Patent Publication Number: US-2003233680-A1

Title: Methods for modifying plant biomass and cell protectant levels

Description:
RELATED APPLICATION INFORMATION  
     [0001] This application is a continuation-in-part of the following U.S. applications: U.S. application Ser. No. 09/773,990, filed: Feb. 1, 2001, which in turn claimed priority from U.S. application Ser. No. 09/580,377, filed: May 26, 2000, which in turn claimed priority from U.S. provisional Application Serial No. 60/165,860, filed: Nov. 16, 1999, now abandoned; U.S. application Ser. No. 09/996,140, filed Nov. 26, 2001; U.S. application Ser. No. 09/601,802, filed: Sep. 15, 2000, which in turn claimed priority from PCT application No.: PCT/US99/01895, filed: Jan. 28, 1999, now abandoned, which in turn claimed priority from U.S. application Ser. No. 09/198,119, filed: Nov. 23, 1998, which issued as U.S. Pat. No. 6,417,428, which, in turn, claimed priority in part to U.S. application Ser. No. 09/018,233, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/017,816, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/018,235, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/017,575, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/018,227, filed: Feb. 3, 1998, now abandoned, and U.S. application Ser. No. 09/018,234, filed: Feb. 3, 1998, now abandoned, all six of which claimed priority in part to U.S. application Ser. No. 08/706,270, filed: Sep. 4, 1996, which issued as U.S. Pat. No. 5,892,009; U.S. application Ser. No. 09/627,348, filed Jul. 28, 2000, which in turn claimed priority from No. 60/148,200 filed Aug. 10, 1999, now abandoned, and U.S. provisional Application Serial No. 60/165,860 filed Nov. 16, 1999, now abandoned; U.S. application Ser. No. 09/713,994, filed: Nov. 16, 2000, which in turn claimed priority from No. 60/197,899 filed Apr. 17, 2000, now abandoned, U.S. provisional Application Serial No. 60/227,439 filed Aug. 22, 2000, now abandoned, and from U.S. provisional Application Serial No. 60/166,228 filed Nov. 17, 1999, now abandoned; each of which are incorporated herein by reference in their entirety. 
    
    
     [0002] This invention was supported by a subcontract under a USDA/CSREES Cooperative Agreement and an NSF/SBIR grant. The US Government has certain rights in this invention. 
    
    
     
       FIELD OF THE INVENTION  
       [0003] The present invention relates to a method for modifying the biomass of a plant. The method is particularly useful for modifying the leaf or root biomass of a plant. The invention is also useful for modifying the levels of a cell protectant, such as a cryoprotectant, an osmoprotectant, or the like, in a cell or a plant to improve its response to environmental stresses, including but not limited to cold or freezing stress, drought stress, or salt stress.  
       BACKGROUND OF THE INVENTION  
       [0004] Increasing the biomass of a plant has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. By increasing plant biomass, increased production levels of the products may be obtained from the plants. Tobacco leaves, in particular, have been employed as plant factories to generate such products. Furthermore, it may be desirable to increase crop yields of plants by increasing total plant photosynthesis. An increase in total plant photosynthesis is typically achieved by increasing leaf area of the plant. Additional photosynthetic capacity may be used to increase the yield derived from particular plant tissue, including the leaves, roots, fruits or seed. In addition, the ability to modify the biomass of the leaves may be useful for permitting the growth of a plant under decreased light intensity or under high light intensity. Modification of the biomass of another tissue, such as roots, may be useful to improve a plant&#39;s ability to grow under harsh environmental conditions, including drought or nutrient deprivation, because the roots may grow deeper into the ground.  
       [0005] Due to the commercial consequences of environmental stress damage to crops, there is an interest in understanding how to improve a plant&#39;s tolerance to environmental stresses. By improving a plant&#39;s performance or survival in response to different environmental stresses, the weather-related losses in productivity and risks to farming can be greatly reduced. Modifying a plant&#39;s tolerance to environmental stresses also allows a plant to be grown in regions where a plant or plant variety is typically unable to grow.  
       [0006] Many biochemical changes occur in a plant when a plant becomes tolerant to an environmental stress. For example, for cold or freezing stress tolerance, it is well documented that lipid composition changes occur during cold acclimation in a wide range of plants and there is compelling data to indicate that this contributes to greater freezing tolerance (Steponkus et al. (1993)  Advances in Low - Temperature Biology,  Vol. 2, P. L. Steponkus, editor, (London: JAI Press), pp. 211-312).  
       [0007] Similarly, the levels of proline and sucrose increase in  Arabidopsis  (McKown et al. (1996)  J. Exp. Bot.  47: 1919-1925; Wanner and Junttila (1999)  Plant Physiol.  120: 391-400) and other plants (Guy et al. (1992)  Plant Physiol.  100: 502-508; Koster and Lynch (1992)  Plant Physiol.  98: 108-113) during cold acclimation and likely have roles in freezing tolerance. There is evidence that proline can protect both membranes and proteins against freeze-induced damage in vitro (Rudolph and Crowe (1985)  Cryobiol.  22: 367-377; Carpenter and Crowe (1988)  Cryobiol.  25: 244-255) and direct evidence that increased levels of proline enhances whole plant freezing tolerance (Nanjo et al. (1999)  FEBS Lett.  461: 205-210).  
       [0008] Sucrose and other simple sugars have also been shown to be effective cryoprotectants in vitro (Strauss et al. (1986)  Proc. Natl. Acad. Sci.  83: 2422-2426; Carpenter and Crowe (1988) supra) and there is correlative evidence indicating a role in freezing tolerance in cold-acclimated plants (Guy et al. (1992) supra; Koster and Lynch (1992) supra; Wanner and Junttila (1999) supra).  
       [0009] Similarly, tolerance to drought or water stress is associated with the accumulation of a variety of osmolytes, including sugar alcohols such as mannitol, amino acids such as proline, and glycine betaine (Greenway et al. (1980)  Ann. Rev. Plant Physiol.  31: 149-190; Yancey et al. (1982)  Science  217: 1214-1222).  
       [0010] Cold acclimation in plants is associated with the expression of cold-regulated (COR) genes that encode many polypeptides of unknown function. One of the more intriguing attributes of the COR genes is that many encode polypeptides that appear to have biochemical similarities with putative cryoprotective proteins (Volger and Heber (1975)  Biochim. Biophys. Acta  412: 335-349). Volger and Heber have reported that the leaves of cold-acclimated cabbage and spinach, but not nonacclimated plants, contain proteins that are effective in protecting isolated thylakoid membranes against in vitro freeze-thaw damage. Subsequently, Hincha et al. (1990) reported that the cryoprotective proteins act by reducing membrane permeability during freezing and increasing membrane expandability during thawing (Hincha et al. (1990)  Planta  180: 416-419). The biochemical properties of highly enriched fractions of the cryoprotective proteins suggest that the cryoprotective proteins have a number of properties in common with the polypeptides encoded by many of the novel COR genes (Artus et al. (1996)  Proc. Natl. Acad. Sci.  93: 13404-13409).  
       [0011] The cold- and drought-regulated COR15a gene of  A. thaliana  encodes a 15-kDa polypeptide, COR15a, that is targeted to the stromal compartment of chloroplasts (Lin and Thomashow (1992)  Plant Physiol.  99: 519-525). During import, COR15a is processed to a mature 9.4-kDa polypeptide, COR15am, that is hydrophilic, remains soluble upon boiling, has a simple amino acid composition (it is rich in both alanine and lysine and devoid of proline, methionine, tryptophan, cysteine, glutamine, arginine, and histidine), is composed largely of a 13-amino acid motif that is repeated four times, and is predicted to form an amphipathic α-helix. The relatively simple amino acid composition of COR15am together with its predicted secondary structure suggests that the polypeptide might have a nonenzymatic function and was shown to have the properties of a cryoprotective protein. (See Artus et al. (1996) supra; Steponkus et al. (1998)  Proc. Natl. Acad. Sci.  95: 14570-14575.)  
       [0012] Thus, the present invention provides a method for modifying the plant biomass by modifying the size or number of leaves or roots of a plant, and for increasing the levels of cell protectants in a cell to allow the cell to tolerate greater environmental stresses.  
       SUMMARY OF THE INVENTION  
       [0013] In one aspect, the present invention provides a method for modifying plant biomass and the level of a cell protectant in a cell or a plant. The method comprises transforming a plant cell or plant with a recombinant polynucleotide comprising a sequence encoding a C-repeat/DRE binding factor (CBF)-related polypeptide and expressing the CBF-related polypeptide in the plant cell. Expression of the CBF-related polypeptide modifies biomass and the level of the cell protectant in the cell or plant. The method may optionally comprise cold-acclimating the cell to increase the levels of cell protectants in the transformed cell or plant even further.  
       [0014] In one embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising the AP2 domain comprising amino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of CBF1 (SEQ ID NO: 2). In a second embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising a CBF-related polypeptide and comprises one or more of the following peptides: PKXXAGR (SEQ ID NO: 319; amino acids 31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRXKF (SEQ ID NO: 320; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In a third embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising a CBF-related polypeptide and comprises one or more of the following peptides: PKKXAGR (SEQ ID NO: 322; amino acids 31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRKXF (SEQ ID NO: 324; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In a fourth embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising a CBF-related polypeptide and comprises one or more of the following peptides: PKKRAGR (SEQ ID NO: 323; amino acids 31-37 of SEQ ID NO. 2) or AGRKKF (SEQ ID NO: 325; amino acids 35-40 of SEQ ID NO. 2) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In a fifth embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising a CBF-related polypeptide and comprises one or more of the following peptides: PKKPAGR (SEQ ID NO: 326; amino acids 31-37 of SEQ ID NO. 2) or AGRKKF (SEQ ID NO: 325; amino acids 35-40 of SEQ ID NO. 2) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In another embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising SEQ ID NO:15. In yet another embodiment, the recombinant polynucleotide comprises SEQ ID NO:14.  
       [0015] Additionally, the recombinant polynucleotide may comprise a regulatory region operably linked to the sequence encoding the CBF-related polypeptide. The regulatory region may comprise a constitutive promoter, an inducible promoter, a tissue specific promoter or a developmental stage specific promoter. The leaf or root biomass of a plant may be modified. Cell protectants whose levels may be modified include proline, sugars, such as sucrose, or lipids, such as fatty acids, or cryoprotective proteins. As a result of the increased levels of any of these cell protectants, or of a combination of any these cell protectants, the environmental stress tolerance of a cell is increased. The environmental stresses may be cold or freezing tolerance, drought tolerance or high salinity tolerance.  
       [0016] In a second aspect, the present invention is another method for modifying the biomass of a plant. This method comprises altering the levels of a polynucleotide comprising a sequence encoding a C-repeat/DRE binding factor (CBF)-related polypeptide in a plant and identifying a plant so modified. The altered polynucleotide expression modifies the biomass of the plant.  
