US20040098764A1

US20040098764A1 - Plant transcriptional regulators of abiotic stress

Info

Publication number: US20040098764A1
Application number: US10/685,922
Authority: US
Inventors: Jacqueline Heard; Jose Riechmann; Robert Creelman; Oliver Ratcliffe; Roderick Kumimoto; Neal Gutterson; T. Reuber; Omaira Pineda; Jeffrey Libby; Bradley Sherman
Original assignee: Mendel Biotechnology Inc
Current assignee: Mendel Biotechnology Inc
Priority date: 2001-03-16
Filing date: 2003-10-14
Publication date: 2004-05-20
Also published as: US20020142281A1; AU2002250342A1; WO2002074917A2; WO2002074917A3; US6835540B2

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, including improved drought and other osmotic stress tolerance, as compared to wild-type or reference plants. 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

This application claims priority from copending U.S. patent application Ser. No. 09/810,836, filed Mar. 16, 2001; U.S. patent application Ser. No. 10/412,699, filed Apr. 10, 2003, which claims priority from U.S. patent application Ser. No. 10/171,468, filed Jun. 14, 2002, U.S. Non-provisional Application No. 09/532,591, filed Mar. 22, 2000, U.S. Non-provisional Application No. 09/533,029, filed Mar. 22, 2000, U.S. Non-provisional Application No. 09/533,392, filed Mar. 22, 2000, which in turned claimed priority from U.S. [0001] Provisional Patent Application 60/125,814, filed Mar. 23, 1999, U.S. Non-provisional Application No. 09/713,994, filed Nov. 16, 2000, which in turn claimed priority from U.S. Provisional Patent Application No. 60/166,228, filed Nov. 17, 1999; U.S. Non-provisional Application No. 09/394,519, filed Sep. 13, 1999, which in turn claimed priority from U.S. Provisional Application No. 60/101,349, filed Sep. 22, 1998, and U.S. Provisional Application No. 60/108,734, filed Nov. 17, 1998; U.S. Non-provisional Application No. 10/374,780, filed Feb. 25, 2003, which claims priority from U.S. Non-provisional Application No. 09/934,455, filed Aug. 22, 2001, which in turn claims priority from U.S. Provisional Application No. 60/227,439, filed Aug. 22, 2000; U.S. Non-provisional Application No. 10/225,068, filed Aug. 9, 2002, which claims priority from U.S. Provisional Application No.60/336,049, filed Nov. 19, 2001 and U.S. Provisional Patent Application No. 60/310,847, filed Aug. 9, 2001; U.S. Non-provisional Application No. 10/225,066, filed Aug. 9, 2002; and U.S. Non-provisional Application No. 10/225,067, filed Aug. 9, 2002; the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

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

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.

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.

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.

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.

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 U.S. 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.

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).

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.

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.

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.

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.).

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.

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.

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 (U.S.) behind only to drought and flooding.

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).

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

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.

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

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

(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);

(b) signal transduction via mitogen-activated and/or calcium dependent protein kinases (CDPKs; see Xiong et al., 2002) and protein phosphatases (Merlot et al. (2001) Plant J. 25:295-303; Tähtiharju and Palva (2001) Plant J. 26:461-470);

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

(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);

(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);

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

(g) increased levels of antioxidants and compatible osmolytes such as proline and soluble sugars (Hasegawa et al. (2000) Annu. Rev. Plant Mol. Plant Physiol. 51:463-499); and

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

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.

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/DREB 1) 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).

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.

We have identified polynucleotides encoding transcription factors, including G867, G9, G993, G1930, 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 drought, excessive salt, cold and heat. 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. We have identified polynucleotides encoding transcription factors, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for their tolerance to abiotic stresses, including those associated with cold or osmotic stresses such as drought and salt tolerance. 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

The invention is directed to nucleic acid sequences that may be used to transform plants and confer abiotic stress tolerance to those plants. These plants have been shown to be tolerant to such diverse abiotic stresses as heat, chilling, cold, drought, and salt stress. The nucleic acid sequences may be incorporated into recombinant polynucleotide constructs prior to their use for transforming plants. These nucleic acid sequences include SEQ ID NO:1 (G867), SEQ ID NO:3 (G9), SEQ ID NO:5 (G993), SEQ ID NO:7 (G1930), and other sequences encoding polypeptides having AP2 and B3 domains that have been shown to confer abiotic stress tolerance in plants. The polypeptides encoded by the nucleic acid sequences of the invention, including SEQ ID NOs:2, 4, 6, 8 and others, have been shown to contain a newly discovered and highly conserved subsequence of about 22 amino acid residues in length, referred to herein as the “DML motif”, and which is always present between the AP2 and B3 domains of the polypeptides of the invention. For example, the “DML motif” of SEQ ID NO 1, (SEQ ID NO 64) is a 22-amino acid residue between the AP2 and may be found in Table 2 and in the Sequence Listing.

The invention includes recombinant polynucleotides comprising a nucleotide sequence that is capable of hybridizing over its full length to the complement of SEQ ID NOs:1, 3, 5, 7, or orthologs of these sequences, under stringent conditions that include two wash steps of 6×SSC and 65° C., each step having a duration of 10-30 minutes. These sequences may be incorporated into expression vectors and host plant cells.

The invention also includes an isolated nucleotide sequence that hybridizes over its full length to the complement of a polynucleotide that encodes the DML motif under stringent conditions that include two wash steps of 6×SSC and 65° C., each step being 10-30 minutes in duration:

The invention also includes transgenic plants that have increased tolerance to abiotic stress. In one iteration, these transgenic plants overexpress a recombinant polynucleotide that hybridizes over its full length to the complement of SEQ ID NO 1 under stringent conditions including two wash steps of 6×SSC and 65° C. for 10-30 minutes, in which instance the transgenic plants have increased abiotic stress tolerance as compared to a non-transformed plant that does not overexpress the polypeptide.

The invention also pertains to transgenic plants that comprise a recombinant polynucleotide that encodes a polypeptide having an AP2 domain and a B3 domain and has the property of the G867 polypeptide (SEQ ID NO:2) of regulating abiotic stress tolerance in a plant when overexpressed. In this instance, the AP2 domain is sufficiently homologous to the AP2 and B3 domains of the G867 polypeptide that the polypeptide binds to a transcription-regulating region comprising the motifs CAACA and CACCTG, respectively. This binding cooperatively enhances the DNA binding affinity of the polypeptide and thus confers increased abiotic stress tolerance in the transgenic plant, as compared to a non-transformed plant that does not overexpress the polypeptide.

The polypeptide of the invention contain a DML motif that is similar or identical in sequence to those found in the Sequence Listing (as a subsequence of any of the listed sequences) or listed in Table 2. These transgenic plants may be characterized by altered responses to high sugar concentrations when grown in Petri plates, which is often indicative of an osmotic stress (one group of abiotic stress) tolerance phenotype.

The invention incorporates methods for producing one or more transgenic plants that have increased tolerance to abiotic stress. This method includes the steps of providing an expression vector comprising any of the recombinant polynucleotides of the invention, introducing this expression vector into a plant cell, and then allowing the plant cell to overexpress a polypeptide encoded by the recombinant polynucleotide in the expression vector. The plant cells so produced may then be cultured and allowed to develop into more mature plant, individuals of which may then be identified as having increased abiotic stress tolerance. The polypeptides of the invention that are expressed in these plants have the property of regulating abiotic stress tolerance in the plant, as determined by comparing the stress tolerance of the plant to that of a non-transformed plant that does not overexpress the polypeptide. Those plants that are so altered and identified as having abiotic stress tolerance may then be selected.

Transgenic plants of the invention that have increased tolerance to abiotic stress may also be produced by the following method. A polynucleotide (or its complement) that encodes a polypeptide having an AP2 domain and a B3 domain is selected. In this case the AP2 domain and B3 domain is sufficiently homologous to the corresponding domains of SEQ ID NO:2 that the polypeptide will bind to a first transcription regulating region comprising the motif CAACA, and a second transcription regulating region comprising the motif CACCTG; respectively. This binding to the transcription regulating regions confers increased abiotic stress tolerance in the transgenic plant when the plant is compared to a nontransformed plant that does not overexpress the polypeptide. The polynucleotides of this aspect of the invention either:(i) comprise SEQ ID NO:1; (ii) encode SEQ ID NO:2; (iii) hybridize to any nucleotide sequence of (i) or (ii) under stringent conditions that include two wash steps of 6×SSC and 65° C. for 10 to 30 minutes; or (iv) encode a polypeptide that comprises AP2 and B3 domains that are substantially identical with the AP2 and B3 domains of SEQ ID NO:2, respectively. These domains may be found in Table 1. The next step of this method is performed by inserting the polynucleotide of (i), (ii), (iii) or (iv) into an expression cassette that also includes a constitutive, inducible, or tissue-specific promoter. The expression cassette is then introduced into a plant or plant cell to overexpress the polynucleotide sequence of (i), (ii), (iii) or (iv), thus producing a transgenic plant having increased tolerance to abiotic stress. This method may also include steps that include identifying transgenic plants so produced that have increased tolerance to abiotic stress, selecting one of these transgenic plants, and crossing the transgenic plant with either itself or another plant. The seed that develops in the plants produced by this crossing may be used to grow a progeny plant, thus producing a transgenic progeny plant that also has increased tolerance to abiotic stress.

The invention is also directed to a method for increasing a plant's tolerance to abiotic stress. In this method, the steps include providing a vector with regulatory elements that are able to control expression of a polynucleotide sequence in a target plant; and a polynucleotide sequence that is flanked by the regulatory elements and that encodes a polypeptide having an AP2 domain and a B3 domain that are sufficiently homologous to the AP2 and B3 domain of SEQ ID NO:1 (G867 polypeptide), respectively, that the polypeptide binds to the transcription regulating regions comprising the motifs CAACA and CACCTG, respectively. This binding is necessary to provide the polypeptide with the same property of SEQ ID NO:2 of regulating abiotic stress tolerance in a plant; that is, the binding confers increased abiotic stress tolerance in the transgenic plant as compared to a non-transformed plant that does not overexpress the polypeptide. A target plant is then transformed with this vector, thus generating a transformed plant with increased tolerance to abiotic stress. The polynucleotide of this aspect of the invention may include, for example:(i ) SEQ ID NO:1; (ii) any nucleotide sequence that encodes SEQ ID NO:2; (iii) any nucleotide sequence that hybridizes to the nucleotide sequence of (i) or (ii) under stringent conditions of 6×SSC and 65° C.; or (iv) a nucleotide sequence encoding a polypeptide comprising has AP2 and B3 domains that are substantially identical with the AP2 and B3 domains of SEQ ID NO:2.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

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. [0042]
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. [0043]
CD-[0044] ROM 1 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 “MBI0049CIP.ST25.txt” and is 139 kilobytes in size. The copies of the Sequence Listing on the CD-ROM disc are hereby incorporated by reference in their entirety.
FIG. 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Angiosperm Phylogeny Group (1998) [0045] 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.
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) [0046] Proc. Natl. Acad. Sci. 97:9121-9126; and Chase et al. (1993) Ann. Missouri Bot. Gard. 80:528-580.
FIG. 3 depicts a phylogenetic tree of several members of the RAV family, identified through BLAST analysis of proprietary (using corn, soy and rice genes) and public data sources (all plant species). This tree was generated as a Clustal X 1.81 alignment:MEGA2 tree, Maximum Parsimony, bootstrap consensus. [0047]
FIGS. [0048] 4A-4J show an alignment of AP2 transcription factors from Arabidopsis, soybean, rice and corn, showing conserved (identical or similar residues) and the AP2 domains, DML motifs, and B3 domains.
In FIG. 5, three G867-overexpressing lines and a wild-type control were germinated on media containing high (150 mM) NaCl. The overexpressing lines showed increased seedling vigor, manifested by increased expansion of the cotyledons, compared to the wild-type controls. [0049]
In FIG. 6, three G867-overexpressing lines and a wild-type control were germinated on media containing high (9.4%) sucrose. Increased seedling vigor was also noted with the overexpressors as compared to the wild-type plants, as indicated by the increased expansion of the cotyledons in the overexpressors. [0050]
FIG. 7 is a photograph of plants grown on MS media; the eight plants to the left of the vertical black line are from three different G9-overexpressing lines and have significantly more root mass and root branching than the four wild-type control plants to the right of the black line. [0051]
FIG. 8 shows that, when grown on media containing 10 μM methyl jasmonate, plants from three different G9-overexpressing lines, as seen with the eight plants to the left of the vertical black line, have more root hairs than the four wild-type control plants to the right of the black line.[0052]

DESCRIPTION OF THE INVENTION

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. [0053]
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. [0054]
Definitions [0055]
“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). [0056]
“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 termns amplimer, primer, oligomer, element, target, and probe and is preferably single stranded. [0057]
“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. [0058]
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) [0059] 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.
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. [0060]
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. [0061]
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. [0062]
“Protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic. [0063]
“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. [0064]
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. [0065]
“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. [0066]
“Hybridization complex” refers to a complex between two nucleic acid molecules by virtue of the formation of hydrogen bonds between purines and pyrimidines. [0067]
“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. [0068]
With regard to polypeptides, the terms “substantial identity” or “substantially identical” may refer to sequences of sufficient similarity and structure to the transcription factors in the Sequence Listing to produce similar function when expressed or overexpressed in a plant; in the present invention, this function is increased tolerance to abiotic stress. Sequences that are at least about 80% identical, to the instant polypeptide sequences, including AP2 and B3 domain sequences, are considered to have “substantial identity” with the latter. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents. The structure required to maintain proper functionality is related to the tertiary structure of the polypeptide. There are discreet domains and motifs within a transcription factor that must be present within the polypeptide to confer function and specificity. These specific structures are required so that interactive sequences will be properly oriented to retain the desired activity. “Substantial identity” may thus also be used with regard to subsequences, for example, motifs, that are of sufficient structure and similarity, being at least about 80% identical to similar motifs in other related sequences so that each confers or is required for increased tolerance to abiotic stress. [0069]
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. [0070]
“Alignment” refers to a number of nucleotide or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those found in FIGS. [0071] 4A-4J may be used to identify AP2, DML and B3 domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MacVector (1999) (Accelrys, Inc., San Diego, Calif.).
A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. AP2 binding domains and B3 domains are examples of conserved domain. [0072]
With respect to polynucleotides encoding presently disclosed transcription factors, a conserved domain is preferably at least 10 base pairs (bp) in length. [0073]
A “conserved domain”, with respect to presently disclosed polypeptides refers to a domain within a transcription factor family that exhibits a higher degree of sequence homology, such as at least 70% sequence similarity, including conservative substitutions, and more preferably at least 79% sequence identity, and even more preferably at least 81%, or at least about 86%, or at least about 87%, or at least about 89%, or at least about 91%, or at least about 95%, or at least about 98% amino acid residue sequence identity 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 subfamily. 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. [0074]
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 (i.e., the AP2 domains) of the AP2 plant transcription factors (Riechmann and Meyerowitz (1998) [0075] Biol. Chem. 379:633-646) may be determined.
The AP2-binding and B3 (or conserved) domains for SEQ ID NO:2, 4, 6, and 8 and numerous orthologs are listed in Table 1. Also, the polypeptides of Table 1 have AP2-binding and B3 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 AP2-binding and B3 domains for any of the polypeptides listed or referred to in this disclosure. [0076]
“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. [0077]
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) [0078] 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.
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, transcription factors having 60% identity, or more preferably greater than about 70% identity, most preferably 72% or greater identity with disclosed transcription factors. [0079]
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. [0080]
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”. [0081]
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. [0082]
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. [0083]
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. [0084]
“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. [0085]
“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. [0086]
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. [0087]
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, for example, replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or 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). [0088]
“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. [0089]
“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. [0090]
The term “plant” includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. (See for example, FIG. 1, adapted from Daly et al. (2001) [0091] 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).
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. [0092]
A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, for example, a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell. [0093]
“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). [0094]
“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. [0095]
“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 an AP2 domain of a transcription factor. [0096]
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 an AP2 binding or a B3 domain of a transcription factor, for example, amino acid residues 59-124 or amino acid residues 187-272 of G867 (SEQ ID NO:2), as noted in Table 1. [0097]
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. [0098]
“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. [0099]
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, for example, 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. [0100]
“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. [0101]
The term “transcript profile” refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell repressing or overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods. [0102]
“Ectopic expression or altered expression” in reference to a polynucleotide indicates that the pattern of expression in, for example, a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term “ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides. [0103]
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 (for example, 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. [0104]
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. [0105]
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. [0106]
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. [0107]
A “sample” with respect to a material containing nucleic acid molecules may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a forensic sample; and the like. In this context “substrate” refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores. A substrate may also refer to a reactant in a chemical or biological reaction, or a substance acted upon (for example, by an enzyme). [0108]
“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. [0109]

DETALED DESCRIPTION

Transcription Factors Modify Expression of Endogenous Genes [0110]
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) [0111] Science 290:2105-2110). The plant transcription factors may belong to the AP2 protein transcription factor family (Riechmann and Meyerowitz (1998) supra).
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. [0112]
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. [0113]
In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, for example, mutation reactions, PCR reactions, or the like; as substrates for cloning for example, including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, for example, genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations. [0114]
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) [0115] 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).
In another example, Mandel et al. (1992) Cell 71-133-143), and Suzuki et al.(2001) [0116] Plant J. 28: 409418 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).
Other examples include Muiller et al. (2001) [0117] 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).
In yet another example, Gilmour et al. (1998) [0118] Plant J. 16:433-442, teach an Arabidopsis AP2 transcription factor, CBF 1 (SEQ ID NO:55), 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 (Jaglo et al. (2001) 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 (for example, by a cascade of transcription factor binding events and transcriptional changes) altered by transcription factor binding. In a global analysis of transcription comparing a standard condition with one in which a transcription factor is overexpressed, the resulting transcript profile associated with transcription factor overexpression is related to the trait or cellular process controlled by that transcription factor. For example, the PAP2 gene (and other genes in the MYB family) have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al. (2000) [0119] Plant Cell, 12:65-79; Borevitz et al. (2000) Plant Cell 12:2383-93). Further, global transcript profiles have been used successfully as diagnostic tools for specific cellular states (for example, cancerous vs. non-cancerous; Bhattacharjee et al. (2001) Proc Natl. Acad. Sci., USA, 98:13790-13795; Xu et al. (2001) Proc. Natl. Acad. Sci., USA, 98:15089-15094). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different transcription factors would indicate similarity of transcription factor function.
Polypeptides and Polynucleotides of the Invention [0120]
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. [0121]
The sequences of G867 and G9 were previously identified in U.S. [0122] provisional patent application 60/101,349, filed Sep. 22, 1998, at which time these sequences were identified as encoding or being transcription factors, which were defined as polypeptides having the ability to effect transcription of a target gene. It is noted that sequences that have gene-regulating activity have been determined to have specific and substantial utility (Federal Register (2001) 66(4):1095). The functions of G867 and G9 were previously disclosed in U.S. provisional patent applications 60/227,439, filed Aug. 22, 2000, and 60/166,228, filed Nov. 17, 1999, respectively. The sequence of G993 was previously identified in U.S. provisional applications 60/108,734, filed Nov. 17, 1998, and 60/125,814, filed Mar. 23, 1999. The function of G993 was implied from its homologous relationship with G867, as disclosed in U.S. non-provisional application 09/934,455, filed Aug. 22, 2001. The sequence of G1930 was previously identified in U.S. non-provisional application 09/934,455, filed Aug. 22, 2001. The functions of G1930 were previously disclosed in U.S. non-provisional patent application 09/934,455, filed Aug. 22, 2001.
In some cases, exemplary polynucleotides encoding the polypeptides of the invention were identified in the [0123] 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.
Additional polynucleotides of the invention were identified by screening [0124] 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.
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. [0125]
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. [0126]
G867, which we have determined to confer osmotic stress tolerance in plants when overexpressed, has been described in the literature as related to ABI3/VP1 (RA VI; Kagaya et al. (1999) [0127] Nucleic Acids Res. 27:470-478) based on the presence of a B3 domain (which is also found in the ABI3/VP1 family of transcription factors). The protein also contains an AP2 domain, and is therefore presently included in the AP2/ERF family of transcription factors. Both the AP2 domain transcription factors and the B3 domain transcription factors are described below.
AP2 domain transcription factors. Ohme-Takagi and Shinshi (1995). [0128] Plant Cell 7, 173-182) determined that the function of the AP2 domain is DNA binding. The AP2 region of the putative tobacco transcription factor EREBP2 is responsible for its binding to the cis-acting ethylene response DNA element referred to as the GCC-repeat. As discussed by Ohme-Takagi and Shinshi (1995) supra), the DNA-binding or AP2 domain of EREBP2 contains no significant amino acid sequence similarities or obvious structural similarities with other known transcription factors or DNA binding motifs beyond AP2 transcription factors. Thus, the domain appears to be a novel DNA-binding motif that, to date, has only been found in plant proteins.
The RAV-like proteins, including G897, G9, G993 and G1930, form a small subgroup in the AP2/ERF family of AP2 transcription factors. This large gene family includes at least 145 transcription factors, and can be further divided in three larger subfamilies: [0129]
(a) The APETALA2 class is characterized by the presence of two AP2 DNA binding domains, and contains fourteen genes. [0130]
(b) The RAV subgroup, which includes six genes, is characterized by the presence of a B3 DNA binding domain in addition to the AP2 DNA binding domain. [0131]
(c) The AP2/ERF subfamily, which is the largest subfamily and includes 125 genes, is characterized by the presence of only one AP2 DNA binding domain, and includes genes that are involved in abiotic and biotic stress responses. This subfamily is composed of two relatively equal size subgroups, the DREB and ERF subgroups (Sakuma et al. (2002) [0132] Biochem and Biophys Res Comm 290:998-1009), which are distinguished on the basis of specific residues in the AP2 DNA binding domain.
The binding characteristics of G867 (RAVI) have been characterized by Kagaya et al. ((1999) [0133] Nucleic Acids Res. 27:470478; see below). There is no published information on the biological function of the RAV-like transcription factors.
B3 domain transcription factors. Transcription factors of the ABI3/[0134] VP 1 family have been implicated in seed maturation processes. AB13 (G621) plays an important role in the acquisition of desiccation tolerance in late embryogenesis. This process is related to dehydration tolerance as evidenced by the protective function of late embryogenesis abundant (LEA) genes such as HVA1 (Xu et al. (1996) Plant Physiol. 110:249-257; Sivamani et al. (2000) Plant Science 155:1-9). Mutants for Arabidopsis ABI3 (Ooms et al. (1993) Plant Physiol. 102:1185-1191) and the maize ortholog VP1 (Carson et al. (1997) Plant J. 12:1231-1240) and references therein) show severe defects in the attainment of desiccation tolerance. Also, 35S::ABI3 overexpression in combination with increased levels of abscisic acid results in an induction of several ABA/cold/drought-responsive genes such as RAB18 and RD29A and increased freezing tolerance in Arabidopsis (Tammninen et al. (2001) Plant J. 25:1-8). This illustrates the relatedness of desiccation and dehydration tolerance and demonstrates that the seed-specific ABI3 transcription factor does not require additional seed-specific proteins to function in vegetative tissues.
Both in Arabidopsis and maize, the B3 domain of [0135] ABI3NP 1 binds the RY/SPH motif (Ezcurra et al. (2000) Plant J. 24:57-66); Carson et al. (1997) supra) while the B2 domain interacts with the ABRE elements in a complex involving bZIP transcription factors (TRAB 1 in maize, Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15348-15353). While in Arabidopsis the B3 domain of ABI3 is essential for abscisic acid dependent activation of late embryogenesis genes (Ezcurra et al. (2000) supra), the B3 domain of VPI is not essential for ABA regulated gene expression in maize seed (Carson et al. (1997) supra; McCarty et al. (1989) Plant Cell 1:523-532). This difference in the regulatory network between Arabidopsis and maize can be explained by differential usage of the RY/SPH versus the ABRE element in the control of seed maturation gene expression (motif (Ezcurra et al. (2000) supra). The RY/SPH element is a key element in gene regulation during late embryogenesis in Arabidopsis (Reidt et al. (2000) Plant J. 21:401408) while it seems to be less important for seed maturation in maize (McCarty et al. (1989) supra).
Mutations in two other B3 domain transcription factors, FUS3 (G1014) and LEC2 (G3035) result in pleiotropic effects. In the case of fus3, these effects are mainly restricted to seed development during late embryogenesis (Keith et al. (1994) [0136] Plant Cell 6:589-600). Overexpression of LEC2 results in somatic embryo formation on the cotyledons (Stone et al. (2001) Proc. Natl. Acad. Sci. USA 98:11806-11811). The FUS3 protein can be considered as a natural truncation of the ABI3 protein (Luerssen et al. (1998) Plant J. 15:755-764); like the latter, it binds to the RY/SPH element, and can activate the expression from target promoters even in non-seed tissues (Reidt et al. (2000) supra).
Singh et al. have recently submitted a polynucleotide sequence (Accession No. CB686050) from a transgenic [0137] Brassica napus (CBF 17) that has been shown to be constitutively frost resistant. The predicted polypeptide sequence has a DML motif that is 90% identical, and a B3 domain that is 95% identical, to the DML motif and B3 domain of G867, respectively. The protein predicted from this sequence does not comprise an AP2 domain.
Binding of G867 and G9 to bipartite recognition sequences by the AP2 and B3 DNA-binding domains. Kagaya et al. ((1999) supra) cloned and characterized G867 (RAV1) and G9 (RAV2) from [0138] Arabidopsis thaliana. The two transcription factors were found to contain two distinct amino acid sequence domains found only in higher plant species, the AP2 and B3 domains. The N-terminal regions of G867 and G9 were shown to be homologous to the AP2 DNA-binding domain present in the Arabidopsis APETALA2 and tobacco EREBP proteins families, while the C-terminal region exhibited homology to the B3 domain of VP 1/ABI3 transcription factors. Binding site selection assays using a recombinant glutathione S-transferase fusion protein revealed that G867 bound specifically to bipartite recognition sequences composed of two unrelated motifs, 5′-CAACA-3′ and 5′-CACCTG-3′, separated by various spacings in two different relative orientations. Analyses using various deletion derivatives of the RAV1 fusion protein showed that the AP2 and B3-like domains of RAV1 bind autonomously to the CAACA and CACCTG motifs, respectively, and together achieve a high affinity and specificity of binding. Kagaya et al. concluded that the AP2 and B3-like domains of RAVI are connected by a highly flexible structure enabling the two domains to bind to the CAACA and CACCTG motifs in various spacings and orientations.
The RAV-like proteins, including G897, G9, G993 and G1930, generally have both AP2 and B3 domains. Within the G867 clade, there is a high degree of conservation of the AP2 and B3 domains in all members of the clade. The proteins in the G867 clade were also found to possess a subsequence with a high degree of conservation between the AP2 and B3 domains. This subsequence was designated DML motif. The DML motif does not appear to be present in transcription factors outside of the G867 clade (more detailed description of the DML motif appears below, and a list of DML motif sequences may be found in Table 2). [0139]

Table 1 shows the polypeptides identified by polypeptide SEQ ID NO and Mendel Gene ID (GID) No., presented in order of similarity to G867 by AP2 domain, and includes the AP2 and B3 binding domains of the polypeptide in amino acid coordinates, the respective AP2 domain sequences, the extent of identity in percentage terms to the AP2 domain of G867, the respective B3 domains, and the extent of identity in percentage terms to the B3 domain of G867.

