gly Val Ala 1 5 10 15 Glu Thr Asn Pro Gly Ser Pro Ser Ser Lys Thr Asn Asn Asn Asn Asn 20 25 30 Asn Asn Lys Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Gly Arg 35 40 45 Ile Met Lys Lys Val Leu Pro Gly Asn Gly Lys Ile Ser Lys Asp Ala 50 55 60 Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr 65 70 75 80 Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile Asn 85 90 95 Gly Asp Asp Ile Ile Trp Ala Ile Thr Thr Leu Gly Phe Glu Asp Tyr 100 105 110 Val Ala Pro Leu Lys Val Tyr Leu Cys Lys Tyr Arg Asp Thr Glu Gly 115 120 125 Glu Lys Val Asn Ser Pro Lys Gln Gln Gln Gln Arg Gln Gln Gln Gln 130 135 140 Gln Ile Gln Gln Gln Asn His His Asn Tyr Gln Phe Gln Glu Gln Asp 145 150 155 160 Gln Asn Asn Asn Asn Met Ser Cys Thr Ser Tyr Ile Ser His His His 165 170 175 Pro Ser Pro Phe Leu Pro Val Asp His Gln Pro Phe Pro Asn Ile Ala 180 185 190 Phe Ser Pro Lys Ser Leu Gln Lys Gln Phe Pro Gln Gln His Asp Asn 195 200 205 Asn Ile Asp Ser Ile His Trp 210 215 73 610 DNA Oryza sativa G3395 73 tggatctagg gtttttggag ggcggcgcgg ggatggcgga cgcggggcac gacgagagcg 60 ggagcccgcc gaggagcggc ggggtgaggg agcaggacag gttcctgccc atcgccaaca 120 tcagccgcat catgaagaag gccgtcccgg cgaacggcaa gatcgccaag gacgccaagg 180 agaccctgca ggagtgcgtc tcggagttca tctccttcgt caccagcgag gcgagcgaca 240 aatgtcagaa ggagaagcgc aagaccatca acggggaaga tctcctcttt gcgatgggta 300 cgcttggctt tgaggagtac gttgatccgt tgaagatcta tttacacaag tacagagaga 360 tggagggtga tagtaagctg tcctcaaagg ctggtgatgg ttcagtaaag aaggatacaa 420 ttggtccgca cagtggcgct agtagctcaa gtgcgcaagg gatggttggg gcttacaccc 480 aagggatggg ttatatgcaa cctcagtatc ataatgggga cacctaaaga tgaggatagt 540 gaaaattttc agtaactggt gtcctctgtg agttattatc catctgttaa ggaagaaccc 600 acattagggc 610 74 164 PRT Oryza sativa G3395 polypeptide 74 Met Ala Asp Ala Gly His Asp Glu Ser Gly Ser Pro Pro Arg Ser Gly 1 5 10 15 Gly Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg 20 25 30 Ile Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala 35 40 45 Lys Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr 50 55 60 Ser Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn 65 70 75 80 Gly Glu Asp Leu Leu Phe Ala Met Gly Thr Leu Gly Phe Glu Glu Tyr 85 90 95 Val Asp Pro Leu Lys Ile Tyr Leu His Lys Tyr Arg Glu Met Glu Gly 100 105 110 Asp Ser Lys Leu Ser Ser Lys Ala Gly Asp Gly Ser Val Lys Lys Asp 115 120 125 Thr Ile Gly Pro His Ser Gly Ala Ser Ser Ser Ser Ala Gln Gly Met 130 135 140 Val Gly Ala Tyr Thr Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr His 145 150 155 160 Asn Gly Asp Thr 75 761 DNA Oryza sativa G3398 AP005193 75 cctctcctct tcgtcttcct cctcgccttc gcttcgactg cttcgatcga gggagatcga 60 ggttgcgatg ccggattcgg acaacgagtc aggggggccg agcaacgcgg gggagtacgc 120 gtcggcgagg gagcaggaca ggttcctgcc gatcgcgaac gtgagcagga tcatgaagag 180 ggcgctcccg gcgaacgcca agatcagcaa ggacgccaag gagacggtgc aggagtgcgt 240 ctcggagttc atctccttca tcaccggcga ggcctccgac aagtgccagc gggagaagcg 300 caagaccatc aacggcgacg acctcctctg ggcgatgacc acgctcggct tcgaggacta 360 catcgacccg ctcaagctct acctccacaa gttccgcgag ctcgagggcg agaaggccat 420 cggcgccgcc ggcagcggcg gcggtggcgc cgcctcctcc ggcggctccg gctccggctc 480 cggctcgcac caccaccagg atgcttcccg gaacaatggc ggatacggca tgtacggcgg 540 cggcggcggc atgatcatga tgatgggaca gcctatgtac ggctcgccgc cggcgtcgtc 600 agctgggtac gcgcagccgc cgccgcccca ccaccaccac caccagatgg tgatgggagg 660 gaaaggtgcg tatggccatg gcggcggcgg cggcggcggg ccctccccgt cgtcgggata 720 cggccggcaa gacaggctat gagcttgctt tcttggttgg t 761 76 224 PRT Oryza sativa G3398 AP005193 polypeptide 76 Met Pro Asp Ser Asp Asn Glu Ser Gly Gly Pro Ser Asn Ala Gly Glu 1 5 10 15 Tyr Ala Ser Ala Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val 20 25 30 Ser Arg Ile Met Lys Arg Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys 35 40 45 Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 50 55 60 Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr 65 70 75 80 Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu 85 90 95 Asp Tyr Ile Asp Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu Leu 100 105 110 Glu Gly Glu Lys Ala Ile Gly Ala Ala Gly Ser Gly Gly Gly Gly Ala 115 120 125 Ala Ser Ser Gly Gly Ser Gly Ser Gly Ser Gly Ser His His His Gln 130 135 140 Asp Ala Ser Arg Asn Asn Gly Gly Tyr Gly Met Tyr Gly Gly Gly Gly 145 150 155 160 Gly Met Ile Met Met Met Gly Gln Pro Met Tyr Gly Ser Pro Pro Ala 165 170 175 Ser Ser Ala Gly Tyr Ala Gln Pro Pro Pro Pro His His His His His 180 185 190 Gln Met Val Met Gly Gly Lys Gly Ala Tyr Gly His Gly Gly Gly Gly 195 200 205 Gly Gly Gly Pro Ser Pro Ser Ser Gly Tyr Gly Arg Gln Asp Arg Leu 210 215 220 77 856 DNA Zea mays G3434 77 ctcccgcccc cttctctccc ctcctcgcct ccccgcgcgc gcgtttttat aagggttgcg 60 gcggaggcgc ccggtcgctg gcgatggccg acgacggcgg gagccacgag ggcagcggcg 120 gcggcggagg cgtccgggag caggaccggt tcctgcccat cgccaacatc agccggatca 180 tgaagaaggc cgtcccggcc aacggcaaga