       [0017] In another aspect, the present invention is a method for improving the tolerance of a cell or plant to an environmental stress. The method comprises transforming the cell or plant with a recombinant polynucleotide comprising a sequence encoding a C-repeat/DRE binding factor (CBF)-related polypeptide and expressing the CBF-related polypeptide in the transformed cell or plant. Expression of the CBF-related polypeptide typically increases cell protectant levels at least 1.5 fold in the transformed cell or plant compared with cell protectant levels in an untransformed cell or plant. The enhanced cell protectant levels improve the environmental stress tolerance of the cell or plant.  
       [0018] In one embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising the AP2 domain comprising amino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of CBF1 (SEQ ID NO: 2). In a second embodiment, the CBF-related polypeptide may comprise one or more of the following peptides: PKXXAGR (SEQ ID NO: 319; amino acids 31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRXKF (SEQ ID NO: 320; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In a third embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising a CBF-related polypeptide and comprises one or more of the following peptides: PKKXAGR (SEQ ID NO: 322; amino acids 31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRKXF (SEQ ID NO: 324; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In a fourth embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising a CBF-related polypeptide and comprises one or more of the following peptides: PKKKAGR (SEQ ID NO: 323; amino acids 31-37 of SEQ ID NO. 2) or AGRKKF (SEQ ID NO: 325; amino acids 35-40 of SEQ ID NO. 2) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO. 2). In another embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising SEQ ID NO:15. In yet another embodiment, the recombinant polynucleotide comprises SEQ ID NO:14.  
       [0019] Additionally, the recombinant polynucleotide may comprise a regulatory region operably linked to the sequence encoding the CBF-related polypeptide. The regulatory region may comprise a constitutive promoter, an inducible promoter, a tissue specific promoter or a developmental stage specific promoter. Cell protectants whose levels may be modified include proline, sugars, such as sucrose, or lipids, such as fatty acids, or crypoprotective proteins. As a result of the increased levels of any of these cell protectants or the combination of any of these cell protectants, the environmental stress tolerance of a cell or plant is improved.  
       [0020] In a further aspect, the present invention is a method for producing a cell protectant. The method comprises transforming a cell or plant with a recombinant polynucleotide comprising a sequence encoding a C-repeat/DRE binding factor (CBF)-related polypeptide, expressing said recombinant polypeptide in the transformed cell so as to increase the levels of the cell protectant in the cell or plant, and then isolating the cell protectant from the transformed cell or plant. In one embodiment, the recombinant polynucleotide encodes a CBF-related polypeptide comprising SEQ ID NO:15. In another embodiment, the recombinant polynucleotide comprises SEQ ID NO:14. 
     
    
    
     BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS  
     [0021] The file of this patent contains at least one drawing executed in clor. Copies of this patent with clor drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.  
     [0022] 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.  
     [0023]FIGS. 1A and 1B show how the yeast reporter strains were constructed.  
     [0024]FIG. 1A is a schematic diagram showing the screening strategy.  
     [0025]FIG. 1B is a chart showing activity of the positive cDNA clones in yeast reporter strains.  
     [0026]FIGS. 2A, 2B,  2 C and  2 D provide an analysis of the pACT-11 cDNA clone.  
     [0027]FIG. 2A is a schematic drawing of the pACT-11 cDNA insert indicating the location and 5′ to 3′ orientation of the 24 kDa polypeptide and 25s rRNA sequences.  
     [0028]FIG. 2B is a DNA and amino acid sequence of the 24 kDa polypeptide (SEQ ID NO: 1 and SEQ ID NO: 2).  
     [0029]FIG. 2C is a schematic drawing indicating the relative positions of the potential nuclear localization signal (NLS), the AP2 domain and the acidic region of the 24 kDa polypeptide.  
     [0030]FIG. 2D is a chart showing comparison of the AP2 domain of the 24 kDa polypeptide (SEQ ID NO:10) with that of the tobacco DNA binding protein EREBP2 (SEQ ID NO:11).  
     [0031]FIG. 3 is a chart showing activation of reporter genes by the 24 kDa polypeptide.  
     [0032]FIG. 4 is a photograph of an electrophoresis gel showing expression of the recombinant 24 kDa polypeptide in  E. coli.    
     [0033]FIG. 5 is a photograph of a gel for shift assays indicating that CBF1 binds to the C-repeat/DRE.  
     [0034]FIG. 6 is a photograph of a Southern blot analysis indicating CBF1 is a unique or low copy number gene.  
     [0035]FIGS. 7A, 7B and  7 C relate to CBF1 transcripts in control and cold-treated Arabidopsis plants.  
     [0036]FIG. 7A is a photograph of a Northern blot analysis of RNA isolated from Arabidopsis plants that were grown at 22° C. (0 h) or grown at 22° C. and transferred to 2.5° C. for the indicated times (2, 4, 8, or 24 h).  
     [0037]FIG. 7B is a graph showing relative transcript levels of CBF1 in control and cold-treated plants.  
     [0038]FIG. 7C is a graph showing relative transcript levels of COR15a in control and cold-treated plants.  
     [0039]FIG. 8 is a Northern blot showing CBF1 and COR transcript levels in RLD and transgenic Arabidopsis plants.  
     [0040]FIG. 9 is an immunoblot showing COR15am protein levels in RLD and transgenic Arabidopsis plants.  
     [0041]FIGS. 10A and 11B are graphs showing freezing tolerance of leaves from RLD and transgenic Arabidopsis plants.  
     [0042]FIG. 11 is a photograph showing freezing survival of RLD and A6 Arabidopsis plants.  
     [0043]FIG. 12 shows the DNA sequence for CBF2 (SEQ ID NO:12) encoding the polypeptide sequence CBF2 (SEQ ID NO:13).  
     [0044]FIG. 13 shows the DNA sequence for CBF3 (SEQ ID NO:14) encoding the polypeptide sequence CBF3 (SEQ ID NO:15).  
     [0045]FIG. 14 shows the amino acid alignment of proteins CBF1 (SEQ ID NO:2), CBF2 (SEQ ID NO:13), and CBF3 (SEQ ID NO:15).  
     [0046]FIG. 15 is a graph showing induction of reporter genes in yeast that carry the C-repeat/DRE regulatory element CBF1, CBF2, or CBF3.  
     [0047]FIG. 16 shows the amino acid sequence of a portion of a canola CBF homolog (SEQ ID NO:17) and its alignment to the amino acid sequence of CBF1 (SEQ ID NO:2).  
     [0048]FIGS. 17A, 17B,  17 C,  17 D,  17 E,  17 F and  17 G show restriction maps of plasmids pMB12008, pMB12009, pMB12010, pMB12011, pMB12012, pMB12013, and pMB12014, respectively.  
     [0049]FIG. 18A shows the DNA sequences for the CBF homologs from  Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Glycine max, Raphanus sativus,  and  Zea mays.    
     [0050]FIG. 18B shows the amino acid sequences (one-letter abbreviations) encoded by the DNA sequences (shown in FIG. 18A) for CBF homologs from  Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Glycine max, Raphanus sativus,  and  Zea mays.    
     [0051]FIG. 19A shows an amino acid alignment of the AP2 domains of several CBF proteins (SEQ ID NOs: 137 through 167) with the consensus sequence between the proteins highlighted as well as a comparison of the AP2 domains with that of the tobacco DNA binding protein EREBp2 (SEQ ID NO: 168).  
     [0052]FIG. 19B shows an amino acid alignment of the AP2 domains of several CBF proteins (SEQ ID NOs: 137 through 167) including dreb2a (SEQ ID NO: 169) and dreb2b (SEQ ID NO: 170) with the consensus sequence between the proteins highlighted.  
     [0053]FIG. 19C shows an amino acid alignment of the AP2 domains of several CBF proteins (SEQ ID NOs: 137 through 167) including dreb2a (SEQ ID NO: 169), dreb2b (SEQ ID NO: 170), and tiny (SEQ ID NO: 171) with the consensus sequence between the proteins highlighted.  
     [0054]FIG. 19D shows a difference between the consensus sequence shown in FIG. 19A (SEQ ID NOs: 137 through 167) and tiny (SEQ ID NO: 171).  
     [0055]FIG. 19E shows a difference between the consensus sequence shown in FIG. 19B (SEQ ID NOs: 137 through 167) and tiny (SEQ ID NO: 171).  
     [0056]FIG. 20 shows an amino acid alignment of the amino terminus of several CBF proteins (SEQ ID NOs: 172 through 194) with their consensus sequence highlighted.  
     [0057]FIG. 21A (SEQ ID NOs: 195 through 266) and FIG. 21B (SEQ ID NOs: 267 through 293) show an amino acid alignment of the carboxy terminus of several CBF proteins, with their consensus sequences highlighted.  
     [0058]FIG. 22 shows the effect of CBF3 expression on proline levels. Free proline levels were determined in leaf tissue from control Ws-2 and B6 plants and CBF3-expressing A40, A30 and A28 plants grown at 20° C. (warm) or plants grown at 20° C. and cold-treated at 5° C. for 7 days (7 d cold).  
     [0059]FIG. 23 shows the effect of CBF3 expression on transcript levels of genes involved in proline and sugar metabolism. Northern analysis of total RNA (20 μg for CBF3; 5 μg for other genes) isolated from control Arabidopsis Ws-2 and B6 plants and from CBF3-expressing A40, A30 and A28 plants. Plants were grown at 20° C., then cold-treated at 5° C. for the times indicated. The blots were hybridized with probes for CBF3, COR78, P5CS2, Suc synthase (SuSy), Suc-phosphate synthase (SPS), and elF4a, a constitutively expressed gene used as a loading control (Metz et al.  Gene  120: 313 (1992)).  
     [0060]FIG. 24 shows the effect of CBF3 Expression on levels of total soluble sugars. Total soluble sugars were determined for leaf tissue from control Ws-2 and B6 plants and CBF3-expressing A40, A30 and A28 plants grown at 20° C. (warm) or plants grown at 20° C. and cold-treated at 5° C. for 7 days (7 d cold).  
     [0061]FIG. 25 shows the effect of CBF3 expression on fatty acid composition. The fatty acid profiles of total lipids extracted from leaf tissue of control Ws-2 plants and CBF3-expressing A28 plants were determined for plants grown at 20° C. (unfilled bars) or at 20° C. followed by 7 days at 5° C. (filled bars).  
     [0062]FIG. 26 shows the effect of CBF3 expression on freezing tolerance. (A) Seedlings of Ws-2 and A30 were grown at 20° C. on solid medium and frozen at −2° C. for 24 hours followed by 24 hours at −6+ C.; (B) Control Ws-2 and CBF3-expressing transgenic A40, A30 and A28 plants were grown at 20° C. and the freezing tolerance of leaves was measured using the electrolyte leakage test; (C and D) Same as (B) except that plants were grown at 20° C. followed by 7 days cold acclimation at 5° C.  