TABLE 1


Gene families and binding domains

		AP2 and B3		% ID to		% ID to
SEQ		Domains in		AP2 Domain		B3 Domain
ID NO:	GID No.	AA Coordinates	AP2 Domain	of G867	B3 Domain	of G867

2	G867	AP2: 59-124	SSKYKGVVPQPNGRWG	100%	LFEKAVTPSDVGKLNRLVIP	100%
		B3: 187-272	AQIYEKHQRVWLGTFN		KHHAEKHFPLPSSNVSVKGV
			EEDEAARAYDVAVHRF		LLNFEDVNGKVWRFRYSYW
			RRRDAVTNFKDVKMDE		NSSQSYVLTKGWSRFVKEK
			DE		NLRAGDVV
6	G993	AP2: 69-134	SSKYKGVVPQPNGRWG	89%	LFEKTVTPSDVGKLNRLVIP	79%
		B3: 194-286	AQIYEKHQRVWLGTFN		KQHAEKHFPLPAMTTAMG
			EEEEAASSYDIAVRRFR		MNPSPTKGVLINLEDRTGKV
			GRDAVTNFKSQVDGND		WRFRYSYWNSSQSYVLTKG
			A		WSRFVKEKNLRAGDVV
42	BZ458719	AP2: 42-107	SSKFKGVVPQPNGRWG	87%	LFEKTVTPSDVGKLNRLVIP	86%
		B3: 172-258	AQIYEKHKRVWLGTFN		KHQAEKHFPLPLTGDVSVR
			EEEEAARVYDVAAHRF		GTLLNFEDVNGKVWRFRYS
			RGSDAVTNFKPDTTFRN		YWNSSQSYVLTKGWSRFVK
			G		EKRLCAGDLI
8	G1930	AP2: 59-124	SSRFKGVVPQPNGRWG	86%	LFEKTVTPSDVGKLNRLVIP	87%
		B3: 182-269	AQIYEKHQRVWLGTFN		KHQAEKHFPLPLGNNNVSV
			EEDEAARAYDVAAHRF		KGMLLNFEDVNGKVWRFR
			RGRDAVTNFKDTTFEEE		YSYWNSSQSYVLTKGWSRF
			V		VKEKRLCAGDLI
36	G3391	AP2: 79-145	SSKFKGVVPQPNGRWG	84%	LFDKTVTPSDVGKLNRLVIP	83%
		B3: 215-302	AQIYERHQRVWLGTFA		KQHAEKHFPLQLPSAGGESK
			GEDDAARAYDVAAQRF		GVLLNFEDAAGKVWRFRYS
			RGRDAVTNFRPLAEADP		YWNSSQSYVLTKGWSRFVK
			DA		EKGLHADGKL
46	BU025988	AP2: 25-90	SSRYKGVVPQPNGRWG	83%	LFQKTVTPSDVGKLNRLVIP	81%
		B3: 152-236	AQIYEKHQRVWLGTFN		KQHAEKHFPVQKGSNSKGV
			DEDEAAKAYDVAVQRF		LLHFEDKGSKVWRFRYSYW
			RGRDAVTNIKQVDADD		NSSQSYVLTKGWSRFVKEK
			KE		NLKAGDSV
22	G3452	AP2: 51-116	SSKYKGVVPQPNGRWG	83%	LFEKTVTPSDVGKLNRLVIP	78%
		B3: 171-266	AQIYEKHQRVWLGTFN		KQHAEKHFPLSGSGDESSPC
			EEDEAARAYDIAALRFR		VAGASAAKGMLLNFEDVGG
			GPDAVTNFKPPAASDDA		KVWRFRYSYWNSSQSYVLT
					KGWSRFVKEKNLRAGDAV
24	G3453	AP2: 57-122	SSKYKGVVPQPNGRWG	83%	LVEKTVTPSDVGKLNRLVIP	77%
		B3: 177-272	AQIYEKHQRVWLGTFN		KQHAEKHFPLSGSGGGALPC
			EEDEAVRAYDIVAHRFR		MAAAAGAKGMLLNFEDVG
			GRDAVTNFKPLAGADD		GKVWRFRYSYWNSSQSYVL
			A		TKGWSRFVKEKNLRAGDAV
38	G3432	AP2: 75-141	SSRYKGVVPQPNGRWG	82%	LFDKTVTPSDVGKLNRLVIP	82%
		B3: 212-299	AQIYERHQRVWLGTFA		KQHAEKHFPLQLPSAGGESK
			GEADAARAYDVAAQRF		GVLLNLEDAAGKVWRFRYS
			RGRDAVTNFRPLADADP		YWNSSQSYVLTKGWSRFVK
			DA		EKGLQAGDVV
32	G3389	AP2: 64-129	SSRYKGVVPQPNGRWG	82%	LFEKAVTPSDVGKLNRLVVP	78%
		B3: 177-266	AQIYERHARVWLGTFPD		KQQAERHFPFPLRRHSSDAA
			EEAAARAYDVAALRFR		GKGVLLNFEDGDGKVWRFR
			GRDAVTNRAPAAEGAS		YSYWNSSQSYVLTKGWSRF
			A		VREKGLRPGDTV
40	G3433	AP2: 80-146	SSRYKGVVPQPNGRWG	82%	MFDKVLTPSDVGKLNRLVV	59%
		B3: 210-291	AQIYERHLRVWLGTFTG		PKQHAERFFPAAGAGSTQLC
			EAEAARAYDVAAQRFR		FQDRGGALWQFRYSYWGSS
			GRDAVTNFRPLAESDLD		QSYVMTKGWSRFVRAARLA
			P		AGDTV
4	G9	AP2: 62-127	SSKYKGVVPQPNGRWG	81%	LFEKAVTPSDVGKLNRLVIP	91%
		B3: 187-273	AQIYEKHQRVWLGTFN		KQHAEKHFPLPSPSPAVTKG
			EQEEAARSYDIAACRFR		VLINFEDVNGKVWRFRYSY
			GRDAVVNFKNVLEDGD		WNSSQSYVLTKGWSRFVKE
			L		KNLRAGDVV
44	BQ971511	AP2: 21-86	SSRYKGVVPQANGRWG	81%	LFQKTVTPSDVGKLNRLVIP	81%
		B3: 147-231	AQIYEKHQRVWLGTFN		KQHAEKHFPVQKGISSKGVL
			DEDEAAKAYDVAVQRF		LHFEDTESKVWRFRYSYWN
			RGRDAVTNFKQLVTDD		SSQSYVLTKGWSRFVKEKN
			NA		LKAGDSV
18	G3451	AP2: 80-146	SSKYKGVVPQPNGRWG	81%	LFEKAVTPSDVGKLNRLVIP	78%
		B3: 209-308	AQIYEKHQRVWLGTFN		KQHAEKHFPLQSSNGVSATT
			EEDEAARAYDIAAQRFR		IAAVTATPTAAKGVLLNFED
			GKDAVTNFKPLAGADD		VGGKVWRFRYSYWNSSQSY
			DD		VLTKGWSRFVKEKNLKAGD
					TV
26	G3454	AP2: 74-139	SSKYKGVVPQPNGRWG	81%	LFEKAVTPSDVWKLNRLVIP	77%
		B3: 203-302	SQIYEKHQRVWLGTFNE		KQHAEKHFPLQSSNGVSATT
			EDEAARAYDVAVQRFR		IAAVTATPTAAKGVLLNFED
			GKDSVTNFKPLAGADD		VGGKVWRFRYSYWNSSQSY
			D		VLTKGWSRFVKEKNLKAGD
					TV
30	G3388	AP2: 66-131	SSRYKGVVPQPNGRWG	78%	LFEKAVTPSDVGKLNRLVVP	76%
		B3: 181-274	AQIYERHARVWLGTFPD		KQHAEKHFPLRRAASSDSAS
			EEAAARAYDVAALRYR		AAATGKGVLLNFEDGEGKV
			GRDAATNFPGAAASAAE		WRFRYSYWNSSQSYVLTKG
					WSRFVREKGLRAGDTI
50	CC616336	AP2: 63-128	SSKYKGVVPQPNGRWG	78%	LFDKTVTPSDVGKLNRLVIP	74%
		B3: 197-291	AQIYERHQRVWLGTFTG		KQHAEKHFPLQLPAAAAAG
			EAEAARAYDVAAQRFR		VGSGGECKGVLLNFEDAAG
			GRDAVTNFRPLAESEPE		KAWRFRYSYWNSSQSYVLT
					KGWSRFVKEKGLHAGDAV
34	G3390	AP2: 66-131	SSKYKGVVPQPNGRWG	77%	LFDKTVTPSDVGKLNRLVIP	70%
		B3: 192-294	AQIYERHQRVWLGTFTG		KQHAEKHFPLQLPPPTTTSS
			EAEAARAYDVAAQRFR		VAAAADAAAGGGDCKGVL
			GRDAVTNFRPLAESDPE		LNFEDAAGKVWKFRYSYW
					NSSQSYVLTKGWSRFVKEK
					GLHAGDAV
52	AAAA01000997	AP2: 66-131	SSKYKGVVPQPNGRWG	77%	LFDKTVTPSDVGKLNRLVIP	69%
		B3: 192-294	AQIYERHQRVWLGTFTG		KQHAEKHFPLQLPPPTTTSS
			EAEAARAYDVAAQRFR		VAAAADAAAGGGECKGVLL
			GRDAVTNFRPLAESDPE		NFEDAAGKVWKFRYSYWN
					SSQSYVLTKGWSRFVKDKG
					LHAGDAV
48	BT009310	AP2: 64-129	SSKYKGVVPQPNGRWG	75%	LFDKTVTPSDVGKLNRLVIP	78%
		B3: 200-291	AQIYERHQRVWLGTFTG		KQHAEKHFPLQLPSAGAAVS
			EAEAARAYDAAAQRFR		GECKGMLLNFDDSAGKVW
			GRDAVTNFRPLTESDPE		RFRYSYWNSSQSYVLTKGW
					SRFVKEKGLHAGDAV

The transcription factors of the present invention possess an AP2 domain and a B3 domain. The present invention also includes fragments of such transcription factors, which may be comprised of both, or only one, of these binding domains. The latter is true for the full-length orthologs of G867 found by BLAST analysis, as described below. Generally, the AP2 domain of the transcription factors will bind to a transcription regulating region comprising the motif CAACA, and the B3 domain of the same transcription factor binds to a second transcription regulating region comprising the motif CACCTG. Each of these transcription factors 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. As shown in Table 1, the AP2 and B3 domains of the transcription factors within the G867 clade are at least 75% (for the AP2 domain) and 69% (for the B3 domain) identical to the corresponding domains of G867, and all four of these transcription factors, which rely on the binding specificity of their conserved AP2 and B3 domains, have very similar or identical functions in plants, conferring increased abiotic, including osmotic, stress tolerance when overexpressed. [0141]
Therefore, the invention provides polynucleotides comprising: Arabidopsis SEQ ID NOs:1, 3, 5, 7, and fragments thereof; and non-Arabidopsis sequences SEQ ID NOs:11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41,43, 45,47, 49, 51, paralogs, orthologs, equivalogs, and fragments thereof. The invention also provides polypeptides and the polynucleotides that encode them, said polypeptides comprising:Arabidopsis SEQ ID NOs:2, 4, 6, 8, and fragments thereof; and non-Arabidopsis SEQ ID NOs:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 53, paralogs, orthologs, equivalogs, and fragments thereof. A number of these polynucleotides have been shown to have a strong association with osmotic stress tolerance, in that plants that overexpress these sequences are more tolerant to these stresses. The invention also encompasses a complement of the polynucleotides. The polynucleotides are useful for screening libraries of molecules or compounds for specific binding and for creating transgenic plants having increased osmotic stress tolerance. [0142]
A number of the polynucleotides of the invention have been, and the remainder of the polynucleotides of the invention may be, 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. [0143]
The polynucleotides are particularly useful when they are hybridizable array elements in a microarray. Such a microarray can be employed to monitor the expression of genes that are differentially expressed in response to osmotic stresses. The microarray can be used in large scale genetic or gene expression analysis of a large number of polynucleotides; or in the diagnosis of osmotic stress before phenotypic symptoms are evident. Furthermore, the microarray can be employed to investigate cellular responses, such as cell proliferation, transformation, and the like. [0144]
When the polynucleotides of the invention may also be used as hybridizable array elements in a microarray, the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular stress, pathology, or treatment. [0145]
The invention also entails an agronomic composition comprising a polynucleotide of the invention in conjunction with a suitable carrier and a method for altering a plant's trait using the composition. [0146]
The invention also encompasses transcription factor polypeptides that comprise the DML motif, which, in the case of G867, is HSKSEIVDMLRKHTYNEELEQS (SEQ ID NO:64), or a motif that has 71% or greater identity to the DML motif of G867, and having substantially similar activity with that of SEQ ID NO:2. [0147]
Identification of Motifs Unique to G867 Dicot Orthologs [0148]
Arabidopsis sequences thought to be paralogous or otherwise highly related evolutionarily to G867 were aligned using Clustal X ([0149] version 1 .81, June 2000). Additionally, by BLASTP analysis of proprietary and public databases with protein sequences of this set, additional sequences were identified with a high degree of sequence relatedness to G867. A number of these sequences, in addition to G867, are known to enhance abiotic stress tolerance, including G9 (SEQ ID NO:3 and 4), G993 (SEQ ID NO:5 and 6), and G1930 (SEQ ID NO:7 and 8). These sequences were then aligned again, and a neighbor-joining algorithm used to generate a phylogenetic tree, using Clustal X v1.81's phylogenetic capabilities. In this alignment, G867 and it paralogs G9, G993 and G1930 appeared in a clade along with two soybean sequences and several rice sequences. Based on the utility of the Arabidopsis sequences, as noted below, and the evolutionary history revealed by analysis of the phylogenetic tree (that the last common ancestor of the monocots and the eudicots had only one gene corresponding to transcription factors of the present invention, which functioned in abiotic stress tolerance), transcription factors of the G867 clade comprise a number of genes involved in the control of abiotic stress tolerance.
Examination of the alignment of only those sequences in the G867 clade (having monocot and dicot subnodes), indicates 1) a high degree of conservation of the AP2 domains in all members of the clade, 2) a high degree of conservation of the B3 domains in all members of the clade; and 3) a high degree of conservation of an additional motif, the DML motif found between the AP2 and B3 domains in all members of the clade:(H/R S K Xa E/G I/V V D M L R K/R H T Y Xa E/D/N E L/F Xa O/H S/N/R/G (where Xa is any amino acid), constituting positions 135-152 in G867, SEQ ID NO:64. As a conserved motif found in G867 and its paralogs, the DML motif was used to identify additional orthologs of SEQ ID 2. A significant number of sequences were found that had a minimum of 71 % identity to the 22 residue DML motif of G867, a number of these motifs are shown in Table 2. [0150]

Upon translation of these nucleotide sequences in a frame that provided the identified conserved motif, all the resulting protein sequences were found to have either a conserved AP2 domain before the DML motif, or a B3 domain after DML motif (i.e., in BU024575, BQ405698, BF424857, BZ458719, AP002913, and AX654438). The protein sequences having conserved AP2 and/or B3 domains in the expected location were aligned with the previously aligned set of AP2 and B3 sequences, and a neighbor-joining algorithm was used to generate a phylogenetic tree, as described above. In this tree, the additional sequences identified through the DML motif all were found within the G867 clade identified previously, indicating that the DML motif was successfully used to identify new orthologs of G867, listed in Table 2.

TABLE 2


Representative ortholog sequences identified using conservation to the DML motif

				Identity (%)
SEQ	Identifier or			with the DML
ID NO:	Accession No.	Species	Subsequence	motif of G867

2	G867	Arabidopsis thaliana	HSKSEIVDMLRKHTYNEELEQS		100%
28	G3455	Glycine max	HSKSEIVDMLRKHTYNDELEQS	95%
	BU024575	Helianthus annuus	HSKSEIVDMLRKHTYNDELEQS	95%
	BQ137035	Medicago truncatula	HSKSEIVDMLRKHTYNDELEQS	95%
	AV412541	Lotus japonicus	HSKSEIVDMLRKHTYNDELEQS	95%
8	G1930	Arabidopsis thaliana	HSKSEIVDMLRKHTYKEELDQR		90%
18	G3451	Glycine max	HSKPEIVDMLRKHTYNDELEQS	90%
42	BZ458719	Brassica oleracea	HSKYEIVDMLRKHTYKEELEQR	90%
	BU871082	Populus balsamifera	HSKAEIVDMLRKHTYNDELEQS		90%
		subsp. Trichocarpa
	BG524914	Stevia rebaudiana	HSKAEIVDMLRKHTYNDELEQS	90%
	BQ405698	Gossypium arboreum	HSKAEIVDMLRKHTYNDELEQS		90%
	BF424857	Glycine max	HSKPEIVDMLRKHTYNDELEQS	90%
	CB686050	Brassica napus	HSKSGIVDMLRKHTYSEELEQS	90%
46	BU025988	Helianthus annuus	HSESEIVDMLRKHTYNDELEQS		90%
	BQ855250	Lactuca sativa	HSKAEIVDMLRKHTYNDELQQS	86%
	BM878902	Ipomoea batatas	HSKAEIVDMLRKHTYADELEQS	86%
	BG590382	Solanum tuberosum	HSKAEIVDMLRKHTYLDELEQS	86%
	BG124312	Lycopersicon esculentum	HSKAEIVDMLRKHTYIDELEQS	86%
26	G3454	Glycine max	HSKPEIVDMLRKHTYNDELEHS	86%
44	BQ971511	Helianthus annuus	HSKSEIVDMLRKHTYNDELEQS	86%
50	CC616336	Zea mays	RSKAEVVDMLRKHTYGEELAHN	83%
4	G9	Arabidopsis thaliana	HSKAEIVDMLRKHTYADELEQN	81%
6	G993	Arabidopsis thaliana	HSKAEIVDMLRKHTYADEFEQS	81%
22	G3452	Glycine max	HSKFEIVDMLRKHTYDDELQQS	81%
24	G3453	Glycine max	HSKSEIVDMLRRHTYDNELQQS	81%
	G3388	Oryza sativa (japonica	HSKAEIVDMLRKHTYADELRQG	80%
	AP002913	cultivar-group)
	CA004137	Hordeum vulgare subsp.	HSKAEIVDMLRKHTYDDELRQG	80%
		vulgare
32	G3389	Oryza sativa (japonica	HSKAEVVDMLRKHTYDDELQQG	76%
		cultivar-group)
34	G3390	Oryza sativa (japonica	RSKAEVVDMLRKHTYLEELTQN	76%
		cultivar-group)
36	G3391	Oryza sativa (japonica	RSKAEVVDMLRKHTYFDELAQS	76%
		cultivar-group)
40	G3433	Zea mays	RSKAEVVDMLRKHTYGEELAQN	76%
	AX654438	Oryza sativa	HSKAEVVDMLRKHTYDDELQQG	76%
52	AAAA01000997	Oryza sativa	RSKAEVVDMLRKHTYLEELTQN	76%
48	BT009310	Triticum aestivum	RSKAEVVDMLRKHTYPDELAQY	75%
38	G3432	Zea mays	RSKAEVVDMLRKHTYFDELAQN	71%

Producing Polypeptides [0152]
The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homolog polypeptides and sequences complementary thereto, as well as unique fragrments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, for example, DNA or RNA, the latter including mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homolog polypeptide, in isolation, in combination with additional coding sequences (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 (for example, introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homolog polypeptide is an endogenous or exogenous gene. [0153]
A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, for example, Berger and Kimmel, [0154] 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”).
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 (for example, 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) [0155] 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.
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) [0156] Tetrahedron Letters 22:1859-1869; and Matthes et al. (1984) EMBO J. 3:801-805. According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.
Homologous Sequences [0157]
Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing, derived from [0158] 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 or fruit trees, 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 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).
Orthologs and Paralogs [0159]
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. [0160]
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. [0161]
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 CLUSTA1. (Thompson et al. (1994) [0162] 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 lade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each lade, but define the functions of these genes; genes within a lade 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.)
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 CLUSTA1. (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. [0163]
Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993) [0164] 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.
Orthologous genes from different organisms have highly conserved functions, and very often essentially identical functions (Lee et al. (2002) [0165] Genome Res. 12:493-502; Remm et al. (2001) J. Mol. Biol. 314:1041-1052). Paralogous genes, which have diverged through gene duplication, may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably with respect to certain embodiments of the instant invention (for example, transgenic expression of a coding sequence). An example of such highly related paralogs is the CBF family, with four well-defined members in Arabidopsis (SEQ ID NOs:54, 56, 58, and GenBank accession number AB015478) and at least one ortholog in Brassica napus, (SEQ ID NO:60), 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).
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. [0166]
(1) The Arabidopsis NPRI gene regulates systemic acquired resistance (SAR) (Cao et al. (1997) Cell 88:57-63); over-expression of NPR1 leads to enhanced resistance in Arabidopsis. When either Arabidopsis NPR1 or the rice NPR1 ortholog was overexpressed in rice (which, as a monocot, is diverse from Arabidopsis), challenge with the rice bacterial blight pathogen [0167] Xanthomonas oryzae pv. Oryzae, the transgenmc 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).
(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) [0168] Plant J. 29:45-59).
(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) [0169] J. Biol. Chem. 277:1689-1694).
(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) [0170] Plant Physiol. 127:1682-1693).
(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) [0171] Transgenic Res. 9:223-227).
(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) [0172] Plant Cell 13:1791-1802).
(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) [0173] Curr. Biol. 10:215-218).
(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) [0174] Plant Cell 12:2383-2394).
(9) Wheat reduced height-I (Rht-B I/Rht-D 1) 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) [0175] Nature 400:256-261).
Transcription factors that are homologous to the listed sequences will typically share at least about 75% and 69% amino acid sequence identity in the AP2 and B3 domains, respectively. More closely related transcription factors can share at least about 79% or about 90% or about 95% or about 98% or more sequence identity with the listed sequences, or with the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site, or with the listed sequences excluding one or all conserved domains. Factors that are most closely related to the listed sequences share, 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. AP2 domains within the AP2 transcription factor family may exhibit a higher degree of sequence homology, such as at least 77% amino acid sequence identity including conservative substitutions, and preferably at least 80% sequence identity, and more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% sequence identity. Transcription factors that are homologous to the listed sequences should share at least 30%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 95% amino acid sequence identity over the entire length of the polypeptide or the homolog. [0176]
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). [0177]
Other techniques for alignment are described in Methods in Enzymology, vol. 266, [0178] 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.
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) [0179] 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 Ser. No. 20010010913).
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. [0180]
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) [0181] 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 28:405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y., p 856-853).
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. [0182]
Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and AP2 binding domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide 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. [0183]
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. [0184]
Identifyring Polynucleotides or Nucleic Acids by Hybridization [0185]
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. [0186]
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) [0187] 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.
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) “[0188] 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.
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[0189] _m) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equations:
(I) DNA-DNA: [0190]
T _m(° C.)=81.5+16.6(log [Na+])+0.4 1 (% G+C)−0.62(% formamide)-31 500/L
(II) DNA-RNA: [0191]
T _m(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)²−0.5(% formnamide)−820/L
(III) RNA-RNA: [0192]
T _m(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)²−0.35(% formamide)−820/L
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° C. is required to reduce the melting temperature for each 1% mismatch. [0193]
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. [0194]
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[0195] _m−5° C. to T_m−20° C., moderate stringency at T_m−20° C. to T_m−35° C. and low stringency at T_m−35° C. to T_m−50° C. for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C. below T_m), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T_m−25° C. for DNA-DNA duplex and T_m−15° 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.
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 filterbased method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5° C. to 20° C. lower than the thermal melting point (T[0196] _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° C. and about 70° 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° 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.
Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., 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° C., more preferably of at least about 37° C., and most preferably of at least about 42° 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. [0197]
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. [0198]
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: [0199]
6×SSC at 65° C.; [0200]
50% formamide, 4×SSC at 42° C.; or [0201]
0.5×SSC, 0.1% SDS at 65° C.; [0202]
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. [0203]
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. [0204]
If desired, one may employ wash steps of even greater stringency, including about 0.2×SSC, 0.1% SDS at 65° C. and washing twice, each wash step being about 30 min, or about 0.1×SSC, 0.1% SDS at 65° C. and washing twice for 30 min. The temperature for the wash solutions will ordinarily be at least about 25° C., and for greater stringency at least about 42° C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C. to about 5° C., and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C. to about 9° C. For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 50° C. [0205]
An example of a low stringency wash step employs a solution and conditions of at least 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42° 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° C. -68° 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 Ser. No. 20010010913). [0206]
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× 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× or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2× 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. [0207]
Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 1 7, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and fragments thereof under various conditions of stringency. (See, e.g., Wahl and Berger (1987) [0208] Methods Enzymol. 152:399407; 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.
Identifyng Polynucleotides or Nucleic Acids with Expression Libraries [0209]
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., [0210] 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, N.Y. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologs, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.
Sequence Variations [0211]
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. [0212]
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. [0213]
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. [0214]
Those skilled in the art would recognize that, for example, G867, SEQ ID NO:2, represents a single transcription factor; allelic variation and alternative splicing may be expected to occur. Allelic variants of SEQ ID NO:1 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: 1, 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:2. 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). [0215]
Thus, in addition to the sequences set forth in the Sequence Listing, the invention also encompasses related nucleic acid molecules that include allelic or splice variants of SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and include sequences which are complementary to any of the above 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 NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 53. Such 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. [0216]

For example, Table 3 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.

TABLE 3


Amino acid	Possible Codons

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

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. [0218]
In addition to silent variations, other conservative variations that alter one, or a few amino acid residues in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention. [0219]
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.) [0220] Methods Enzymol. (1993) vol. 217, Academic Press) or the other methods noted below. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In 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.

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 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 conservative substitutions.

	TABLE 4


	Residue	Conservative Substitutions

	Ala	Ser
	Arg	Lys
	Asn	Gln; His
	Asp	Glu
	Gln	Asn
	Cys	Ser
	Glu	Asp
	Gly	Pro
	His	Asn; Gln
	Ile	Leu, Val
	Leu	Ile; Val
	Lys	Arg; Gln
	Met	Leu; Ile
	Phe	Met; Leu; Tyr
	Ser	Thr; Gly
	Thr	Ser; Val
	Trp	Tyr
	Tyr	Trp; Phe
	Val	Ile; Leu

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 5 when it is desired to maintain the activity of the protein. Table 5 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 5 may be substituted with a residue in column 2; in addition, a residue in column 2 of Table 5 may be substituted with the residue of column 1.

	TABLE 5


	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

Substitutions that are less conservative than those in Table 5 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. [0223]
Further Modifying Sequences of the Invention—Mutation/Forced Evolution [0224]
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. [0225]
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) [0226] 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.
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. [0227]
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. [0228]
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 [0229] E. coli prefer to use TAA as the stop codon.
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. [0230]
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 VP 16 or GAL4 (Moore et al. (1998) [0231] Proc. Natl. Acad. Sci. 95:376-381; Aoyama et al. (1995) Plant Cell 7:1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51:113-119) and synthetic peptides (Giniger and Ptashne (1987) Nature 330:670-672).
Expression and Modification of Polypeptides [0232]
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. [0233]
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. [0234]
Vectors, Promoters, and Expression Systems [0235]
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. [0236]
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) [0237] 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.
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) [0238] 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).
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. [0239]
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. [0240]
The promoter sequences can be isolated according to methods known to one skilled in the art. [0241]
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) [0242] 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).
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 drought, wounding, heat, cold, 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 [0243] dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A1 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), flowerspecific (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, Schafffier and Sheen (1991) Plant Cell 3:997-1012); wounding (e.g., wunI, Siebertz et al. (1989) Plant Cell 1:961-968); pathogens (such as the PR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40:387-396, and the PDF1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38:1071-1080), and chemicals such as methyl jasmonate or salicylic acid (Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (Gan and Amasino (1995) Science 270:1986-1988); or late seed development (Odell et al. (1994) Plant Physiol. 106:447-458).
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. [0244]
Additional Expression Elements [0245]
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. [0246]
Expression Hosts [0247]
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. [0248]
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) [0249] 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).
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. [0250]
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 understcod 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. [0251]
Modified Amino Acid Residues [0252]
Polypeptides of the invention may contain one or more modified amino acid residues. The presence of modified amino acids may be advantageous in, for example, increasing polypeptide half-life, reducing polypeptide antigenicity or toxicity, increasing polypeptide storage stability, or the like. Amino acid residue(s) are modified, for example, co-translationally or post-translationally during recombinant production or modified by synthetic or chemical means. [0253]
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., famesylated, 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. [0254]
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. [0255]
Identification of Additional Protein Factors [0256]
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. 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) [0257] Nature Biotechnol. 17:573-577).
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. [0258]
The two-hybrid system detects protein interactions in vivo and is described in Chien et al. ((1991) [0259] 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. 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.
Subsequences [0260]
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. [0261]
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. [0262]
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 polynucieotides 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. [0263]
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. [0264]
Production of Transzenic Plants [0265]
Modification of Traits [0266]
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. [0267]
Arabidopsis as a Model System [0268]
[0269] 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).
Arabidopsis Genes in Transgenic Plants. [0270]
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 [0271] 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 377:482483); and Weigel and Nilsson (1995, Nature 377:482-500).
Homologous Genes Introduced into Transgenic Plants. [0272]
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. [0273]
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. [0274]
Transcription Factors of Interest for the Modification of Plant Traits [0275]
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. drought tolerance) 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. [0276]
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. [0277]
Genes, Traits and Utilities that Affect Plant Characteristics [0278]
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. [0279]
Sugar Sensing. [0280]
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) [0281] 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.
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 [0282] The Arabidopsis Book, Editors: Somerville and Meyerowitz (American Society of Plant Biologists, Rockville, Md.).
This potentially implicates G867, G9, G993 and G1930 in hormone signaling based on the sucrose sugar sensing phenotype of 35S::G867, 35S::G9, 35S::G993 and 35S::G1930 transgenic lines (see Example VIII, below). On the other hand, the sucrose treatment used in these experiments (9.4% w/v) could also be an osmotic stress. Therefore, one could interpret these data as an indication that these transgenic lines are more tolerant to osmotic stress. However, it is well known that plant responses to ABA, osmotic and other stress may be linked, and these different treatments may even act in a synergistic manner to increase the degree of a response. For example, Xiong, Ishitani, and Zhu ((1999) [0283] Plant Physiol. 119:205-212) have shown that genetic and molecular studies may be used to show extensive interaction between osmotic stress, temperature stress, and ABA responses in plants. These investigators analyzed the expression of RD29A-LUC in response to various treatment regimes in Arabidopsis. The RD29A promoter contains both the ABA-responsive and the dehydration-responsive element—also termed the C-repeat—and can be activated by osmotic stress, low temperature, or ABA treatment; transcription of the RD29A gene in response to osmotic and cold stresses is mediated by both ABA-dependent and ABA-independent pathways (Xiong, Ishitani, and Zhu (1999) supra). LUC refers to the firefly luciferase coding sequence, which, in this case, was driven by the stress responsive RD29A promoter. The results revealed both positive and negative interactions, depending on the nature and duration of the treatments. Low temperature stress was found to impair osmotic signaling but moderate heat stress strongly enhanced osmotic,stress induction, thus acting synergistically with osmotic signaling pathways. In this study, the authors reported that osmotic stress and ABA can act synergistically by showing that the treatments simultaneously induced transgene and endogenous gene expression. Similar results were reported by Bostock and Quatrano ((1992) Plant Physiol. 98:1356-1363), who found that osmotic stress and ABA act synergistically and induce maize Em gene expression. Ishitani et al (1997) Plant Cell 9:1935-1949) isolated a group of Arabidopsis single-gene mutations that confer enhanced responses to both osmotic stress and ABA. The nature of the recovery of these mutants from osmotic stress and ABA treatment suggested that although separate signaling pathways exist for osmotic stress and ABA, the pathways share a number of components; these common components may mediate synergistic interactions between osmotic stress and ABA. Thus, contrary to the previously-held belief that ABA-dependent and ABA-independent stress signaling pathways act in a parallel manner, our data reveal that these pathways cross-talk and converge to activate stress gene expression.
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. [0284]
Thus, the presently disclosed transcription factor genes that manipulate the sugar signal transduction pathway, including, for example, G867, G9, G993 and G1930, along with their 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. [0285]
Abiotic stress:drought and low humidity tolerance. Exposure to dehydration invokes similar survival strategies in plants as does freezing stress (see, for example, Yelenosky (1989) [0286] 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 coldacclimation proteins, strategies that allow plants to survive in low water conditions may include, for example, reduced surface area, or surface oil or wax production. Modifying the expression of a number of presently disclosed transcription factor genes, such as G867, 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.
Osmotic stress. Modification of the expression of a number of presently disclosed transcription factor genes, e.g., G867, G1930, and their equivalogs, may be used to increase germination rate or growth under adverse osmotic conditions, which could impact survival and yield of seeds and plants. Osmotic stresses may be regulated by specific molecular control mechanisms that include genes controlling water and ion movements, functional and structural stress-induced proteins, signal perception and transduction, and free radical scavenging, and many others (Wang et al. (2001) [0287] Acta Hort. (ISHS) 560:285-292). Instigators of osmotic stress include freezing, drought and high salinity, each of which are discussed in more detail below.
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. [0288]
Salt and Drought Tolerance [0289]
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) [0290] 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).
Exposure to dehydration invokes similar survival strategies in plants as does freezing stress (see, for example, Yelenosky (1989) [0291] 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.
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. [0292]
The genes of the sequence listing, including, for example, G867, G1930, and their equivalogs, that provide tolerance to salt may be used to engineer salt tolerant crops and trees that can flourish in soils with high saline content or under drought conditions. In particular, increased salt tolerance during the germination stage of a plant enhances survival and yield. Presently disclosed transcription factor genes that provide increased salt tolerance during germination, the seedling stage, and throughout a plant's life cycle, would find particular value for imparting survival and yield in areas where a particular crop would not normally prosper. [0293]
Root growth and vigor. Some of the genes in the Sequence Listing, e.g., G9, have been shown to increase root growth and to produce hairy roots on media containing methyl jasmonate. Thus, these genes could potentially be used to increase root growth and vigor, which might in turn allow better plant growth during periods of osmotic stress, or limited nutrient availability. [0294]
Summary of altered plant characteristics. A clade of structurally and functionally related sequences that derive from a wide range of plants, including polynucleotide [0295] SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51, polynucleotides that encode polypeptide SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 53, 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, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 53, 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 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 abiotic stress tolerance.
Antisense and Co-Suppression [0296]
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) [0297] 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; Rosenberget 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.
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. [0298]
Suppression of endogenous transcription factor gene expression can also be achieved using RNA interference , or RNAi. RNAi is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to incite degradation of messenger RNA (mRNA) containing the same sequence as the dsRNA (Constans, (2002) [0299] The Scientist 16:36). Small interfering RNAs, or siRNAs are produced in at least two steps:an endogenous ribonuclease cleaves longer dsRNA into shorter, 21-23 nucleotide-long RNAs. The siRNA segments then mediate the degradation of the target mRNA (Zamore, (2001) Nature Struct. Biol., 8:746-50). RNAi has been used for gene function determination in a manner similar to antisense oligonucleotides (Constans, (2002) The Scientist 16:36). Expression vectors that continually express siRNAs in transiently and stably transfected 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.
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) [0300] 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.).
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) [0301] Nature 389:802-803).
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. [0302]
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) [0303] 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).
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. [0304]
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. [0305]
The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbellhferae (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) [0306] Handbook of Plant Cell Culture—Crop Species, Macmillan Publ. Co., New York, N.Y.; Shimamoto et al. (1989) Nature 338:274-276; Fromin et al. (1990) Bio/Technol. 8:833-839; and Vasil et al. (1990) Bio/Technol. 8:429434.
Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to:electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and [0307] 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.
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. [0308]
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. [0309]
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. [0310]
Integrated Systems—Sequence Identity [0311]
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. [0312]
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. [0313]
Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (1981) [0314] Adv. Appl. Math. 2:482-489, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Ausubel et al. supra.
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. [0315]
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) [0316] J. Mol. Biol. 215:403410. 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 U.S. 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).
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. [0317] 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.
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. [0318]
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. [0319]
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. [0320]
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. [0321]
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) [0322] 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).