tcgccaagga cgctaaggag accctgcagg 240 agtgcgtctc cgagttcata tcattcgtga ccagcgaggc cagcgacaaa tgccagaagg 300 agaaacgaaa gacaatcaac ggggacgatt tgctctgggc gatggccact ttaggattcg 360 aggagtacgt cgagcctctc aagatttacc tacaaaagta caaagagatg gagggtgata 420 gcaagctgtc tacaaaggct ggcgagggct ctgtaaagaa ggatgcaatt agtccccatg 480 gtggcaccag tagctcaagt aatcagttgg ttcagcatgg agtctacaac caagggatgg 540 gctatatgca gccacagtac cacaatgggg aaacctaata aagggctaat acagcagcaa 600 tttatgctag ggaagtctct gcattgctta ccatgtgtat tggcagaaaa caggaggcac 660 ttacaaaggg tgttaatctc tgcgatggct gcctctcagg tgtaaattgg cttcggttta 720 gcgctgcttt tgtccgtata tttaggatga tttgactgtt gctacttttg gcaacctttt 780 acatttacag atatgtatta ttcagcataa atataatata gtagtcctag gcctaaataa 840 tggtgattaa aaaaaa 856 78 164 PRT Zea mays G3434 polypeptide 78 Met Ala Asp Asp Gly Gly Ser His Glu Gly Ser Gly Gly Gly Gly Gly 1 5 10 15 Val Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile Ser Arg Ile 20 25 30 Met Lys Lys Ala Val Pro Ala Asn Gly Lys Ile Ala Lys Asp Ala Lys 35 40 45 Glu Thr Leu Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val Thr Ser 50 55 60 Glu Ala Ser Asp Lys Cys Gln Lys Glu Lys Arg Lys Thr Ile Asn Gly 65 70 75 80 Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Glu Tyr Val 85 90 95 Glu Pro Leu Lys Ile Tyr Leu Gln Lys Tyr Lys Glu Met Glu Gly Asp 100 105 110 Ser Lys Leu Ser Thr Lys Ala Gly Glu Gly Ser Val Lys Lys Asp Ala 115 120 125 Ile Ser Pro His Gly Gly Thr Ser Ser Ser Ser Asn Gln Leu Val Gln 130 135 140 His Gly Val Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro Gln Tyr His 145 150 155 160 Asn Gly Glu Thr 79 772 DNA Glycine max G3472 79 agactttagc tttacacaac atattattgt aaggctagct agctagccat ggctgagtcg 60 gacaacgagt ccggaggtca cacggggaac gcaagcggaa gcaacgaatt ctccggttgc 120 agggagcaag acaggttcct tccgatagcg aacgtgagca ggatcatgaa gaaggcgttg 180 ccggcgaacg cgaagatctc gaaggaggcg aaggagacgg tgcaggagtg cgtgtcggag 240 ttcatcagct tcataacagg agaagcgtcc gataagtgcc agaaggagaa gaggaagacg 300 atcaacggcg atgatctgct gtgggccatg accacgctgg gattcgagga gtacgtggag 360 cctctcaagg tttatctgca taagtatagg gagctggaag gggagaaaac tgctatgatg 420 ggaaggccac atgagaggga tgagggttat ggtcatgcaa ctcctatgat gatcatgatg 480 gggcatcaac agcagcagca tcagggacac gtgtatggat ctggaactac tactggatca 540 gcatcttctg caagaactag ataacaggtt tatgcatgtg ttatctcatc tgtttaagct 600 tattaattga ttactataag gatggtgata tttgatttat attctgtttg attttagaaa 660 cacacccgct ccagcttgta attgttgctt gaaacttcgt tgttgagaga atatagacat 720 tattgtggat ggtgatgtga catgcacaga atttttgtat tcttctttct tt 772 80 171 PRT Glycine max G3472 polypeptide 80 Met Ala Glu Ser Asp Asn Glu Ser Gly Gly His Thr Gly Asn Ala Ser 1 5 10 15 Gly Ser Asn Glu Phe Ser Gly Cys Arg Glu Gln Asp Arg Phe Leu Pro 20 25 30 Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala 35 40 45 Lys Ile Ser Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu 50 55 60 Phe Ile Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Lys Glu 65 70 75 80 Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr 85 90 95 Leu Gly Phe Glu Glu Tyr Val Glu Pro Leu Lys Val Tyr Leu His Lys 100 105 110 Tyr Arg Glu Leu Glu Gly Glu Lys Thr Ala Met Met Gly Arg Pro His 115 120 125 Glu Arg Asp Glu Gly Tyr Gly His Ala Thr Pro Met Met Ile Met Met 130 135 140 Gly His Gln Gln Gln Gln His Gln Gly His Val Tyr Gly Ser Gly Thr 145 150 155 160 Thr Thr Gly Ser Ala Ser Ser Ala Arg Thr Arg 165 170 81 1000 DNA Glycine max G3474 81 taataaggtt gtatatggtt tggtgggatg gctcgagagt ctttagaaaa gatatccatg 60 gctgagtccg acaacgagtc aggaggtcac acggggaacg cgagcgggag caacgagttg 120 tccggttgca gggagcaaga caggttcctc ccaatagcaa acgtgagcag gatcatgaag 180 aaggcgttgc cggcgaacgc gaagatatcg aaggaggcga aggagacggt gcaggagtgc 240 gtgtcggagt tcatcagctt cataacagga gaggcttccg ataagtgcca gaaggagaag 300 aggaagacga tcaacggcga cgatcttctc tgggccatga ctaccctggg cttcgaggac 360 tacgtggatc ctctcaagat ttacctgcac aagtataggg agatggaggg ggagaaaacc 420 gctatgatgg gaaggccaca tgagagggat gagggttatg gccatggcca tggtcatgca 480 actcctatga tgacgatgat gatggggcat cagccccagc accagcacca gcaccagcac 540 cagcaccagc accagggaca cgtgtatgga tctggatcag catcttctgc aagaactaga 600 tagcatgtgt catctgttta agcttaattg attttattat gaggatgata tgatataaga 660 tttatattcg tatatgtttg gttttagaaa tacaccagct ccagcttgta attgcttgaa 720 acttccttgt tgagagaata tagacattat tgtggatggt gatgtggcat atgtggcata 780 cacagaattt ttgtattctt ctttctctct atggattttt gtgtaagggc aggactatgg 840 ctttgtttgc tgatcgtata gctagtatgg tgctatctag gttcggattt ttttcttttt 900 catgtataat gaaaaattaa cggaggaaat tactcttacg ttactttgaa attaattaac 960 taaatcccgc ttctgccttt ttttttttct cctttctgag 1000 82 181 PRT Glycine max G3474 polypeptide 82 Met Ala Glu Ser Asp Asn Glu Ser Gly Gly His Thr Gly Asn Ala Ser 1 5 10 15 Gly Ser Asn Glu Leu Ser Gly Cys Arg Glu Gln Asp Arg Phe Leu Pro 20 25 30 Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu

Pro Ala Asn Ala 35 40 45 Lys Ile Ser Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu 50 55 60 Phe Ile Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Lys Glu 65 70 75 80 Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr 85 90 95 Leu Gly Phe Glu Asp Tyr Val Asp Pro Leu Lys Ile Tyr Leu His Lys 100 105 110 Tyr Arg Glu Met Glu Gly Glu Lys Thr Ala Met Met Gly Arg Pro His 115 120 125 Glu Arg Asp Glu Gly Tyr Gly His Gly His Gly His Ala Thr Pro Met 130 135 140 Met Thr Met Met Met Gly His Gln Pro Gln His Gln His Gln His Gln 145 150 155 160 His Gln His Gln His Gln Gly His Val Tyr Gly Ser Gly Ser Ala Ser 165 170 175 Ser Ala Arg Thr Arg 180 83 967 DNA Glycine max G3477 83 tcccctcaat ttttttcact tccctctcat ctcccataat acatgtttct tctataaaca 60 tcatcatcaa caacaaacaa aggtgcattg gtggttggtt tgtgagaaat cagaaatatt 120 ttgtattgta atttgtaggg tttgtgagat gtcggatgca ccggcgagtc cgagtcacga 180 gagtggtggc gagcagagcc ctcgcggctc gttgtccggc gcggctagag agcaggaccg 240 gtaccttccc attgccaaca tcagccgcat catgaagaag gctctgcctc ccaatggcaa 300 gattgcgaag gatgcaaaag acacaatgca agaatgcgtt tctgaattca tcagcttcat 360 taccagcgag gcgagtgaga aatgccagaa ggagaagaga aagacaatca atggagacga 420 tttactatgg gccatggcaa ctttagggtt tgaagactac attgagccgc ttaaggtgta 480 cctggctagg tacagagagg cggagggtga cactaaagga tctgctagaa gtggtgatgg 540 atctgctaca ccagatcaag ttggccttgc aggtcaaaat tctcagcttg ttcatcaggg 600 ttcgctgaac tatattggtt tgcaggtgca accacaacat ctggttatgc cttcaatgca 660 aagccatgaa tagtttagat gcttctacgc atcttattta tttcccttta atgcttgtac 720 gcatggcatg ggtggaaaca attgtctggt gatttaatat ttaggttctc gtgtagaagg 780 gtgtcagaat tttgttacgg tactaatgta gatttttatt aatacatgtc ttatttagct 840 ttgtaatacc tactcaaggg agagatgtgt ttagggttat gctagtgatt cgccatgtag 900 cttgtcaggg tgagaagcac ttgcttttag agttttcttt agattattat ataatatata 960 atatttg 967 84 174 PRT Glycine max G3477 polypeptide 84 Met Ser Asp Ala Pro Ala Ser Pro Ser His Glu Ser Gly Gly Glu Gln 1 5 10 15 Ser Pro Arg Gly Ser Leu Ser Gly Ala Ala Arg Glu Gln Asp Arg Tyr 20 25 30 Leu Pro Ile Ala Asn Ile Ser Arg Ile Met Lys Lys Ala Leu Pro Pro 35 40 45 Asn Gly Lys Ile Ala Lys Asp Ala Lys Asp Thr Met Gln Glu Cys Val 50 55 60 Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala Ser Glu Lys Cys Gln 65 70 75 80 Lys Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met 85 90 95 Ala Thr Leu Gly Phe Glu Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu 100 105 110 Ala Arg Tyr Arg Glu Ala Glu Gly Asp Thr Lys Gly Ser Ala Arg Ser 115 120 125 Gly Asp Gly Ser Ala Thr Pro Asp Gln Val Gly Leu Ala Gly Gln Asn 130 135 140 Ser Gln Leu Val His Gln Gly Ser Leu Asn Tyr Ile Gly Leu Gln Val 145 150 155 160 Gln Pro Gln His Leu Val Met Pro Ser Met Gln Ser His Glu 165 170 85 864 DNA Glycine max G3478 85 gattcaaatc gatttcgcga actcgtccga tattttccct gcactgactt agtgattcgt 60 ttcatctttc tcagcgcgtc ttcgattttt ccgttagtcg atggcggact ccgacaacga 120 ctccggcggc gcgcacaacg gcggcaaggg gagcgagatg tcgccgcggg agcaggaccg 180 gtttctcccg atcgcgaacg tgagccgcat catgaagaag gcgctgccgg cgaacgcgaa 240 gatctcgaag gacgcgaagg agacggtgca ggagtgcgtg tcagagttca tcagcttcat 300 caccggcgag gcctccgaca agtgccagcg cgagaagcgc aagacgatca acggcgacga 360 cctgctctgg gcgatgacca ctctgggctt cgaggactac gtggagcctc tcaaaggcta 420 cctccagcgc ttccgagaaa tggaaggaga gaagaccgtg gcggcgcgtg acaaggacgc 480 gcctcctctt acgaatgcta ccaacagtgc ctacgagagt gctaattatg ctgctgctgc 540 tgctgttcct ggtggaatca tgatgcatca gggacacgtg tacggttctg ccggcttcca 600 tcaagtggct ggcggggcta taaagggtgg gcctgcttat cctgggcctg gatccaatgc 660 cggtaggccc agataaatga gcctattatt attagtagta agttaaaaag aaaaatgtga 720 tatagtggtg attagactga actagtttca acaaggtcta atttgattgg taaagaatga 780 tgcatcacct cttcatctct attcgattct tattgataaa aaaaaaatta gagtgaacta 840 gtataataat tctgagagag ttgg 864 86 191 PRT Glycine max G3478 polypeptide 86 Met Ala Asp Ser Asp Asn Asp Ser Gly Gly Ala His Asn Gly Gly Lys 1 5 10 15 Gly Ser Glu Met Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala 20 25 30 Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile 35 40 45 Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile 50 55 60 Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg 65 70 75 80 Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly 85 90 95 Phe Glu Asp Tyr Val Glu Pro Leu Lys Gly Tyr Leu Gln Arg Phe Arg 100 105 110 Glu Met Glu Gly Glu Lys Thr Val Ala Ala Arg Asp Lys Asp Ala Pro 115 120 125 Pro Leu Thr Asn Ala Thr Asn Ser Ala Tyr Glu Ser Ala Asn Tyr Ala 130 135 140 Ala Ala Ala Ala Val Pro Gly Gly Ile Met Met His Gln Gly His Val 145 150 155 160 Tyr Gly Ser Ala Gly Phe His Gln Val Ala Gly Gly Ala Ile Lys Gly 165 170 175 Gly Pro Ala Tyr Pro Gly Pro Gly Ser Asn Ala Gly Arg Pro Arg 180 185 190 87 1231 DNA Oryza sativa G3394 Cl26105_1 87 ggccgcttct cttctccagc