     [0063] FIGS.  27 A-C show a comparison of leaf number to bolting time (A), biomass to bolting time (B) and days to bolt (C) for  Arabidopsis thaliana  plants overexpressing a CBF-related polypeptide (13-4, 2-1, 16-3, 16-2, 17-10) compared with a wild-type plant (wt-control).  
     [0064]FIG. 28 shows a comparison of the leaves of two plants one overexpressing a polynucleotide encoding a CBF-related polypeptide (A) and a second plant not overexpressing the polynucleotide (B).  
     [0065]FIG. 29 shows a comparison of the roots of two plants one overexpressing a polynucleotide encoding a CBF-related polypeptide (A) and a second plant not overexpressing the polynucleotide (B).  
     [0066]FIG. 30 shows a comparison of the % electrolyte leakage for a plant overexpressing CBF3 (SEQ ID NO: 15) (A40a2, A28a3, A28a3) or a control plant (WS-2), none of which have been cold acclimated.  
     [0067]FIG. 31 shows a comparison of the % electrolyte leakage for a plant overexpressing CBF3 (SEQ ID NO: 15) (A28a3, A30a1) or a control plant (WS-2) after cold acclimation.  
     [0068]FIG. 32 shows a comparison of the % electrolyte leakage for a plant overexpressing CBF2 (SEQ ID NO: 13) (E24, E8 or E2) or a control plant (WS-2, B6), none of which have been cold acclimated.  
     [0069]FIG. 33 shows a comparison of the % electrolyte leakage for a plant overexpressing CBF2 (SEQ ID NO: 13) (E24, E8 or E2) or a control plant (WS-2, B6), after cold acclimation.  
     [0070]FIGS. 34A through 34D shows an alignment of CBF sequences from Arabidopsis (SEQ ID NOs: 2, 15, 13, and 97) and from  Medicago trunculata  (SEQ ID NOs: 294 through 300). Amino acid residues with identity between aligned sequences are boxed. The consensus sequence is shown below each set of alignments.  
     [0071]FIGS. 35A through 35D shows an alignment of CBF sequences from Arabidopsis (SEQ ID NOs: 2, 15, 13, and 97) and from  Oryza sativa  (SEQ ID NOs: 301 through 310). Amino acid residues with identity between aligned sequences are boxed. The consensus sequence is shown below each set of alignments.  
     [0072]FIGS. 36A through 36C shows an alignment of CBF sequences from Arabidopsis (SEQ ID NOs: 2, 15, 13, and 97) and from  Zea mays  (SEQ ID NOs: 311 through 315). Amino acid residues with identity between aligned sequences are boxed. The consensus sequence is shown below each set of alignments.  
     [0073]FIG. 37 shows that CBFs from  Medicago trunculata, Oryza sativa,  and Arabidopsis (CBF3) activate transcription from the Arabidopsis COR gene promoter from the RD29a gene, as measured by fold-increase over control (pMEN65 plasmid control=1 unit). The CBF sequences are: Arabidopsis CBF3 (SEQ ID NO:15; CBF3),  Medicago trunculata  CBF (SEQ ID NO:294; mt G3362),  Medicago trunculata  CBF (SEQ ID NO:295; mt G3364),  Medicago trunculata  CBF (SEQ ID NO:296; mt G3365),  Medicago trunculata  CBF (SEQ ID NO:297; mt G3366),  Medicago trunculata  CBF (SEQ ID NO:298; mt G3367),  Medicago trunculata  CBF (SEQ ID NO:299; mt G3368),  Medicago trunculata  CBF (SEQ ID NO:300; mt G3369),  Oryza sativa  CBF (SEQ ID NO:301; os G3370),  Oryza sativa  CBF (SEQ ID NO:302; os G3371),  Oryza sativa  CBF (SEQ ID NO:303; os G3372),  Oryza sativa  CBF (SEQ ID NO:304; os G3373),  Oryza sativa  CBF (SEQ ID NO:308; os G3377),  Oryza sativa  CBF (SEQ ID NO:309; os G3378), and  Oryza sativa  CBF (SEQ ID NO:310; os G3379). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0074] Definitions  
     [0075] “Environmental stress tolerance” refers to a decrease in the extent of a cell&#39;s injury or growth inhibition or an increase in survival rate after exposure to cold or freezing temperatures, drought conditions, high salinity environments or the like.  
     [0076] A “cell protectant” refers to a compound that improves the environmental stress tolerance of a cell. The cell protectant may be a cryoprotectant or an osmoprotectant. The cell protectant may be proline or any metabolically related compound, sugars or any metabolically related compound, and a variety of lipids, including fatty acids, which protect a cell&#39;s integrity during an environmental stress.  
     [0077] A “polynucleotide” is a nucleotide sequence comprising a gene coding sequence or a fragment thereof (comprising at least 18 consecutive nucleotides, preferably at least 30 consecutive nucleotides, and more preferably at least 50 consecutive nucleotides). 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 may comprise single stranded or double stranded DNA or RNA. The polynucleotide may comprise modified bases or a modified backbone. The polynucleotide may be genomic, a transcript (such as an mRNA) or a processed nucleotide sequence (such as a cDNA). The polynucleotide may comprise a sequence in either sense or antisense orientations.  
     [0078] A “recombinant polynucleotide” is a polynucleotide that is not in its native state, for example, the polynucleotide is comprised of a nucleotide sequence not found in nature or the polynucleotide is separated from nucleotide sequences with which it typically is in proximity or is next to nucleotide sequences with which it typically is not in proximity or is expressed at different levels.  
     [0079] A “consensus sequence”, with regard to nucleotide sequences, refers to a nucleotide sequence that serves to represent a family of similar, experimentally-derived sequences. Each position in the consensus sequence is assigned a base that corresponds to the most frequently occurring nucleotide in the experimentally-derived sequences, when sequences are compared in an alignment. A “consensus sequence”, with regard to polypeptide sequences, refers to a polypeptide sequence that serves to represent a family of similar, experimentally-derived sequences. Each position in the consensus sequence is assigned an amino acid residue that corresponds to the most frequently occurring amino acid residue in the experimentally-derived sequences, when sequences are compared in an alignment.  
     [0080] A “transformed plant” refers to a plant that contains genetic material not normally found in a wild type plant and which has been introduced into a plant by human manipulation. A transformed plant is a plant that may contain an expression vector or cassette. The expression cassette comprises a gene coding sequence and allows for the expression of the gene coding sequence. The expression cassette may be introduced into a plant by transformation or by breeding after transformation of a parent cell. In particular, the transformed plant may refer to a whole plant as well as to a plant part, such as flower, seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, and progeny thereof.  
     [0081] A “transformed cell” refers to a cell that contains genetic material not normally found in a wild type cell and which has been introduced into the cell by human manipulation. A transformed cell is a cell that may contain an expression vector or cassette. The expression cassette comprises a gene coding sequence and allows for the expression of the gene coding sequence. The expression cassette may be introduced into a cell by transformation or by breeding after transformation of a parent cell. A transformed cell may refer to a cell from any organism, including mammalian cells, plant cells, bacterial cells, and the like. In particular, the transformed cell is a plant cell and may refer 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, and progeny thereof.  
     [0082] The term “modified expression” in reference to polynucleotide or polypeptide expression refers to an expression pattern in a transformed cell or plant that is different from the expression pattern in the wild type cell or plant; for example, by expression in a cell type or plant tissue other than a cell type or plant tissue in which the polynucleotide or polypeptide is naturally expressed, or by expression at a time other than at the time the polynucleotide or polypeptide is expressed in the wild type cell or 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 to those observed a wild type cell or plant. The term may also refer to lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern may be transient or stable, constitutive or inducible.  
     [0083] A “CBF-related polypeptide” or “CBF” is a protein transcription factor that binds to a promoter comprising a cold- and dehydration-responsive DNA regulatory element known as the CRT (C-repeat)/DRE (dehydration responsive element) (Baker et al. (1994) i Plant. Mol. Biol. 24: 701-713; Yamaguchi-Shinozaki and Shinozaki (1994)  Plant Cell  6: 251-264). The terms “CBF-related polypeptide” and “CBF” are interchangeable. These proteins comprise an AP2/EREBP DNA binding motif (Riechmann and Meyerowitz (1998)  Biol. Chem.  379: 633-646) and are transcription factors (Stockinger et al. (1997)  Proc. Natl. Acad. Sci.  94: 1035-1040). The AP2 domain, which may also be referred to as the ERF domain (Hao et al. (1998)  J. Biol. Chem  273: 26857-26861) may comprise amino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of CBF1 (G40; SEQ ID NO: 2). Additionally, the CBF-related polypeptide may comprise one or more of the following peptides: PKXXAGR (SEQ ID NO: 319; amino acids 31-37 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or AGRXKF (SEQ ID NO: 320; amino acids 35-40 of SEQ ID NO. 2; ‘X’ may represent any amino acid) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO: 2).  
     [0084] A “regulatory region” is a region that can regulate the transcription of a gene coding sequence. The regulatory region may be a promoter or an enhancer. The regulatory region sequence is “operably linked” when it is placed into a functional relationship with the gene coding sequence. For example, a promoter or enhancer is operably linked to a gene coding sequence if the presence of the promoter or enhancer increases the level of expression of the gene coding sequence. Promoters may increase transcription of a gene at all times (constitutive promoter), increase transcription only in the presence of specific agents or events (inducible promoter), increase transcription in specific tissue(s) (tissue specific promoter) or during specific stages of cell or tissue or organism development (developmental stage specific promoter).  
     [0085] “Plant biomass modification” refers to a detectable difference in the size of one or more plant tissues, such as the leaves, roots, stem, fruit or flowers of a transformed plant expressing a polynucleotide or polypeptide of the present invention compared with a plant not doing so. The modification may entail at least a 5% increase or decrease in an observed trait (difference), at least a 10% difference, at least a 20% difference, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater difference. It is known that there may be a natural variation in plant biomass. Therefore, the modification observed entails a change in the normal distribution of the trait in transformed plants compared with the distribution observed in wild type plants.  
     [0086] “Cell protectant level modification” refers to a detectable difference in cell protectant levels in a transformed cell expressing a polynucleotide or polypeptide of the present invention compared with a cell not doing so, such as a wild type cell. The trait modification may entail at least a 5% increase or decrease in an observed trait (difference), at least a 10% difference, at least a 20% difference, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater difference. It is known that there may be a natural variation in the modified cell protectant levels. Therefore, the cell protectant level modification observed entails a change in the normal distribution of the levels in transformed cells compared with the distribution observed in wild type cells.  