Table 6 lists sequences discovered to be orthologous to a number of representative transcription factors of the present invention, in decreasing order of similarity to G867. The column headings include the transcription factors listed by (a) the SEQ ID NO:of the homolog (paralog or ortholog) or the nucleotide encoding the homolog; (b) the GID sequence identifier; (c) the Sequence Identifier or GenBank Accession Number; (d) the species from which the homologs (orthologs or paralogs) to the transcription factors are derived; and (e) the smallest sum probability relationship to G867 determined by BLAST analysis.

TABLE 6


Homologs of Representative Arabidopsis Transcription Factor
Genes Identified using BLAST

SEQ ID NO:		Smallest
of Homolog		Sum
or Nucleotide		Probability

Encoding	GID	Sequence Identifier or	Species from Which	to
Homolog	No.	Accession Number	Homolog is Derived	G867

1	G867		Arabidopsis thaliana	0.0
7	G1930		Arabidopsis thaliana	1.00E−132
3	G9		Arabidopsis thaliana	1.00E−115
5	G993		Arabidopsis thaliana	1.00E−115
41		BZ458719	Brassica oleracea	1.00E−113
17	G3451	GLYMA-28NOV01-	Glycine max	1.00E−110
		CLUSTER19062_3
25	G3454		Glycine max	1.00E−109
21	G3452	GLYMA-28NOV01-	Glycine max	2.00E−99
		CLUSTER19062_7
23	G3453		Glycine max	3.0E−98
		CB686050	Brassica napus	1.00E−97
43		BQ971511	Helianthus annuus	2.00E−94
45		BU025988	Helianthus annuus	3.00E−92
		BQ971525	Helianthus annuus	2.00E−92
37	G3432		Zea mays	1.00E−87
35		AP003450	Oryza sativa	9.00E−85
		gi18565433	Oryza sativa (japonica	3.00E−85
			cultivar-group)
33	G3390	AC130725	Oryza sativa	8.00E−84
47		BT009310	Triticum aestivum	4.00E−82
35	G3391	AP003450	Oryza sativa	1.00E−82
29	G3388	OSC21673.C1.p5.fg	Oryza sativa	2.00E−80
		AP002913
49		CC616336	Zea mays	2.00E−80
33		AC130725	Oryza sativa (japonica	1.00E−80
			cultivar-group)
		AC136492	Oryza sativa (japonica	1.00E−80
			cultivar-group)
51		AAAA01000997	Oryza sativa (indica	1.00E−79
			cultivar-group)
31	G3389	OSC21674.C1.p12.fg	Oryza sativa	1.00E−79
		AP002913
		BQ405698	Gossypium arboreum	2.00E−77
39	G3433		Zea mays	2.00E−73
27	G3455	GLYMA-28NOV01-	Glycine max	3.00E−70
		CLUSTER19062_5
		BZ015521	Brassica oleracea	5.00E−69
		BF520598	Medicago truncatula	2.00E−66
		BU994579	Hordeum vulgare	5.00E−64
			subsp. vulgare
		CD814840	Brassica napus	4.00E−64
		CB894555	Medicago truncatula	3.00E−64
		BF424857	Glycine max	2.00E−62
		BU871082	Populus balsamifera	2.00E−61
			subsp. trichocarpa
		BQ855250	Lactuca sativa	7.00E−61

Molecular Modeling [0324]
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 commericallly 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, O'Neil et al. ((1990) [0325] 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 substitued 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.
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. [0326]

EXAMPLES

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. [0327]
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 AP2 binding domains and B3 domains of the polypeptide encoded by the polynucleotide is fully disclosed in Table 1. [0328]

Example I

Full Length Gene Identification and Cloning [0329]
Putative transcription factor sequences (genomic or ESTs) related to known transcription factors were identified in the [0330] 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.
Alternatively, [0331] 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° C.) and labeled with 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 NaPO₄pH 7.0, 7% SDS, 1% w/v bovine serum albumin) and hybridized overnight at 60° C. with shaking. Filters were washed two times for 45 to 60 minutes with 1×SCC, 1% SDS at 60° C.
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, Calfi.). 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. [0332]
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. [0333]

Example II

Construction of Expression Vectors [0334]
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) [0335] 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° 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° 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° C. Plasmid DNA was purified by using Qiaquick Mini Prep kits (Qiagen).

Example III

Transformation of Agrobacterium with the Expression Vector [0336]
After the plasmid vector containing the gene was constructed, the vector was used to transform [0337] Agrobacterium tumefaciens cells expressing the gene products. The stock of Agrobacterium tumefaciens cells for transformation were 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° C. with shaking until an absorbance over 1 cm at 600 nm (A₆₀₀) of 0.5-1.0 was reached. Cells were harvested by centrifugation at 4,000×g for 15 min at 4° C. Cells were then resuspended in 250 μl chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 μl chilled buffer. Cells were then centrifuged and resuspended two more times in the same HEPES buffer as described above at a volume of 100 μl and 750 μl, respectively. Resuspended cells were then distributed into 40 μl aliquots, quickly frozen in liquid nitrogen, and stored at −80° C.
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-HCI, 1 mM EDTA, pH 8.0) was mixed with 40 μl of Agrobacterium cells. The DNA/cell mixture was then transferred to a chilled cuvette with a 2mm electrode gap and subject to a 2.5 kV charge dissipated at 25 μF. and 200 μF. 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° C. in a shaking incubator. After recovery, cells were plated onto selective medium of LB broth containing 100 μg/ml spectinomycin (Sigma) and incubated for 24-48 hours at 28° 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. [0338]

Example IV

Transformation of Arabidopsis Plants with Agrobacterium tumefaciens with Expression Vector [0339]
After transformation of [0340] 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° C. with shaking for 2 days until an optical absorbance at 600 nm wavelength over 1 cm (A₆₀₀) of >2.0 is reached. Cells were then harvested by centrifugation at 4,000×g for 10 min, and resuspended in infiltration medium (½×Murashige and Skoog salts (Sigma), 1×Gamborg's B-5 vitamins (Sigma), 5.0% (w/v) sucrose (Sigma), 0.044 μM benzylamino purine (Sigma), 200 μl/l Silwet L-77 (Lehle Seeds) until an A₆₀₀of 0.8 was reached.
Prior to transformation, [0341] Arabidopsis thaliana seeds (ecotype Columbia) were sown at a density of ˜10 plants per 4″ pot onto Pro-Mix BX potting medium (Hummert International) covered with fiberglass mesh (18 mm×16 mm). Plants were grown under continuous illumination (50-75 μE/m²/sec) at 22-23° 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.
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′×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. [0342]

Example V

Identification of Arabidopsis Primary Transformants [0343]
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° C. for 2 days in the dark before being plated onto antibiotic selection medium (1×Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH), 1×Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin). Seeds were germinated under continuous illumination (50-75 μE/m[0344] ²/sec) at 22-23° 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).
Primary transformants were crossed and progeny seeds (T[0345] ₂) collected; kanamycin resistant seedlings were selected and analyzed. The expression levels of the recombinant polynucleotides in the transformants varies from about a 5% expression level increase to a least a 100% expression level increase. Similar observations are made with respect to polypeptide level expression.

Example VI

Identification of Arabidopsis Plants with Transcription Factor Gene Knockouts [0346]
The screening of insertion mutagenized Arabidopsis collections for null mutants in a known target gene was essentially as described in Krysan et al. (1999) [0347] 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 transpose ends (the “right” and “left” borders). All possible combinations of gene specific and T-DNA/transpose 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/transpose 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. [0348]
Experiments were performed to identify those transformants or knockouts that exhibited modified biochemical characteristics. [0349]
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. [0350]
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. [0351]
In some instances, expression patterns of the stress-induced genes may be monitored by microarray experiments. In these experiments, cDNAs are generated by PCR and resuspended at a final concentration of -100 ng/ul in 3×SSC or 150 mM Na-phosphate (Eisen and Brown (1999) [0352] 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).
Sample total RNA (10 μg) samples are labeled using fluorescent Cy3 and Cy5 dyes. Labeled samples are resuspended in 4×SSC/0.03% SDS/4 μg salmon sperm DNA/2 μg tRNA/50 mM Na-pyrophosphate, heated for 95° 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° 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.). [0353]
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. [0354]
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. [0355]
For RT PCR, cDNA template was mixed with corresponding primers and Taq DNA polymerase. Each reaction consisted of 0.2 μl cDNA template, 2 [0356] μl 10×Tricine buffer, 2 μl 10×Tricine buffer and 16.8 μl water, 0.05 μl Primer 1, 0.05 μl, Primer 2, 0.3 μl Taq DNA polymerase and 8.6 μl water.
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: [0357]
Step 1: 93° C. for 3 min; [0358]
Step 2: 93° C. for 30 sec; [0359]
Step 3: 65° C. for 1 min; [0360]
Step 4: 72° C. for 2 min; [0361]
Steps 2, 3 and 4 are repeated for 28 cycles; [0362]
Step 5: 72° C. for 5 min; and [0363]
Step 6 4° C. [0364]
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. [0365]
Step 2 93° C. for 30 sec; [0366]
Step 3 65° C. for 1 min; [0367]
Step 4 72° C. for 2 min, repeated for 8 cycles; and [0368]
Step 5 4° C. [0369]
Eight microliters of PCR product and 1.5 μl 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. [0370]
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. [0371]
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. [0372]

Example VIII

Genes that Confer Significant Improvements to Plants [0373]
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. [0374]
This example provides experimental evidence for increased biomass and abiotic stress tolerance controlled by the transcription factor polypeptides and polypeptides of the invention. [0375]
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. Plants differ in their tolerance to NaCl depending on their stage of development, therefore seed germination, seedling vigor, and plant growth responses are evaluated. [0376]
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. [0377]
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. [0378]
Temperature stress assays are intended to find genes that confer better germination, seedling vigor or plant growth under temperature stress (cold, freezing and heat). [0379]
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). [0380]
Germination assays followed modifications of the same basic protocol. Sterile seeds were sown on the conditional media listed below. Plates were incubated at 22° C. under 24-hour light (120-130 μEin/m[0381] 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.
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 μM ABA, 9.4% sucrose, or 5% glucose. [0382]
Temperature stress cold germination experiments were carried out at 8 ° C. Heat stress germination experiments were conducted at 32 ° C. to 37° C. for 6 hours of exposure. [0383]
For stress experiments conducted with more mature plants, seeds were germinated and grown for seven days on MS+vitamins+1% sucrose at 22° C. and then transferred to chilling and heat stress conditions. The plants were either exposed to chilling stress (6 hour exposure to 4-8° C.), or heat stress (32° C. was applied for five days, after which the plants were transferred back 22° C. for recovery and evaluated after 5 days relative to controls not exposed to the depressed or elevated temperature). [0384]
Results: [0385]
As noted below, G867, G9, G993, and G1930 overexpression has been shown to increase osmotic stress tolerance. [0386]
G867 (Polynucleotide SEQ ID NO:1) [0387]
Published Information [0388]
There are six RAV-like proteins in Arabidopsis. One of them, G867, has been described in the literature as related to ABBI3/VPI (RAV1; Kagaya et al. (1999) [0389] Nucleic Acids Res. 27:470478) based on the presence of a B3 domain (which is also found in the ABI3/VP1 family of transcription factors). G867/RAV1 belongs to a small subgroup within the AP2/EREBP family of transcription factors, whose distinguishing characteristic is that its members contain a second DNA-binding domain, in addition to the conserved AP2 domain, that is related to the B3 domain of VP1/ABI3 (Kagaya et al., 1999) supra). Analyses using various deletion derivatives of the RAV1 fusion protein showed that the two DNA-binding domains of G867, the AP2 and B3 domains, separately recognize each of two motifs that constitute a bipartite binding sequence, CAACA and CACCTG, respectively, and together cooperatively enhance the DNA-binding affinity and specificity of the transcription factor (Kagaya et al., 1999) supra). No functional data are available for G867/RAV1.
Experimental Observations [0390]
G867 was initially identified as a public Arabidopsis EST. G867 appears to be constitutively expressed at medium levels. [0391]
G867 was first characterized using a line that contained a T-DNA insertion in the gene. The insertion in that line resides immediately downstream of the conserved AP2 domain, and would therefore be expected to result in a severe or null mutation. G867 knockout mutant plants do not show significant changes in overall plant morphology, neither has a significant difference between these plants and control plants been detected in any of the assays that have been performed so far. [0392]
The function of G867 was also analyzed using transgenic plants in which this gene was expressed under the control of the 35S promoter. G867 overexpressing lines are morphologically wild-type. Increased seedling vigor, manifested by increased expansion of the cotyledons, was observed in germination assays on both high salt (150 mM salt; FIG. 5) and media containing high sucrose (9.4%; FIG. 6), as compared to wild-type controls. Subsequently, G867-overexpressing Arabidopsis plants were shown to be more tolerant of drought in a soil-based assay, as compared to wild-type plants. [0393]
G867 overexpressing plants exposed to chilling conditions (6 h at 4° -8° C.) were more vigorous than control plants exposed to the same chilling conditions. [0394]
Several G867 overexpressing lines were found to be more sensitive to 0.3 μM ABA. [0395]
Utilities [0396]
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, and thus increased ABA sensitivity is a likely indicator of an enhanced stress response. These observation, and those in salt and sucrose tolerance assays, indicate that G867 or its equivalogs can be used to increase or facilitate seed germination and seedling or plant growth under adverse conditions such osmotic stresses, including drought and salt stress. [0397]
The enhanced performance of 35S::G867 seedlings under chilling conditions indicates that the gene or its equivalogs might be applied to engineer crops that show better growth under cold conditions, which may extend a crops planting season or range, or improve yield or performance. [0398]
G9 (Polynucleotide SEQ ID NO:3) [0399]
Published Information [0400]
G9 was first identified in a partial cDNA clone, and the corresponding gene named RAP2.8 (Okamuro et al., 1997). It has also been named RAV2 (Kagaya et al. (1999) [0401] Nucleic Acids Res. 27:470-478). G91RAV21RAP2.8 belongs to a small subgroup within the AP2/EREBP family of transcription factors, whose distinguishing characteristic is that its members contain a second DNA-binding domain, in addition to the conserved AP2 domain, that is related to the B3 domain of VP I/ABI3 (Kagaya et al., 1999) supra). It has been shown that the two DNA-binding domains of RAV1 (another member of this subgroup of proteins) can separately recognize each of two motifs that constitute a bipartite binding sequence and together cooperatively enhance its DNA-binding affinity and specificity (Kagaya et al., 1999) supra). No functional data are available for G9/RAV2/RAP2.8 or RAV1.
Experimental Observations [0402]
The complete sequence of G9 was determined. G9 appeared to be constitutively expressed. However, overexpression of G9 caused phenotypic changes in the roots: more root growth on MS plates (FIG. 7), and hairy roots on media containing 10 μM methyl jasmonate (MeJ; FIG. 8). [0403]
Increased seedling vigor, manifested by increased expansion of the cotyledons of G9 overexpressing plants, was observed in germination assays on both high salt (150 mM NaCl) and high sucrose-containing media (9.4% sucrose), as compared to wild-type controls. [0404]
35S::G9 transgenic plants were more tolerant to chilling (4° -8° C. for 6 h) compared to the wild-type controls in seedling growth assays. [0405]
Several G9 overexpressing lines were found to be more sensitive to 0.3 μM ABA. [0406]
Utilities [0407]
G9 or its equivalogs could potentially be used to increase root growth/vigor, which might in turn allow better plant growth under adverse conditions (for example, limited water or nutrient availability). [0408]
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, and thus increased ABA sensitivity is a likely indicator of an enhanced stress response. These observations, coupled with the root growth results and the salt and sucrose tolerance assays, indicate that G9 or its equivalogs could potentially be used to increase or facilitate seed germination and seedling or plant growth under adverse conditions such osmotic stresses, including drought and salt stress. [0409]
The enhanced performance of 35S::G9 seedlings under chilling conditions indicates that the gene or its equivalogs might be applied to engineer crops that show better growth under cold conditions, which may extend a crops planting season or range, or improve yield or performance. [0410]
G993 (Polynucleotide SEQ ID NO:5) [0411]
Published Information [0412]
G993 corresponds to gene F2J7.3 (AAG12735). No informnation is available about the function(s) of G993. [0413]
Closely Related Genes from Other Species [0414]
G993 shows some sequence similarity, outside of the conserved AP2/EREBP and B3 domains, to other RAV proteins from different species, such as a putative DNA binding protein RAV2 from Oryza sativa (GenBank accession number gil2328560). [0415]
Experimental Observations [0416]
The function of G993 was studied using transgenic plants in which the gene was expressed under the control of the 35S promoter. [0417]
Overexpression of G993 produced highly pleiotropic effects on plant development and influenced growth rate, overall plant size, branching pattern and fertility. 35S::G993 seedlings were small, developed slowly, and produced inflorescences markedly later than wild-type controls. They also showed a reduction in apical dominance and disorganized rosettes, as multiple axillary shoots developed simultaneously. Inflorescence stems were generally shorter than wild type, and produced an increased number of cauline leaf nodes leading to a leafy, bushy, appearance. In addition, the seed yield of 35S::G993 plants was generally very poor, and senescence occurred later than in wild-type controls. The transformation rate attained with the G993 construct was relatively low, suggesting that high levels of G993 activity might produce lethal effects. No alterations were detected in 35S::G993 plants in the biochemical analyses that were performed. [0418]
G993 is ubiquitously expressed and does not appear to be significantly induced by any of the conditions tested. [0419]
Increased seedling vigor, manifested by increased expansion of the cotyledons of G993 overexpressing plants, was observed in germination assays on both high salt (150 mM) and high sucrose (9.4%) containing-media, as compared to wild-type controls. [0420]
In addition, several 35S::G993 transgenic lines were more tolerant to cold germination (8° C.) and numerous lines were more tolerant to chilling (4° -8° C. for 6 h) compared to the wild-type controls, in both germination as well as seedling growth assays, respectively. [0421]
Utilities [0422]
The salt and sucrose tolerance assays indicate that G993 or its equivalogs could potentially be used to increase or facilitate seed germination and seedling or plant growth under adverse conditions such osmotic stresses, including drought and salt stress. [0423]
The enhanced performance of 35S::G993 seedlings under cold germination and chilling conditions indicates that the gene or its equivalogs might be applied to engineer crops that show better germination and growth under cold conditions, which may extend a crops planting season or range, or improve yield or performance. [0424]
G1930 (Polynucleotide SEQ ID NO:7) [0425]
Published Information [0426]
G1930 was identified in the sequence of PI clone K13N2 (gene K13N2.7, GenBank protein accession number BAA95760). No information is available about the function(s) of G1930. [0427]
Closely Related Genes from Other Sipecies [0428]
G1930 shows sequence similarity, outside of the conserved AP2 and ABI3 domains, to a predicted rice protein (GenBank accession number BAB21218). [0429]
Experimental Observations [0430]
G1930 is ubiquitously expressed and does not appear to be induced by any of the conditions tested. [0431]
The function of G1930 was studied using transgenic plants in which this gene was expressed under the control of the 35S promoter. [0432]
35S::G1930 TI plants were generally small and developed spindly inflorescences. The fertility of these plants was low and flowers often failed to open or pollinate. [0433]
G1930 overexpressors were more tolerant to osmotic stress conditions. The plants responded to high NaCl (150 mM) and high sucrose (9.4%) on plates with more seedling vigor compared to wild-type control plants. In addition, an increase in the amount of chlorophylls a and b in seeds of two T2 lines was detected. [0434]
In addition, 35S::several G1930 transgenic lines were more tolerant to cold germination conditions (8° C. for 6 h) and numerous G1930 transgenic lines were more tolerant to chilling (4° -8° C. for 6 h) compared to the wild-type controls, in both germination as well as seedling growth assays, respectively. [0435]
Several G1930 overexpressing lines were found to be more sensitive to 0.3 μM ABA. [0436]
Utilities [0437]
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, and thus increased ABA sensitivity is a likely indicator of an enhanced stress response. These observations, coupled with the root growth results and the salt and sucrose tolerance assays, indicate that G1930 or its equivalogs could potentially be used to increase or facilitate seed germination and seedling or plant growth under adverse conditions such osmotic stresses, including drought and salt stress. [0438]
The enhanced performance of 35S::G1930 seedlings under cold germination and chilling conditions indicates that the gene or its equivalogs might be applied to engineer crops that show better germination and/or growth under cold conditions, which may extend a crop's planting season or range, or improve yield or performance. [0439]

Table 7 provides a summary of the data collected from one series of experiments conducted with plants overexpressing G867 or a paralog of G867. In each case the promoter used for regulating the introduced transcription factor was the cauliflower mosaic virus 35S transcription initiation region. The column headings include the transcription factors used to transform the Arabidopsis plants listed by Gene ID (GIID) numbers, the corresponding polypeptide SEQ ID NO; the transformation system used in these assays, and the ratio of lines determine to have one of the enhanced abiotic stress phenotypes listed over the number of lines tested. In Table 9, “Direct promoter fusion” refers to a transgenic plant generated by transforming wild type Arabidopsis with a DNA construct in which the CaMV35S promoter is directly linked to the transcription factor gene and used drive expression of the latter. “supTfn” refers to a transgenic plant generated by transforming an Arabidopsis line, containing a transactivator construct, incorporating a LexA DNA binding domain, programmed for expression using the promoter indicated in the next column, with a DNA construct in which a LexA operator promoter region is fused to the transcription factor gene.

TABLE 7


Summary of results of physiological assays.

Overexpressor lines showing phenotype/No. lines tested

				Improved	Improved		Improved	Improved	Improved
	SEQ	One or two Component		Germination	Germination in	ABA	Germination	Germination	Chilling
GID	ID NO:	Transformation System	Promoter	in High NaCl	High Sucrose	sensitivity	in Cold	in Heat	Tolerance

G867	2	Direct	35S	5/10	4/10	5/10	0/10	0/10	6/10
		promoter-fusion
G867	2	2-components-	CUT1	1/10	1/10	0/10	2/10	1/10	0/10
		supTfn
G9	4	Direct	35S		10/10	6/10	3/10	0/10	0/10	6/10
		promoter-fusion
G993	6	Direct	35S	6/10	5/10	0/10	3/10	0/10	6/10
		promoter-fusion
G1930	8	Direct	35S	6/10	5/10	0/10	0/10	0/10	7/10
		promoter-fusion
G1930	8	2-components-	35S	10/10	8/10	5/10	0/10	0/10	6/10
		supTfn

Example IX

Identification of Homologous Sequences [0441]
This example describes identification of genes that are orthologous to [0442] Arabidopsis thaliana transcription factors from a computer homology search.
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) [0443] 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 [0444] 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).
These sequences are compared to sequences representing genes of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, using the Washington University TBLASTX algorithm (version 2.0a19MP) at the default settings using gapped alignments with the filter “off”. For each gene of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, individual comparisons were ordered by probability score (P-value), where the score reflects the probability that a particular alignment occurred by chance. For example, a score of 3.6E-40 is 3.6×10-40. 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 6. The percent sequence identity among these sequences can be as low as 47%, or even lower sequence identity. [0445]
Candidate paralogous sequences were identified among Arabidopsis transcription factors through alignment, identity, and phylogenic relationships. Paralogs of G867 determined in this manner include G9, G993 and G1930. Candidate orthologous sequences were identified from proprietary unigene sets of plant gene sequences in [0446] 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 6.

Example X

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. [0447]
The “one-hybrid” strategy (Li and Herskowitz (1993) [0448] 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 GAL1. 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-β-D-galactoside; Invitrogen Corporation, Carlsbad Calif.). Alternatively, the strains are transformed with a cDNA polynucleotide encoding a known transcription factor DNA binding domain polypeptide sequence.
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. [0449]
Upon screening about 2×10[0450] ⁶yeast 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 XI

Gel Shift Assays. [0451]
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 [0452] E. coli or isolated from plant material. Total soluble protein, including transcription factor, (40 ng) is incubated at room temperature in 10 μl of 1×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 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 pUC118 (Vieira et al. (1987) Methods Enzymol. 153:3-11). Orientation and concatenation number of the inserts are determined by dideoxy DNA sequence analysis (Sambrook et al. supra). Inserts are recovered after restriction digestion with EcoRI and HindIII and fractionation on polyacrylamide gels (12% w/v) (Sambrook et al. supra).