gtccgatctc cccctctcgc ctctccgcct cacctccgct 60 ccgcttccca cccccgcttc ctctctctct cctctccccc cccctctctc tctctctctc 120 tctctctctc tcctcctcgc ttcaccacct cgcgcccaac ccccctctct ctcctctcca 180 cgtcgcgccc tctccgcgcg cgcccgcgct tctatataag gaggggggag gtgggatggc 240 ggatgggccg gggagcccgg ggggaggagg ggggagccac gagagcggga gcccgagggg 300 gggaggggga ggagggggag gtgggggtgg ggggggccca ctcgtccggc aggacaggtt 360 cctccccatc gccaacatca gccgcatcat gaagaaggcc atcccggcca acgggaagat 420 cgccaaggac gccaaggaga ccgtgcagga gtgcgtctcc gagttcatct ccttcatcac 480 cagcgaggcg agcgataaat gccagaggga gaagcgcaag accatcaacg gcgacgactt 540 gctgtgggcg atggccacgc tgggcttcga ggactacatc gagcccctca aggtctacct 600 gcagaagtac agagagatgg agggtgatag taaattaact gcaaaggctg gtgatggctc 660 tgtgaaaaag gatgtacttg gttctcatgg aggaagcagt tcaagtgccc aagggatggg 720 ccaacaagca gcatacaatc aaggaatggg ttatatgcaa cctcagtacc ataatgggga 780 tgtctcaaac tgaagatagg accttttcat gcaactgttg ctaggtggat tttatttggt 840 gctgcagtcg ttagctaata tatataccta cacctcatgg tgagcagtga aggaagtaac 900 ttgctaccac ctctaggtcc catgtttgtc aaccaggaac tgatgctgct tggaagcgtc 960 gagccaaggc tgcttctcag atgtaaatta ctccccgtga ggatagtttc ggttcgtggt 1020 ctagctctgt tgttgtatgt atattcagga taatttaaca attggtcttt ggctgtcatt 1080 cggttccata taatctgtat tgggaggcat aaatattcat gttgtatttc gtcctgaact 1140 agcgtgttgt actattgaga aatagatgct ctctgtaatg gtagcaattt tactctgatt 1200 cccaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1231 88 185 PRT Oryza sativa G3394 Cl26105_1 polypeptide 88 Met Ala Asp Gly Pro Gly Ser Pro Gly Gly Gly Gly Gly Ser His Glu 1 5 10 15 Ser Gly Ser Pro Arg Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Pro Leu Val Arg Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile 35 40 45 Ser Arg Ile Met Lys Lys Ala Ile Pro Ala Asn Gly Lys Ile Ala Lys 50 55 60 Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 65 70 75 80 Ile Thr Ser Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr 85 90 95 Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu 100 105 110 Asp Tyr Ile Glu Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Met 115 120 125 Glu Gly Asp Ser Lys Leu Thr Ala Lys Ala Gly Asp Gly Ser Val Lys 130 135 140 Lys Asp Val Leu Gly Ser His Gly Gly Ser Ser Ser Ser Ala Gln Gly 145 150 155 160 Met Gly Gln Gln Ala Ala Tyr Asn Gln Gly Met Gly Tyr Met Gln Pro 165 170 175 Gln Tyr His Asn Gly Asp Val Ser Asn 180 185 89 837 DNA Oryza sativa G3396 89 gtcgagatcc ggcggccggt ggcgtcctcc tccctctccc tcctccccaa ccaacggcgc 60 tgatcccctc cgccatctcc gtccatctcc gcctaaaaaa actaagcgat gtcggagggg 120 ttcgacggga cggagaacgg cggcggcggc ggcggaggcg gagtagggaa ggagcaggac 180 cggttcctgc cgatcgccaa catcggccgc atcatgcgcc gggccgtgcc ggagaacggc 240 aagatcgcca aggactccaa ggagtccgtc caggagtgcg tctccgagtt catcagcttc 300 atcaccagcg aagcaagcga caagtgcctc aaggagaagc gcaagaccat caatggggac 360 gacctgatct ggtcaatggg cacgctcgga ttcgaggact atgtcgagcc tctcaagctc 420 tacctcaggc tctaccggga gacggagggt gacacaaagg gttcaagagc ttctgaactg 480 ccagtaaaga aagatgttgt acttaatgga gatcctggat catcgtttga aggcatgtag 540 gacgaggagt gtgatagcat ctaggaagga gaaccatcgt ttttagggaa agaacgctcc 600 agcatcctgt tatgttgtaa gcaggatgct tctaaagttc caataccttg ttaccacgaa 660 tgttagtcgt cgttcttttt gaaatgttct tgtgttagcc aggatgtcca aatttgttgt 720 aggttctagt tcagtcgtgt gttgtgtggt tgtgtctaac catatttggc cgtttccggc 780 tgtcctgcat atgctaaatt cagaggggta aagagatcta agaaaaaaaa aaaaaaa 837 90 143 PRT Oryza sativa G3396 polypeptide 90 Met Ser Glu Gly Phe Asp Gly Thr Glu Asn Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Val Gly Lys Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Ile 20 25 30 Gly Arg Ile Met Arg Arg Ala Val Pro Glu Asn Gly Lys Ile Ala Lys 35 40 45 Asp Ser Lys Glu Ser Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe 50 55 60 Ile Thr Ser Glu Ala Ser Asp Lys Cys Leu Lys Glu Lys Arg Lys Thr 65 70 75 80 Ile Asn Gly Asp Asp Leu Ile Trp Ser Met Gly Thr Leu Gly Phe Glu 85 90 95 Asp Tyr Val Glu Pro Leu Lys Leu Tyr Leu Arg Leu Tyr Arg Glu Thr 100 105 110 Glu Gly Asp Thr Lys Gly Ser Arg Ala Ser Glu Leu Pro Val Lys Lys 115 120 125 Asp Val Val Leu Asn Gly Asp Pro Gly Ser Ser Phe Glu Gly Met 130 135 140 91 720 DNA Oryza sativa G3397 AC120529 91 gcgtctgatt tgctgaagag gaggaggagg atgccggact cggacaacga ctccggcggg 60 ccgagcaact acgcgggagg ggagctgtcg tcgccgcggg agcaggacag gttcctgccg 120 atcgcgaacg tgagcaggat catgaagaag gcgctgccgg cgaacgccaa gatcagcaag 180 gacgccaagg agacggtgca ggagtgcgtc tccgagttca tctccttcat caccggcgag 240 gcctccgaca agtgccagcg cgagaagcgc aagaccatca acggcgacga cctgctctgg 300 gccatgacca ccctcggctt cgaggactac gtcgaccccc tcaagcacta cctccacaag 360 ttccgcgaga tcgagggcga gcgcgccgcc gcctccacca ccggcgccgg caccagcgcc 420 gcctccacca cgccgccgca gcagcagcac accgccaatg ccgccggcgg ctacgccggg 480 