     [0087] “Cold-acclimating a cell” and “cold acclimation” refers to a process whereby a transformed cell is exposed to temperatures below about 12° C. for different periods of time to elicit higher levels of a cell protectant compared with cell protectant levels in a cell that has not been cold acclimated.  
     [0088] Traits that May Be Modified  
     [0089] Trait modifications of particular interest include those to seed (such as embryo or endosperm), fruit, root, flower, leaf, stem, shoot, seedling or the like, including: enhanced tolerance to environmental conditions including freezing, chilling, heat, drought, water saturation, radiation and ozone; improved tolerance to microbial, fungal or viral diseases; improved tolerance to pest infestations, including nematodes, mollicutes, parasitic higher plants or the like; decreased herbicide sensitivity; improved tolerance of heavy metals or enhanced ability to take up heavy metals; improved growth under poor photoconditions (for example, low light and/or short day length), or changes in expression levels of genes of interest. Other phenotype that can be modified relate to the production of plant metabolites, such as variations in the production of taxol, tocopherol, tocotrienol, sterols, phytosterols, vitamins, wax monomers, anti-oxidants, amino acids, lignins, cellulose, tannins, prenyllipids (such as chlorophylls and carotenoids), glucosinolates, and terpenoids, enhanced or compositionally altered protein or oil production (especially in seeds), or modified sugar (insoluble or soluble) and/or starch composition. Physical plant characteristics or traits that can be modified include cell development (such as the number of trichomes), fruit and seed size and number, yields of plant parts such as stems, leaves, inflorescences, and roots, the stability of the seeds during storage, characteristics of the seed pod (for example, susceptibility to shattering), root hair length and quantity, internode distances, or the quality of seed coat. Plant growth characteristics that can be modified include growth rate, germination rate of seeds, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, rate of nitrogen uptake, osmotic sensitivity to soluble sugar concentrations, biomass or transpiration characteristics, as well as plant architecture characteristics such as apical dominance, branching patterns, number of organs, organ identity, organ shape or size.  
     [0090] Transcription Factors Modify Expression of Endogenous Genes  
     [0091] Expression of genes that encode transcription factors that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997)  Genes and Development  11: 3194-3205) and Peng et al. (1999)  Nature  400: 256-261). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001)  Plant Cell  13: 1791-1802); Nandi et al. (2000)  Curr. Biol.  10: 215-218); Coupland (1995)  Nature  377: 482-483); and Weigel et al. (1995)  Nature  377: 482-500).  
     [0092] In another example, Mandel et al. (1992)  Cell  71: 133-143) and Suzuki et al. (2001) i Plant J. 28: 409-418) teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis. Other examples can be found in the teachings of Müller et al. (2001,  Plant J.  28: 169-179); Kim et al. (2001)  Plant J.  25: 247-259); Kyozuka and Shimamoto (2002)  Plant Cell Physiol.  43: 130-135); Boss and Thomas (2002)  Nature,  416: 847-850); He et al. (2000)  Transgenic Res.  9: 223-227); and Robson et al. (2001)  Plant J.  28: 619-631).  
     [0093] In yet another example, Gilmour et al. (1998)  Plant J.  16: 433-442) and Jaglo-Ottosen et al. (1998)  Science  280: 104-106) teach an Arabidopsis AP2 transcription factor, CBF1, which, when overexpressed in transgenic plants, increases plant freezing tolerance. An alignment of the CBF proteins from Arabidopsis,  B. napus, l wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that flank the AP 2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family.  
     [0094] Polypeptides and Polynucleotides of the Invention  
     [0095] 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 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&#39;s characteristic.  
     [0096] Exemplary polynucleotides encoding the polypeptides of the invention were identified by screening an Arabidopsis cDNA expression library for clones encoding C-repeat/DRE binding domains (Stockinger et al. (1997)  Proc. Natl. Acad. Sci.  94: 1035-1040). The cDNA library harbored in  Escherichia coli  BNN132 was amplified, and plasmid DNA was isolated and transformed into yeast GGY1 reporter strains. CBF genes were expressed in  E. coli  and yeast, and gel shift assays, in which orientation and concatenation number of inserts were determined by dideoxy DNA sequence analysis, were conducted to confirm that the CBF gene products bound to the C-repeat/DRE (Stockinger (1997) supra). 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.  
     [0097] Additional polynucleotides of the invention were identified by screening  Arabidopsis thaliana  and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure, using a commercially available kit according to the manufacturer&#39;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.  
     [0098] 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)characteristics or traits of the plants observed. Therefore, the polynucleotides and polypeptides can be employed to improve the characteristics or traits of plants.  
     [0099] 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.  
     [0100] Producing Polypeptides  
     [0101] The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homolog polypeptides and sequences complementary thereto, as well as unique fragments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, for example, DNA or RNA, for example, mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homolog polypeptide, in isolation, in combination with additional coding sequences (for example, a purification tag, a localization signal, as a fusion-protein, as a pre-protein, or the like), in combination with non-coding sequences (for example, introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homolog polypeptide is an endogenous or exogenous gene.  
     [0102] A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, for example, Berger and Kimmel,  Guide to Molecular Cloning Techniques Methods in Enzymology  volume 152; Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al.  Molecular Cloning—A Laboratory Manual  (2nd edition), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) (“Sambrook”) and  Current Protocols in Molecular Biology,  F. M. Ausubel et al., editors,  Current Protocols,  a joint venture between Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., (supplemented through 2000) (“Ausubel”).  
     [0103] 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), for example, for the production of the homologous nucleic acids of the invention are found in Berger (supra), Sambrook (supra) and Ausubel (supra), as well as Mullis et al. (1987)  PCR Protocols A Guide to Methods and Applications,  Innis et al., editors, (1990) Academic Press Inc. San Diego, Calif. (“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, for example, Ausubel, Sambrook and Berger, all supra.  
     [0104] Alternatively, polynucleotides and oligonucleotides of the invention can be assembled from fragments produced by solid-phase synthesis methods. Typically, fragments of up to approximately 100 bases are individually synthesized and then enzymatically or chemically ligated to produce a desired sequence, for example, a polynucleotide encoding all or part of a transcription factor. For example, chemical synthesis using the phosphoramidite method is described, for example, by Beaucage et al. (1981)  Tetrahedron Letters  22: 1859-1869; and Matthes et al. (1984)  EMBO J.  3: 801-805. According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.  
     [0105] Homologous Sequences  
     [0106] Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing, derived from  Arabidopsis thaliana  or from other plants of choice are also an aspect of the invention. Homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn, potato, cotton, rice, rape, oilseed rape (including rapeseed and canola), sunflower, alfalfa, sugarcane and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops, fruits and vegetables whose phenotype can be changed 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.  
     [0107] Orthologs and Paralogs  
     [0108] 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 paralogs and orthologs are described; a paralog or ortholog or homolog may be identified by one or more of the methods described below.  
     [0109] 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 and that are derived by a duplication event.  
     [0110] 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 similar function known as paralogs. A paralog is therefore a similar gene with a similar function within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994)  Nucleic Acids Res.  22: 4673-4680; Higgins et al. (1996)  Methods Enzymol.  266 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng et al. (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) supra). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the lade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function. (See also, for example, Mount, D. W. (2001)  Bioinformatics: Sequence and Genome Analysis  Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., page 543.)  
     [0111] Speciation, the production of new species from a parental species, can also give rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994)  Nucleic Acids Res.  22: 4673-4680; Higgins et al. (1996)  Methods Enzymol.  266: 383-402), potential orthologous sequences can placed into the phylogenetic tree and its relationship to genes from the species of interest can be determined. Once the ortholog pair has been identified, the function of the test ortholog can be determined by determining the function of the reference ortholog.  
     [0112] Transcription factors that are homologous to the listed sequences will typically share at least about 30% amino acid sequence identity, or at least about 30% amino acid sequence identity outside of a known consensus sequence or consensus DNA-binding site. More closely related transcription factors can share at least about 50%, about 60%, about 65%, about 70%, about 75% or about 80% or about 90% or about 95% or about 98% or more sequence identity with the listed sequences, or with the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site, or with the listed sequences excluding one or all conserved domain. Factors that are most closely related to the listed sequences share, for example, at least about 85%, about 90% or about 95% or more % sequence identity to the listed sequences, or to the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site or outside one or all conserved domain. At the nucleotide level, the sequences will typically share at least about 40% nucleotide sequence identity, preferably at least about 50%, about 60%, about 70% or about 80% sequence identity, and more preferably about 85%, about 90%, about 95% or about 97% or more sequence identity to one or more of the listed sequences, or to a listed sequence but excluding or outside a known consensus sequence or consensus DNA-binding site, or outside one or all conserved domain. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein. Conserved domains within a transcription factor family may exhibit a higher degree of sequence homology, such as at least 65% 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. In addition, 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 similarity over the entire length of the polypeptide or the homolog.  
     [0113] Percent identity can be determined electronically, for example, by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins et al. (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. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, for example, each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).  
     [0114] Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), editor, Doolittle, Academic Press, Inc., San Diego, Calif. 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  Methods Mol. Biol.  70: 173-187 (1997). Also, the CLUSTALW alignment algorithm (for example, in the MACVECTOR 6.0 or MACVECTOR 6.5 applications, Accelrys, San Diego Calif.) may be utilized to align sequences. 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.  
     [0115] The percentage similarity between two polypeptide sequences, for example, sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, for example, the Jotun Hein method (see, for example, Hein (1990)  Methods Enzymol.  183: 626-645). Identity between sequences can also be determined by other methods known in the art, for example, by varying hybridization conditions (see U.S. patent application No. 20010010913).  
     [0116] 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 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.  
     [0117] In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997)  Nucleic Acids Res.  25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992)  Protein Eng.  5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul, S. F. (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 &amp; Sons, New York N.Y., unit 7.7) and in Meyers (1995;  Molecular Biology and Biotechnology,  Wiley VCH, New York N.Y., pages 856-853).  
     [0118] 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 conserved 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.  
     [0119] CBF Genes and Related Sequences  
     [0120] Many plants, including Arabidopsis, show increased resistance to freezing after they have been exposed to low, non-freezing temperatures. This cold-acclimation response is associated with the induction of COR (cold-regulated) genes mediated by the C-repeat/drought-responsive element (CRT/DRE) DNA regulatory element) (Baker et al. (1994)  Plant. Mol. Biol.  24: 701-713; Yamaguchi-Shinozaki and Shinozaki (1994)  Plant Cell  6: 251-264). Increased expression of Arabidopsis CBF genes (transcriptional activators that bind to the CRT/DRE sequence), induce COR gene expression and increase the freezing tolerance of non-cold acclimated Arabidopsis plants. CBF genes are thus regulators of the cold acclimation response, and act by controlling the level of COR gene expression, which in turn promotes tolerance to freezing. CBF genes have also been shown to influence cell protectant levels such as proline in plants, which can lead to increased plant biomass (Kishor et al. (1995)  Plant Physiol.  108: 1387-1394).  