Example XII

Introduction of Polynucleotides into Dicotyledonous Plants [0453]
SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, or polynucleotide sequences encoding SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 53, 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 [0454] 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 XIII

Transformation of Cereal Plants with an Expression Vector [0455]
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 [0456] 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.
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 [0457] 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).
Vectors according to the present invention may be transformed into corn embryogenic cells derived from immature scutellar tissue by using microprojectile bombardment, with the A188XB73 genotype as the preferred genotype (Fromm et al. (1990) [0458] 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-Kanmr 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).
The plasmids prepared as described above can also be used to produce transgenic wheat and rice plants (Christou (1991) [0459] 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 XIV

Transformation of Tomato and Soy Plants [0460]
Numerous protocols for the transformation of tomato and soy plants have been previously described, and are well known in the art. Gruber et al. ((1993) in [0461] Methods in Plant Molecular Biology and Biotechnology, p. 89-119, Glick and Thompson, eds., CRC Press, Inc., Boca Raton) describe several expression vectors and culture methods that may be used for cell or tissue transformation and subsequent regeneration. For soybean transformation, methods are described by Miki et al. (1993) in Methods in Plant Molecular Biology and Biotechnology, p. 67-88, Glick and Thompson, eds., CRC Press, Inc., Boca Raton; and U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.
There are a substantial number of alternatives to Agrobacterium-mediated transformation protocols, other methods for the purpose of transferring exogenous genes into soybeans or tomatoes. One such method is microprojectile-mediated transformation, in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al., (1987) [0462] Part. Sci. Technol. 5:27-37; Christou et al. (1992) Plant. J. 2:275-281; Sanford (1993) Methods Enzymol. 217:483-509; Klein et al. (1987) Nature 327:70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21, 1994.
Alternatively, sonication methods (see, for example, Zhang et al. (1991) [0463] Bio/Technology 9:996-997); direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine (see, for example, Hain et al. (1985) Mol. Gen. Genet. 199:161-168; Draper et al., Plant Cell Physiol. 23:451-458 (1982)); liposome or spheroplast fusion (see, for example, Deshayes et al. (1985) EMBO J., 4:2731-2737; Christou et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966); and electroporation of protoplasts and whole cells and tissues (see, for example, Donn et al.(1990) in Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38:53; D'Halluin et al. (1992) Plant Cell 4:1495-1505;and Spencer et al. (1994) Plant Mol. Biol. 24:51-61) have been used to introduce foreign DNA and expression vectors into plants.
After plants or plant cells are transformed (and the latter regenerated into plants) the transgenic plant thus generated may be crossed with itself (“selfing”) or a plant from the same line, a non-transformed or wild-type plant, or another transformed plant from a different transgenic line of plants. Crossing provides the advantages of being able to produce new and perhaps stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art. Transformation of tomato plants may be conducted using the protocols of Koornneef et al (1986) In [0464] Tomato Biotechnology: Alan R. Liss, Inc., 169-178,and in U.S. Pat. No. 6,613,962, the latter method described in brief here. Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 μM α-naphthalene acetic acid and 4.4 μM 6-benzylaminopurine. The explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the invention for 5-10 minutes, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates. Culture conditions are as described above. Overnight cultures of Agrobacterium tumefaciens are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7) to an OD₆₀₀of 0.8.
Following the cocultivation, the cotyledon explants are transferred to Petri dishes with selective medium consisting of MS medium supplemented with 4.56 μM zeatin, 67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulfate, and cultured under the culture conditions described above. The explants are subcultured every three weeks onto fresh medium. Emerging shoots are dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of roots in a medium containing kanamycin sulfate is regarded as a positive indication of a successful transformation. [0465]
Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Pat. No. 5,563,055 (Townsend et al., issued Oct. 8, 1996), described in brief here. In this method soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on {fraction (1/10)} strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons. [0466]
Overnight cultures of [0467] Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the invention are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium which has been solidified. Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Pat. No. 5,563,055).
The explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium. [0468]

Example XV

Genes that Confer Significant Improvements to Non-Arabidopsis Species [0469]
The function of specific orthologs of G867 may be analyzed through their ectopic overexpression in plants, using the CaMV 35S or other appropriate promoter, identified above. These genes, which include polynucleotide sequences found in the Sequence Listing, Table 6 and FIG. 3, encode members of the AP2 transcription factors, such as those found in [0470] Oryza sativa (SEQ ID NO:20, 30, 32, 34, 36, 52, and 53), Arabidopsis thaliana (SEQ ID NO 2, 4, 6, 8), Glycine max (SEQ ID NO:18, 22, 24, 26, 28), Zea mays (SEQ ID NO:38, 40, 50), Triticum aestivum (SEQ ID NO:48), Brassica oleracea (SEQ ID NO:42), and Helianthus annuus (SEQ ID NO:44 and 46). The polynucleotide and polypeptide sequences derived from monocots may be used to transform both monocot and dicot plants, and those derived from dicots may be used to transform either group, although some of these sequences will function best if the gene is transformed into a plant from the same group as that from which the sequence is derived.
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° C. under 24-hour light, 120-130 μEin/m[0471] 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 G867, G9, G993 and/or G1930 orthologs are involved in sucrose-specific sugar sensing.
Plants overexpressing these orthologs may also be subjected to soil-based drought assays to identify those lines that are more tolerant to water deprivation than wild-type control plants. Generally, 35S:G867, G9, G993 and/or G1930 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. [0472]

Example XVI

Identification of Orthologous and Paralogous Sequences [0473]
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 CBFI (polynucleotide SEQ ID NO:54, encoded polypeptide SEQ ID NO:55), 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 ([0474] Brassica napus) using polymerase chain reaction (PCR).
Degenerate primers were designed for regions of AP2 binding domain and outside of the AP2 (carboxyl terminal domain): [0475]

Mol 368 (reverse)

5′-CAY CCN ATH TAY MGN GGN GT-3′ (SEQ ID NO: 62)

Mol 378 (forward)

5′-GGN ARN ARC ATN CCY TCN GCC-3′ (SEQ ID NO: 63)
(Y: C/T, N: AC/CI/T, H: A/C/T, M: A/C, R: A/G) [0476]
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). [0477]
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° C.; 35 cycles of 93° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min; and a final incubation of 7 min at 72° C. at the end of cycling. [0478]
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 [0479] E. coli strain TOP10 (Invitrogen).
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. [0480]
The nucleic acid sequence and amino acid sequence of one canola ortholog found in this manner (bnCBF1; polynucleotide SEQ ID NO:60 and polypeptide SEQ ID NO:61) identified by this process is shown in the Sequence Listing. [0481]
The aligned amino acid sequences show that the bnCBFl 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. [0482]
Similarly, paralogous sequences to Arabidopsis genes, such as CBF1, may also be identified. [0483]
Two paralogs of CBF1 from [0484] Arabidopsis thaliana: CBF2 and CBF3. CBF2 and CBF3 have been cloned and sequenced as described below. The sequences of the DNA SEQ ID NO:54, 56 and 58 and encoded proteins SEQ ID NO:55, 57 and 59 are set forth in the Sequence Listing.
A lambda cDNA library prepared from RNA isolated from [0485] 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). CBF 1 was 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×SSPE buffer, 60° C. for hybridization and 0.1×SSPE buffer and 60° 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:54, 56, 58 and SEQ ID NOs:55, 57, and 59, respectively). The nucleic acid sequences and predicted protein coding sequence for Brassica napus CBF ortholog is listed in the Sequence Listing (SEQ ID NOs:60 and 61, respectively).
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 8. [0486]

TABLE 8

Percent identity^a

DNA^b Polypeptide

cbf1/cbf2 85 86

cbf1/cbf3 83 84

cbf2/cbf3 84 85
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 [0487] CBF 1 serves as an activation domain in both yeast and Arabidopsis (not shown).
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. [0488]

Example XVII

Transformation of Canola with a Plasmid Containing CBF1, CBF2, or CBF3 [0489]
After identifying homologous genes to CBF1, canola was transformed with a plasmid containing the Arabidopsis CBF1, CBF2, or CBF3 genes cloned into the vector pGA643 (An (1987) [0490] 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 GV3 101. Transformed Agrobacteria were grown for 2 days in minimal AB medium containing appropriate antibiotics.
Spring canola ([0491] 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° C. under 60-80 μE/m²s light using al 6 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.
The transformed plants were then analyzed for the presence of the NPTII gene/kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, Colo.). Approximately 70% of the screened plants were NPTII positive. Only those plants were further analyzed. [0492]
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° C. Spring canola transformed with eieitr CBFl or CBF2 showed a 50% leakage at −6 to −7° C. Spring canola transformed with CBF3 shows a 50% leakage at about −10 to −15° C. Winter canola transformed with CBF3 may show a 50% leakage at about −16 to −20° 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° C. [0493]
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. [0494]
These results demonstrate that homologs of Arabidopsis transcription factors can be identified and shown to confer similar functions in non-Arabidopsis plant species. [0495]