tacgccgccc cgggagccgg ccccggcggc atgatgatga tgatggggca gcccatgtac 540 ggctcgccgc caccgccgcc acagcagcag cagcagcaac accaccacat ggcaatggga 600 ggaagaggcg gcttcggtca tcatcccggc ggcggcggcg gcgggtcgtc gtcgtcgtcg 660 gggcacggtc ggcaaaacag gggcgcttga catcgctccg agacgagtag catgcaccat 720 92 219 PRT Oryza sativa G3397 AC120529 polypeptide 92 Met Pro Asp Ser Asp Asn Asp Ser Gly Gly Pro Ser Asn Tyr Ala Gly 1 5 10 15 Gly Glu Leu Ser Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala 20 25 30 Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile 35 40 45 Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile 50 55 60 Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg 65 70 75 80 Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly 85 90 95 Phe Glu Asp Tyr Val Asp Pro Leu Lys His Tyr Leu His Lys Phe Arg 100 105 110 Glu Ile Glu Gly Glu Arg Ala Ala Ala Ser Thr Thr Gly Ala Gly Thr 115 120 125 Ser Ala Ala Ser Thr Thr Pro Pro Gln Gln Gln His Thr Ala Asn Ala 130 135 140 Ala Gly Gly Tyr Ala Gly Tyr Ala Ala Pro Gly Ala Gly Pro Gly Gly 145 150 155 160 Met Met Met Met Met Gly Gln Pro Met Tyr Gly Ser Pro Pro Pro Pro 165 170 175 Pro Gln Gln Gln Gln Gln Gln His His His Met Ala Met Gly Gly Arg 180 185 190 Gly Gly Phe Gly His His Pro Gly Gly Gly Gly Gly Gly Ser Ser Ser 195 200 205 Ser Ser Gly His Gly Arg Gln Asn Arg Gly Ala 210 215 93 1322 DNA Zea mays G3437 93 tattgtctat gagggaagca gatcctctac gctgcaattg ggccactgac atgtgggacc 60 aggtctagat tggacccaca catcaatgac cgaaatgcag aagagggtct cgttgccact 120 gtaagctatc tcctagagtt cagagcaggg caagaatctt gcaatgctca catgaacata 180 atataatcgt tgtgttagct atgcgtcggc atcactaccg tcctcccact ggcatctccc 240 gtctactatt ttgggacgaa cagaacagag acactagcta actagcttat tagcttgctc 300 ccctccttcc tttcaagctt taaaaggaga ccatctcttg caccacctct tcatccatcc 360 ggccaagcaa ggggcatgaa gaacaggaag ggctacgggc accagggcca cctgctgagc 420 cccgtgggca gcccgctgtc ggacaacgag tccggcgccg cggcagcggc cggcggcggc 480 gggtgcggga gcagcgtggg gtactgcggc ggcggcggcg gtgagtcgcc ggccaaggag 540 caagaccggt tcctgccgat cgccaacgtg tcgcgcatca tgaagcgctc cctgccggcg 600 aacgccaaga tctccaagga ggccaaggag acggtgcagg agtgcgtgtc cgagttcatc 660 agcttcgtca cgggggaggc ctccgacaag tgccagcgcg agaagcgcaa gaccatcaac 720 ggcgacgacc tgctctgggc catgaccacg ctcggcttcg aggcctacgt cgccccgctc 780 aagtcctacc tcaaccgcta ccgcgaggcc gagggcgaga aggccgccgt gctcggcggc 840 ggcgcgcgcc acggcgacgg cgcggcgcgg cggacgacgc cggcccactc gccgcgcaat 900 ggcgcgggcg ggcccgtcgg gggatacggc atgtacggcg gcgcgggcgg gggaagcggt 960 atgatcatga tgatggggca gcccatgtac ggcggctccc cgccggccgc gtcgtccggg 1020 tcgtacccgc accaccagat ggccatgggc ggaaaaggtg gcgcctatgg ctacggcgga 1080 ggctcgtcgt cgtcgccgtc agggctcggc aggtaggcca ggttgtgacc gtcgccgtcc 1140 atgcttgcat ggccatggca tggctcagtc ccgccgccgg cttcttgctt ggtgtcggta 1200 attagcgctg gttaagttaa ccttcggttt ttcccccctt ttcttttcgt ggtaagtaat 1260 gttgtgctga atggagccag tgatatggtt aagatagctc cataacctct cggtaaaaaa 1320 aa 1322 94 246 PRT Zea mays G3437 polypeptide 94 Met Lys Asn Arg Lys Gly Tyr Gly His Gln Gly His Leu Leu Ser Pro 1 5 10 15 Val Gly Ser Pro Leu Ser Asp Asn Glu Ser Gly Ala Ala Ala Ala Ala 20 25 30 Gly Gly Gly Gly Cys Gly Ser Ser Val Gly Tyr Cys Gly Gly Gly Gly 35 40 45 Gly Glu Ser Pro Ala Lys Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn 50 55 60 Val Ser Arg Ile Met Lys Arg Ser Leu Pro Ala Asn Ala Lys Ile Ser 65 70 75 80 Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser 85 90 95 Phe Val Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys 100 105 110 Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe 115 120 125 Glu Ala Tyr Val Ala Pro Leu Lys Ser Tyr Leu Asn Arg Tyr Arg Glu 130 135 140 Ala Glu Gly Glu Lys Ala Ala Val Leu Gly Gly Gly Ala Arg His Gly 145 150 155 160 Asp Gly Ala Ala Arg Arg Thr Thr Pro Ala His Ser Pro Arg Asn Gly 165 170 175 Ala Gly Gly Pro Val Gly Gly Tyr Gly Met Tyr Gly Gly Ala Gly Gly 180 185 190 Gly Ser Gly Met Ile Met Met Met Gly Gln Pro Met Tyr Gly Gly Ser 195 200 205 Pro Pro Ala Ala Ser Ser Gly Ser Tyr Pro His His Gln Met Ala Met 210 215 220 Gly Gly Lys Gly Gly Ala Tyr Gly Tyr Gly Gly Gly Ser Ser Ser Ser 225 230 235 240 Pro Ser Gly Leu Gly Arg 245 95 929 DNA Arabidopsis thaliana CBF1 95 cttgaaaaag aatctacctg aaaagaaaaa aaagagagag agatataaat agctttacca 60 agacagatat actatctttt attaatccaa aaagactgag aactctagta actacgtact 120 acttaaacct tatccagttt cttgaaacag agtactctga tcaatgaact cattttcagc 180 tttttctgaa atgtttggct ccgattacga gcctcaaggc ggagattatt gtccgacgtt 240 ggccacgagt tgtccgaaga aaccggcggg ccgtaagaag tttcgtgaga ctcgtcaccc 300 aatttacaga ggagttcgtc aaagaaactc cggtaagtgg gtttctgaag tgagagagcc 360 aaacaagaaa accaggattt ggctcgggac tttccaaacc gctgagatgg cagctcgtgc 420 tcacgacgtc gctgcattag ccctccgtgg ccgatcagca tgtctcaact tcgctgactc