     [0121] It is believed that a significant class of environmental stress tolerance and biomass regulatory genes encode for binding proteins with an AP2 domain capable of binding to a DNA regulatory sequence. Each of the presently disclosed CBF gene sequences encodes a binding protein that includes an AP2 domain, the latter being a DNA-binding motif similar to those present in Arabidopsis proteins APETALA2, AINTEGUMENTA and TINY, the tobacco ethylene response element binding proteins, and numerous other plant proteins. The AP2 domains of CBF binding proteins in general, including the CBF binding proteins described herein, share significant homology, and comprise a consensus sequence sufficiently homologous to any one of the consensus sequences shown in FIG. 19A, 19B, or  19 C that the binding protein is capable of binding to a CCG regulatory sequence, preferably a CCGAC regulatory sequence. Specifically, CBF proteins comprise an AP2/EREBP DNA binding motif (Riechmann and Meyerowitz (1998)  Biol. Chem.  379: 633-646) and are transcription factors (Stockinger et al. (1997) supra). The AP2 domain of CBF proteins may comprise amino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of CBF1 (G40; SEQ ID NO: 2), or the consensus sequence:  
     [0122] H P X n  Y X n  G V R X n  R X n  W V X n  E X n  R E X n  N K X n  R I W X n  G T F X n  T X n  E X n  A A R A H D V A A X n  A L R G X n  A X n  L N X n  A D S  
     [0123] where X is any amino acid residue and n is any number of amino acid residues.  
     [0124] CBF1 (G40; SEQ ID NO: 2), CBF2 (G41; SEQ ID NO: 13) and CBF3 (G42; SEQ ID NO: 15) have similar sequences, particularly in the AP2 domain (defined by the consensus sequence defined above and bounded by: HP X n  Y X n  GVR X n  ADS). CBF2 has 95% sequence identity with CBF1 in the AP2 domain, and CBF3 shares 96% sequence identity with CBF1 in the AP2 domain. These three and related genes can be used to prepare transgenic plants and plants with altered traits. CBF1, for example, was studied using transgenic plants in which the gene encoding the protein was expressed under the control of the 35S promoter (for a more complete discussion of studies involving CBF1, CBF2 and CBF3 experiments, see examples below). CBF1 was shown to improve tolerance to freezing and salt stress in Arabidopsis and Canola. CBF1 could thus be used to manipulate those tolerances, and to generate plants that might germinate and survive under such adverse conditions. For example, evaporation from the soil surface causes upward water movement and salt accumulation in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt concentration in the whole soil profile. Increased salt tolerance during the germination stage of a crop plant would impact survivability and yield. If the activity of CBF1 is regulated at a post-translational level (for example, by being phosphorylated), it might be possible to engineer constitutively active versions of the protein that protect the plant under adverse environmental conditions As seen in FIGS. 27A through 27C, CBF1-overexpressing plants grew more slowly, than the control plants. However, as the transgenic plants flowered later they grew for a longer period of time prior to bolting; at this developmental stage the transgenic plants had more leaves and higher biomass than the control plants at the same developmental stage (which the control plant reached a few days earlier).  
     [0125] Similarly, overexpression of CBF3 in transformed plants increased the time to flowering; the plants grew for a longer period of time prior to bolting and increased the number of rosette leaves per plant (see Example 22, below). Thus, CBF polypeptide overexpression could be used to manipulate time to flowering, leaf number, and plant biomass. If the activity of CBF polypeptides is regulated at a post-translational level (for example, by being phosphorylated), it might be possible to engineer constitutively active versions of the protein that increase leaf number and/or biomass.  
     [0126] G912 (CBF4; SEQ ID NO: 97). G912 was recognized by Applicants as the AP2/EREBP gene most closely related to Arabidopsis CBF1, CBF2, and CBF3 (SEQ ID NO: 2, 13, and 15, respectively) (Haake et al. (2002)  Plant Physiol.  130: 639-648; Stockinger et al. (1997) supra); Gilmour et al. (1998) supra), G912 shares 93%, 91 and 93% sequence identity with CBF1, CBF2 and CBF3, respectively, in the AP2 domain. G912 sequence similarity with CBF1, 2 and 3 extends beyond the conserved AP2 domain. This AP2/EREBP transcription factor is also closely related to the members of the CBF-like subgroup of AP2/EREBP proteins from other plants, such as AF084185  Brassica napus  dehydration responsive element binding protein. G912 was identified in the sequence of P1 clone MSG15 (GenBank accession number AB015478; gene MSG15.6; no published information is available about the functions of G912).  
     [0127] G912 expression appears to be induced by cold, drought, and osmotic stress. The function of G912 was studied using transgenic plants in which this gene was expressed under the control of the 35S promoter. As with plants overexpressing CBF1, CBF2 and CBF3, plants overexpressing G912 were dark green, and flowered later than non-transformed control plants. Plants overexpressing G912 were more freezing and drought tolerant than the wild-type controls.  
     [0128] All these results mirror the extensive body of work presented herein that has shown that related genes CBF1, CBF2, and CBF3 are involved in the control of the low-temperature response in Arabidopsis, and that those genes can be used to improve freezing, drought, and salt tolerance in plants (Stockinger et al. (1997) supra; Gilmour et al. (1998) supra; Jaglo-Ottosen et al. (1998)  Science  280: 104-106; Liu et al. (1998)  Plant Cell  10: 1391-1406, Kasuga et al. (1999)  Nature Biotechnol.  17: 287-291). In addition, G912 overexpressing plants also exhibit a sugar sensing phenotype: i.e., reduced seedling vigor and cotyledon expansion upon germination on high glucose media.  
     [0129] Polypeptide Transcription Factors from other Plant Species (Odd Numbered SEQ ID NO: 39-95 and Even numbered 116-128).  
     [0130] As described in more detail in Example 15, below, a PCR strategy was used to isolate CBF homologs from a number of species of plants both related and diverse from Arabidopsis. These species included  Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Glycine max, Raphanus sativus, Secale cereale, Triticum aestivum,  and  Zea mays.  The nucleotide (for example, bjCBF1) and peptide sequences (for example, BJCBF 1-PEP) of these isolated CBF homologs are shown in FIGS. 18A and 18B, respectively. Table 11 (which may be found in Example 15) lists the sequence names and SEQ ID NO: of these isolated CBF homologs. The percentage sequence identity of each of the AP2 domains of the sequences from other species with the Arabadopsis CBF1 AP2 domain (subsequence of G40, SEQ ID NO: 2) is also shown and is 80% identity for the  Zea mays  AP2 domain and from 85-93% for the other six species.  
     [0131] SEQ ID NOs: 39-45 are from  B. juncea.  The AP2 regions present in each of these sequences, and which may be found as subsequences in the corresponding sequences in the Sequence Listing, are:  
     [0132] SEQ ID NO: 39 (percent sequence identity with CBF1: 87% (50/57)): PGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRAACLNFADS  
     [0133] SEQ ID NO: 143 (percent sequence identity with CBF1: 85% (53/62)): HPIYRGVRLRKSGKWVCEVREPNKRSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS  
     [0134] SEQ ID NO: 145 (percent sequence identity with CBF1: 85% (53/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFLTAEIAARAHDVAAIALRGKSACLNFADS  
     [0135] SEQ ID NO: 140 (percent sequence identity with CBF1: 93% (58/62)): HPIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFADS  
     [0136] SEQ ID NOs: 47-63 are from  B. napus.  The AP2 regions present in each of these sequences are:  
     [0137] SEQ ID NO: 148 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0138] SEQ ID NO: 146 (percent sequence identity with CBF1: 87% (54/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0139] SEQ ID NO: 147 (percent sequence identity with CBF1: 87% (54/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0140] SEQ ID NO: 153 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0141] SEQ ID NO: 154 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0142] SEQ ID NO: 149 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0143] SEQ ID NO: 144 (percent sequence identity with CBF1: 87% (54/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS  
     [0144] SEQ ID NO: 150 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0145] SEQ ID NO: 165 (percent sequence identity with CBF1: 85% (53/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWPGTFKTAEMAARAHDVAALALRGRGARLNYADS  
     [0146] SEQ ID NOs: 65-73 are from  B. oleracea.  The AP2 regions present in each of these sequences are:  
     [0147] SEQ ID NO: 163 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRAACLNFADS  
     [0148] SEQ ID NO: 141 (percent sequence identity with CBF1: 87% (54/62)): HPVYRGVRLRNSGKWVCEVREPNKKSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS  
     [0149] SEQ ID NO: 151 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0150] SEQ ID NO: 155 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0151] SEQ ID NO: 164 (percent sequence identity with CBF1: 87% (54/62)): HPIYRGVRLRKSGKWVCEVRELNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0152] SEQ ID NOs: 75-87 are from  B. rapa.  The AP2 regions present in each of these sequences are:  
     [0153] SEQ ID NO: 156 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0154] SEQ ID NO: 152 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADS  
     [0155] SEQ ID NO: 157 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0156] SEQ ID NO: 158 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0157] SEQ ID NO: 159 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0158] SEQ ID NO: 160 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0159] SEQ ID NO: 161 (percent sequence identity with CBF1: 88% (55/62)): HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0160] SEQ ID NO: 89 is from  Glycine max.  The AP2 region present in this sequence is:  
     [0161] SEQ ID NO: 167 (percent sequence identity with CBF1: 87%): HPIYSGVRRRNTDKWVSEVREPNKKTRIWLGTFPTPEMAARAHDVAAMALRGRYACLNFADS  
     [0162] SEQ ID NO: 91-93 are from  Raphanus sativus.  The AP2 regions present in each of these sequences are:  
     [0163] SEQ ID NO: 162 (percent sequence identity with CBF1: HPIYRGVRLRKSGKWVCEVREPNKKSRIWLGTFKTAEIAARAHDVAALALRGRGACLNFADS  
     [0164] SEQ ID NO: 142 (percent sequence identity with CBF1: 88% (55/62)):  
     [0165] HPIYRGVRLRNSGKWVCEVREPNKKSRIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADS  
     [0166] SEQ ID NO: 95 is from  Zea mays.  The AP2 region present in this sequence is:  
     [0167] SEQ ID NO: 166 (percent sequence identity with CBF1: 80% (51/63)): HPVYRGVRRRGPAGRWVCEVREPNKKSRIWLGTFATPEAAARAHDVAALALRGRAACLNFADS  
     [0168] SEQ ID NO: 116-124 are from  Secale cereale.  The AP2 regions present in each of these sequences are:  
     [0169] SEQ ID NO: 116 (percent sequence identity with CBF1: 71% (37/52)) ATAQDGEVGAAGRWVCEVRVLGMRGSRLWLGTFVTAEMAARAHDAAVLALSGRKACLNFADS  
     [0170] SEQ ID NO: 118 (percent sequence identity with CBF1: 72% (46/64)) HPLYRGVRRRGRVGQWVCEVRVPGIKGSRLWLGTFNTAEMAARAHDAAVLALSCRAACLNFADS  
     [0171] SEQ ID NO: 120 (percent sequence identity with CBF1: 73% (47/64)) HPLYRGVRRRGRVGQWVCEVRVPGIKGSRLWLGTFNTAEMAARAHDAAVLALSGRKACLNFADS  
     [0172] SEQ ID NO: 122 (percent sequence identity with CBF1: 69%(44/64)) HPLYRGVRRRGRLGQWVCEVRVRGAQGYRLWLGTFTTAEMAARAHDSAVLALLDRAACLNFADS  
     [0173] SEQ ID NO: 124 (percent sequence identity with CBF1: (73% (47/64) HPLYRGVRRRGRVGQWVCEVRVPGIKGSRLWLGTFNTAEMAARAHDAAVLALSGRAACLNFADS  
     [0174] SEQ ID NO: 126 is from  Triticum aestivum.  The AP2 region present in this sequence is:  
     [0175] SEQ ID NO: 126 (percent sequence identity with CBF1: (73% (47/64) HPLYRGVRRRGRVGQWVCEVRVPGVKGSRLWLGTFTTAEMAARAHDAAVLALSGRAACLNFADS  
     [0176] SEQ ID NO: 128 is found in  Glycine max.  The AP2 region present in this sequence is:  
     [0177] SEQ ID NO: 128 (percent sequence identity with CBF1: (89% (57/64) HPVYRGVRRRNSDKWVCEVREPNKKTRIWLGTFPTPEMAARAHDVAAMALRGRYACLNFADS  
     [0178] Other Arabidopsis Homologs  
     [0179] G2513 (SEQ ID NO: 99) G2513 is also closely related to CBF1, CBF2, and CBF3 (SEQ ID NO: 2, 13, and 15, respectively). G2513 shares 73% sequence identity with CBF1, 73% sequence identity with CBF2, and 52%% sequence identity with CBF3. In the AP2 domain G2513 shares 77%, 75%, and 80% identity with CBF1, CBF2 and CBF3, respectively.  