Example XVIII

Cloning of Transcription Factor Promoters [0496]
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, and varied hormone concentrations (ABA, GA, auxin, cytokinin, salicylic acid, brassinosteroid). 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. [0497]
Transcription factor promoters for the genes disclosed herein are obtained by cloning 1.5 kb to 2.0 kb of genomic sequence immediately upstream of the translation start codon for the coding sequence of the encoded transcription factor protein. This region includes the 5′-UTR of the transcription factor gene, which can comprise regulatory elements. The 1.5 kb to 2.0 kb region is cloned through PCR methods, using primers that include one in the 3′ direction located at the translation start codon (including appropriate adaptor sequence), and one in the 5′ direction located from 1.5 kb to 2.0 kb upstream of the translation start codon (including appropriate adaptor sequence). The desired fragments are PCR-amplified from Arabidopsis Col-0 genomic DNA using high-fidelity Taq DNA polymerase to minimize the incorporation of point mutation(s). The cloning primers incorporate two rare restriction sites, such as NotI and Sfi1, found at low frequency throughout the Arabidopsis genome. Additional restriction sites are used in the instances where a NotI or Sfi1 restriction site is present within the promoter. [0498]
The 1.5-2.0 kb fragment upstream from the translation start codon, including the 5′-untranslated region of the transcription factor, is cloned in a binary transformation vector immediately upstream of a suitable reporter gene, or a transactivator gene that is capable of programming expression of a reporter gene in a second gene construct. Reporter genes used include green fluorescent protein (and related fluorescent protein color variants), beta-glucuronidase, and luciferase. Suitable transactivator genes include LexA-GAL4, along with a transactivatable reporter in a second binary plasmid (as disclosed in U.S. patent application Ser. No. 09/958,131, incorporated herein by reference). The binary plasmid(s) is transferred into Agrobacterium and the structure of the plasmid confirmed by PCR. These strains are introduced into Arabidopsis plants as described in other examples, and gene expression patterns determined according to standard methods know to one skilled in the art for monitoring GFP fluorescence, beta-glucuronidase activity, or luminescence. [0499]
All references, publications, patent documents, web pages, and other documents cited or mentioned herein are hereby incorporated by reference in their entirety for all purposes. Although the invention has been described with reference to specific embodiments and examples, it should be understood that one of ordinary skill can make various modifications without departing from the spirit of the invention. The scope of the invention is not limited to the specific embodiments and examples provided. [0500]
1 64 1 1281 DNA Arabidopsis thaliana G867 Predicted polypeptide sequence is paralogous to G9, G993, G1930 1 cacaacacaa acacatttct gttttctcca ttgtttcaaa ccataaaaaa aaacacagat 60 taaatggaat cgagtagcgt tgatgagagt actacaagta caggttccat ctgtgaaacc 120 ccggcgataa ctccggcgaa aaagtcgtcg gtaggtaact tatacaggat gggaagcgga 180 tcaagcgttg tgttagattc agagaacggc gtagaagctg aatctaggaa gcttccgtcg 240 tcaaaataca aaggtgtggt gccacaacca aacggaagat ggggagctca gatttacgag 300 aaacaccagc gcgtgtggct cgggacattc aacgaagaag acgaagccgc tcgtgcctac 360 gacgtcgcgg ttcacaggtt ccgtcgccgt gacgccgtca caaatttcaa agacgtgaag 420 atggacgaag acgaggtcga tttcttgaat tctcattcga aatctgagat cgttgatatg 480 ttgaggaaac atacttataa cgaagagtta gagcagagta aacggcgtcg taatggtaac 540 ggaaacatga ctaggacgtt gttaacgtcg gggttgagta atgatggtgt ttctacgacg 600 gggtttagat cggcggaggc actgtttgag aaagcggtaa cgccaagcga cgttgggaag 660 ctaaaccgtt tggttatacc gaaacatcac gcagagaaac attttccgtt accgtcaagt 720 aacgtttccg tgaaaggagt gttgttgaac tttgaggacg ttaacgggaa agtgtggagg 780 ttccgttact cgtattggaa cagtagtcag agttatgttt tgactaaagg ttggagcagg 840 ttcgttaagg agaagaatct acgtgctggt gacgtggtta gtttcagtag atctaacggt 900 caggatcaac agttgtacat tgggtggaag tcgagatccg ggtcagattt agatgcgggt 960 cgggttttga gattgttcgg agttaacatt tcaccggaga gttcaagaaa cgacgtcgta 1020 ggaaacaaaa gagtgaacga tactgagatg ttatcgttgg tgtgtagcaa gaagcaacgc 1080 atctttcacg cctcgtaaca actcttcttc tttttttttc ttttgttgtt ttaataattt 1140 ttaaaaactc cattttcgtt ttctttattt gcatcggttt ctttcttctt gtttaccaaa 1200 ggttcatgag ttgtttttgt tgtattgatg aactgtaaat tttatttata ggataaattt 1260 taaaaaaaaa aaaaaaaaaa a 1281 2 344 PRT Arabidopsis thaliana G867 polypeptide Paralogous to G9, G993, G1930 2 Met Glu Ser Ser Ser Val Asp Glu Ser Thr Thr Ser Thr Gly Ser Ile 1 5 10 15 Cys Glu Thr Pro Ala Ile Thr Pro Ala Lys Lys Ser Ser Val Gly Asn 20 25 30 Leu Tyr Arg Met Gly Ser Gly Ser Ser Val Val Leu Asp Ser Glu Asn 35 40 45 Gly Val Glu Ala Glu Ser Arg Lys Leu Pro Ser Ser Lys Tyr Lys Gly 50 55 60 Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys 65 70 75 80 His Gln Arg Val Trp Leu Gly Thr Phe Asn Glu Glu Asp Glu Ala Ala 85 90 95 Arg Ala Tyr Asp Val Ala Val His Arg Phe Arg Arg Arg Asp Ala Val 100 105 110 Thr Asn Phe Lys Asp Val Lys Met Asp Glu Asp Glu Val Asp Phe Leu 115 120 125 Asn Ser His Ser Lys Ser Glu Ile Val Asp Met Leu Arg Lys His Thr 130 135 140 Tyr Asn Glu Glu Leu Glu Gln Ser Lys Arg Arg Arg Asn Gly Asn Gly 145 150 155 160 Asn Met Thr Arg Thr Leu Leu Thr Ser Gly Leu Ser Asn Asp Gly Val 165 170 175 Ser Thr Thr Gly Phe Arg Ser Ala Glu Ala Leu Phe Glu Lys Ala Val 180 185 190 Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys His 195 200 205 His Ala Glu Lys His Phe Pro Leu Pro Ser Ser Asn Val Ser Val Lys 210 215 220 Gly Val Leu Leu Asn Phe Glu Asp Val Asn Gly Lys Val Trp Arg Phe 225 230 235 240 Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly 245 250 255 Trp Ser Arg Phe Val Lys Glu Lys Asn Leu Arg Ala Gly Asp Val Val 260 265 270 Ser Phe Ser Arg Ser Asn Gly Gln Asp Gln Gln Leu Tyr Ile Gly Trp 275 280 285 Lys Ser Arg Ser Gly Ser Asp Leu Asp Ala Gly Arg Val Leu Arg Leu 290 295 300 Phe Gly Val Asn Ile Ser Pro Glu Ser Ser Arg Asn Asp Val Val Gly 305 310 315 320 Asn Lys Arg Val Asn Asp Thr Glu Met Leu Ser Leu Val Cys Ser Lys 325 330 335 Lys Gln Arg Ile Phe His Ala Ser 340 3 1246 DNA Arabidopsis thaliana G9 Predicted polypeptide sequence is paralogous to G867, G993, G1930 3 gtgtttcttc tttctgctaa aaggttataa tttttgtttc ttggtttggt gagaatcttc 60 aagaaactga aacaaagaaa atggattcta gttgcataga cgagataagt tcctccactt 120 cagaatcttt ctccgccacc accgccaaga agctctctcc tcctcccgcg gcggcgttac 180 gcctctaccg gatgggaagc ggcgggagca gcgtcgtgtt ggatcccgag aacggcctag 240 agacggagtc acgaaagcta ccatcttcaa aatacaaagg tgttgttcct cagcctaacg 300 gaagatgggg agctcagatc tacgagaagc accaacgagt atggctcggg actttcaacg 360 agcaagaaga agctgctcgt tcctacgaca tcgcagcttg tagattccgt ggccgcgacg 420 ccgtcgtcaa cttcaagaac gttctggaag acggcgattt agcttttctt gaagctcact 480 caaaggccga gatcgtcgac atgttgagaa aacacactta cgccgacgag cttgaacaga 540 acaataaacg gcagttgttt ctctccgtcg acgctaacgg aaaacgtaac ggatcgagta 600 ctactcaaaa cgacaaagtt ttaaagacgt gtgaagttct tttcgagaag gctgttacac 660 ctagcgacgt tgggaagcta aaccgtctcg tgatacctaa acaacacgcc gagaaacact 720 ttccgttacc gtcaccgtca ccggcagtga ctaaaggagt tttgatcaac ttcgaagacg 780 ttaacggtaa agtgtggagg ttccgttact catactggaa cagtagtcaa agttacgtgt 840 tgaccaaggg atggagtcga ttcgtcaagg agaagaatct tcgagccggt gatgttgtta 900 ctttcgagag atcgaccgga ctagagcggc agttatatat tgattggaaa gttcggtctg 960 gtccgagaga aaacccggtt caggtggtgg ttcggctttt cggagttgat atctttaatg 1020 tgaccaccgt gaagccaaac gacgtcgtgg ccgtttgcgg tggaaagaga tctcgagatg 1080 ttgatgatat gtttgcgtta cggtgttcca agaagcaggc gataatcaat gctttgtgac 1140 atatttcctt ttccgatttt atgctttcgt tttttaattt ttttttttgt caagttgtgt 1200 aggttgtgat tcatgctagg ttgtatttag gaaaagagat aagacc 1246 4 352 PRT Arabidopsis thaliana G9 polypeptide Paralogous to G867, G993, G1930 4 Met Asp Ser Ser Cys Ile Asp Glu Ile Ser Ser Ser Thr Ser Glu Ser 1 5 10 15 Phe Ser Ala Thr Thr Ala Lys Lys Leu Ser Pro Pro Pro Ala Ala Ala 20 25 30 Leu Arg Leu Tyr Arg Met Gly Ser Gly Gly Ser Ser Val Val Leu Asp 35 40 45 Pro Glu Asn Gly Leu Glu Thr Glu Ser Arg Lys Leu Pro Ser Ser Lys 50 55 60 Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile 65 70 75 80 Tyr Glu Lys His Gln Arg Val Trp Leu Gly Thr Phe Asn Glu Gln Glu 85 90 95 Glu Ala Ala Arg Ser Tyr Asp Ile Ala Ala Cys Arg Phe Arg Gly Arg 100 105 110 Asp Ala Val Val Asn Phe Lys Asn Val Leu Glu Asp Gly Asp Leu Ala 115 120 125 Phe Leu Glu Ala His Ser Lys Ala Glu Ile Val Asp Met Leu Arg Lys 130 135 140 His Thr Tyr Ala Asp Glu Leu Glu Gln Asn Asn Lys Arg Gln Leu Phe 145 150 155 160 Leu Ser Val Asp Ala Asn Gly Lys Arg Asn Gly Ser Ser Thr Thr Gln 165 170 175 Asn Asp Lys Val Leu Lys Thr Cys Glu Val Leu Phe Glu Lys Ala Val 180 185 190 Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln 195 200 205 His Ala Glu Lys His Phe Pro Leu Pro Ser Pro Ser Pro Ala Val Thr 210 215 220 Lys Gly Val Leu Ile Asn Phe Glu Asp Val Asn Gly Lys Val Trp Arg 225 230 235 240 Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys 245 250 255 Gly Trp Ser Arg Phe Val Lys Glu Lys Asn Leu Arg Ala Gly Asp Val 260 265 270 Val Thr Phe Glu Arg Ser Thr Gly Leu Glu Arg Gln Leu Tyr Ile Asp 275 280 285 Trp Lys Val Arg Ser Gly Pro Arg Glu Asn Pro Val Gln Val Val Val 290 295 300 Arg Leu Phe Gly Val Asp Ile Phe Asn Val Thr Thr Val Lys Pro Asn 305 310 315 320 Asp Val Val Ala Val Cys Gly Gly Lys Arg Ser Arg Asp Val Asp Asp 325 330 335 Met Phe Ala Leu Arg Cys Ser Lys Lys Gln Ala Ile Ile Asn Ala Leu 340 345 350 5 1239 DNA Arabidopsis thaliana G993 Predicted polypeptide sequence is paralogous to G867, G9, G1930 5 caaatatgga atacagctgt gtagacgaca gtagtacaac gtcagaatct ctctccatct 60 ctactactcc aaagccgaca acgacgacgg agaagaaact ctcttctccg ccggcgacgt 120 cgatgcgtct ctacagaatg ggaagcggcg gaagcagcgt cgttttggat tcagagaacg 180 gcgtcgagac cgagtcacgt aagcttcctt cgtcgaaata taaaggcgtt gtgcctcagc 240 ctaacggaag atggggagct cagatttacg agaagcatca gcgagtttgg ctcggtactt 300 tcaacgagga agaagaagct gcgtcttctt acgacatcgc cgtgaggaga ttccgcggcc 360 gcgacgccgt cactaacttc aaatctcaag ttgatggaaa cgacgccgaa tcggcttttc 420 ttgacgctca ttctaaagct gagatcgtgg atatgttgag gaaacacact tacgccgatg 480 agtttgagca gagtagacgg aagtttgtta acggcgacgg aaaacgctct gggttggaga 540 cggcgacgta cggaaacgac gctgttttga gagcgcgtga ggttttgttc gagaagactg 600 ttacgccgag cgacgtcggg aagctgaacc gtttagtgat accgaaacaa cacgcggaga 660 agcattttcc gttaccggcg atgacgacgg cgatggggat gaatccgtct ccgacgaaag 720 gcgttttgat taacttggaa gatagaacag ggaaagtgtg gcggttccgt tacagttact 780 ggaacagcag tcaaagttac gtgttgacca agggctggag ccggttcgtt aaagagaaga 840 atcttcgagc cggtgatgtg gtttgtttcg agagatcaac cggaccagac cggcaattgt 900 atatccactg gaaagtccgg tctagtccgg ttcagactgt ggttaggcta ttcggagtca 960 acattttcaa tgtgagtaac gagaaaccaa acgacgtcgc agtagagtgt gttggcaaga 1020 agagatctcg ggaagatgat ttgttttcgt tagggtgttc caagaagcag gcgattatca 1080 acatcttgtg acaaattctt tttttttggt ttttttcttc aatttgtttc tcctttttca 1140 atattttgta ttgaaatgac aagttgtaaa ttaggacaag acaagaaaaa atgacaacta 1200 gacaaaatag tttttgttta aaaaaaaaaa aaaaaaaaa 1239 6 361 PRT Arabidopsis thaliana G993 polypeptide Paralogous to G867, G9, G1930 6 Met Glu Tyr Ser Cys Val Asp Asp Ser Ser Thr Thr Ser Glu Ser Leu 1 5 10 15 Ser Ile Ser Thr Thr Pro Lys Pro Thr Thr Thr Thr Glu Lys Lys Leu 20 25 30 Ser Ser Pro Pro Ala Thr Ser Met Arg Leu Tyr Arg Met Gly Ser Gly 35 40 45 Gly Ser Ser Val Val Leu Asp Ser Glu Asn Gly Val Glu Thr Glu Ser 50 55 60 Arg Lys Leu Pro Ser Ser Lys Tyr Lys Gly Val Val Pro Gln Pro Asn 65 70 75 80 Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys His Gln Arg Val Trp Leu 85 90 95 Gly Thr Phe Asn Glu Glu Glu Glu Ala Ala Ser Ser Tyr Asp Ile Ala 100 105 110 Val Arg Arg Phe Arg Gly Arg Asp Ala Val Thr Asn Phe Lys Ser Gln 115 120 125 Val Asp Gly Asn Asp Ala Glu Ser Ala Phe Leu Asp Ala His Ser Lys 130 135 140 Ala Glu Ile Val Asp Met Leu Arg Lys His Thr Tyr Ala Asp Glu Phe 145 150 155 160 Glu Gln Ser Arg Arg Lys Phe Val Asn Gly Asp Gly Lys Arg Ser Gly 165 170 175 Leu Glu Thr Ala Thr Tyr Gly Asn Asp Ala Val Leu Arg Ala Arg Glu 180 185 190 Val Leu Phe Glu Lys Thr Val Thr Pro Ser Asp Val Gly Lys Leu Asn 195 200 205 Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro Leu Pro 210 215 220 Ala Met Thr Thr Ala Met Gly Met Asn Pro Ser Pro Thr Lys Gly Val 225 230 235 240 Leu Ile Asn Leu Glu Asp Arg Thr Gly Lys Val Trp Arg Phe Arg Tyr 245 250 255 Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser 260 265 270 Arg Phe Val Lys Glu Lys Asn Leu Arg Ala Gly Asp Val Val Cys Phe 275 280 285 Glu Arg Ser Thr Gly Pro Asp Arg Gln Leu Tyr Ile His Trp Lys Val 290 295 300 Arg Ser Ser Pro Val Gln Thr Val Val Arg Leu Phe Gly Val Asn Ile 305 310 315 320 Phe Asn Val Ser Asn Glu Lys Pro Asn Asp Val Ala Val Glu Cys Val 325 330 335 Gly Lys Lys Arg Ser Arg Glu Asp Asp Leu Phe Ser Leu Gly Cys Ser 340 345 350 Lys Lys Gln Ala Ile Ile Asn Ile Leu 355 360 7 1155 DNA Arabidopsis thaliana G1930 Predicted polypeptide sequence is paralogous to G867, G9, G993 7 attcacatta ctaatctctc aagatttcac aattttcttg tgattttctc tcagtttctt 60 atttcgtttc ataacatgga tgccatgagt agcgtagacg agagctctac aactacagat 120 tccattccgg cgagaaagtc atcgtctccg gcgagtttac tatatagaat gggaagcgga 180 acaagcgtgg tacttgattc agagaacggt gtcgaagtcg aagtcgaagc cgaatcaaga 240 aagcttcctt cttcaagatt caaaggtgtt gttcctcaac caaatggaag atggggagct 300 cagatttacg agaaacatca acgcgtgtgg cttggtactt tcaacgagga agacgaagca 360 gctcgtgctt acgacgtcgc ggctcaccgt ttccgtggcc gcgatgccgt tactaatttc 420 aaagacacga cgttcgaaga agaggttgag ttcttaaacg cgcattcgaa atcagagatc 480 gtagatatgt tgagaaaaca cacttacaaa gaagagttag accaaaggaa acgtaaccgt 540 gacggtaacg gaaaagagac gacggcgttt gctttggctt cgatggtggt tatgacgggg 600 tttaaaacgg cggagttact gtttgagaaa acggtaacgc caagtgacgt cgggaaacta 660 aaccgtttag ttataccaaa acaccaagcg gagaaacatt ttccgttacc gttaggtaat 720 aataacgtct ccgttaaagg tatgctgttg aatttcgaag acgttaacgg gaaagtgtgg 780 aggttccgtt actcttattg gaatagtagt caaagttatg tgttgaccaa aggttggagt 840 agattcgtta aagagaagag actttgtgct ggtgatttga tcagttttaa aagatccaac 900 gatcaagatc aaaaattctt tatcgggtgg aaatcgaaat ccgggttgga tctagagacg 960 ggtcgggtta tgagattgtt tggggttgat atttctttaa acgccgtcgt tgtagtgaag 1020 gaaacaacgg aggtgttaat gtcgtcgtta aggtgtaaga agcaacgagt tttgtaataa 1080 caatttaaca acttgggaaa gaaaaaaaag ctttttgatt ttaatttctc ttcaacgtta 1140 atcttgctga gatta 1155 8 333 PRT Arabidopsis thaliana G1930 polypeptide Paralogous to G867, G9, G993 8 Met Asp Ala Met Ser Ser Val Asp Glu Ser Ser Thr Thr Thr Asp Ser 1 5 10 15 Ile Pro Ala Arg Lys Ser Ser Ser Pro Ala Ser Leu Leu Tyr Arg Met 20 25 30 Gly Ser Gly Thr Ser Val Val Leu Asp Ser Glu Asn Gly Val Glu Val 35 40 45 Glu Val Glu Ala Glu Ser Arg Lys Leu Pro Ser Ser Arg Phe Lys Gly 50 55 60 Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys 65 70 75 80 His Gln Arg Val Trp Leu Gly Thr Phe Asn Glu Glu Asp Glu Ala Ala 85 90 95 Arg Ala Tyr Asp Val Ala Ala His Arg Phe Arg Gly Arg Asp Ala Val 100 105 110 Thr Asn Phe Lys Asp Thr Thr Phe Glu Glu Glu Val Glu Phe Leu Asn 115 120 125 Ala His Ser Lys Ser Glu Ile Val Asp Met Leu Arg Lys His Thr Tyr 130 135 140 Lys Glu Glu Leu Asp Gln Arg Lys Arg Asn Arg Asp Gly Asn Gly Lys 145 150 155 160 Glu Thr Thr Ala Phe Ala Leu Ala Ser Met Val Val Met Thr Gly Phe 165 170 175 Lys Thr Ala Glu Leu Leu Phe Glu Lys Thr Val Thr Pro Ser Asp Val 180 185 190 Gly Lys Leu Asn Arg Leu Val Ile Pro Lys His Gln Ala Glu Lys His 195 200 205 Phe Pro Leu Pro Leu Gly Asn Asn Asn Val Ser Val Lys Gly Met Leu 210 215 220 Leu Asn Phe Glu Asp Val Asn Gly Lys Val Trp Arg Phe Arg Tyr Ser 225 230 235 240 Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg 245 250 255 Phe Val Lys Glu Lys Arg Leu Cys Ala Gly Asp Leu Ile Ser Phe Lys 260 265 270 Arg Ser Asn Asp Gln Asp Gln Lys Phe Phe Ile Gly Trp Lys Ser Lys 275 280 285 Ser Gly Leu Asp Leu Glu Thr Gly Arg Val Met Arg Leu Phe Gly Val 290 295 300 Asp Ile Ser Leu Asn Ala Val Val Val Val Lys Glu Thr Thr Glu Val 305 310 315 320 Leu Met Ser Ser Leu Arg Cys Lys Lys Gln Arg Val Leu 325 330 9 1194 DNA Arabidopsis thaliana G2687 9 ctctgtctct cgtatctttc tactactctg tttcttgaat tctaatgaac aacatcgacg 60 acgcaaagac ggagacttca gtgtcttcag gttcaagcga ctctttcttg cctctcaaga 120 aacgcatgag acttgatgac gaaccagaaa acgccctagt ggtttcgtct tcaccaaaga 180 cggttgtggc ttctggcaat gtcaagtaca aaggagtcgt tcagcaacag aacggtcatt 240 ggggtgccca gatttacgca gaccacaaaa ggatttggct tggaactttc aaatccgctg 300 atgaagccgc cacggcttac gatagtgcat ctatcaaact ccgaagcttt gacgctaact 360 cgcaccggaa cttcccttgg tctacaatca ctctcaacga accagacttt caaaattgct 420 acacaacaga gactgtgttg aacatgatca gagacggttc gtaccaacac aaattcagag 480 attttctcag aatcagatct cagattgttg cgagtatcaa catcggggga ccaaaacaag 540 cccgaggaga agtgaatcaa gaatcagaca agtgtttttc ttgcacacag ctttttcaga 600 aggaattgac accgagcgat gtagggaaac taaataggct tgtgatacct aaaaagtatg 660 cagtgaagta tatgcctttc ataagcgctg atcaaagcga gaaagaagag ggtgaaatag 720 taggatctgt ggaagatgtg gaggttgtgt tttacgacag agcaatgaga caatggaagt 780 ttaggtattg ttactggaaa agtagccaga gctttgtctt caccagagga tggaatagtt 840 tcgtgaagga gaagaatctc aaggagaagg atgttattgc cttctacact tgcgatgtcc 900 cgaacaatgt gaagacatta gaaggtcaaa gaaagaactt cttgatgatc gatgttcatt 960 gcttttcaga caacggttcc gtggtagctg aggaagtaag tatgacggtt catgacagtt 1020 cagtgcaagt aaagaaaaca gaaaacttgg ttagctccat gttagaagat aaagaaacca 1080 aatcagagga gaacaaagga gggtttatgc tgtttggtgt aaggatcgaa tgtccttagg 1140 gaatttttct ttaaaagttt cttacttcaa ctagaacttg ttttacttgt acct 1194 10 363 PRT Arabidopsis thaliana G2687 polypeptide 10 Met Asn Asn Ile Asp Asp Ala Lys Thr Glu Thr Ser Val Ser Ser Gly 1 5 10 15 Ser Ser Asp Ser Phe Leu Pro Leu Lys Lys Arg Met Arg Leu Asp Asp 20 25 30 Glu Pro Glu Asn Ala Leu Val Val Ser Ser Ser Pro Lys Thr Val Val 35 40 45 Ala Ser Gly Asn Val Lys Tyr Lys Gly Val Val Gln Gln Gln Asn Gly 50 55 60 His Trp Gly Ala Gln Ile Tyr Ala Asp His Lys Arg Ile Trp Leu Gly 65 70 75 80 Thr Phe Lys Ser Ala Asp Glu Ala Ala Thr Ala Tyr Asp Ser Ala Ser 85 90 95 Ile Lys Leu Arg Ser Phe Asp Ala Asn Ser His Arg Asn Phe Pro Trp 100 105 110 Ser Thr Ile Thr Leu Asn Glu Pro Asp Phe Gln Asn Cys Tyr Thr Thr 115 120 125 Glu Thr Val Leu Asn Met Ile Arg Asp Gly Ser Tyr Gln His Lys Phe 130 135 140 Arg Asp Phe Leu Arg Ile Arg Ser Gln Ile Val Ala Ser Ile Asn Ile 145 150 155 160 Gly Gly Pro Lys Gln Ala Arg Gly Glu Val Asn Gln Glu Ser Asp Lys 165 170 175 Cys Phe Ser Cys Thr Gln Leu Phe Gln Lys Glu Leu Thr Pro Ser Asp 180 185 190 Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Lys Tyr Ala Val Lys 195 200 205 Tyr Met Pro Phe Ile Ser Ala Asp Gln Ser Glu Lys Glu Glu Gly Glu 210 215 220 Ile Val Gly Ser Val Glu Asp Val Glu Val Val Phe Tyr Asp Arg Ala 225 230 235 240 Met Arg Gln Trp Lys Phe Arg Tyr Cys Tyr Trp Lys Ser Ser Gln Ser 245 250 255 Phe Val Phe Thr Arg Gly Trp Asn Ser Phe Val Lys Glu Lys Asn Leu 260 265 270 Lys Glu Lys Asp Val Ile Ala Phe Tyr Thr Cys Asp Val Pro Asn Asn 275 280 285 Val Lys Thr Leu Glu Gly Gln Arg Lys Asn Phe Leu Met Ile Asp Val 290 295 300 His Cys Phe Ser Asp Asn Gly Ser Val Val Ala Glu Glu Val Ser Met 305 310 315 320 Thr Val His Asp Ser Ser Val Gln Val Lys Lys Thr Glu Asn Leu Val 325 330 335 Ser Ser Met Leu Glu Asp Lys Glu Thr Lys Ser Glu Glu Asn Lys Gly 340 345 350 Gly Phe Met Leu Phe Gly Val Arg Ile Glu Cys 355 360 11 1216 DNA Arabidopsis thaliana G1957 11 caagaaccat ctcgtaaatc aagatttctc caaggaaaat cagataagtc ataatggatc 60 tatccctggc tccgacaaca acaacaagtt ccgaccaaga acaagacaga gaccaagaat 120 taacctccaa catcggagca agcagcagct ccggtcccag cggaaacaac aacaaccttc 180 cgatgatgat gattccacct ccggagaaag aacacatgtt cgacaaagtg gtaacaccaa 240 gcgacgtcgg aaaactcaac agactcgtga tccctaaaca acacgctgag aggtatttcc 300 ctctagactc ctcaaacaac caaaacggca cgcttttgaa cttccaagac agaaacggca 360 agatgtggag attccgttac tcgtattgga actctagcca gagctacgtt atgaccaaag 420 gatggagccg tttcgtcaaa gagaaaaagc tcgatgcagg agacattgtc tctttccaac 480 gaggcatcgg agatgagtca gaaagatcca aactttacat agattggagg catagacccg 540 acatgagcct cgttcaagca catcagtttg gtaattttgg tttcaatttc aatttcccga 600 ccacttctca atattccaac agatttcatc cattgccaga atataactcc gtcccgattc 660 accggggctt aaacatcgga aatcaccaac gttcctatta taacacccag cgtcaagagt 720 tcgtagggta tggttatggg aatttagctg gaaggtgtta ctacacggga tcaccgttgg 780 atcataggaa cattgttgga tcagagccgt tggttataga ctcagtccct gtggttcccg 840 ggagattaac tccggtgatg ttaccgccgc ttcctccgcc tccttctacg gcgggaaaga 900 gactaaggct ctttggggtg aatatggaat gtggcaatga ctataatcaa caagaagagt 960 catggttggt gccacgtggc gaaattggtg catcttcttc ttcttcttca gctctacgac 1020 taaatttatc gactgatcat gatgatgata atgatgatgg tgatgatggc gatgatgatc 1080 aatttgctaa gaaagggaag tcttcacttt ctctcaattt caatccatga gaagtttcat 1140 catcttcttg ttttgaatct ctctttatat tgtttccatt agtaattttt actaagggta 1200 ttagattcta gctagt 1216 12 358 PRT Arabidopsis thaliana G1957 polypeptide 12 Met Asp Leu Ser Leu Ala Pro Thr Thr Thr Thr Ser Ser Asp Gln Glu 1 5 10 15 Gln Asp Arg Asp Gln Glu Leu Thr Ser Asn Ile Gly Ala Ser Ser Ser 20 25 30 Ser Gly Pro Ser Gly Asn Asn Asn Asn Leu Pro Met Met Met Ile Pro 35 40 45 Pro Pro Glu Lys Glu His Met Phe Asp Lys Val Val Thr Pro Ser Asp 50 55 60 Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Arg 65 70 75 80 Tyr Phe Pro Leu Asp Ser Ser Asn Asn Gln Asn Gly Thr Leu Leu Asn 85 90 95 Phe Gln Asp Arg Asn Gly Lys Met Trp Arg Phe Arg Tyr Ser Tyr Trp 100 105 110 Asn Ser Ser Gln Ser Tyr Val Met Thr Lys Gly Trp Ser Arg Phe Val 115 120 125 Lys Glu Lys Lys Leu Asp Ala Gly Asp Ile Val Ser Phe Gln Arg Gly 130 135 140 Ile Gly Asp Glu Ser Glu Arg Ser Lys Leu Tyr Ile Asp Trp Arg His 145 150 155 160 Arg Pro Asp Met Ser Leu Val Gln Ala His Gln Phe Gly Asn Phe Gly 165 170 175 Phe Asn Phe Asn Phe Pro Thr Thr Ser Gln Tyr Ser Asn Arg Phe His 180 185 190 Pro Leu Pro Glu Tyr Asn Ser Val Pro Ile His Arg Gly Leu Asn Ile 195 200 205 Gly Asn His Gln Arg Ser Tyr Tyr Asn Thr Gln Arg Gln Glu Phe Val 210 215 220 Gly Tyr Gly Tyr Gly Asn Leu Ala Gly Arg Cys Tyr Tyr Thr Gly Ser 225 230 235 240 Pro Leu Asp His Arg Asn Ile Val Gly Ser Glu Pro Leu Val Ile Asp 245 250 255 Ser Val Pro Val Val Pro Gly Arg Leu Thr Pro Val Met Leu Pro Pro 260 265 270 Leu Pro Pro Pro Pro Ser Thr Ala Gly Lys Arg Leu Arg Leu Phe Gly 275 280 285 Val Asn Met Glu Cys Gly Asn Asp Tyr Asn Gln Gln Glu Glu Ser Trp 290 295 300 Leu Val Pro Arg Gly Glu Ile Gly Ala Ser Ser Ser Ser Ser Ser Ala 305 310 315 320 Leu Arg Leu Asn Leu Ser Thr Asp His Asp Asp Asp Asn Asp Asp Gly 325 330 335 Asp Asp Gly Asp Asp Asp Gln Phe Ala Lys Lys Gly Lys Ser Ser Leu 340 345 350 Ser Leu Asn Phe Asn Pro 355 13 1368 DNA Arabidopsis thaliana G1010 13 attcttcttc taaaaaatct tgacaacttt ttgtttttgt tttctttctc tgaatttttt 60 aaaagagaga gagctatgta gctatgaaac agtaagagat atagatatag agagacagag 120 aaagatgatg atcagtgaag ttaggctaaa cccactttct atttatgtat aattaggtca 180 atcacatcac caatctcctc ctccaattct cctcctctcc ttccaaattc tagggttttg 240 cttgtatctc accccctttc tcaattccct agggaaactg tgaatttcat caaattccat 300 tattttttgg tcacaccctt aaagagatct gagagttcta aagatgatga cagatttatc 360 tctcacgaga gatgaagatg aagaagaagc aaagccctta gcagaagaag aaggagcgcg 420 tgaagtagca gacagagagc acatgttcga caaagttgtg actccaagtg atgtcggaaa 480 actaaaccga cttgtgatcc caaagcaaca cgcagagaga ttcttccctt tagattcatc 540 ttcaaacgag aaaggtttgc ttttaaactt cgaagatctc actggcaaat cttggaggtt 600 ccgttactct tactggaaca gtagtcaaag ctatgtcatg actaaaggtt ggagcagatt 660 cgttaaagac aaaaagcttg acgccggaga tattgtctct ttccaaagat gtgtcggaga 720 ttcaggaaga gatagccgtt tgtttattga ttggaggaga agacctaaag tccctgacca 780 tcctcatttc gccgccggag ctatgttccc taggttttac agctttcctt cgaccaatta 840 cagtctttat aatcatcagc agcaacgtca tcatcacagt ggtggtggtt ataattatca 900 tcaaattccg agagaatttg gttatggtta cttcgttagg tcagtggatc agaggaacaa 960 tcctgcggct gcggtggctg atccgttggt gattgaatct gtgccggtga tgatgcacgg 1020 gagagctaat caggaacttg ttggaacggc cgggaagaga ctgaggcttt ttggagttga 1080 tatggaatgc ggcgagagcg gaatgaccaa cagtacggag gaggaatcat catcttccgg 1140 tggaagtttg ccacgtggag gcggtggtgg tgcttcatct tcctctttct ttcagctgag 1200 acttggaagc agcagtgaag atgatcactt cactaagaaa ggaaagtctt cattgtcttt 1260 tgatttggat caataataat gatgatgatg aaattagttg gtattttaag aaaaaaaaca 1320 tacatatata attctatata tatgacaaca taatgcattg atttcctt 1368 14 310 PRT Arabidopsis thaliana G1010 polypeptide 14 Met Met Thr Asp Leu Ser Leu Thr Arg Asp Glu Asp Glu Glu Glu Ala 1 5 10 15 Lys Pro Leu Ala Glu Glu Glu Gly Ala Arg Glu Val Ala Asp Arg Glu 20 25 30 His Met Phe Asp Lys Val Val Thr Pro Ser Asp Val Gly Lys Leu Asn 35 40 45 Arg Leu Val Ile Pro Lys Gln His Ala Glu Arg Phe Phe Pro Leu Asp 50 55 60 Ser Ser Ser Asn Glu Lys Gly Leu Leu Leu Asn Phe Glu Asp Leu Thr 65 70 75 80 Gly Lys Ser Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser 85 90 95 Tyr Val Met Thr Lys Gly Trp Ser Arg Phe Val Lys Asp Lys Lys Leu 100 105 110 Asp Ala Gly Asp Ile Val Ser Phe Gln Arg Cys Val Gly Asp Ser Gly 115 120 125 Arg Asp Ser Arg Leu Phe Ile Asp Trp Arg Arg Arg Pro Lys Val Pro 130 135 140 Asp His Pro His Phe Ala Ala Gly Ala Met Phe Pro Arg Phe Tyr Ser 145 150 155 160 Phe Pro Ser Thr Asn Tyr Ser Leu Tyr Asn His Gln Gln Gln Arg His 165 170 175 His His Ser Gly Gly Gly Tyr Asn Tyr His Gln Ile Pro Arg Glu Phe 180 185 190 Gly Tyr Gly Tyr Phe Val Arg Ser Val Asp Gln Arg Asn Asn Pro Ala 195 200 205 Ala Ala Val Ala Asp Pro Leu Val Ile Glu Ser Val Pro Val Met Met 210 215 220 His Gly Arg Ala Asn Gln Glu Leu Val Gly Thr Ala Gly Lys Arg Leu 225 230 235 240 Arg Leu Phe Gly Val Asp Met Glu Cys Gly Glu Ser Gly Met Thr Asn 245 250 255 Ser Thr Glu Glu Glu Ser Ser Ser Ser Gly Gly Ser Leu Pro Arg Gly 260 265 270 Gly Gly Gly Gly Ala Ser Ser Ser Ser Phe Phe Gln Leu Arg Leu Gly 275 280 285 Ser Ser Ser Glu Asp Asp His Phe Thr Lys Lys Gly Lys Ser Ser Leu 290 295 300 Ser Phe Asp Leu Asp Gln 305 310 15 1065 DNA Arabidopsis thaliana G2690 15 atggatatgg acgagatgag caatgtagcc aagacaacga cagagacttc aggcttaact 60 gactctgtct tgagcctcac gaaacgcatg aaacctactg aggttacgac caccacaaaa 120 cctgccttgt ccaacacgac gaaattcaaa ggagttgttc agcaacagaa cggtcattgg 180 ggtgctcaga tttacgcaga ccatcgaagg atttggcttg gaactttcaa atccgctcat 240 gaagccgctg ctgcttacga tagcgcatcg attaagcttc gaagctttga tgctaactcg 300 caccggaact tcccttggtc tgattttacc ctccatgaac cggactttca agagtgctac 360 acgacagaag ctgtgttgaa catgatcaga gacggttctt atcaacacaa gttcagagat 420 tttctcagaa tccggtctca gattgttgcg aatatcaaca tcgtgggatc aaaacaagtc 480 ttaggaggag gagaaggtgg tcaagaatcg aacaagtgtt tctcgtgcac gcagcttttt 540 cagaaagaac tgacaccgag cgatgtaggg aaactgaata ggcttgtgat acctaagaag 600 tatgcagtga agtatatgcc tttcataagc gatgatcaaa gcgagaaaga gacgagtgaa 660 ggagtagaag atgtggaggt tgtcttttac gacagagcaa tgagacaatg gaagtttagg 720 tattgttact ggagaagtag ccagagcttt gtcttcacca gaggatggaa tggtttcgtg 780 aaggagaaga atctcaagga gaaagatatt attgtctttt acacttgcga tgtccccaac 840 aatgtgaaga cattagaagg ccaaagcaag accttcttga tgattgatgt tcatcacttt 900 tcaggcaacg gtttcgtggt tcccgaggaa gtaaacaaga cggttcatga gatttctgat 960 gaagagatga aaacagaaac cctctttacc tcgaaggtag aagaagaaac caaatcagag 1020 gagaagaaag gagggtttat gctgtttggt gttaggatcc aatag 1065 16 354 PRT Arabidopsis thaliana G2690 polypeptide 16 Met Asp Met Asp Glu Met Ser Asn Val Ala Lys Thr Thr Thr Glu Thr 1 5 10 15 Ser Gly Leu Thr Asp Ser Val Leu Ser Leu Thr Lys Arg Met Lys Pro 20 25 30 Thr Glu Val Thr Thr Thr Thr Lys Pro Ala Leu Ser Asn Thr Thr Lys 35 40 45 Phe Lys Gly Val Val Gln Gln Gln Asn Gly His Trp Gly Ala Gln Ile 50 55 60 Tyr Ala Asp His Arg Arg Ile Trp Leu Gly Thr Phe Lys Ser Ala His 65 70 75 80 Glu Ala Ala Ala Ala Tyr Asp Ser Ala Ser Ile Lys Leu Arg Ser Phe 85 90 95 Asp Ala Asn Ser His Arg Asn Phe Pro Trp Ser Asp Phe Thr Leu His 100 105 110 Glu Pro Asp Phe Gln Glu Cys Tyr Thr Thr Glu Ala Val Leu Asn Met 115 120 125 Ile Arg Asp Gly Ser Tyr Gln His Lys Phe Arg Asp Phe Leu Arg Ile 130 135 140 Arg Ser Gln Ile Val Ala Asn Ile Asn Ile Val Gly Ser Lys Gln Val 145 150 155 160 Leu Gly Gly Gly Glu Gly Gly Gln Glu Ser Asn Lys Cys Phe Ser Cys 165 170 175 Thr Gln Leu Phe Gln Lys Glu Leu Thr Pro Ser Asp Val Gly Lys Leu 180 185 190 Asn Arg Leu Val Ile Pro Lys Lys Tyr Ala Val Lys Tyr Met Pro Phe 195 200 205 Ile Ser Asp Asp Gln Ser Glu Lys Glu Thr Ser Glu Gly Val Glu Asp 210 215 220 Val Glu Val Val Phe Tyr Asp Arg Ala Met Arg Gln Trp Lys Phe Arg 225 230 235 240 Tyr Cys Tyr Trp Arg Ser Ser Gln Ser Phe Val Phe Thr Arg Gly Trp 245 250 255 Asn Gly Phe Val Lys Glu Lys Asn Leu Lys Glu Lys Asp Ile Ile Val 260 265 270 Phe Tyr Thr Cys Asp Val Pro Asn Asn Val Lys Thr Leu Glu Gly Gln 275 280 285 Ser Lys Thr Phe Leu Met Ile Asp Val His His Phe Ser Gly Asn Gly 290 295 300 Phe Val Val Pro Glu Glu Val Asn Lys Thr Val His Glu Ile Ser Asp 305 310 315 320 Glu Glu Met Lys Thr Glu Thr Leu Phe Thr Ser Lys Val Glu Glu Glu 325 330 335 Thr Lys Ser Glu Glu Lys Lys Gly Gly Phe Met Leu Phe Gly Val Arg 340 345 350 Ile Gln 17 1253 DNA Glycine max G3451 GLYMA-28NOV01-CLUSTER19062_3 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 17 ctagaatccg tacaatctaa tcaacataac aaaaatggat gcaattagtt gcatggatga 60 gagcaccacc actgagtcac tctctataag tctttctccg acgtcatcgt cggagaaagc 120 gaagccttct tcgatgatta catcgtcgga gaaggtttct ctgtccccgc cgccgtcaaa 180 cagactatgc cgtgttggaa gcggcgcgag cgcagtcgtg gatcctgatg gcggcggcag 240 cggcgctgag gtagagtcgc ggaaactccc ctcgtcgaag tacaagggcg tggtgcccca 300 gcccaacggc cgctggggtg cgcagattta cgagaagcac cagcgcgtgt ggcttggaac 360 gttcaacgag gaagacgagg cggcgcgtgc gtacgacatc gccgcgcagc ggttccgcgg 420 caaggacgcc gtcacgaact tcaagccgct cgccggcgcc gacgacgacg acggagaatc 480 ggagtttctc aactcgcatt ccaaacccga gatcgtcgac atgctgcgaa agcacacgta 540 caatgacgag ctggagcaga gcaagcgcag ccgcggcgtc gtccggcggc gaggctccgc 600 cgccgccggc accgcaaact caatttccgg cgcgtgcttt actaaggcac gtgagcagct 660 attcgagaag gctgttacgc cgagcgacgt