480 ggcttggcgg ctacgaatcc cggagtcaac atgcgccaag gatatccaaa aagcggctgc 540 tgaagcggcg ttggcttttc aagatgagac gtgtgatacg acgaccacga atcatggcct 600 ggacatggag gagacgatgg tggaagctat ttatacaccg gaacagagcg aaggtgcgtt 660 ttatatggat gaggagacaa tgtttgggat gccgactttg ttggataata tggctgaagg 720 catgctttta ccgccgccgt ctgttcaatg gaatcataat tatgacggcg aaggagatgg 780 tgacgtgtcg ctttggagtt actaatattc gatagtcgtt tccatttttg tactatagtt 840 tgaaaatatt ctagttcctt tttttagaat ggttccttca ttttatttta ttttattgtt 900 gtagaaacga gtggaaaata attcaatac 929 96 213 PRT Arabidopsis thaliana CBF1 polypeptide 96 Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Pro Gln Gly Gly Asp Tyr Cys Pro Thr Leu Ala Thr Ser Cys Pro Lys 20 25 30 Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile Tyr 35 40 45 Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val Ser Glu Val Arg 50 55 60 Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln Thr Ala 65 70 75 80 Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg Gly 85 90 95 Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg Ile 100 105 110 Pro Glu Ser Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu Ala 115 120 125 Ala Leu Ala Phe Gln Asp Glu Thr Cys Asp Thr Thr Thr Thr Asn His 130 135 140 Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala Ile Tyr Thr Pro Glu 145 150 155 160 Gln Ser Glu Gly Ala Phe Tyr Met Asp Glu Glu Thr Met Phe Gly Met 165 170 175 Pro Thr Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu Pro Pro Pro 180 185 190 Ser Val Gln Trp Asn His Asn Tyr Asp Gly Glu Gly Asp Gly Asp Val 195 200 205 Ser Leu Trp Ser Tyr 210 97 803 DNA Arabidopsis thaliana CBF2 97 ctgatcaatg aactcatttt ctgccttttc tgaaatgttt ggctccgatt acgagtctcc 60 ggtttcctca ggcggtgatt acagtccgaa gcttgccacg agctgcccca agaaaccagc 120 gggaaggaag aagtttcgtg agactcgtca cccaatttac agaggagttc gtcaaagaaa 180 ctccggtaag tgggtgtgtg agttgagaga gccaaacaag aaaacgagga tttggctcgg 240 gactttccaa accgctgaga tggcagctcg tgctcacgac gtcgccgcca tagctctccg 300 tggcagatct gcctgtctca atttcgctga ctcggcttgg cggctacgaa tcccggaatc 360 aacctgtgcc aaggaaatcc aaaaggcggc ggctgaagcc gcgttgaatt ttcaagatga 420 gatgtgtcat atgacgacgg atgctcatgg tcttgacatg gaggagacct tggtggaggc 480 tatttatacg ccggaacaga gccaagatgc gttttatatg gatgaagagg cgatgttggg 540 gatgtctagt ttgttggata acatggccga agggatgctt ttaccgtcgc cgtcggttca 600 atggaactat aattttgatg tcgagggaga tgatgacgtg tccttatgga gctattaaaa 660 ttcgattttt atttccattt ttggtattat agctttttat acatttgatc cttttttaga 720 atggatcttc ttcttttttt ggttgtgaga aacgaatgta aatggtaaaa gttgttgtca 780 aatgcaaatg tttttgagtg cag 803 98 207 PRT Arabidopsis thaliana CBF2 polypeptide 98 Met Phe Gly Ser Asp Tyr Glu Ser Pro Val Ser Ser Gly Gly Asp Tyr 1 5 10 15 Ser Pro Lys Leu Ala Thr Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys 20 25 30 Lys Phe Arg Glu Thr Arg His Pro Ile Tyr Arg Gly Val Arg Gln Arg 35 40 45 Asn Ser Gly Lys Trp Val Cys Glu Leu Arg Glu Pro Asn Lys Lys Thr 50 55 60 Arg Ile Trp Leu Gly Thr Phe Gln Thr Ala Glu Met Ala Ala Arg Ala 65 70 75 80 His Asp Val Ala Ala Ile Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn 85 90 95 Phe Ala Asp Ser Ala Trp Arg Leu Arg Ile Pro Glu Ser Thr Cys Ala 100 105 110 Lys Glu Ile Gln Lys Ala Ala Ala Glu Ala Ala Leu Asn Phe Gln Asp 115 120 125 Glu Met Cys His Met Thr Thr Asp Ala His Gly Leu Asp Met Glu Glu 130 135 140 Thr Leu Val Glu Ala Ile Tyr Thr Pro Glu Gln Ser Gln Asp Ala Phe 145 150 155 160 Tyr Met Asp Glu Glu Ala Met Leu Gly Met Ser Ser Leu Leu Asp Asn 165 170 175 Met Ala Glu Gly Met Leu Leu Pro Ser Pro Ser Val Gln Trp Asn Tyr 180 185 190 Asn Phe Asp Val Glu Gly Asp Asp Asp Val Ser Leu Trp Ser Tyr 195 200 205 99 908 DNA Arabidopsis thaliana misc_feature (851)..