     [0180] G2513 corresponds to gene T12C24.14 (AAF88096). G2513 shows sequence similarity, outside of the conserved AP2 domain, with a protein from Nicotiana tabacum (gi12003384 AF211531 — 1 Avr9/Cf-9 rapidly elicited protein 111B [ Nicotiana tabacum ]). No published information is available about the functions of G2513. G2513-overexpressing plants were initially small with narrow dark green leaves, grew slowly and initiated floral buds several weeks later than in wild-type controls.  
     [0181] G2513 forms part of a monophyletic group within the Arabidopsis AP2/EREBP family that also includes G40 (SEQ ID NO: 2), G41 (SEQ ID NO: 13), G41 (SEQ ID NO: 15), and G912 (SEQ ID NO: 97), (CBF1, CBF2, CBF3 and CBF4, respectively). However, the clade is divided into two subgroups, one comprised by the four CBF genes (SEQ ID NOs: 2, 13, 15, and 97) and the other by G2107 (SEQ ID NO:101) and G2513 (SEQ ID NO:99).  
     [0182] G2513 is ubiquitously expressed, at significantly higher levels in rosette leaves, flower, and embryo tissues. Because of its phylogenetic relationship to the CBF genes and the delay in floral bud development, G2513 may be used to delay flowering and increase plant biomass relative to control after the plant flowers.  
     [0183] G2107 (SEQ ID NO: 101) shares 44% sequence identity with CBF1 (SEQ ID NO: 2), 45% sequence identity with CBF2 (SEQ ID NO: 13), and 51%% sequence identity with CBF3 (SEQ ID NO: 15). In the AP2 domain G2107 shares 75%, 74%, and 79% sequence identity with CBF1, CBF2, and CBF3, respectively. G2107 shows sequence similarity, outside of the conserved AP2 domain, with a protein from  Nicotiana tabacum  (gi12003384 AF211531 — 1 Avr9/Cf-9 rapidly elicited protein 111B [ Nicotiana tabacum ]). G2107 corresponds to gene F16M19.17 (AAF18701). No published information is available about the function of G2107.  
     [0184] G2107 expression is detected in floral tissues (including embryo and silique), as well as in rosette leaves, but not in roots or germinating seedlings. Because of its phylogenetic relationship to the CBF genes and the delay in floral bud development, G2107 may be used to delay flowering and increase plant biomass relative to control after the latter flowers or when environmental conditions induce stress in control plants.  
     [0185] G21 (SEQ ID NO: 103) corresponds to gene At2g44940 (AAD32841). G21 corresponds to gene At2g44940 (AAD32841). G2107 shares 57% sequence identity with CBF1 (SEQ ID NO: 2), 58% sequence identity with CBF2 (SEQ ID NO: 13), and 53%% sequence identity with CBF3 (SEQ ID NO: 15). In the AP2 domain G21 shares 76%, 72%, and 74% sequence identity with CBF1, CBF2, and CBF3, respectively. G21 does not show extensive sequence similarity with known genes from other plant species outside of the conserved AP2/EREBP domain.  
     [0186] Overexpression of G21 caused alterations in plant growth and development: 35S::G21 plants were smaller than wild type, often possessed curled, darker green leaves, and showed reduced fertility. No alterations were detected in 35S::G21 plants in the physiological and biochemical analyses that were performed.  
     [0187] G21 is ubiquitously expressed, and appears to be induced by several environmental or physiological conditions, in particular cold and abscisic acid. Because of its phylogenetic relationship to the CBF genes and the delay in floral bud development, G21 may also be used to delay flowering and increase plant biomass relative to control after the latter flowers, or when environmental conditions induce stress in control plants.  
     [0188] Summing up the presently disclosed means by which the levels of a cell protectant in a plant cell or plant may be modified, it has been shown that:  
     [0189] a) CBF1, CBF2, CBF3 are expressed in response to environmental stresses. For example, CBF1 expression increases in response to cold stress.  
     [0190] b) CBF1, CBF2, CBF3 have been shown to modify the levels of cell protectants in plant cells. These cell protectants include proline, sugars and fatty acids.  
     [0191] c) CBF1, CBF2, CBF3 have been shown to confer tolerance to environmental stresses. In Arabidopsis, for example, overexpression of any of these polypeptides results in improved tolerance to cold, high salt and drought.  
     [0192] d) A variety of plant genera and species may be transformed with a recombinant polynucleotide encoding a C-repeat/DRE binding factor (CBF)-related polypeptide; these species include  Arabidopsis thaliana,  leaf mustard ( Brassica juncea ),  Brassica oleracea  (including cabbage, Brussels sprouts, broccoli, kohlrabi, cauliflower, and kale),  Brassica rapa  (including turnip greens, turnip rape, and field mustard), rapeseed and canola ( Brassica rapa, Brassica campestris L.,  and  Brassica napus  L),  Brassica napus  (in addition to rapeseed and canola, also includes rutabaga and Swedish turnip), soybean ( Glycine max ), radish and clover radish ( Raphanus sativus ), corn ( Zea mays ), wheat (Triticum), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor  and  Sorghum vulgare,  and barley ( Hordeum vulgare ).  
     [0193] e) Different plants may be made more tolerant to environmental stresses. Overexpression of the paralogous genes CBF1, CBF2, and CBF3 in different plants, including Arabidopsis and canola, resulted in increased tolerance to several environmental stresses, including freezing, salt, and drought tolerance.  
     [0194] f) Orthologous sequences to CBF genes have been identified in canola, soybeans, rice, corn and other diverse plant species. This demonstrates that CBF genes are present and likely function in a similar manner in diverse species.  
     [0195] g) Overexpression of paralogs of the CBF genes, including CBF4 (G912), have also been shown to confer improved tolerance to environmental stress, which demonstrates that genes encoding polypeptides with the AP2 domain or a similarly functioning variant are able to confer improved stress resistance.  
     [0196] h) Gao et al. ((2002)  Plant Mol. Biol.  49 (5), 459-471) have characterized four CBF transcription factors from  Brassica napus  that function in a similar manner and show a high degree of sequence similarity to Arabidopsis CBF.  
     [0197] i) Zhou et al. have identified a dehydration responsive element binding protein GenBank Accession No. AAD45623) that shares 88% identity with the conserved domain of CBF1.  
     [0198] j) Durrant et al. (2002; unpublished; GenBank Accession No. AAG43548) have identified Avr9/Cf-9 (rapidly elicited protein 111B) in  Nicotiana tabacum,  the cDNA expression profiling of which reveals rapid, resistance gene-dependent, active oxygen-independent, gene induction during the plant defense response; this protein shares 64% identity with the CBF1 sequence and 85% (53/62 residues) with the conserved domain of CBF1.  
     [0199] Identifying Polynucleotides or Nucleic Acids by Hybridization  
     [0200] Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, for example, by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited above.  
     [0201] Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed transcription factor polynucleotide sequences, and, in particular, to those shown in SEQ ID NO: 1, 12, 14, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, and 127, and fragments thereof under various conditions of stringency. (See, for example, Wahl et al. (1987)  Methods Enzymol.  152: 399-407; Kimmel (1987)  Methods Enzymol.  152: 507-511.) In addition to the nucleotide sequences listed in Tables 4 and 5, 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.  
     [0202] With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989) “ Molecular Cloning: A Laboratory Manual ” (2nd ed., Cold Spring Harbor Laboratory); Berger and Kimmel, editors, (1987) “Guide to Molecular Cloning Techniques”, In  Methods in Enzymology:  152: 467-469; and Anderson and Young (1985) “Quantitative Filter Hybridisation.” In: Hames and Higgins, editors,  Nucleic Acid Hybridisation, A Practical Approach.  Oxford, IRL Press, pages 73-111.  
     [0203] 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 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:  
     [0204] (I) DNA-DNA: 
     T m (° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−0.62(% formamide−500/L 
     [0205] (II) DNA-RNA: 
     T m (° C.)=79.8+18.5(log[Na+])+0.58(% G+C)+0.12(% G+C) 2 −0.5(% formamide)−820/L 
     [0206] (III) RNA-RNA: 
     T m (° C.)=79.8+18.5(log[Na+])+0.58(% G+C)+0.12(% G+C) 2 −0.35(% formamide)−820/L 
     [0207] 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.  
     [0208] 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&#39;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.  
     [0209] 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 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 &gt;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.  