tgggaaattg aaccgtttgg tgataccgaa 720 gcagcacgcg gagaagcact ttccgttaca gagctctaac ggcgttagcg cgacgacgat 780 agcggcggtg acggcgacgc cgacggcggc gaagggcgtt ttgttgaact tcgaagacgt 840 tggagggaaa gtgtggcggt ttcgttactc gtattggaac agtagccaga gttacgtctt 900 aaccaaaggt tggagccggt tcgttaagga gaagaatctg aaagctggtg acacggtttg 960 ttttcaccgg tccactggac cggacaagca gctttacatc gattggaaga cgaggaatgt 1020 tgttaacaac gaggtcgcgt tgttcggacc ggtcggaccg gttgtcgaac cgatccagat 1080 ggttcggctc tttggggtta acattttgaa actacccggt tcagatacta ttgttggcaa 1140 taacaataat gcaagtgggt gctgcaatgg caagagaaga gaaatggaac tgttctcgtt 1200 agagtgtagc aagaaaccta agattattgg tgctttgtaa cgttacgtta ggc 1253 18 401 PRT Glycine max G3451 polypeptide GLYMA-28NOV01-CLUSTER19062_3 Orthologous to G867, G9, G993, G1930 18 Met Asp Ala Ile Ser Cys Met Asp Glu Ser Thr Thr Thr Glu Ser Leu 1 5 10 15 Ser Ile Ser Leu Ser Pro Thr Ser Ser Ser Glu Lys Ala Lys Pro Ser 20 25 30 Ser Met Ile Thr Ser Ser Glu Lys Val Ser Leu Ser Pro Pro Pro Ser 35 40 45 Asn Arg Leu Cys Arg Val Gly Ser Gly Ala Ser Ala Val Val Asp Pro 50 55 60 Asp Gly Gly Gly Ser Gly Ala Glu Val Glu Ser Arg Lys Leu Pro Ser 65 70 75 80 Ser Lys Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala 85 90 95 Gln Ile Tyr Glu Lys His Gln Arg Val Trp Leu Gly Thr Phe Asn Glu 100 105 110 Glu Asp Glu Ala Ala Arg Ala Tyr Asp Ile Ala Ala Gln Arg Phe Arg 115 120 125 Gly Lys Asp Ala Val Thr Asn Phe Lys Pro Leu Ala Gly Ala Asp Asp 130 135 140 Asp Asp Gly Glu Ser Glu Phe Leu Asn Ser His Ser Lys Pro Glu Ile 145 150 155 160 Val Asp Met Leu Arg Lys His Thr Tyr Asn Asp Glu Leu Glu Gln Ser 165 170 175 Lys Arg Ser Arg Gly Val Val Arg Arg Arg Gly Ser Ala Ala Ala Gly 180 185 190 Thr Ala Asn Ser Ile Ser Gly Ala Cys Phe Thr Lys Ala Arg Glu Gln 195 200 205 Leu Phe Glu Lys Ala Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg 210 215 220 Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro Leu Gln Ser 225 230 235 240 Ser Asn Gly Val Ser Ala Thr Thr Ile Ala Ala Val Thr Ala Thr Pro 245 250 255 Thr Ala Ala Lys Gly Val Leu Leu Asn Phe Glu Asp Val Gly Gly Lys 260 265 270 Val Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val 275 280 285 Leu Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Asn Leu Lys Ala 290 295 300 Gly Asp Thr Val Cys Phe His Arg Ser Thr Gly Pro Asp Lys Gln Leu 305 310 315 320 Tyr Ile Asp Trp Lys Thr Arg Asn Val Val Asn Asn Glu Val Ala Leu 325 330 335 Phe Gly Pro Val Gly Pro Val Val Glu Pro Ile Gln Met Val Arg Leu 340 345 350 Phe Gly Val Asn Ile Leu Lys Leu Pro Gly Ser Asp Thr Ile Val Gly 355 360 365 Asn Asn Asn Asn Ala Ser Gly Cys Cys Asn Gly Lys Arg Arg Glu Met 370 375 380 Glu Leu Phe Ser Leu Glu Cys Ser Lys Lys Pro Lys Ile Ile Gly Ala 385 390 395 400 Leu 19 1067 DNA Oryza sativa ORYSA-22JAN02-CLUSTER46187_2 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 19 gcacgagcac ctgctttggc ttctccctct tcactgccct aattccttgc ttctctctcc 60 tctcctctct ctctctctct ctctctcgct gcagccatag cttagctttc ttggtgccaa 120 gatgggggtg gtcagcttct cctcgacttc ctccggcgcg tccactgcca ccaccgagtc 180 cggcggcgcc gtgcggatgt cgccggagcc ggtggtggcg gtggcggcgg cggctcaaca 240 gctgccggtg gtgaagggag ttgactcggc ggatgaggtg gtgacgtcga agcccgcagc 300 ggcggcggtg gcgcagcagt cgtcgaggta caagggggtg gtgccgcagc cgaacgggcg 360 gtggggggcg cagatctacg agcgccacgc gcgggtgtgg ctcgggacgt tccccgacga 420 ggaggcggcg gcgcgggcct acgacgtggc ggcgctccgg taccgggggc gcgacgcggc 480 caccaacttc cccggggccg cggcgtcggc ggccgagctc gcgttcctcg ccgcgcactc 540 caaggccgag atcgtcgaca tgctgcggaa gcacacctac gccgacgagc tccgccaggg 600 cctccgccgc ggccgcggca tgggcgcccg cgcccagccc acgccatcgt gggcgcgcga 660 gccgctgttc gagaaggccg tgacgcccag cgacgtcggc aagctcaacc gcctcgtggt 720 gcccaagcag cacgccgaga agcacttccc gctccgccgc gcggcgagct ccgactccgc 780 ctccgccgcc gccaccggca agggcgtgct cctcaacttc gaggacggcg agggcaaggt 840 gtggcgattc cggtactcgt actggaacag cagccagagc tacgtgctga ccaaggggtg 900 gagccgattc gtgagggaga agggcctccg cgccggcgac accattgtct tctccccgct 960 cggcgtacgg ccccgacaag ctgctcttca tcgactgcaa gaagaacaac gcggcggtgg 1020 cggcgaccac cacctgcgcc ggcgacgaga ggccaaccac aaccaca 1067 20 315 PRT Oryza sativa ORYSA-22JAN02-CLUSTER46187_2 polypeptide Orthologous to G867, G9, G993, G1930 20 Met Gly Val Val Ser Phe Ser Ser Thr Ser Ser Gly Ala Ser Thr Ala 1 5 10 15 Thr Thr Glu Ser Gly Gly Ala Val Arg Met Ser Pro Glu Pro Val Val 20 25 30 Ala Val Ala Ala Ala Ala Gln Gln Leu Pro Val Val Lys Gly Val Asp 35 40 45 Ser Ala Asp Glu Val Val Thr Ser Lys Pro Ala Ala Ala Ala Val Ala 50 55 60 Gln Gln Ser Ser Arg Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg 65 70 75 80 Trp Gly Ala Gln Ile Tyr Glu Arg His Ala Arg Val Trp Leu Gly Thr 85 90 95 Phe Pro Asp Glu Glu Ala Ala Ala Arg Ala Tyr Asp Val Ala Ala Leu 100 105 110 Arg Tyr Arg Gly Arg Asp Ala Ala Thr Asn Phe Pro Gly Ala Ala Ala 115 120 125 Ser Ala Ala Glu Leu Ala Phe Leu Ala Ala His Ser Lys Ala Glu Ile 130 135 140 Val Asp Met Leu Arg Lys His Thr Tyr Ala Asp Glu Leu Arg Gln Gly 145 150 155 160 Leu Arg Arg Gly Arg Gly Met Gly Ala Arg Ala Gln Pro Thr Pro Ser 165 170 175 Trp Ala Arg Glu Pro Leu Phe Glu Lys Ala Val Thr Pro Ser Asp Val 180 185 190 Gly Lys Leu Asn Arg Leu Val Val Pro Lys Gln His Ala Glu Lys His 195 200 205 Phe Pro Leu Arg Arg Ala Ala Ser Ser Asp Ser Ala Ser Ala Ala Ala 210 215 220 Thr Gly Lys Gly Val Leu Leu Asn Phe Glu Asp Gly Glu Gly Lys Val 225 230 235 240 Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu 245 250 255 Thr Lys Gly Trp Ser Arg Phe Val Arg Glu Lys Gly Leu Arg Ala Gly 260 265 270 Asp Thr Ile Val Phe Ser Pro Leu Gly Val Arg Pro Arg Gln Ala Ala 275 280 285 Leu His Arg Leu Gln Glu Glu Gln Arg Gly Gly Gly Gly Asp His His 290 295 300 Leu Arg Arg Arg Arg Glu Ala Asn His Asn His 305 310 315 21 1081 DNA Glycine max G3452 CLUSTER19062_7 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 21 caccaacaca aaatggatgg aggctgtgtc acagacgaaa ccaccacatc cagcgactct 60 ctttccgttc cgccgcccag ccgcgtcggc agcgttgcaa gcgccgtcgt cgaccccgac 120 ggttgttgcg tttccggcga ggccgaatcc cggaaactcc cttcgtcgaa atacaaaggc 180 gtggtgccgc aaccgaacgg tcgctgggga gctcagattt acgagaagca ccagcgcgtg 240 tggctcggca ctttcaacga ggaagacgaa gccgccagag cctacgacat cgccgcgctg 300 cgcttccgcg gccccgacgc cgtcaccaac ttcaagcctc ccgccgcctc cgacgacgcc 360 gagtccgagt tcctcaactc gcattccaag ttcgagatcg tcgacatgct ccgcaagcac 420 acctacgacg acgagctcca gcagagcacg cgcggtggta ggcgccgcct cgacgctgac 480 accgcgtcga gcggtgtgtt cgacgcgaaa gcgcgtgagc agctgttcga gaaaacggtt 540 acgccgagcg acgtcgggaa gctgaatcga ttagtgatac cgaagcagca cgcggagaag 600 cactttccgt taagcggatc cggcgacgaa agctcgccgt gcgtggcggg ggcttcggcg 660 gcgaagggaa tgttgttgaa ctttgaggac gttggaggga aagtgtggcg gtttcgttac 720 tcttattgga acagtagcca gagctacgtg cttaccaaag gatggagccg gttcgttaag 780 gagaagaatc ttcgagccgg tgacgcggtt cagttcttca agtcgaccgg accggaccgg 840 cagctatata tagactgcaa ggcgaggagt ggtgaggtta acaataatgc tggcggtttg 900 tttgttccga ttggaccggt cgttgagccg gttcagatgg ttcggctttt cggggtcaac 960 cttttgaaac tacccgtacc cggttcggat ggtgtaggga agagaaaaga gatggaactg 1020 tttgcatttg aatgttgcaa gaagttaaaa gtaattggag ctttgtaaca ttacatagtg 1080 c 1081 22 351 PRT Glycine max G3452 polypeptide CLUSTER19062_7 Orthologous to G867, G9, G993, G1930 22 Met Asp Gly Gly Cys Val Thr Asp Glu Thr Thr Thr Ser Ser Asp Ser 1 5 10 15 Leu Ser Val Pro Pro Pro Ser Arg Val Gly Ser Val Ala Ser Ala Val 20 25 30 Val Asp Pro Asp Gly Cys Cys Val Ser Gly Glu Ala Glu Ser Arg Lys 35 40 45 Leu Pro Ser Ser Lys Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg 50 55 60 Trp Gly Ala Gln Ile Tyr Glu Lys His Gln Arg Val Trp Leu Gly Thr 65 70 75 80 Phe Asn Glu Glu Asp Glu Ala Ala Arg Ala Tyr Asp Ile Ala Ala Leu 85 90 95 Arg Phe Arg Gly Pro Asp Ala Val Thr Asn Phe Lys Pro Pro Ala Ala 100 105 110 Ser Asp Asp Ala Glu Ser Glu Phe Leu Asn Ser His Ser Lys Phe Glu 115 120 125 Ile Val Asp Met Leu Arg Lys His Thr Tyr Asp Asp Glu Leu Gln Gln 130 135 140 Ser Thr Arg Gly Gly Arg Arg Arg Leu Asp Ala Asp Thr Ala Ser Ser 145 150 155 160 Gly Val Phe Asp Ala Lys Ala Arg Glu Gln Leu Phe Glu Lys Thr Val 165 170 175 Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln 180 185 190 His Ala Glu Lys His Phe Pro Leu Ser Gly Ser Gly Asp Glu Ser Ser 195 200 205 Pro Cys Val Ala Gly Ala Ser Ala Ala Lys Gly Met Leu Leu Asn Phe 210 215 220 Glu Asp Val Gly Gly Lys Val Trp Arg Phe Arg Tyr Ser Tyr Trp Asn 225 230 235 240 Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val Lys 245 250 255 Glu Lys Asn Leu Arg Ala Gly Asp Ala Val Gln Phe Phe Lys Ser Thr 260 265 270 Gly Pro Asp Arg Gln Leu Tyr Ile Asp Cys Lys Ala Arg Ser Gly Glu 275 280 285 Val Asn Asn Asn Ala Gly Gly Leu Phe Val Pro Ile Gly Pro Val Val 290 295 300 Glu Pro Val Gln Met Val Arg Leu Phe Gly Val Asn Leu Leu Lys Leu 305 310 315 320 Pro Val Pro Gly Ser Asp Gly Val Gly Lys Arg Lys Glu Met Glu Leu 325 330 335 Phe Ala Phe Glu Cys Cys Lys Lys Leu Lys Val Ile Gly Ala Leu 340 345 350 23 1089 DNA Glycine max G3453 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 23 atggatggag gcagtgtcac agacgaaacc accacaacca gcaactctct ttcggttccg 60 gcgaatctat ctccgccgcc tctcagcctt gacggaagcg gcgcaaccgc cgtcgtctac 120 cccgacggtt gttgcgtctc cggcgaagcc gaatcccgga aactcccgtc ctcgaaatac 180 aaaggcgtgg tgccgcaacc gaacggtcgt tggggagctc agatttacga gaagcaccag 240 cgcgtgtggc tcggcacctt caacgaggaa gacgaagccg tcagagccta cgacatcgtc 300 gcgcatcgct tccgcggccg cgacgccgtc actaacttca agcctctcgc cggcgccgac 360 gacgccgaag ccgagttcct cagcacgcat tccaagtccg agatcgtcga catgctccgc 420 aggcacacct acgacaacga gctccagcag agcacccgcg gcggcaggcg ccgccgggac 480 gccgaaaccg cgtcgagcgg cgcgttcgac gcgaaggcgc gtgagcagct ggtcgagaaa 540 accgttacgc cgagcgacgt cgggaagctg aaccgattag tgataccaaa gcagcacgcg 600 gagaagcact ttccgttaag cggatccggc ggcggagcct tgccgtgcat ggcggcggct 660 gcgggggcga aaggaatgtt gctgaacttt gaggacgttg gagggaaagt gtggcggttc 720 cgttactcgt attggaacag tagccagagc tacgtgctta ccaaaggatg gagccggttc 780 gttaaggaga agaatcttcg agctggtgac gcggttcagt tcttcaagtc gaccggactg 840 gaccggcaac tatatataga ctgcaaggcg aggagtggta aggttaacaa taatgctgcc 900 ggtttgttta ttccggttgg accggttgtt gagccggttc agatggtacg gcttttcggg 960 gtcgaccttt tgaaactacc cgtacccggt tcggatggta ttggggttgg ctgtgacggg 1020 aagagaaaag agatggagct gtttgcattt gaatgtagca agaagttaaa agtaattgga 1080 gctttgtaa 1089 24 362 PRT Glycine max G3453 polypeptide Orthologous to G867, G9, G993, G1930 24 Met Asp Gly Gly Ser Val Thr Asp Glu Thr Thr Thr Thr Ser Asn Ser 1 5 10 15 Leu Ser Val Pro Ala Asn Leu Ser Pro Pro Pro Leu Ser Leu Asp Gly 20 25 30 Ser Gly Ala Thr Ala Val Val Tyr Pro Asp Gly Cys Cys Val Ser Gly 35 40 45 Glu Ala Glu Ser Arg Lys Leu Pro Ser Ser Lys Tyr Lys Gly Val Val 50 55 60 Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys His Gln 65 70 75 80 Arg Val Trp Leu Gly Thr Phe Asn Glu Glu Asp Glu Ala Val Arg Ala 85 90 95 Tyr Asp Ile Val Ala His Arg Phe Arg Gly Arg Asp Ala Val Thr Asn 100 105 110 Phe Lys Pro Leu Ala Gly Ala Asp Asp Ala Glu Ala Glu Phe Leu Ser 115 120 125 Thr His Ser Lys Ser Glu Ile Val Asp Met Leu Arg Arg His Thr Tyr 130 135 140 Asp Asn Glu Leu Gln Gln Ser Thr Arg Gly Gly Arg Arg Arg Arg Asp 145 150 155 160 Ala Glu Thr Ala Ser Ser Gly Ala Phe Asp Ala Lys Ala Arg Glu Gln 165 170 175 Leu Val Glu Lys Thr Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg 180 185 190 Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro Leu Ser Gly 195 200 205 Ser Gly Gly Gly Ala Leu Pro Cys Met Ala Ala Ala Ala Gly Ala Lys 210 215 220 Gly Met Leu Leu Asn Phe Glu Asp Val Gly Gly Lys Val Trp Arg Phe 225 230 235 240 Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly 245 250 255 Trp Ser Arg Phe Val Lys Glu Lys Asn Leu Arg Ala Gly Asp Ala Val 260 265 270 Gln Phe Phe Lys Ser Thr Gly Leu Asp Arg Gln Leu Tyr Ile Asp Cys 275 280 285 Lys Ala Arg Ser Gly Lys Val Asn Asn Asn Ala Ala Gly Leu Phe Ile 290 295 300 Pro Val Gly Pro Val Val Glu Pro Val Gln Met Val Arg Leu Phe Gly 305 310 315 320 Val Asp Leu Leu Lys Leu Pro Val Pro Gly Ser Asp Gly Ile Gly Val 325 330 335 Gly Cys Asp Gly Lys Arg Lys Glu Met Glu Leu Phe Ala Phe Glu Cys 340 345 350 Ser Lys Lys Leu Lys Val Ile Gly Ala Leu 355 360 25 1188 DNA Glycine max G3454 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 25 atggatgcaa ttagttgcat ggatgagagc accaccaccg agtcactctc cataagtcag 60 gcgaagcctt cttcgacgat tatgtcgtcc gagaaggctt ctccttcccc gccgccgccg 120 aacaggctgt gccgcgtcgg tagcggtgct agcgcagtcg tggattccga cggcggcggc 180 gggggtggca gcaccgaggt ggagtcgcgg aagctcccct cgtccaagta taagggcgtc 240 gtgccccagc ccaacggccg ctggggctcg cagatttacg agaagcacca gcgcgtgtgg 300 ctgggaacgt tcaacgagga agacgaggcg gcgcgtgcgt acgacgtcgc cgtgcagcga 360 ttccgcggca aggactccgt cacgaacttc aagccgctcg ccggcgccga cgacgacgac 420 ggagaatcgg agtttctcaa ctcgcattcc aaacccgaga tcgtcgacat gctgcgaaag 480 cacacgtaca atgacgagct ggagcatagc aagcgcaacc gcggcgtcgt ccggcggcga 540 ggctccgccg ccgtcggcac cgcagactca atttccggcg cgtgctttac taatgcacgt 600 gagcagctat tcgagaaagc tgttacgccg agcgacgttt ggaaattgaa ccgtttggtg 660 ataccgaagc agcacgcgga gaagcacttt ccgttacaga gctctaacgg cgttagcgcg 720 acgacgatag cggcggtgac ggcgacgccg acggcggcga agggcgtttt gttgaacttc 780 gaagacgttg gagggaaagt gtggcggttt cgttactcgt attggaacag tagccagagt 840 tacgtcttaa ccaaaggttg gagccggttc gttaaggaga agaatctgaa agctggtgac 900 acggtttgtt ttcaccggtc cactggaccg gacaagcagc tttacatcga ttggaagacg 960 aggaatgttg ttaacaacga ggtcgcgttg ttcggaccgg tcggaccggt tgtcgaaccg 1020 atccagatgg ttcggctctt tggggttaac attttgaaac tacccggttc agatactatt 1080 gttggcaata acaataatgc aagtgggtgc tgcaatggca agagaagaga aatggaactg 1140 ttctcgttag agtgtagcaa gaaacctaag attattggtg ctttgtaa 1188 26 395 PRT Glycine max G3454 polypeptide Orthologous to G867, G9, G993, G1930 26 Met Asp Ala Ile Ser Cys Met Asp Glu Ser Thr Thr Thr Glu Ser Leu 1 5 10 15 Ser Ile Ser Gln Ala Lys Pro Ser Ser Thr Ile Met Ser Ser Glu Lys 20 25 30 Ala Ser Pro Ser Pro Pro Pro Pro Asn Arg Leu Cys Arg Val Gly Ser 35 40 45 Gly Ala Ser Ala Val Val Asp Ser Asp Gly Gly Gly Gly Gly Gly Ser 50 55 60 Thr Glu Val Glu Ser Arg Lys Leu Pro Ser Ser Lys Tyr Lys Gly Val 65 70 75 80 Val Pro Gln Pro Asn Gly Arg Trp Gly Ser Gln Ile Tyr Glu Lys His 85 90 95 Gln Arg Val Trp Leu Gly Thr Phe Asn Glu Glu Asp Glu Ala Ala Arg 100 105 110 Ala Tyr Asp Val Ala Val Gln Arg Phe Arg Gly Lys Asp Ser Val Thr 115 120 125 Asn Phe Lys Pro Leu Ala Gly Ala Asp Asp Asp Asp Gly Glu Ser Glu 130 135 140 Phe Leu Asn Ser His Ser Lys Pro Glu Ile Val Asp Met Leu Arg Lys 145 150 155 160 His Thr Tyr Asn Asp Glu Leu Glu His Ser Lys Arg Asn Arg Gly Val 165 170 175 Val Arg Arg Arg Gly Ser Ala Ala Val Gly Thr Ala Asp Ser Ile Ser 180 185 190 Gly Ala Cys Phe Thr Asn Ala Arg Glu Gln Leu Phe Glu Lys Ala Val 195 200 205 Thr Pro Ser Asp Val Trp Lys Leu Asn Arg Leu Val Ile Pro Lys Gln 210 215 220 His Ala Glu Lys His Phe Pro Leu Gln Ser Ser Asn Gly Val Ser Ala 225 230 235 240 Thr Thr Ile Ala Ala Val Thr Ala Thr Pro Thr Ala Ala Lys Gly Val 245 250 255 Leu Leu Asn Phe Glu Asp Val Gly Gly Lys Val Trp Arg Phe Arg Tyr 260 265 270 Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser 275 280 285 Arg Phe Val Lys Glu Lys Asn Leu Lys Ala Gly Asp Thr Val Cys Phe 290 295 300 His Arg Ser Thr Gly Pro Asp Lys Gln Leu Tyr Ile Asp Trp Lys Thr 305 310 315 320 Arg Asn Val Val Asn Asn Glu Val Ala Leu Phe Gly Pro Val Gly Pro 325 330 335 Val Val Glu Pro Ile Gln Met Val Arg Leu Phe Gly Val Asn Ile Leu 340 345 350 Lys Leu Pro Gly Ser Asp Thr Ile Val Gly Asn Asn Asn Asn Ala Ser 355 360 365 Gly Cys Cys Asn Gly Lys Arg Arg Glu Met Glu Leu Phe Ser Leu Glu 370 375 380 Cys Ser Lys Lys Pro Lys Ile Ile Gly Ala Leu 385 390 395 27 1160 DNA Glycine max G3455 GLYMA-28NOV01-CLUSTER19062_5 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 27 atggatgcaa ttagttgcct ggatgagagc accaccaccg agtcactctc cataagtcag 60 gcgaagcctt cttcgacgat tatgtcgtcc gagaaggctt ctccttcccc gccgccgccg 120 aacaggctgt gccgcgtcgg tagcggtgct agcgcagtcg tggattccga cggcggcggc 180 gggggtggca gcaccgaggt ggagtcgcgg aagctcccct cgtccaagta taagggcgtc 240 gtgccccagc ccaacggccg ctggggctcg cagatttacg agaagcacca gcgcgtgtgg 300 ctgggaacgt tcaacgagga agacgaggcg gcgcgtgcgt acgacgtcgc cgtgcagcga 360 ttccgcggca aggacgccgt cacaaacttc aagccgctct ccggcaccga cgacgacgac 420 ggggaatcgg agtttctcaa ctcgcattcg aaatccgaga tcgtcgacat gctgcgtaag 480 catacgtaca atgacgagct ggaacaaagc aagcgcagcc gcggctttcg tacgttcgcc 540 gcggctccgc cggccggcgc cggaaacgga aactcaatct ccggcgcgtg tgttatgaag 600 gcgcgtgagc agctattcca gaaggccgtt acgccgagcg acgttgggaa actgaaccgt 660 ttggtgatac cgaagcagca cgcggagaag cactttcctt tacagagcgc tgctaacggc 720 gtttagcgcg acggcgaacg gcggcgaagg gcgttttgtt gaacttcgaa gacgttggag 780 ggaaagtgtg gcggtttcgt tactcgtatt ggaacagtag ccagagttac gtcttgacca 840 aaggttggag ccggttcgtt aaggagaaga atctgaaagc cggtgacacg gtttgttttc 900 aacggtccac tggaccggac aggcagcttt acatcgattg gaagacgagg aatgttgtta 960 acgaggtcgc gttgttcgga ccggttgtcg aaccgatcca gatggttcgg ctctttggtg 1020 ttaacatttt gaaactaccc ggttcagatt ctatcgccaa taacaataat gcaagtgggt 1080 gctgcaatgg caagagaaga gaaatggaac tcttttcatt agagtgtagc aagaaaccta 1140 agattattgg tgctttgtag 1160 28 241 PRT Glycine max G3455 polypeptide GLYMA-28NOV01-CLUSTER19062_5 Orthologous to G867, G9, G993, G1930 28 Met Asp Ala Ile Ser Cys Leu Asp Glu Ser Thr Thr Thr Glu Ser Leu 1 5 10 15 Ser Ile Ser Gln Ala Lys Pro Ser Ser Thr Ile Met Ser Ser Glu Lys 20 25 30 Ala Ser Pro Ser Pro Pro Pro Pro Asn Arg Leu Cys Arg Val Gly Ser 35 40 45 Gly Ala Ser Ala Val Val Asp Ser Asp Gly Gly Gly Gly Gly Gly Ser 50 55 60 Thr Glu Val Glu Ser Arg Lys Leu Pro Ser Ser Lys Tyr Lys Gly Val 65 70 75 80 Val Pro Gln Pro Asn Gly Arg Trp Gly Ser Gln Ile Tyr Glu Lys His 85 90 95 Gln Arg Val Trp Leu Gly Thr Phe Asn Glu Glu Asp Glu Ala Ala Arg 100 105 110 Ala Tyr Asp Val Ala Val Gln Arg Phe Arg Gly Lys Asp Ala Val Thr 115 120 125 Asn Phe Lys Pro Leu Ser Gly Thr Asp Asp Asp Asp Gly Glu Ser Glu 130 135 140 Phe Leu Asn Ser His Ser Lys Ser Glu Ile Val Asp Met Leu Arg Lys 145 150 155 160 His Thr Tyr Asn Asp Glu Leu Glu Gln Ser Lys Arg Ser Arg Gly Phe 165 170 175 Arg Thr Phe Ala Ala Ala Pro Pro Ala Gly Ala Gly Asn Gly Asn Ser 180 185 190 Ile Ser Gly Ala Cys Val Met Lys Ala Arg Glu Gln Leu Phe Gln Lys 195 200 205 Ala Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro 210 215 220 Lys Gln His Ala Glu Lys His Phe Pro Leu Gln Ser Ala Ala Asn Gly 225 230 235 240 Val 29 1291 DNA Oryza sativa G3388 AP002913b GI12328560 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 29 ctagacactg ccctaattac aacccatttg cttatctctc tcctctctct ctctctctcg 60 ctgcagccat agcttagcta gagctagagc tttcttggtg ccgagatggg ggtggtcagc 120 ttctcctcga cttcctccgg cgcgtccacg gccaccaccg agtccggcgg cgccgtgcgg 180 atgtcgccgg agccggtggt ggcggtggcg gcggcggctc aacagctacc ggtggtgaag 240 ggagttgact cggcggatga ggtggtgacg tcgaggccgg cggcggcggc ggcgcagcag 300 tcgtcgcggt acaagggggt ggtgccgcag ccgaacggga ggtggggggc gcagatctac 360 gagcggcacg cgcgggtgtg gctcgggacg ttccccgacg aggaggcggc ggcgcgggcc 420 tacgacgtgg cggcgctccg gtaccggggg cgcgacgcgg ccaccaactt ccccggggcc 480 gcggcgtcgg ccgccgagct cgcgttcctc gccgcgcact ccaaggccga gatcgtcgac 540 atgctccgga agcacaccta cgccgacgag ctccgccagg ggctccgccg cggccgcggc 600 atgggcgccc gcgcccagcc cacgccgtcg tgggcgcgcg agccgctgtt cgagaaggcc 660 gtgacgccca gcgacgtcgg caagctcaac cgcctcgtgg tgcccaagca gcacgccgag 720 aagcacttcc cgctccgccg cgcggcgagc tccgactccg cctccgccgc cgccaccggc 780 aagggcgtgc tcctcaactt cgaggacggc gaggggaagg tgtggcgatt ccggtactcg 840 tactggaaca gcagccagag ctacgtgctg accaaggggt ggagccgatt cgtgagggag 900 aagggcctcc gcgccggcga caccatagtc ttctcccgct cggcgtacgg ccccgacaag 960 ctgctcttca tcgactgcaa gaagaacaac gcggcggcgg cgaccaccac ctgcgccggc 1020 gacgagaggc caaccacaag cggcgccgaa ccacgcgtcg tgaggctctt cggcgtcgac 1080 atcgccggcg gcgattgccg gaagcgggag agggcggtgg agatggggca agaggtcttc 1140 ctactgaaga ggcaatgcgt ggttcatcag cgtactcctg ccctaggtgc cctgctgtta 1200 tagcatcaaa tcaaattcat atatagatca aatcaaatct tcttctcttc catctttttt 1260 gttgttcatc gtctgttgtt tcatcttcga g 1291 30 365 PRT Oryza sativa G3388 polypeptide OSC21673.C1.p5.fg GI12328560 Orthologous to G867, G9, G993, G1930 30 Met Gly Val Val Ser Phe Ser Ser Thr Ser Ser Gly Ala Ser Thr Ala 1 5 10 15 Thr Thr Glu Ser Gly Gly Ala Val Arg Met Ser Pro Glu Pro Val Val 20 25 30 Ala Val Ala Ala Ala Ala Gln Gln Leu Pro Val Val Lys Gly Val Asp 35 40 45 Ser Ala Asp Glu Val Val Thr Ser Arg Pro Ala Ala Ala Ala Ala Gln 50 55 60 Gln Ser Ser Arg Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp 65 70 75 80 Gly Ala Gln Ile Tyr Glu Arg His Ala Arg Val Trp Leu Gly Thr Phe 85 90 95 Pro Asp Glu Glu Ala Ala Ala Arg Ala Tyr Asp Val Ala Ala Leu Arg 100 105 110 Tyr Arg Gly Arg Asp Ala Ala Thr Asn Phe Pro Gly Ala Ala Ala Ser 115 120 125 Ala Ala Glu Leu Ala Phe Leu Ala Ala His Ser Lys Ala Glu Ile Val 130 135 140 Asp Met Leu Arg Lys His Thr Tyr Ala Asp Glu Leu Arg Gln Gly Leu 145 150 155 160 Arg Arg Gly Arg Gly Met Gly Ala Arg Ala Gln Pro Thr Pro Ser Trp 165 170 175 Ala Arg Glu Pro Leu Phe Glu Lys Ala Val Thr Pro Ser Asp Val Gly 180 185 190 Lys Leu Asn Arg Leu Val Val Pro Lys Gln His Ala Glu Lys His Phe 195 200 205 Pro Leu Arg Arg Ala Ala Ser Ser Asp Ser Ala Ser Ala Ala Ala Thr 210 215 220 Gly Lys Gly Val Leu Leu Asn Phe Glu Asp Gly Glu Gly Lys Val Trp 225 230 235 240 Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr 245 250 255 Lys Gly Trp Ser Arg Phe Val Arg Glu Lys Gly Leu Arg Ala Gly Asp 260 265 270 Thr Ile Val Phe Ser Arg Ser Ala Tyr Gly Pro Asp Lys Leu Leu Phe 275 280 285 Ile Asp Cys Lys Lys Asn Asn Ala Ala Ala Ala Thr Thr Thr Cys Ala 290 295 300 Gly Asp Glu Arg Pro Thr Thr Ser Gly Ala Glu Pro Arg Val Val Arg 305 310 315 320 Leu Phe Gly Val Asp Ile Ala Gly Gly Asp Cys Arg Lys Arg Glu Arg 325 330 335 Ala Val Glu Met Gly Gln Glu Val Phe Leu Leu Lys Arg Gln Cys Val 340 345 350 Val His Gln Arg Thr Pro Ala Leu Gly Ala Leu Leu Leu 355 360 365 31 1068 DNA Oryza sativa G3389 AP002913 OSC21674.C1.p12.fg Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 31 gctaggctgt tctctctttc cattcgtcaa gaactaccac gacgtcgaca tcataatcgt 60 cagagagctc gagaaatttt ttagtatctt tgatccagat cgatcatgga gcaagaagct 120 gccatggtcg tcttctcctg caactccggc tccggtgggt cgtcgtcgac gaccgattca 180 aagcaagagg aggaggagga ggaggagttg gccgcaatgg aggaagacga gttgatccac 240 gtcgtccagg cggcggagct gcggctgccg tcgtcgacga cggcgacgcg gccgtcgtcg 300 cggtacaagg gggtggtgcc gcagccgaac gggcggtggg gggcgcagat ctacgagcgg 360 cacgcgcggg tgtggctcgg gacgttcccc gacgaggagg cggcggcgcg cgcctacgac 420 gtggcggcgc tccgcttccg ggggcgcgac gccgtcacca accgcgcccc ggcggcggag 480 ggcgcgtccg ccggcgagct cgcgttcctg gccgcgcact ccaaggcgga ggtcgtggac 540 atgctgcgga agcacaccta cgacgacgag ctccagcagg gcctccgccg cggctcgcgc 600 gcgcagccga cgccgcggtg ggcgcgcgag ccgctgttcg agaaggccgt gacgccgagc 660 gacgtcggca agctcaaccg cctcgtggtg cccaagcagc aggccgagag gcatttcccg 720 ttcccgctcc gccgccacag ctccgacgcc gccggcaagg gcgtgctcct caacttcgag 780 gacggcgacg gcaaggtgtg gcgattccgg tactcgtact ggaacagcag ccagagttac 840 gtgctcacca aggggtggag ccgattcgtg agggagaagg gcctccgacc aggcgacacc 900 gtggccttct cccggtcggc ggcggcgtgg gggacggaga agcacctcct catcgactgc 960 aagaagatgg agaggaacaa cctggcaacc gtcgacgacg atgcccgtgt cgtcgtcaag 1020 ctgttcggcg ttgacatcgc cggagacaag acgaggtaac acgcaagc 1068 32 317 PRT Oryza sativa G3389 polypeptide BAB21211.1 OSC21674.C1.p12.fg Orthologous to G867, G9, G993, G1930 32 Met Glu Gln Glu Ala Ala Met Val Val Phe Ser Cys Asn Ser Gly Ser 1 5 10 15 Gly Gly Ser Ser Ser Thr Thr Asp Ser Lys Gln Glu Glu Glu Glu Glu 20 25 30 Glu Glu Leu Ala Ala Met Glu Glu Asp Glu Leu Ile His Val Val Gln 35 40 45 Ala Ala Glu Leu Arg Leu Pro Ser Ser Thr Thr Ala Thr Arg Pro Ser 50 55 60 Ser Arg Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala 65 70 75 80 Gln Ile Tyr Glu Arg His Ala Arg Val Trp Leu Gly Thr Phe Pro Asp 85 90 95 Glu Glu Ala Ala Ala Arg Ala Tyr Asp Val Ala Ala Leu Arg Phe Arg 100 105 110 Gly Arg Asp Ala Val Thr Asn Arg Ala Pro Ala Ala Glu Gly Ala Ser 115 120 125 Ala Gly Glu Leu Ala Phe Leu Ala Ala His Ser Lys Ala Glu Val Val 130 135 140 Asp Met Leu Arg Lys His Thr Tyr Asp Asp Glu Leu Gln Gln Gly Leu 145 150 155 160 Arg Arg Gly Ser Arg Ala Gln Pro Thr Pro Arg Trp Ala Arg Glu Pro 165 170 175 Leu Phe Glu Lys Ala Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg 180 185 190 Leu Val Val Pro Lys Gln Gln Ala Glu Arg His Phe Pro Phe Pro Leu 195 200 205 Arg Arg His Ser Ser Asp Ala Ala Gly Lys Gly Val Leu Leu Asn Phe 210 215 220 Glu Asp Gly Asp Gly Lys Val Trp Arg Phe Arg Tyr Ser Tyr Trp Asn 225 230 235 240 Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val Arg 245 250 255 Glu Lys Gly Leu Arg Pro Gly Asp Thr Val Ala Phe Ser Arg Ser Ala 260 265 270 Ala Ala Trp Gly Thr Glu Lys His Leu Leu Ile Asp Cys Lys Lys Met 275 280 285 Glu Arg Asn Asn Leu Ala Thr Val Asp Asp Asp Ala Arg Val Val Val 290 295 300 Lys Leu Phe Gly Val Asp Ile Ala Gly Asp Lys Thr Arg 305 310 315 33 1348 DNA Oryza sativa G3390 AC130725 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 33 cttcagaggc ttcacctttc atcagcttag ctagctagct gctcgatccg gcggcgtgat 60 cgatcgatct ctctgattct atcaggtgtt cgaccagatt ccatcgatgg acagcacgag 120 ctgtctcttg gacgacgcga gcagcggcgc gtccacgggc aagaaggcgg cggcggcggc 180 ggcgtcgaag gcgctgcagc gcgtgggcag cggcgccagc gcggtgatgg acgcggccga 240 gcctggcgcc gaggcggact cgggcggcga gcggcgcggc ggcggcggcg ggaagctgcc 300 gtcgtccaag tacaagggcg tggtgccgca accgaacggg cggtggggcg cgcagatata 360 cgagcggcac cagcgggtgt ggctcggcac gttcaccggc gaggcggagg cggcgcgcgc 420 ctacgacgtg gcggcgcagc ggttccgcgg ccgcgacgcc gtcaccaact tccgcccgct 480 cgccgagtcc gacccggagg ccgccgtcga gctccgcttc ctcgcgtccc gctccaaggc 540 cgaggtcgtc gacatgctcc gcaagcacac ctacctcgag gagctcacgc agaacaagcg 600 cgccttcgcc gccatctccc cgccgccccc caagcacccc gcctcctctc cgacgtcctc 660 ctccgccgcg cgcgagcacc tgttcgacaa gacggtgacg cccagcgacg tcgggaagct 720 gaaccggctg gtgatcccca agcagcacgc cgagaagcac ttcccgctcc agctccctcc 780 ccctaccaca acctcctccg tcgccgccgc cgccgacgcc gccgccggcg gcggcgattg 840 caagggcgtc ctcctcaact tcgaggacgc cgccgggaag gtgtggaaat tccggtactc 900 ctactggaac agcagccaga gctacgtgct caccaagggg tggagccgct tcgtcaagga 960 gaaggggctc cacgccggcg acgccgtcgg cttctaccgc gccgccggta agaacgcgca 1020 gctcttcatc gactgcaagg tccgggcaaa acccaccacc gccgccgccg ccgccgcctt 1080 cctcagcgcg gtggccgccg ccgccgcgcc gccacccgcc gtgaaggcta tcaggctgtt 1140 cggtgtcgac ctgctcacgg cggcggcgcc ggagctgcag gacgccggcg gcgccgccat 1200 gaccaagagc aagagagcca tggacgccat ggctgagtca caagcacacg tggtttttaa 1260 gaagcaatgc atagagcttg cgctaaccta gctagcacgc tgatgcagct agcgtttttt 1320 ttgctcattc gcttgcttgc ttaattat 1348 34 394 PRT Oryza sativa G3390 polypeptide Orthologous to G867, G9, G993, G1930 34 Met Asp Ser Thr Ser Cys Leu Leu Asp Asp Ala Ser Ser Gly Ala Ser 1 5 10 15 Thr Gly Lys Lys Ala Ala Ala Ala Ala Ala Ser Lys Ala Leu Gln Arg 20 25 30 Val Gly Ser Gly Ala Ser Ala Val Met Asp Ala Ala Glu Pro Gly Ala 35 40 45 Glu Ala Asp Ser Gly Gly Glu Arg Arg Gly Gly Gly Gly Gly Lys Leu 50 55 60 Pro Ser Ser Lys Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp 65 70 75 80 Gly Ala Gln Ile Tyr Glu Arg His Gln Arg Val Trp Leu Gly Thr Phe 85 90 95 Thr Gly Glu Ala Glu Ala Ala Arg Ala Tyr Asp Val Ala Ala Gln Arg 100 105 110 Phe Arg Gly Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Ala Glu Ser 115 120 125 Asp Pro Glu Ala Ala Val Glu Leu Arg Phe Leu Ala Ser Arg Ser Lys 130 135 140 Ala Glu Val Val Asp Met Leu Arg Lys His Thr Tyr Leu Glu Glu Leu 145 150 155 160 Thr Gln Asn Lys Arg Ala Phe Ala Ala Ile Ser Pro Pro Pro Pro Lys 165 170 175 His Pro Ala Ser Ser Pro Thr Ser Ser Ser Ala Ala Arg Glu His Leu 180 185 190 Phe Asp Lys Thr Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu 195 200 205 Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro Leu Gln Leu Pro 210 215 220 Pro Pro Thr Thr Thr Ser Ser Val Ala Ala Ala Ala Asp Ala Ala Ala 225 230 235 240 Gly Gly Gly Asp Cys Lys Gly Val Leu Leu Asn Phe Glu Asp Ala Ala 245 250 255 Gly Lys Val Trp Lys Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser 260 265 270 Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Gly Leu 275 280 285 His Ala Gly Asp Ala Val Gly Phe Tyr Arg Ala Ala Gly Lys Asn Ala 290 295 300 Gln Leu Phe Ile Asp Cys Lys Val Arg Ala Lys Pro Thr Thr Ala Ala 305 310 315 320 Ala Ala Ala Ala Phe Leu Ser Ala Val Ala Ala Ala Ala Ala Pro Pro 325 330 335 Pro Ala Val Lys Ala Ile Arg Leu Phe Gly Val Asp Leu Leu Thr Ala 340 345 350 Ala Ala Pro Glu Leu Gln Asp Ala Gly Gly Ala Ala Met Thr Lys Ser 355 360 365 Lys Arg Ala Met Asp Ala Met Ala Glu Ser Gln Ala His Val Val Phe 370 375 380 Lys Lys Gln Cys Ile Glu Leu Ala Leu Thr 385 390 35 1338 DNA Oryza sativa G3391 AP003450 OSC26104.C1.p13.fg Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 35 ggagagtagg agtgtgctag tgtgtgaggt ctactgaaat ggacagctcc agctgcctgg 60 tggatgatac caacagcggc ggctcgtcca cggacaagct gagggcgttg gccgccgcgg 120 cggcggagac ggcgccgctg gagcgcatgg ggagcggggc gagcgcggtg gtggacgcgg 180 ccgagcctgg cgcggaggcg gactccgggt ccgggggacg tgtgtgcggc ggcggcggcg 240 gcggtgccgg cggtgcggga gggaagctgc cgtcgtccaa gttcaagggc gtcgtgccgc 300 agcccaacgg gaggtggggc gcgcagatct acgagcggca ccagcgggtg tggctcggca 360 cgttcgccgg ggaggacgac gccgcgcgcg cctacgacgt cgccgcgcag cgcttccgcg 420 gccgcgacgc cgtcaccaac ttccgcccgc tcgccgaggc cgacccggac gccgccgccg 480 agcttcgctt cctcgccacg cgctccaagg ccgaggtcgt cgacatgctc cgcaagcaca 540 cctacttcga cgagctcgcg cagagcaagc gcaccttcgc cgcctccacg ccgtcggccg 600 cgaccaccac cgcctccctc tccaacggcc acctctcgtc gccccgctcc cccttcgcgc 660 ccgccgcggc gcgcgaccac ctgttcgaca agacggtcac cccgagcgac gtgggcaagc 720 tgaacaggct cgtcataccg aagcagcacg ccgagaagca cttcccgcta cagctcccgt 780 ccgccggcgg cgagagcaag ggtgtcctcc tcaacttcga ggacgccgcc ggcaaggtgt 840 ggcggttccg gtactcgtac tggaacagca gccagagcta cgtgctaacc aagggctgga 900 gccgcttcgt caaggagaag ggtctccacg ccgacggcaa gctcttcatc gactgcaagt 960 tagtacggtc gaccggcgcc gccctcgcgt cgcccgctga tcagccagcg ccgtcgccgg 1020 tgaaggccgt caggctcttc ggcgtggacc tgctcacggc