(851) n is a, c, g, or t 99 cctgaactag aacagaaaga gagagaaact attatttcag caaaccatac caacaaaaaa 60 gacagagatc ttttagttac cttatccagt ttcttgaaac agagtactct tctgatcaat 120 gaactcattt tctgcttttt ctgaaatgtt tggctccgat tacgagtctt cggtttcctc 180 aggcggtgat tatattccga cgcttgcgag cagctgcccc aagaaaccgg cgggtcgtaa 240 gaagtttcgt gagactcgtc acccaatata cagaggagtt cgtcggagaa actccggtaa 300 gtgggtttgt gaggttagag aaccaaacaa gaaaacaagg atttggctcg gaacatttca 360 aaccgctgag atggcagctc gagctcacga cgttgccgct ttagcccttc gtggccgatc 420 agcctgtctc aatttcgctg actcggcttg gagactccga atcccggaat caacttgcgc 480 taaggacatc caaaaggcgg cggctgaagc tgcgttggcg tttcaggatg agatgtgtga 540 tgcgacgacg gatcatggct tcgacatgga ggagacgttg gtggaggcta tttacacggc 600 ggaacagagc gaaaatgcgt tttatatgca cgatgaggcg atgtttgaga tgccgagttt 660 gttggctaat atggcagaag ggatgctttt gccgcttccg tccgtacagt ggaatcataa 720 tcatgaagtc gacggcgatg atgacgacgt atcgttatgg agttattaaa actcagatta 780 ttatttccat ttttagtacg atacttttta ttttattatt atttttagat ccttttttag 840 aatggaatct ncattatgtt tgtaaaactg agaaacgagt gtaaattaaa ttgattcagt 900 ttcagtat 908 100 216 PRT Arabidopsis thaliana CBF3 polypeptide 100 Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Ser Ser Val Ser Ser Gly Gly Asp Tyr Ile Pro Thr Leu Ala Ser Ser 20 25 30 Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His 35 40 45 Pro Ile Tyr Arg Gly Val Arg Arg Arg Asn Ser Gly Lys Trp Val Cys 50 55 60 Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe 65 70 75 80 Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 85 90 95 Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 100 105 110 Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala 115 120 125 Ala Glu Ala Ala Leu Ala Phe Gln Asp Glu Met Cys Asp Ala Thr Thr 130 135 140 Asp His Gly Phe Asp Met Glu Glu Thr Leu Val Glu Ala Ile Tyr Thr 145 150 155 160 Ala Glu Gln Ser Glu Asn Ala Phe Tyr Met His Asp Glu Ala Met Phe 165 170 175 Glu Met Pro Ser Leu Leu Ala Asn Met Ala Glu Gly Met Leu Leu Pro 180 185 190 Leu Pro Ser Val Gln Trp Asn His Asn His Glu Val Asp Gly Asp Asp 195 200 205 Asp Asp Val Ser Leu Trp Ser Tyr 210 215 101 632 DNA Brassica napus bnCBF1 101 cacccgatat accggggagt tcgtctgaga aagtcaggta agtgggtgtg tgaagtgagg 60 gaaccaaaca agaaatctag aatttggctt ggaactttca aaacagctga gatggcagct 120 cgtgctcacg acgtcgctgc cctagccctc cgtggaagag gcgcctgcct caattatgcg 180 gactcggctt ggcggctccg catcccggag acaacctgcc acaaggatat ccagaaggct 240 gctgctgaag ccgcattggc ttttgaggct gagaaaagtg atgtgacgat gcaaaatggc 300 cagaacatgg aggagacgac ggcggtggct tctcaggctg aagtgaatga cacgacgaca 360 gaacatggca tgaacatgga ggaggcaacg gcagtggctt ctcaggctga ggtgaatgac 420 acgacgacgg atcatggcgt agacatggag gagacaatgg tggaggctgt ttttactggg 480 gaacaaagtg aagggtttaa catggcgaag gagtcgacgg tggaggctgc tgttgttacg 540 gaggaaccga gcaaaggatc ttacatggac gaggagtgga tgctcgagat gccgaccttg 600 ttggctgata tggcagaagg gatgctcctg cc 632 102 208 PRT Brassica napus bnCBF1 polypeptide 102 His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val 1 5 10 15 Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr 20 25 30 Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 35 40 45 Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala Trp 50 55 60 Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys Ala 65 70 75 80 Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val Thr 85 90 95 Met Gln Asn Gly Gln Asn Met Glu Glu Thr Thr Ala Val Ala Ser Gln 100 105 110 Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 115 120 125 Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 130 135 140 His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 145 150 155 160 Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala 165 170 175 Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu 180 185 190 Trp Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met 195 200 205 103 20 DNA artificial sequence Artificial sequence 103 cayccnatht aymgnggngt 20 104 21 DNA artificial sequence Artificial sequence 104 ggnarnarca tnccytcngc c 21