     [0210] High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5° C. to 20° C. lower than the thermal melting point (T 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, for example, to a unique subsequence, of the DNA.  
     [0211] Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, for example, formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° 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, for example, sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.  
     [0212] 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.  
     [0213] 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:  
     [0214] 6×SSC at 65° C.;  
     [0215] 50% formamide, 4×SSC at 42° C.; or  
     [0216] 0.5×SSC, 0.1% SDS at 65° C.;  
     [0217] 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.  
     [0218] 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.  
     [0219] 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 homolog, wash steps may be performed at a lower temperature, for example, 50° C.  
     [0220] 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 No. 20010010913).  
     [0221] 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, for example, a fluorescent label, a colorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.  
     [0222] Identifying Polynucleotides or Nucleic Acids with Expression Libraries  
     [0223] In addition to hybridization methods, transcription factor homolog polypeptides can be obtained by screening an expression library using antibodies specific for one or more transcription factors. With the provision herein of the disclosed transcription factor, and transcription factor homolog nucleic acid sequences, the encoded polypeptide(s) can be expressed and purified in a heterologous expression system (for example,  E. coli ) and used to raise antibodies (monoclonal or polyclonal) specific for the polypeptide(s) in question. Antibodies can also be raised against synthetic peptides derived from transcription factor, or transcription factor homolog, amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988),  Antibodies: A Laboratory Manual,  Cold Spring Harbor Laboratory, New York. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologs, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.  
     [0224] Sequence Variations  
     [0225] 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. A “variant” of a transcription factor may have an amino acid sequence that is different by one or more deletions, insertions, or substitutions of amino acid residues which produce a silent change 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. Variant 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.  
     [0226] The polypeptide 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 2). 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. 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).  
     [0227] Altered or variant 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.  
     [0228] 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.  
     [0229] Those skilled in the art would recognize that SEQ ID NO: 2, for example, 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).  
     [0230] For example, Table 1 illustrates, for example, that the codons AGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine. Accordingly, at each position in the sequence where there is a codon encoding serine, any of the above trinucleotide sequences can be used without altering the encoded polypeptide.  
                   TABLE 1                       Amino acid   Possible Codons                                                Alanine   Ala   A   GCA GCC GCG GCU               Cysteine   Cys   C   TGC TGT               Aspartic acid   Asp   D   GAC GAT               Glutamic acid   Glu   E   GAA GAG               Phenylalanine   Phe   F   TTC TTT               Glycine   Gly   G   GGA GGC GGG GGT               Histidine   His   H   CAC CAT               Isoleucine Ile   I   ATA ATC ATT               Lysine   Lys   K   AAA AAG               Leucine   Leu   L   TTA TTG CTA CTC CTG CTT               Methionine Met   M   ATG               Asparagine Asn   N   AAC AAT               Proline   Pro   P   CCA CCC CCG CCT               Glutamine   Gin   Q   CAA CAG               Arginine   Arg   R   AGA AGG CGA CGC CGG CGT               Serine   Ser   S   AGC AGT TCA TCC TCG TCT               Threonine   Thr   T   ACA ACC ACG ACT               Valine   Val   V   GTA GTC GTG GTT               Tryptophan Trp   W   TGG               Tyrosine   Tyr   Y   TAC TAT                  
 
     [0231] Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, for example, site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.  
     [0232] In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention.  
     [0233] 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 (editor)  Meth. 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, for example, a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.  
     [0234] 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 2 when it is desired to maintain the activity of the protein. Table 2 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.  
                   TABLE 2                       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                  
 
     [0235] 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 3 when it is desired to maintain the activity of the protein. Table 3 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as structural and functional substitutions. For example, a residue in column 1 of Table 3 may be substituted with residue in column 2; in addition, a residue in column 2 of Table 3 may be substituted with the residue of column 1.  
                           TABLE 3                                   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; Giy; 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                      
 
     [0236] Substitutions that are less conservative than those in Table 2 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.  
     [0237] Further Modifying Sequences of the Invention—Mutation/Forced Evolution  
     [0238] 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.  
     [0239] Thus, in one embodiment, given nucleic acid sequences are modified, for example, according to standard mutagenesis or artificial evolution methods to produce modified sequences. The modified sequences may be created using purified natural polynucleotides isolated from any organism or may be synthesized from purified compositions and chemicals using chemical means well know to those of skill in the art. For example, Ausubel, supra, provides additional details on mutagenesis methods. Artificial forced evolution methods are described, for example, by Stemmer (1994)  Nature  370: 389-391, Stemmer (1994)  Proc. Natl. Acad. Sci.  91: 10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and 6,242,568. Methods for engineering synthetic transcription factors and other polypeptides are described, for example, by Zhang et al. (2000)  J. Biol. Chem.  275: 33850-33860, Liu et al. (2001)  J. Biol. Chem.  276: 11323-11334, and Isalan et al. (2001)  Nature Biotechnol.  19: 656-660. Many other mutation and evolution methods are also available and expected to be within the skill of the practitioner.  
     [0240] 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.  
     [0241] Accordingly, the invention provides for modification of any given nucleic acid by mutation, evolution, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, for example, using the sequences herein as a starting substrate for the various modification approaches.  
     [0242] For example, optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be used for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced using a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for  Saccharomyces cerevisiae  and mammals are TAA and TGA, respectively. The preferred stop codon for monocotyledonous plants is TGA, whereas insects and  E. coli  prefer to use TAA as the stop codon.  
     [0243] The polynucleotide sequences of the present invention can also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the sequence to facilitate cloning, processing and/or expression of the gene product. For example, alterations are optionally introduced using techniques which are well known in the art, for example, site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.  
     [0244] Furthermore, a fragment or domain derived from any of the polypeptides of the invention can be combined with domains derived from other transcription factors or synthetic domains to modify the biological activity of a transcription factor. For instance, a DNA-binding domain derived from a transcription factor of the invention can be combined with the activation domain of another transcription factor or with a synthetic activation domain. A transcription activation domain assists in initiating transcription from a DNA-binding site. Examples include the transcription activation region of VP16 or GAL4 (Moore et al. (1998)  Proc. Natl. Acad. Sci.  95: 376-381; and 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).  
     [0245] Expression and Modification of Polypeptides  
     [0246] 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.  
     [0247] Vectors, Promoters, and Expression Systems  
     [0248] The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (for example, a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.  
     [0249] 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 and Ausubel, supra. Any of the identified sequences can be incorporated into a cassette or vector, for example, for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach (1989)  Methods for Plant Molecular Biology,  Academic Press, and Gelvin et al. (1990)  Plant Molecular Biology Manual,  Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of  Agrobacterium tumefaciens,  as well as those disclosed by Herrera-Estrella et al. (1983)  Nature  303: 209, Bevan (1984)  Nucl Acid Res.  12: 8711-8721, Klee (1985)  Bio/Technology  3: 637-642, for dicotyledonous plants.  
     [0250] Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991)  Bio/Technology  9: 957-962) and corn (Gordon-Kamm (1990)  Plant Cell  2: 603-618) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993)  Plant Physiol.  102: 1077-1084; Vasil (1993)  Bio/Technology  10: 667-674; Wan and Lemeaux (1994)  Plant Physiol.  104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996)  Nature Biotechnol.  14: 745-750).  
     [0251] Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (for example, a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.  
     [0252] 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, for example, Odell et al. (1985)  Nature  313: 810-812); the nopaline synthase promoter (An et al. (1988)  Plant Physiol.  88: 547-552); and the octopine synthase promoter (Fromm et al. (1989)  Plant Cell  1: 977-984).  
     [0253] A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a TF sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (for example, seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (for example, in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like. Numerous known promoters have been characterized and can favorably be employed to promote expression of a polynucleotide of the invention in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988)  Plant Mol. Biol.  11: 651-662), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active in vascular tissue (Ringli et al. (1998)  Plant Mol. Biol.  37: 977-988), flower-specific (Kaiser et al, (1995)  Plant Mol. Biol.  28: 231-243), pollen (Baerson et al. (1994)  Plant Mol. Biol.  26: 1947-1959), carpels (Ohl et al. (1990)  Plant Cell  2: 837-848), pollen and ovules (Baerson et al. (1993)  Plant Mol. Biol.  22: 255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999)  Plant Mol. Biol.  39: 979-990 or Baumann et al. (1999)  Plant Cell  11: 323-334), cytokinin-inducible promoter (Guevara-Garcia (1998)  Plant Mol. Biol.  38: 743-753), promoters responsive to gibberellin (Shi et al. (1998)  Plant Mol. Biol.  38: 1053-1060, Willmott et al. (1998) 38: 817-825) and the like. Additional promoters are those that elicit expression in response to heat (Ainley et al. (1993)  Plant Mol. Biol.  22: 13-23), light (for example, the pea rbcS-3A promoter, Kuhlemeier et al. (1989)  Plant Cell  1: 471-478, and the maize rbcS promoter, Schaffner and Sheen (1991)  Plant Cell  3: 997-1012); wounding (for example, wunI, Siebertz et al. (1989)  Plant Cell  1: 961-968); pathogens (such as the PR-1 promoter described in Buchel et al. (1999)  Plant Mol. Biol.  40: 387-396, and the PDF1.2 promoter described in Manners et al. (1998)  Plant Mol. Biol.  38: 1071-1080), and chemicals such as methyl jasmonate or salicylic acid (Gatz et al. (1997)  Ann. 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 et al. (1995)  Science  270: 1986-1988); or late seed development (Odell et al. (1994)  Plant Physiol.  106: 447-458).  
     [0254] Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3′-untranslated region of plant genes, for example, a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.  
     [0255] Additional Expression Elements  
     [0256] Specific initiation signals can aid in efficient translation of coding sequences. These signals can include, for example, 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 (for example, a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.  
     [0257] Expression Hosts  
     [0258] The present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (i.e., nucleic acids are introduced, for example, transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector comprising the relevant nucleic acids herein. The vector is optionally a plasmid, a viral particle, a phage, a naked nucleic acid, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the relevant gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook and Ausubel.  
     [0259] The host cell can be a eukaryotic cell, such as a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Plant protoplasts are also suitable for some applications. For example, the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (Fromm et al. (1985)  Proc. Natl. Acad. Sci.  82: 5824, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982)  Molecular Biology of Plant Tumors,  (Academic Press, New York) 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).  
     [0260] The cell can include a nucleic acid of the invention that encodes a polypeptide, wherein the cell expresses a polypeptide of the invention. The cell can also include vector sequences, or the like. Furthermore, cells and transgenic plants that include any polypeptide or nucleic acid above or throughout this specification, for example, produced by transduction of a vector of the invention, are an additional feature of the invention.  
     [0261] Modified Amino Acid Residues  
     [0262] 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.  