gccggcgccg gtcgaacaga 1080 tggccgggtg caagagagcc agggacttgg cggcgacgac gcctccacaa gcggcggcgt 1140 tcaagaagca atgcatagag ctggcactag tatagagtta gcactattag ctcgatcttc 1200 tctagctagt gtcttttttg ctcccatgca tcataattca ggtggtagct agcttagtcc 1260 cttgttgatc ctatctacta atctcacttg gttttttttg ttaatttatt cgcccatgtt 1320 cttgcttgct ttgctgta 1338 36 378 PRT Oryza sativa G3391 polypeptide AP003450 OSC26104.C1.p13.fg Orthologous to G867, G9, G993, G1930 36 Met Asp Ser Ser Ser Cys Leu Val Asp Asp Thr Asn Ser Gly Gly Ser 1 5 10 15 Ser Thr Asp Lys Leu Arg Ala Leu Ala Ala Ala Ala Ala Glu Thr Ala 20 25 30 Pro Leu Glu Arg Met Gly Ser Gly Ala Ser Ala Val Val Asp Ala Ala 35 40 45 Glu Pro Gly Ala Glu Ala Asp Ser Gly Ser Gly Gly Arg Val Cys Gly 50 55 60 Gly Gly Gly Gly Gly Ala Gly Gly Ala Gly Gly Lys Leu Pro Ser Ser 65 70 75 80 Lys Phe Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln 85 90 95 Ile Tyr Glu Arg His Gln Arg Val Trp Leu Gly Thr Phe Ala Gly Glu 100 105 110 Asp Asp Ala Ala Arg Ala Tyr Asp Val Ala Ala Gln Arg Phe Arg Gly 115 120 125 Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Ala Glu Ala Asp Pro Asp 130 135 140 Ala Ala Ala Glu Leu Arg Phe Leu Ala Thr Arg Ser Lys Ala Glu Val 145 150 155 160 Val Asp Met Leu Arg Lys His Thr Tyr Phe Asp Glu Leu Ala Gln Ser 165 170 175 Lys Arg Thr Phe Ala Ala Ser Thr Pro Ser Ala Ala Thr Thr Thr Ala 180 185 190 Ser Leu Ser Asn Gly His Leu Ser Ser Pro Arg Ser Pro Phe Ala Pro 195 200 205 Ala Ala Ala Arg Asp His Leu Phe Asp Lys Thr Val Thr Pro Ser Asp 210 215 220 Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys 225 230 235 240 His Phe Pro Leu Gln Leu Pro Ser Ala Gly Gly Glu Ser Lys Gly Val 245 250 255 Leu Leu Asn Phe Glu Asp Ala Ala Gly Lys Val Trp Arg Phe Arg Tyr 260 265 270 Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser 275 280 285 Arg Phe Val Lys Glu Lys Gly Leu His Ala Asp Gly Lys Leu Phe Ile 290 295 300 Asp Cys Lys Leu Val Arg Ser Thr Gly Ala Ala Leu Ala Ser Pro Ala 305 310 315 320 Asp Gln Pro Ala Pro Ser Pro Val Lys Ala Val Arg Leu Phe Gly Val 325 330 335 Asp Leu Leu Thr Ala Pro Ala Pro Val Glu Gln Met Ala Gly Cys Lys 340 345 350 Arg Ala Arg Asp Leu Ala Ala Thr Thr Pro Pro Gln Ala Ala Ala Phe 355 360 365 Lys Lys Gln Cys Ile Glu Leu Ala Leu Val 370 375 37 1290 DNA Zea mays G3432 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 37 ctatagctag cactagcagt ggtgcacact gaaatggaca gcgccagcag cctcgtggac 60 gacaccagta gcggtggcgg cggcggcgcg tccacggaca agctaagggc tctggccgtc 120 ttcgccgccg cctcggggac gccgctggag cgcatgggca gcggcgccag cgcggtcgtg 180 gacgcggccg agccgggcgc cgaggcggac tccggttccg gtgccgccgc ggtgagcgtc 240 ggcgggaagc tgccgtcgtc aaggtacaag ggcgtggtgc cgcagcccaa cgggcggtgg 300 ggcgcgcaga tctacgagcg ccaccagcgc gtgtggctcg gcaccttcgc gggcgaggcc 360 gacgcggcgc gcgcctacga cgtcgcggcg cagcggttcc gcggccgcga cgcggtcacc 420 aacttccgcc cgctcgcgga cgccgacccg gacgccgccg ccgagctccg gttcctggcg 480 tcccgctcca aggccgaggt cgtcgacatg ctccgcaagc acacctactt cgacgagctc 540 gcgcagaaca agcgcgcctt cgccgccgcg tccgcgtccg cggccaccgc ctcgtcgctg 600 gccaacaacc cttcttccta cgcgtcgctc tcccccgcga ccgcgacggc cgccgcgcgg 660 gagcacctct tcgacaagac ggtcaccccc agcgacgtgg gcaagctgaa ccggctggtg 720 atcccgaagc agcacgccga gaagcacttc ccgctgcagc tcccatccgc cggcggcgag 780 agcaagggcg tgctcctcaa cctggaggac gccgcgggca aggtgtggcg gttccgctac 840 tcgtactgga acagcagcca gagctacgtg ctcaccaagg gctggagccg cttcgtcaag 900 gagaagggcc tccaagccgg cgacgtcgtc ggcttctacc gctccgctgc cggcgccgac 960 accaagctct tcatcgactg caagctgcgg cccaacagcg tcgtcgtcgc ctcgacggca 1020 ggcccgtcgc ctccggcgcc ggtggcgaag gccgtgcgtc tcttcggcgt cgacctgctg 1080 acggcaccgg ccaccgccgc ggcgccggcg gaggccgtgg ccgggtgcaa gagagccagg 1140 gacttgggtt cgcccccgca ggcggcgttc aagaagcagc tcgtggagct ggcactagtg 1200 tagattaatg ctacggagcg atcgatcttt ccctggctag ctagtctttt ttttttttgc 1260 tcgatcgctc aactcagatg gtagcatcat 1290 38 389 PRT Zea mays G3432 polypeptide Orthologous to G867, G9, G993, G1930 38 Met Asp Ser Ala Ser Ser Leu Val Asp Asp Thr Ser Ser Gly Gly Gly 1 5 10 15 Gly Gly Ala Ser Thr Asp Lys Leu Arg Ala Leu Ala Val Phe Ala Ala 20 25 30 Ala Ser Gly Thr Pro Leu Glu Arg Met Gly Ser Gly Ala Ser Ala Val 35 40 45 Val Asp Ala Ala Glu Pro Gly Ala Glu Ala Asp Ser Gly Ser Gly Ala 50 55 60 Ala Ala Val Ser Val Gly Gly Lys Leu Pro Ser Ser Arg Tyr Lys Gly 65 70 75 80 Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile Tyr Glu Arg 85 90 95 His Gln Arg Val Trp Leu Gly Thr Phe Ala Gly Glu Ala Asp Ala Ala 100 105 110 Arg Ala Tyr Asp Val Ala Ala Gln Arg Phe Arg Gly Arg Asp Ala Val 115 120 125 Thr Asn Phe Arg Pro Leu Ala Asp Ala Asp Pro Asp Ala Ala Ala Glu 130 135 140 Leu Arg Phe Leu Ala Ser Arg Ser Lys Ala Glu Val Val Asp Met Leu 145 150 155 160 Arg Lys His Thr Tyr Phe Asp Glu Leu Ala Gln Asn Lys Arg Ala Phe 165 170 175 Ala Ala Ala Ser Ala Ser Ala Ala Thr Ala Ser Ser Leu Ala Asn Asn 180 185 190 Pro Ser Ser Tyr Ala Ser Leu Ser Pro Ala Thr Ala Thr Ala Ala Ala 195 200 205 Arg Glu His Leu Phe Asp Lys Thr Val Thr Pro Ser Asp Val Gly Lys 210 215 220 Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro 225 230 235 240 Leu Gln Leu Pro Ser Ala Gly Gly Glu Ser Lys Gly Val Leu Leu Asn 245 250 255 Leu Glu Asp Ala Ala Gly Lys Val Trp Arg Phe Arg Tyr Ser Tyr Trp 260 265 270 Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val 275 280 285 Lys Glu Lys Gly Leu Gln Ala Gly Asp Val Val Gly Phe Tyr Arg Ser 290 295 300 Ala Ala Gly Ala Asp Thr Lys Leu Phe Ile Asp Cys Lys Leu Arg Pro 305 310 315 320 Asn Ser Val Val Val Ala Ser Thr Ala Gly Pro Ser Pro Pro Ala Pro 325 330 335 Val Ala Lys Ala Val Arg Leu Phe Gly Val Asp Leu Leu Thr Ala Pro 340 345 350 Ala Thr Ala Ala Ala Pro Ala Glu Ala Val Ala Gly Cys Lys Arg Ala 355 360 365 Arg Asp Leu Gly Ser Pro Pro Gln Ala Ala Phe Lys Lys Gln Leu Val 370 375 380 Glu Leu Ala Leu Val 385 39 1200 DNA Zea mays G3433 Predicted polypeptide sequence is orthologous to G867, G9, G993, G1930 39 atggacagcg ccagcagcct cgtggacgac accagcggca gcggcggcgg cgcgtgcacg 60 gacaagctaa gggctttggc cgccgccgcc gcctccgcct cggggccacc gccggagcgc 120 atgggcagcg gagccagcgc ggtcgtggac gcggccgagc cgggcgccga ggcggactcc 180 ggctccgccc cggcctccgt cgccgccgtc gcggcgggcg tgggcgggaa gctgccgtcg 240 tccaggtaca agggcgtggt gccgcagccc aacgggcggt ggggcgcgca gatctacgag 300 cgccacctgc gcgtgtggct cggcaccttc acgggcgagg ccgaggccgc gcgcgcctac 360 gacgtggccg cgcagcggtt ccgggggcgc gacgccgtca ccaacttccg cccgctcgcg 420 gagtcggact tggacccgga cgccgccgcc gagctccggt tcctcgcgtc ccggtccaag 480 gccgaggtcg tcgacatgct ccgcaagcac acctacggcg aggagctcgc gcagaacagg 540 cgcgccttcg ccgctgcggc ggcgtccctg gcctcgccgc agctgccgcc ggccaagaac 600 actagcccgg cggcggcgcg cgagcacatg ttcgacaagg tgctgacccc gagcgacgta 660 ggcaagctca accggctggt ggtgccaaag cagcacgcgg agcggttctt cccggcggcc 720 ggcgccgggt cgacgcagct gtgcttccag gaccgcggcg gggcgctgtg gcagttccgc 780 tactcctact gggggagcag ccagagctat gtcatgacca aggggtggag ccgcttcgtc 840 cgcgccgcac gacttgccgc gggggacacc gtcaccttct cccgcagcgg cggcggccga 900 tacttcatcg agtaccgcca ctgccagcgc cggcgccgcg acgtcgatat cagcttcggc 960 gacgctgcca ccgtgccggc gtggccgagg ccgatagtta tcggaaccgc ggccatgaat 1020 aatgggggtg caacggtggc gtccgccacc atcgccggcc atgacatcga ggtggcagtg 1080 gcaccctcgg gggcgaggag cttcaggctc ttcgggttca atgttgagtg cagcggcgac 1140 gatgcaccgg caccggcacc tgctcccgcc gaagtggagt atgtcgacgg cgacacctag 1200 40 399 PRT Zea mays G3433 polypeptide Orthologous to G867, G9, G993, G1930 40 Met Asp Ser Ala Ser Ser Leu Val Asp Asp Thr Ser Gly Ser Gly Gly 1 5 10 15 Gly Ala Cys Thr Asp Lys Leu Arg Ala Leu Ala Ala Ala Ala Ala Ser 20 25 30 Ala Ser Gly Pro Pro Pro Glu Arg Met Gly Ser Gly Ala Ser Ala Val 35 40 45 Val Asp Ala Ala Glu Pro Gly Ala Glu Ala Asp Ser Gly Ser Ala Pro 50 55 60 Ala Ser Val Ala Ala Val Ala Ala Gly Val Gly Gly Lys Leu Pro Ser 65 70 75 80 Ser Arg Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala 85 90 95 Gln Ile Tyr Glu Arg His Leu Arg Val Trp Leu Gly Thr Phe Thr Gly 100 105 110 Glu Ala Glu Ala Ala Arg Ala Tyr Asp Val Ala Ala Gln Arg Phe Arg 115 120 125 Gly Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Ala Glu Ser Asp Leu 130 135 140 Asp Pro Asp Ala Ala Ala Glu Leu Arg Phe Leu Ala Ser Arg Ser Lys 145 150 155 160 Ala Glu Val Val Asp Met Leu Arg Lys His Thr Tyr Gly Glu Glu Leu 165 170 175 Ala Gln Asn Arg Arg Ala Phe Ala Ala Ala Ala Ala Ser Leu Ala Ser 180 185 190 Pro Gln Leu Pro Pro Ala Lys Asn Thr Ser Pro Ala Ala Ala Arg Glu 195 200 205 His Met Phe Asp Lys Val Leu Thr Pro Ser Asp Val Gly Lys Leu Asn 210 215 220 Arg Leu Val Val Pro Lys Gln His Ala Glu Arg Phe Phe Pro Ala Ala 225 230 235 240 Gly Ala Gly Ser Thr Gln Leu Cys Phe Gln Asp Arg Gly Gly Ala Leu 245 250 255 Trp Gln Phe Arg Tyr Ser Tyr Trp Gly Ser Ser Gln Ser Tyr Val Met 260 265 270 Thr Lys Gly Trp Ser Arg Phe Val Arg Ala Ala Arg Leu Ala Ala Gly 275 280 285 Asp Thr Val Thr Phe Ser Arg Ser Gly Gly Gly Arg Tyr Phe Ile Glu 290 295 300 Tyr Arg His Cys Gln Arg Arg Arg Arg Asp Val Asp Ile Ser Phe Gly 305 310 315 320 Asp Ala Ala Thr Val Pro Ala Trp Pro Arg Pro Ile Val Ile Gly Thr 325 330 335 Ala Ala Met Asn Asn Gly Gly Ala Thr Val Ala Ser Ala Thr Ile Ala 340 345 350 Gly His Asp Ile Glu Val Ala Val Ala Pro Ser Gly Ala Arg Ser Phe 355 360 365 Arg Leu Phe Gly Phe Asn Val Glu Cys Ser Gly Asp Asp Ala Pro Ala 370 375 380 Pro Ala Pro Ala Pro Ala Glu Val Glu Tyr Val Asp Gly Asp Thr 385 390 395 41 969 DNA Brassica oleraceae BZ458719 Predicted sequence is orthologous to G867, G9, G993, G1930 41 caacccggcc cgtatcctgt tccaacccag cctttgtact tccacacaat atacaactgt 60 tgatcctgac cgttagatct tttaaaactg atcaaatcac cggcacagag tctcttctct 120 ttaacgaacc tgctccaacc tttagtcaac acgtagcttt gactactgtt ccaatacgag 180 taacggaacc tccacacttt cccgttaacg tcttcgaaat tcaacagcgt ccctctcacg 240 gagacgtcgc cggttaacgg taacggaaaa tgtttctccg cttggtgttt cggtatcact 300 aaacggttta gcttcccgac gtcactcggc gttaccgttt tctcaaacag acactccgcc 360 gttttaaacc ccgtcacaac cgtaacgtta gcaaacgccg tctcttccgt gtctccgtta 420 ccaccgttac gtttgcgttt cctctgctct aactcttctt tgtaagtgtg tttcctcaac 480 atatcaacga tctcatattt cgaatgtgcg tttaagaact ttacttcgtc tccgtcacct 540 tcaccgttac ggaacgtcgt gtctggtttg aaattagtga cggcgtcaga gccgcggaaa 600 cggtgagcgg cgacgtcgta gacacgcgcc gcttcttctt cttcgttgaa tgtcccgagc 660 caaacgcgct tgtgcttctc gtatatctga gctccccatc ttccgtttgg ctgagggacg 720 acgcctttga attttgacga cgggagcttt cttgattctg cttcgacgcc gttctgggaa 780 tcgagaacca cgcttgaacc gcttcccatt ctgtataaac tcgccggaga tgacgacttc 840 gccgtcttcg gcggcggtgt atcttagccg gagtgtggat ggaaactgta cttgttgagc 900 tctccatcac actactcaca gctttcatat tagagaaatc acaagaaagt tgtgaaattt 960 gagaatgaa 969 42 288 PRT Brassica oleraceae misc_feature (288)..(288) Xaa can be any naturally occurring amino acid 42 Asp Thr Pro Pro Pro Lys Thr Ala Lys Ser Ser Ser Pro Ala Ser Leu 1 5 10 15 Tyr Arg Met Gly Ser Gly Ser Ser Val Val Leu Asp Ser Gln Asn Gly 20 25 30 Val Glu Ala Glu Ser Arg Lys Leu Pro Ser Ser Lys Phe Lys Gly Val 35 40 45 Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys His 50 55 60 Lys Arg Val Trp Leu Gly Thr Phe Asn Glu Glu Glu Glu Ala Ala Arg 65 70 75 80 Val Tyr Asp Val Ala Ala His Arg Phe Arg Gly Ser Asp Ala Val Thr 85 90 95 Asn Phe Lys Pro Asp Thr Thr Phe Arg Asn Gly Glu Gly Asp Gly Asp 100 105 110 Glu Val Lys Phe Leu Asn Ala His Ser Lys Tyr Glu Ile Val Asp Met 115 120 125 Leu Arg Lys His Thr Tyr Lys Glu Glu Leu Glu Gln Arg Lys Arg Lys 130 135 140 Arg Asn Gly Gly Asn Gly Asp Thr Glu Glu Thr Ala Phe Ala Asn Val 145 150 155 160 Thr Val Val Thr Gly Phe Lys Thr Ala Glu Cys Leu Phe Glu Lys Thr 165 170 175 Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys 180 185 190 His Gln Ala Glu Lys His Phe Pro Leu Pro Leu Thr Gly Asp Val Ser 195 200 205 Val Arg Gly Thr Leu Leu Asn Phe Glu Asp Val Asn Gly Lys Val Trp 210 215 220 Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr 225 230 235 240 Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Arg Leu Cys Ala Gly Asp 245 250 255 Leu Ile Ser Phe Lys Arg Ser Asn Gly Gln Asp Gln Gln Leu Tyr Ile 260 265 270 Val Trp Lys Tyr Lys Gly Trp Val Gly Thr Gly Tyr Gly Pro Gly Xaa 275 280 285 43 756 DNA Helianthus annuus BQ971511 Predicted sequence is orthologous to G867, G9, G993, G1930 43 ttggaagcgg agccagcgtg gtttctgacc cggaagtgga agccttgtcg aggaagctac 60 cgtcgtcgag atataaaggc gttgttccgc aagcgaatgg ccgttgggga gctcagattt 120 atgagaaaca tcaaagggta tggcttggca cgtttaacga cgaagacgaa gccgcgaaag 180 cgtacgacgt cgcggtccaa cgctttcgcg gacgagacgc agtaacaaac ttcaagcaac 240 tcgtcaccga cgacaacgcc gctgcctttg aagcaacttt cttaaaccgt cactcaaaat 300 ccgaaatagt tgacatgcta agaaaacaca catacaatga cgagttagaa caaagcaaaa 360 gaaccatcaa cacacacaaa accctatttc aaaccgggtt caaccttccc ggaccgggtt 420 gcaccatgcc acgcgaacac ctcttccaaa aaaccgtcac accaagcgac gttggcaaac 480 taaaccggct cgtgatacca aaacaacatg ctgaaaaaca ctttccggtt caaaaaggca 540 tcagttcaaa gggagttttg ttacacttcg aagataccga gtcaaaagtt tggcgatttc 600 ggtattcata ttggaatagt agccagagtt atgtgttaac caaagggtgg agccggtttg 660 ttaaagaaaa gaaccttaaa gccggtgata gcgttagctt tcacagctcg accggaacgg 720 ataagcagtt ttacattcac tgggagtcaa aaaccg 756 44 252 PRT Helianthus annuus misc_feature (252)..(252) Xaa can be any naturally occurring amino acid 44 Gly Ser Gly Ala Ser Val Val Ser Asp Pro Glu Val Glu Ala Leu Ser 1 5 10 15 Arg Lys Leu Pro Ser Ser Arg Tyr Lys Gly Val Val Pro Gln Ala Asn 20 25 30 Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys His Gln Arg Val Trp Leu 35 40 45 Gly Thr Phe Asn Asp Glu Asp Glu Ala Ala Lys Ala Tyr Asp Val Ala 50 55 60 Val Gln Arg Phe Arg Gly Arg Asp Ala Val Thr Asn Phe Lys Gln Leu 65 70 75 80 Val Thr Asp Asp Asn Ala Ala Ala Phe Glu Ala Thr Phe Leu Asn Arg 85 90 95 His Ser Lys Ser Glu Ile Val Asp Met Leu Arg Lys His Thr Tyr Asn 100 105 110 Asp Glu Leu Glu Gln Ser Lys Arg Thr Ile Asn Thr His Lys Thr Leu 115 120 125 Phe Gln Thr Gly Phe Asn Leu Pro Gly Pro Gly Cys Thr Met Pro Arg 130 135 140 Glu His Leu Phe Gln Lys Thr Val Thr Pro Ser Asp Val Gly Lys Leu 145 150 155 160 Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro Val 165 170 175 Gln Lys Gly Ile Ser Ser Lys Gly Val Leu Leu His Phe Glu Asp Thr 180 185 190 Glu Ser Lys Val Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln 195 200 205 Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Asn 210 215 220 Leu Lys Ala Gly Asp Ser Val Ser Phe His Ser Ser Thr Gly Thr Asp 225 230 235 240 Lys Gln Phe Tyr Ile His Trp Glu Ser Lys Thr Xaa 245 250 45 743 DNA Helianthus annuus BU025988 Predicted sequence is orthologous to G867, G9, G993, G1930 45 ttggaagcgg atccagcgtg gttcttgacc cagaaggagg cgtggaagtt gaagctcagt 60 cgagaaagct accctcgtcg cgatacaaag gtgtcgttcc acaaccgaat ggccgttggg 120 gagctcagat ttacgagaaa caccaaaggg tatggttagg tacgttcaac gacgaagatg 180 aagctgcaaa ggcgtacgat gttgccgtac aacgcttccg cggccgagac gcggtcacaa 240 acattaagca ggttgatgcc gacgataaag aggccgcgat ggaagcaagt ttcttaagcc 300 gccattcgga gtcagaaatt gttgacatgc ttagaaaaca cacatacaat gacgagctag 360 aacaaagcaa aagaagctgc acctcacacc aaaccctttc tcaaaccggt ttaaccaaca 420 ccacccgttt agtctccatg aagccacgcg aacacctctt ccagaaaacc gtgaccccta 480 gcgacgtagg aaagctgaac cggctcgtta taccaaaaca acacgcggag aaacacttcc 540 cggttcaaaa agggagcaat tcaaaaggag ttcttttaca tttcgaagat aaagggtcaa 600 aagtatggag atttcgttac tcttactgga acagtagcca gagttatgtt ttaaccaaag 660 gctggagccg gttcgtgaaa gaaaaaaatc taaaagccgg agatagcgtc agctttcaaa 720 gctcaaccgg accggataag cag 743 46 247 PRT Helianthus annuus BU025988 polypeptide Orthologous to G867, G9, G993, G1930 46 Gly Ser Gly Ser Ser Val Val Leu Asp Pro Glu Gly Gly Val Glu Val 1 5 10 15 Glu Ala Gln Ser Arg Lys Leu Pro Ser Ser Arg Tyr Lys Gly Val Val 20 25 30 Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln Ile Tyr Glu Lys His Gln 35 40 45 Arg Val Trp Leu Gly Thr Phe Asn Asp Glu Asp Glu Ala Ala Lys Ala 50 55 60 Tyr Asp Val Ala Val Gln Arg Phe Arg Gly Arg Asp Ala Val Thr Asn 65 70 75 80 Ile Lys Gln Val Asp Ala Asp Asp Lys Glu Ala Ala Met Glu Ala Ser 85 90 95 Phe Leu Ser Arg His Ser Glu Ser Glu Ile Val Asp Met Leu Arg Lys 100 105 110 His Thr Tyr Asn Asp Glu Leu Glu Gln Ser Lys Arg Ser Cys Thr Ser 115 120 125 His Gln Thr Leu Ser Gln Thr Gly Leu Thr Asn Thr Thr Arg Leu Val 130 135 140 Ser Met Lys Pro Arg Glu His Leu Phe Gln Lys Thr Val Thr Pro Ser 145 150 155 160 Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu 165 170 175 Lys His Phe Pro Val Gln Lys Gly Ser Asn Ser Lys Gly Val Leu Leu 180 185 190 His Phe Glu Asp Lys Gly Ser Lys Val Trp Arg Phe Arg Tyr Ser Tyr 195 200 205 Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe 210 215 220 Val Lys Glu Lys Asn Leu Lys Ala Gly Asp Ser Val Ser Phe Gln Ser 225 230 235 240 Ser Thr Gly Pro Asp Lys Gln 245 47 1470 DNA Triticum aestivum BT009310 Predicted sequence is orthologous to G867, G9, G993, G1930 47 gcacgaggct agcttcagct tttagctaag ctctacttcc ctcccgagct aagcatcttc 60 ttgatttctc ggtgatcgga ttcggatgga cagcgcaaga agctgcctcg tggacgacgt 120 gagcagcggc gcgtccacgg gcaagaaggc ctctccgtcc ccggccgcgc cggcgaccaa 180 gccgctgcag cgcgtgggca gcggggccag cgcggtcatg gacgcgccgg agcccggcgc 240 cgaggcggac tccggccgcg tcggcaggct gccgtcctcc aagtacaagg gcgtggtgcc 300 gcagcccaac gggcgctggg gcgcgcagat ctacgagcgc caccagcgcg tctggctcgg 360 caccttcacg ggggaggccg aggctgcgcg cgcctacgac gcggcggcgc agcgcttccg 420 cggccgcgac gcagtcacca acttccgccc gctcaccgag tccgacccgg aggacgccgc 480 cgagctccgc ttcctcgctg cccgctccaa ggccgaggtc gtcgacatgc tgcgcaagca 540 cacctatccc gacgagctcg ctcagtacaa gcgcgcctac ttcgccgccg ctgcggcgtc 600 ctcccctaca tcgtcctcgg tgcctcccgc ctcgtcgccc tcttcggcgg cttcgccctc 660 gccggcggcg cggcgcgagc acctgttcga caagacggtc acgcccagcg acgtggggaa 720 gctgaaccgg cttgtgatac cgaagcagca cgccgagaag cactttcctc tccagcttcc 780 ttccgccggc gccgccgtgt ccggcgagtg caagggcatg cttctcaact tcgacgactc 840 ggccggcaag gtgtggaggt tccggtactc gtactggaac agcagccaga gctacgtgct 900 caccaagggc tggagccgct ttgtcaagga gaagggcctg cacgccggcg acgccgtcgg 960 gttctaccgc tctgcctcag gcagcaacca gctcttcatc gactgcaagc tccggtccaa 1020 gaccacgacg atgacgacga ctttcgtcaa cgcggcggcc gccccgtcgc cggcacccgt 1080 gatgaggacc gtgcgactct tcggcgtcga ccttctcacg gcgccggcgc cgagtcacgc 1140 gcccgagcac gaggactgca gcatggtgcc caagacaagc aagagatcca tggacgccaa 1200 cgcagcggcc actccggcgc acgcggtctg gaagaagcgg tgcatagact tcgcgctgac 1260 ctagccagct agcgtttttc ctccatggtt gctttgcttg cctccaaatt tccatgttag 1320 tagcttagag ctcttgatcg gtccaagtgt ttgccttttt tttcctcttc ttctcataca 1380 caagttagct ctaaatccag tcttctttaa ttaatctact gtaaattaag cccgttctcg 1440 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1470 48 392 PRT Triticum aestivum BT009310 polypeptide Orthologous to G867, G9, G993, G1930 48 Met Asp Ser Ala Arg Ser Cys Leu Val Asp Asp Val Ser Ser Gly Ala 1 5 10 15 Ser Thr Gly Lys Lys Ala Ser Pro Ser Pro Ala Ala Pro Ala Thr Lys 20 25 30 Pro Leu Gln Arg Val Gly Ser Gly Ala Ser Ala Val Met Asp Ala Pro 35 40 45 Glu Pro Gly Ala Glu Ala Asp Ser Gly Arg Val Gly Arg Leu Pro Ser 50 55 60 Ser Lys Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala 65 70 75 80 Gln Ile Tyr Glu Arg His Gln Arg Val Trp Leu Gly Thr Phe Thr Gly 85 90 95 Glu Ala Glu Ala Ala Arg Ala Tyr Asp Ala Ala Ala Gln Arg Phe Arg 100 105 110 Gly Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Thr Glu Ser Asp Pro 115 120 125 Glu Asp Ala Ala Glu Leu Arg Phe Leu Ala Ala Arg Ser Lys Ala Glu 130 135 140 Val Val Asp Met Leu Arg Lys His Thr Tyr Pro Asp Glu Leu Ala Gln 145 150 155 160 Tyr Lys Arg Ala Tyr Phe Ala Ala Ala Ala Ala Ser Ser Pro Thr Ser 165 170 175 Ser Ser Val Pro Pro Ala Ser Ser Pro Ser Ser Ala Ala Ser Pro Ser 180 185 190 Pro Ala Ala Arg Arg Glu His Leu Phe Asp Lys Thr Val Thr Pro Ser 195 200 205 Asp Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu 210 215 220 Lys His Phe Pro Leu Gln Leu Pro Ser Ala Gly Ala Ala Val Ser Gly 225 230 235 240 Glu Cys Lys Gly Met Leu Leu Asn Phe Asp Asp Ser Ala Gly Lys Val 245 250 255 Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu 260 265 270 Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Gly Leu His Ala Gly 275 280 285 Asp Ala Val Gly Phe Tyr Arg Ser Ala Ser Gly Ser Asn Gln Leu Phe 290 295 300 Ile Asp Cys Lys Leu Arg Ser Lys Thr Thr Thr Met Thr Thr Thr Phe 305 310 315 320 Val Asn Ala Ala Ala Ala Pro Ser Pro Ala Pro Val Met Arg Thr Val 325 330 335 Arg Leu Phe Gly Val Asp Leu Leu Thr Ala Pro Ala Pro Ser His Ala 340 345 350 Pro Glu His Glu Asp Cys Ser Met Val Pro Lys Thr Ser Lys Arg Ser 355 360 365 Met Asp Ala Asn Ala Ala Ala Thr Pro Ala His Ala Val Trp Lys Lys 370 375 380 Arg Cys Ile Asp Phe Ala Leu Thr 385 390 49 914 DNA Zea mays CC616336 Predicted sequence is orthologous to G867, G9, G993, G1930 49 gaagagctgc tgcttgccgc cggcggaacg gtagaacccg acggcgtcgc cggcgtggag 60 ccccttctcc ttgacgaagc ggctccaccc cttggtcagc acgtagctct ggctgctgtt 120 ccagtacgag taccggaacc tccacgcctt cccggcggcg tcctcgaagt tgaggagcac 180 gcccttgcac tcgccgccac taccgacgcc cgccgccgcc gccgccggga gctgcagcgg 240 gaagtgcttc tccgcgtgct gcttcggtat caccagccgg ttcagcttcc ccacgtcgct 300 cggcgtcacc gtcttgtcga agaggtgctc gcgcgccgcc gcgggcgacg acgacgacga 360 ggcggcgggc ggacggcggt tattctcggg cggcggcgag gcggccgggg acgccgcggc 420 gaaggcgcgc ctgttgtgcg cgagctcctc gccgtaggtg tgcttgcgga gcatgtcgac 480 gacctcggcc ttggaccggg acgcgaggaa ccggagctcg acggcggcct ccggctccga 540 ctccgccagc gggcggaagt tggtgacggc gtcgcggccc cggaaccgct gcgcggccac 600 gtcgtaggcg cgcgcggcct cggcctcgcc cgtgaacgtg ccgagccaca cgcgctggtg 660 ccgctcgtag atctgcgcgc cccaccgccc gttgggctgt ggcaccacgc ccttgtactt 720 ggacgacggc agcttcccgc tgaccccgcc gggggcccgc cccgcgccgc ccgagtctgc 780 ctcggcgccc ggctccgccg cgtccatcac cgcgctggtg ccgctgccca cgcgctgtag 840 cggcttgccg gtcgccgcag ccggagccgg cgccggtttc ttgcccgtgg acgcgccgct 900 gctcgcgtcg tcca 914 50 304 PRT Zea mays CC616336 polypeptide Orthologous to G867, G9, G993, G1930 50 Asp Asp Ala Ser Ser Gly Ala Ser Thr Gly Lys Lys Pro Ala Pro Ala 1 5 10 15 Pro Ala Ala Ala Thr Gly Lys Pro Leu Gln Arg Val Gly Ser Gly Thr 20 25 30 Ser Ala Val Met Asp Ala Ala Glu Pro Gly Ala Glu Ala Asp Ser Gly 35 40 45 Gly Ala Gly Arg Ala Pro Gly Gly Val Ser Gly Lys Leu Pro Ser Ser 50 55 60 Lys Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln 65 70 75 80 Ile Tyr Glu Arg His Gln Arg Val Trp Leu Gly Thr Phe Thr Gly Glu 85 90 95 Ala Glu Ala Ala Arg Ala Tyr Asp Val Ala Ala Gln Arg Phe Arg Gly 100 105 110 Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Ala Glu Ser Glu Pro Glu 115 120 125 Ala Ala Val Glu Leu Arg Phe Leu Ala Ser Arg Ser Lys Ala Glu Val 130 135 140 Val Asp Met Leu Arg Lys His Thr Tyr Gly Glu Glu Leu Ala His Asn 145 150 155 160 Arg Arg Ala Phe Ala Ala Ala Ser Pro Ala Ala Ser Pro Pro Pro Glu 165 170 175 Asn Asn Arg Arg Pro Pro Ala Ala Ser Ser Ser Ser Ser Pro Ala Ala 180 185 190 Ala Arg Glu His Leu Phe Asp Lys Thr Val Thr Pro Ser Asp Val Gly 195 200 205 Lys Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe 210 215 220 Pro Leu Gln Leu Pro Ala Ala Ala Ala Ala Gly Val Gly Ser Gly Gly 225 230 235 240 Glu Cys Lys Gly Val Leu Leu Asn Phe Glu Asp Ala Ala Gly Lys Ala 245 250 255 Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu 260 265 270 Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Gly Leu His Ala Gly 275 280 285 Asp Ala Val Gly Phe Tyr Arg Ser Ala Gly Gly Lys Gln Gln Leu Phe 290 295 300 51 1182 DNA Oryza sativa AAAA01000997 Predicted sequence is orthologous to G867, G9, G993, G1930 51 atggacagca cgagctgtct cttggacgac gcgagcagcg gcgcgtccac gggcaagaag 60 gcggcggcgg cggcggcgtc gaaggcgctg cagcgcgtgg gcagcggcgc cagcgcggtg 120 atggacgcgg ccgagcctgg cgccgaggcg gactcgggcg gcgagcggcg cggcggcggc 180 ggcgggaagc tgccgtcgtc caagtacaag ggcgtggtgc cgcaaccgaa cgggcggtgg 240 ggcgcgcaga tatacgagcg gcaccagcgg gtgtggctcg gcacgttcac cggcgaggcg 300 gaggcggcgc gcgcctacga cgtggcggcg cagcggttcc gcggccgcga cgccgtcacc 360 aacttccgcc cgctcgccga gtccgacccg gaggccgctg tcgagctccg cttcctcgcg 420 tcccgctcca aggccgaggt cgtcgacatg ctccgcaagc acacctacct cgaggagctc 480 acgcagaaca agcgcgcctt cgccgccatc tccccgccgc cccccaagca ccccgcctcc 540 tctccgccgt cctccgccgc cgcgcgcgag cacctgttcg acaagacggt gacgcccagc 600 gacgtcggga agctgaaccg gctggtgatc cccaagcagc acgccgagaa gcacttcccg 660 ctccagctcc ctccccctac cacaacctcc tccgtcgccg ccgccgccga cgccgccgcc 720 ggcggcggcg agtgcaaggg agtcctcctc aacttcgagg acgccgccgg gaaggtgtgg 780 aaattccggt actcctactg gaacagcagc cagagctacg tgctcaccaa ggggtggagc 840 cgcttcgtca aggacaaggg gctccacgcc ggcgacgccg tcggcttcta ccgcgccgcc 900 ggtaagaacg cgcagctctt catcgactgc aaggtccggg caaaacccac caccgccgcc 960 gccgccgccg ccttcctcag cgcggtggcc gccgccgccg cgccgccacc cgccgtgaag 1020 gctatcaggc tgttcggtgt cgacctgctc acggcggcgg cgccggagct gcaggacgcc 1080 ggcggcgccg ccatgaccaa gagcaagaga gccatggacg ccatggctga gtcacaagcg 1140 cacgtggttt ttaagaagca atgcatagag ctcgcgctaa cc 1182 52 394 PRT Oryza sativa AAAA01000997 polypeptide Orthologous to G867, G9, G993, G1930 52 Met Asp Ser Thr Ser Cys Leu Leu Asp Asp Ala Ser Ser Gly Ala Ser 1 5 10 15 Thr Gly Lys Lys Ala Ala Ala Ala Ala Ala Ser Lys Ala Leu Gln Arg 20 25 30 Val Gly Ser Gly Ala Ser Ala Val Met Asp Ala Ala Glu Pro Gly Ala 35 40 45 Glu Ala Asp Ser Gly Gly Glu Arg Arg Gly Gly Gly Gly Gly Lys Leu 50 55 60 Pro Ser Ser Lys Tyr Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp 65 70 75 80 Gly Ala Gln Ile Tyr Glu Arg His Gln Arg Val Trp Leu Gly Thr Phe 85 90 95 Thr Gly Glu Ala Glu Ala Ala Arg Ala Tyr Asp Val Ala Ala Gln Arg 100 105 110 Phe Arg Gly Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Ala Glu Ser 115 120 125 Asp Pro Glu Ala Ala Val Glu Leu Arg Phe Leu Ala Ser Arg Ser Lys 130 135 140 Ala Glu Val Val Asp Met Leu Arg Lys His Thr Tyr Leu Glu Glu Leu 145 150 155 160 Thr Gln Asn Lys Arg Ala Phe Ala Ala Ile Ser Pro Pro Pro Pro Lys 165 170 175 His Pro Ala Ser Ser Pro Pro Ser Ser Ala Ala Ala Arg Glu His Leu 180 185 190 Phe Asp Lys Thr Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu 195 200 205 Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro Leu Gln Leu Pro 210 215 220 Pro Pro Thr Thr Thr Ser Ser Val Ala Ala Ala Ala Asp Ala Ala Ala 225 230 235 240 Gly Gly Gly Glu Cys Lys Gly Val Leu Leu Asn Phe Glu Asp Ala Ala 245 250 255 Gly Lys Val Trp Lys Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser 260 265 270 Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val Lys Asp Lys Gly Leu 275 280 285 His Ala Gly Asp Ala Val Gly Phe Tyr Arg Ala Ala Gly Lys Asn Ala 290 295 300 Gln Leu Phe Ile Asp Cys Lys Val Arg Ala Lys Pro Thr Thr Ala Ala 305 310 315 320 Ala Ala Ala Ala Phe Leu Ser Ala Val Ala Ala Ala Ala Ala Pro Pro 325 330 335 Pro Ala Val Lys Ala Ile Arg Leu Phe Gly Val Asp Leu Leu Thr Ala 340 345 350 Ala Ala Pro Glu Leu Gln Asp Ala Gly Gly Ala Ala Met Thr Lys Ser 355 360 365 Lys Arg Ala Met Asp Ala Met Ala Glu Ser Gln Ala His Val Val Phe 370 375 380 Lys Lys Gln Cys Ile Glu Leu Ala Leu Thr 385 390 53 393 PRT Oryza sativa OSC26104.C1.p13.fg polypeptide Orthologous to G867, G9, G993, G1930 53 Met Asp Ser Ser Ser Cys Leu Val Asp Asp Thr Asn Ser Gly Gly Ser 1 5 10 15 Ser Thr Asp Lys Leu Arg Ala Leu Ala Ala Ala Ala Ala Glu Thr Ala 20 25 30 Pro Leu Glu Arg Met Gly Ser Gly Ala Ser Ala Val Val Asp Ala Ala 35 40 45 Glu Pro Gly Ala Glu Ala Asp Ser Gly Ser Gly Gly Arg Val Cys Gly 50 55 60 Gly Gly Gly Gly Gly Ala Gly Gly Ala Gly Gly Lys Leu Pro Ser Ser 65 70 75 80 Lys Phe Lys Gly Val Val Pro Gln Pro Asn Gly Arg Trp Gly Ala Gln 85 90 95 Ile Tyr Glu Arg His Gln Arg Val Trp Leu Gly Thr Phe Ala Gly Glu 100 105 110 Asp Asp Ala Ala Arg Ala Tyr Asp Val Ala Ala Gln Arg Phe Arg Gly 115 120 125 Arg Asp Ala Val Thr Asn Phe Arg Pro Leu Ala Glu Ala Asp Pro Asp 130 135 140 Ala Ala Ala Glu Leu Arg Phe Leu Ala Thr Arg Ser Lys Ala Glu Val 145 150 155 160 Val Asp Met Leu Arg Lys His Thr Tyr Phe Asp Glu Leu Ala Gln Ser 165 170 175 Lys Arg Thr Phe Ala Ala Ser Thr Pro Ser Ala Ala Thr Thr Thr Ala 180 185 190 Ser Leu Ser Asn Gly His Leu Ser Ser Pro Arg Ser Pro Phe Ala Pro 195 200 205 Ala Ala Ala Arg Asp His Leu Phe Asp Lys Thr Val Thr Pro Ser Asp 210 215 220 Val Gly Lys Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys 225 230 235 240 His Phe Pro Leu Gln Leu Pro Ser Ala Gly Gly Glu Ser Lys Gly Val 245 250 255 Leu Leu Asn Phe Glu Asp Ala Ala Gly Lys Val Trp Arg Phe Arg Tyr 260 265 270 Ser Tyr Trp Asn Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser 275 280 285 Arg Phe Val Lys Glu Lys Gly Leu His Ala Gly Asp Val Val Gly Phe 290 295 300 Tyr Arg Ser Ala Ala Ser Ala Gly Asp Asp Gly Lys Leu Phe Ile Asp 305 310 315 320 Cys Lys Leu Val Arg Ser Thr Gly Ala Ala Leu Ala Ser Pro Ala Asp 325 330 335 Gln Pro Ala Pro Ser Pro Val Lys Ala Val Arg Leu Phe Gly Val Asp 340 345 350 Leu Leu Thr Ala Pro Ala Pro Val Glu Gln Met Ala Gly Cys Lys Arg 355 360 365 Ala Arg Asp Leu Ala Ala Thr Thr Pro Pro Gln Ala Ala Ala Phe Lys 370 375 380 Lys Gln Cys Ile Glu Leu Ala Leu Val 385 390 54 929 DNA Arabidopsis thaliana CBF1 G40 54 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 55 213 PRT Arabidopsis thaliana CBF1 polypeptide G40 55 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 56 803 DNA Arabidopsis thaliana CBF2 G41 56 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 57 207 PRT Arabidopsis thaliana CBF2 polypeptide G41 57 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 58 908 DNA Arabidopsis thaliana misc_feature (851)..(851) n is a, c, g, or t 58 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 59 216 PRT Arabidopsis thaliana CBF3 polypeptide G42 59 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 60 632 DNA Brassica napus bnCBF1 60 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 61 208 PRT Brassica napus bnCBF1 polypeptide 61 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 62 20 DNA artificial sequence Artificial sequence 62 cayccnatht aymgnggngt 20 63 21 DNA artificial sequence Artificial sequence 63 ggnarnarca tnccytcngc c 21 64 22 PRT Arabidopsis thaliana DML motif of G867 64 His Ser Lys Ser Glu Ile Val Asp Met Leu Arg Lys His Thr Tyr Asn 1 5 10 15 Glu Glu Leu Glu Gln Ser 20