Claims

What is claimed is:

1. A transgenic plant comprising a recombinant polynucleotide encoding a polypeptide having a HAP3 subfamily B domain, wherein said polypeptide has the property of SEQ ID NO: 4 of regulating abiotic stress tolerance in a plant when said polypeptide is overexpressed, and wherein: the HAP3 subfamily B domain is sufficiently homologous to the B domain of SEQ ID NO: 4 that the polypeptide binds to a transcription regulating region comprising the motif CCAAT; and wherein said binding confers increased abiotic stress tolerance in said transgenic plant as compared to a non-transformed plant that does not overexpress the polypeptide.

2. The transgenic plant of claim 1, wherein the HAP3 subfamily B domain is at least 83% identical in amino acid sequence to the B domain of SEQ ID NO: 4, and wherein said HAP3 subfamily B domain comprises:

12 Asn-(Xaa).sub.4-Lys-(Xaa).sub.33-34-Asn-Gly;

where Xaa is any amino acid residue; and overexpression of said polypeptide confers increased abiotic stress tolerance in said transgenic plant as compared to a non-transformed plant that does not overexpress the polypeptide.

3. The transgenic plant of claim 2, wherein said HAP3 subfamily B domain comprises:

13 Ser-(Xaa).sub.9-Asn-(Xaa).sub.4-Lys-(Xaa).sub.33-34-Asn-Gly;

where Xaa is any amino acid residue; and overexpression of said polypeptide confers increased abiotic stress tolerance in said transgenic plant as compared to a non-transformed plant that does not overexpress the polypeptide.

4. The transgenic plant of claim 1, wherein said polypeptide comprises SEQ ID NO: 4.

5. The transgenic plant of claim 1, wherein said recombinant polynucleotide has a nucleotide sequence that hybridizes over its full length to the complement of SEQ ID NO:3 under stringent conditions including two wash steps of 6.times.SSC and 65.degree. C. for 10-30 minutes.

6. The transgenic plant of claim 5, wherein said nucleotide sequence comprises SEQ ID NO: 3.

7. The transgenic plant of claim 1, wherein said abiotic stress tolerance is selected from the group consisting of heat tolerance, drought stress and salt stress.

8. The transgenic plant of claim 1, wherein said transgenic plant is characterized by altered sugar sensing as compared to a non-transformed plant that does not overexpress the recombinant polynucleotide.

9. The transgenic plant of claim 1, wherein the plant is selected from the group consisting of: soybean, wheat, corn, potato, cotton, rice, oilseed rape, sunflower, alfalfa, clover, sugarcane, turf, banana, blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, mint and other labiates, fruit trees, rosaceous fruits, citrus, and brassicas.

10. The transgenic plant of claim 1, further comprising a constitutive, inducible, or tissue-specific promoter operably linked to said recombinant polynucleotide.

11. The transgenic plant of claim 1, wherein said HAP3 subfamily B domain is at least 83% identical with the B domain of SEQ If) NO: 4.

12. A method for producing a transgenic plant having increased tolerance to osmotic stress, the method steps comprising: (a) providing an expression vector comprising (i) a nucleotide sequence that encodes a polypeptide having a B domain that is sufficiently homologous to the B domain of SEQ ID NO: 3 that the polypeptide binds to a transcription regulating region comprising the motif CCAAT and has the property of regulating abiotic stress tolerance in a plant as compared to a non-transformed plant that does not overexpress the polypeptide; wherein said nucleotide sequence comprises a B domain that is at least 83% identical with the B domain of SEQ ID NO: 4; and (ii) regulatory elements flanking the nucleotide sequence, said regulatory elements being effective to control expression of said nucleotide sequence in a target plant; (b) introducing the expression vector into a plant cell; (c) growing the plant cell into a plant and allowing the plant to overexpress said polypeptide; and; (d) identifying one or more abiotic stress tolerant plants so produced with increased abiotic stress tolerance by comparing said one or more abiotic stress tolerant plants with one or more non-transformed plants that do not overexpress the polypeptide.

13. The method of claim 12, wherein said nucleotide sequence hybridizes over its full length to the complement of SEQ ID NO: 3 under stringent conditions including two wash steps of 6.times.SSC and 65.degree. C. for 10-30 minutes.

14. The method of claim 12, wherein said abiotic stress tolerance is selected from the group consisting of heat tolerance, drought stress and salt stress.

15. The method of claim 12, the method steps further comprising: (e) crossing one of said abiotic stress tolerant plants with itself or another plant; (f) selecting seed that develops as a result of said crossing; and growing a progeny plant from the seed, thus producing a transgenic progeny plant having increased tolerance to abiotic stress.

16. The method of claim 15, wherein: said progeny plant expresses mRNA that encodes a DNA-binding protein that binds to a CCAAT DNA regulatory sequence and induces expression of a plant trait gene; and said mRNA is expressed at a level greater than a non-transformed plant that does not overexpress said DNA-binding protein.

17. A method for increasing a plant's tolerance to abiotic stress, said method comprising: (a) providing a vector comprising: regulatory elements flanking the polynucleotide sequence, said regulatory elements being effective to control expression of said polynucleotide sequence in a target plant; and a polynucleotide sequence that encodes a polypeptide having a B domain sufficiently homologous to the B domain of SEQ ID NO: 3 that the polypeptide binds to a transcription regulating region comprising the motif CCAAT and has the property of SEQ ID NO:4 of regulating abiotic stress tolerance in a plant, wherein said binding confers increased abiotic stress tolerance in said transgenic plant as compared to a non-transformed plant that does not overexpress the polypeptide; and (b) transforming the target plant with said vector to generate a transformed plant with increased tolerance to osmotic stress.

18. The method of claim 17, wherein said polynucleotide comprises: (i) SEQ ID NO: 3; (ii) a nucleotide sequence that encodes SEQ ID NO: 4; (iii) a nucleotide sequence that hybridizes to the nucleotide sequence of (i) or (ii) under stringent conditions including two wash steps of 6.times.SSC and 65.degree. C. for 10-30 minutes; or (iv) a nucleotide sequence encoding a polypeptide that comprises a B domain that is at least 83% identical with the B domain of SEQ ID NO: 4.

19. The method of claim 17, wherein said abiotic stress tolerance is selected from the group consisting of heat tolerance, cold germination, drought stress and salt stress.

20. A recombinant polynucleotide comprising a nucleotide sequence at least 99.6% identical to SEQ ID NO: 3.

21. The recombinant polynucleotide of claim 20, wherein said recombinant polynucleotide comprises SEQ ID NO: 3.

22. The recombinant polynucleotide of claim 20, wherein said recombinant polynucleotide is incorporated into an expression vector comprising one or more regulatory elements that are effective to control expression of said recombinant polynucleotide in a target plant

23. The recombinant polynucleotide of claim 22, wherein said recombinant polynucleotide is incorporated into a cultured host cell.