Claims

What is claimed is:

1. A recombinant polynucleotide comprising a nucleotide sequence that hybridizes over its full length to the complement of SEQ ID NO 1 under stringent conditions that include two wash steps of 6x SSC and 65° C., each step being 10-30 minutes in duration.

2. The recombinant polynucleotide of claim 1, wherein the polynucleotide is operably linked to at least one regulatory element being effective in controlling expression of said isolated polynucleotide when said isolated polynucleotide is transformed into a plant.

3. The recombinant polynucleotide of claim 1, wherein said recombinant polynucleotide is incorporated into an expression vector.

4. The recombinant polynucleotide of claim 3, wherein said recombinant polynucleotide is incorporated into a cultured host cell.

5. The recombinant polynucleotide of claim 1, wherein the recombinant polynucleotide encodes a polypeptide comprising the AP2 domain, the DML motif, and the B3 domain of SEQ ID NO:2.

6. The recombinant polynucleotide of claim 1, wherein said recombinant polynucleotide encodes a polypeptide comprising SEQ ID NO:2.

7. The recombinant polynucleotide of claim 1, wherein said recombinant polynucleotide comprises SEQ ID NO:1.

8. An isolated nucleotide sequence that hybridizes over its full length to the complement of a polynucleotide encoding SEQ ID NO 64, which represents the DML motif of SEQ ID NO:1, under stringent conditions that include two wash steps of 6×SSC and 65° C., each step being 10-30 minutes in duration.

9. The isolated nucleotide sequence of claim 8, wherein the isolated nucleotide sequence encodes a polypeptide comprising a DML motif that is substantially identical to SEQ ID NO 64.

10. The isolated nucleotide sequence of claim 8, wherein said isolated nucleotide sequence is incorporated into an expression vector.

11. The isolated nucleotide sequence of claim 8, wherein said isolated nucleotide sequence is incorporated into a cultured host cell.

12. A transgenic plant that overexpresses the recombinant polynucleotide according to claim 1, wherein said transgenic plant has increased abiotic stress tolerance as compared to a non-transformed plant that does not overexpress a polypeptide encoded by the recombinant polynucleotide.

13. A transgenic plant comprising a recombinant polynucleotide encoding a polypeptide having an AP2 domain and a B3 domain, wherein the polypeptide has the property of SEQ ID NO:2 of regulating abiotic stress tolerance in a plant when said polypeptide is overexpressed, wherein:

the AP2 domain is sufficiently homologous to the AP2 domain of SEQ ID NO:2 that the polypeptide binds to a transcription-regulating region comprising the motif CAACA;

the B3 domain is sufficiently homologous to the B3 domain of SEQ ID NO:2 that the polypeptide binds to a transcription regulating region comprising the motif CACCTG; and

wherein said binding cooperatively enhances DNA binding affinity of said polypeptide 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.

14. The transgenic plant of claim 13, wherein said polypeptide comprises SEQ ID NO:2.

15. The transgenic plant of claim 13, wherein said recombinant polynucleotide comprises SEQ ID NO:1.

16. The transgenic plant of claim 13, wherein said recombinant polynucleotide comprises SEQ ID NO:64, which represents the DML motif of SEQ ID NO:1.

17. The transgenic plant of claim 13, wherein said abiotic stress tolerance is selected from the group consisting of drought tolerance, heat tolerance, and salt stress tolerance.

18. The transgenic plant of claim 13, 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, citrus, fruit trees, rosaceous fruits, and brassicas.

19. The transgenic plant of claim 13, further comprising a constitutive, inducible, or tissue-specific promoter operably linked to said polynucleotide sequence.

20. The transgenic plant of claim 13, wherein said polypeptide is selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, and 53.

21. A method for producing a transgenic plant having increased tolerance to abiotic stress, the method steps comprising:

(a) providing the expression vector according to claim 3;

(b) introducing the expression vector into a plant cell, and allowing the plant cell to overexpress a polypeptide encoded by the recombinant polynucleotide, said polypeptide having the property of regulating abiotic stress tolerance in a plant as compared to a non-transformed plant that does not overexpress the polypeptide;

(c) growing the plant cell into a plant; and

(d) identifying an abiotic stress tolerant plant so produced with increased abiotic stress tolerance by comparing said abiotic stress tolerant plant with one or more non-transformed plants that do not overexpress the polypeptide.

22. The method of claim 21, the method steps further comprising:

(e) selfing or crossing said abiotic stress tolerant plant with itself or another plant, respectively, to produce seed; and

(f) growing a progeny plant from the seed, thus producing a transgenic progeny plant having increased tolerance to abiotic stress.

23. The method of claim 22, wherein:

said progeny plant expresses MRNA that encodes a DNA-binding protein having an AP2 domain that binds to a DNA molecule, regulates expression of said DNA molecule, 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.

24. The method of claim 21, wherein said abiotic stress tolerance is selected from the group consisting of heat tolerance, chilling tolerance, germination in heat, germination in cold, drought stress tolerance, and salt stress tolerance.

25. The method of claim 21, wherein the polypeptide comprises an AP2 domain, a DML motif, and a B3 domain, in order from N-terminal to C-terminal, respectively.

26. A method for increasing a plant's tolerance to abiotic stress, said method comprising:

(a) providing a vector comprising:

(i) regulatory elements flanking the polynucleotide sequence, said regulatory elements being effective to control expression of said polynucleotide sequence in a target plant; and

(ii) a polynucleotide sequence that encodes a polypeptide having an AP2 domain that is sufficiently homologous to the AP2 domain of SEQ ID NO:2 that the polypeptide binds to a first transcription regulating region comprising the motif CAACA, and the B3 domain is sufficiently homologous to the B3 domain of SEQ ID NO:2, that the polypeptide binds to a second transcription regulating region comprising the motif CACCTG, and the polypeptide has the property of SEQ ID NO:2 of regulating abiotic stress tolerance in a plant, wherein said binding of said first and second transcription regulating regions 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 abiotic stress.

27. The method of claim 26, wherein said polynucleotide comprises:

(i) SEQ ID NO:1;

(ii) a nucleotide sequence that encodes SEQ ID NO:2;

(iii) a nucleotide sequence that hybridizes to the nucleotide sequence of (i) or (ii) under stringent conditions of two washes of 6×SSC and 65° C., each step being 10-30 minutes in duration;

(iv) a nucleotide sequence encoding a polypeptide comprising an AP2 domain and a B3 domain that are substantially identical with the AP2 and B3 domains of SEQ ID NO:2, respectively; or

(v) a nucleotide sequence encoding a polypeptide comprising a DML motif that is substantially identical to SEQ ID NO:64, the DML motif of SEQ ID NO:2.

28. The method of claim 26, wherein said abiotic stress tolerance is selected from the group consisting of heat tolerance, chilling tolerance, germination in heat, germination in cold, drought stress tolerance, and salt stress tolerance.