     [0263] Non-limiting examples of a modified amino acid residue include incorporation or other use of acetylated amino acids, glycosylated amino acids, sulfated amino acids, prenylated (for example, farnesylated, geranylgeranylated) amino acids, PEG modified (for example, “PEGylated”) amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References adequate to guide one of skill in the modification of amino acid residues are replete throughout the literature.  
     [0264] 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.  
     [0265] Identification of Additional Factors  
     [0266] A transcription factor provided by the present invention can also be used to identify additional endogenous or exogenous molecules that can affect a phentoype or trait of interest. On the one hand, such molecules include organic (small or large molecules) and/or inorganic compounds that affect expression of (i.e., regulate) a particular transcription factor. Alternatively, such molecules include endogenous molecules that are acted upon either at a transcriptional level by a transcription factor of the invention to modify a phenotype as desired. For example, the transcription factors can be employed to identify one or more downstream gene with which is subject to a regulatory effect of the transcription factor. In one approach, a transcription factor or transcription factor homolog of the invention is expressed in a host cell, for example, a transgenic plant cell, tissue or explant, and expression products, either RNA or protein, of likely or random targets are monitored, for example, by hybridization to a microarray of nucleic acid probes corresponding to genes expressed in a tissue or cell type of interest, by two-dimensional gel electrophoresis of protein products, or by any other method known in the art for assessing expression of gene products at the level of RNA or protein. Alternatively, a transcription factor of the invention can be used to identify promoter sequences (i.e., binding sites) involved in the regulation of a downstream target. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence can be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA-binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences can be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the transcription factors with their promoters (Bulyk et al. (1999)  Nature Biotechnol.  17: 573-577).  
     [0267] 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.  
     [0268] The two-hybrid system detects protein interactions in vivo and is described in Chien et al. ((1991),  Proc. Natl. Acad. Sci.  88: 9578-9582) and is commercially available from Clontech (Palo Alto, Calif.). In such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the TF polypeptide and the other consists of the transcription activator protein&#39;s activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast  Saccharomyces cerevisiae  that contains a reporter gene (for example, lacZ) whose regulatory region contains the transcription activator&#39;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.  
     [0269] Identification of Modulators  
     [0270] In addition to the intracellular molecules described above, extracellular molecules that alter activity or expression of a transcription factor, either directly or indirectly, can be identified. For example, the methods can entail first placing a candidate molecule in contact with a plant or plant cell. The molecule can be introduced by topical administration, such as spraying or soaking of a plant, and then the molecule&#39;s effect on the expression or activity of the TF polypeptide or the expression of the polynucleotide monitored. Changes in the expression of the TF polypeptide can be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis or the like. Changes in the expression of the corresponding polynucleotide sequence can be detected by use of microarrays, Northerns, quantitative PCR, or any other technique for monitoring changes in mRNA expression. These techniques are exemplified in Ausubel et al., editors,  Current Protocols in Molecular Biology,  John Wiley &amp; Sons (1998, and supplements through 2001). Such changes in the expression levels can be correlated with modified plant traits and thus identified molecules can be useful for soaking or spraying on fruit, vegetable and grain crops to modify traits in plants.  
     [0271] Essentially any available composition can be tested for modulatory activity of expression or activity of any nucleic acid or polypeptide herein. Thus, available libraries of compounds such as chemicals, polypeptides, nucleic acids and the like can be tested for modulatory activity. Often, potential modulator compounds can be dissolved in aqueous or organic (for example, DMSO-based) solutions for easy delivery to the cell or plant of interest in which the activity of the modulator is to be tested. Optionally, the assays are designed to screen large modulator composition libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (for example, in microtiter formats on microtiter plates in robotic assays).  
     [0272] In one embodiment, high throughput screening methods involve providing a combinatorial library containing a large number of potential compounds (potential modulator compounds). Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as target compounds.  
     [0273] A combinatorial chemical library can be, for example, a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis. For example, a combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (for example, in one example, amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound of a set length). Exemplary libraries include peptide libraries, nucleic acid libraries, antibody libraries (see, for example, Vaughn et al. (1996)  Nature Biotechnol.  14: 309-314 and PCT/US96/10287), carbohydrate libraries (see, for example, Liang et al.  Science  (1996) 274: 1520-1522 and U.S. Pat. No. 5,593,853), peptide nucleic acid libraries (see, for example, U.S. Pat. No. 5,539,083), and small organic molecule libraries (see, for example, benzodiazepines, Baum (1993)  Chem Eng. News  January 18, p. 33; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337) and the like.  
     [0274] Preparation and screening of combinatorial or other libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, for example, U.S. Pat. No. 5,010,175; Furka, (1991)  Int. J. Pept. Prot. Res.  37: 487-493; and Houghton et al. (1991)  Nature  354: 84-88). Other chemistries for generating chemical diversity libraries can also be used.  
     [0275] In addition, as noted, compound screening equipment for high-throughput screening is generally available, for example, using any of a number of well known robotic systems that have also been developed for solution phase chemistries useful in assay systems. These systems include automated workstations including an automated synthesis apparatus and robotic systems utilizing robotic arms. Any of the above devices are suitable for use with the present invention, for example, for high-throughput screening of potential modulators. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.  
     [0276] Indeed, entire high-throughput screening systems are commercially available. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. Similarly, microfluidic implementations of screening are also commercially available.  
     [0277] The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. The integrated systems herein, in addition to providing for sequence alignment and, optionally, synthesis of relevant nucleic acids, can include such screening apparatus to identify modulators that have an effect on one or more polynucleotides or polypeptides according to the present invention.  
     [0278] In some assays it is desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. That is, known transcriptional activators or inhibitors can be incubated with cells/plants/etc. in one sample of the assay, and the resulting increase/decrease in transcription can be detected by measuring the resulting increase in RNA/protein expression, etc., according to the methods herein. It will be appreciated that modulators can also be combined with transcriptional activators or inhibitors to find modulators that inhibit transcriptional activation or transcriptional repression. Either expression of the nucleic acids and proteins herein or any additional nucleic acids or proteins activated by the nucleic acids or proteins herein, or both, can be monitored.  
     [0279] In an embodiment, the invention provides a method for identifying compositions that modulate the activity or expression of a polynucleotide or polypeptide of the invention. For example, a test compound, whether a small or large molecule, is placed in contact with a cell, plant (or plant tissue or explant), or composition comprising the polynucleotide or polypeptide of interest and a resulting effect on the cell, plant, (or tissue or explant) or composition is evaluated by monitoring, either directly or indirectly, one or more of: expression level of the polynucleotide or polypeptide, activity (or modulation of the activity) of the polynucleotide or polypeptide. In some cases, an alteration in a plant phenotype can be detected following contact of a plant (or plant cell, or tissue or explant) with the putative modulator, for example, by modulation of expression or activity of a polynucleotide or polypeptide of the invention. Modulation of expression or activity of a polynucleotide or polypeptide of the invention may also be caused by molecular elements in a signal transduction second messenger pathway and such modulation can affect similar elements in the same or another signal transduction second messenger pathway.  
     [0280] Subsequences  
     [0281] 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.  
     [0282] Subsequences of the polynucleotides of the invention, including polynucleotide fragments and oligonucleotides are useful as nucleic acid probes and primers. An oligonucleotide suitable for use as a probe or primer is at least about 15 nucleotides in length, more often at least about 18 nucleotides, often at least about 21 nucleotides, frequently at least about 30 nucleotides, or about 40 nucleotides, or more in length. A nucleic acid probe is useful in hybridization protocols, for example, to identify additional polypeptide homologs of the invention, including protocols for microarray experiments. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods. See Sambrook and Ausubel, supra.  
     [0283] In addition, the invention includes an isolated or recombinant polypeptide including a subsequence of at least about 15 contiguous amino acids encoded by the recombinant or isolated polynucleotides of the invention. For example, such polypeptides, or domains or fragments thereof, can be used as immunogens, for example, to produce antibodies specific for the polypeptide sequence, or as probes for detecting a sequence of interest. A subsequence can range in size from about 15 amino acids in length up to and including the full length of the polypeptide.  
     [0284] 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 binds to a specific DNA promoter region, an activation domain or a domain for protein-protein interactions.  
     [0285] 2. Description of the Invention  
     [0286] The present invention relates to a method for modifying the biomass of a plant. By modifying a plant&#39;s biomass, any of a number of desirable plant traits can be modified, including plant yield, larger root mass to increase the tolerance to certain environmental stresses, modified leaf area in response to different light intensities, increased leaf mass for human or animal consumption, or the like.  
     [0287] The present invention also relates to a method for increasing the levels of a cell protectant in a cell. By increasing the levels of the cell protectant, a cell&#39;s or plant&#39;s response to a variety of environmental stresses can be improved. The type of environmental stress that can be modified includes the cold or freezing tolerance of the cell or the drought or salinity tolerance of the cell or plant. The cell protectant may be a cryoprotectant, an osmoprotectant or the like. Exemplary cell protectants include proline, sugars, lipids or the like. The method can also be used to increase levels of a number of cell protectants simultaneously.  
     [0288] The method entails altering the levels of a polynucleotide encoding a CBF-related polypeptide in a transformed plant. The transformed plant may be generated by transforming a plant with an expression vector comprising a polynucleotide sequence encoding a CBF-related polypeptide or by breeding after the initial transformation of a parental plant comprising the expression vector. Transformed plants are then selected that express the polynucleotide. The resulting plants are plants with increased biomass, including, for example, larger leaves and/or larger root systems, or plants that produce higher levels of cell protectants.  
     [0289] The method also entails generating a transformed cell or plant that overexpresses a recombinant polynucleotide encoding a CBF-related polypeptide. The transformed cell or plant may be generated by transforming a cell or plant with an expression vector comprising a polynucleotide sequence encoding a cold-regulatable polypeptide or by breeding after the initial transformation of a parental cell comprising the expression vector. Transformed cells or plants are then selected for expression of the polynucleotide and grown. The resulting cells or plants produce higher levels of cell protectants. Higher levels of cell protectants can be detected either in the absence of or after exposure to cold temperatures (cold acclimation). However, higher levels of cell protectants are typically observed in cells or plants that have been cold acclimated compared with levels observed in cells or plants that have not been cold-acclimated.  
     [0290] By increasing the levels of a single cell protectant or a combination of cell protectants simultaneously in the cell, the cell&#39;s tolerance to environmental stresses can be substantially improved, as measured for example by a plant&#39;s survival rate after exposure to freezing temperatures or the growth of a plant&#39;s roots after exposure to drought conditions or high salinity.  
     [0291] The present invention also relates to a method for producing a cell protectant from a cell or plant. The method entails generating a transformed cell that overexpresses a recombinant polynucleotide encoding a CBF-related polypeptide. Expression of the recombinant polynucleotide in the cell or plant derived from the transformed cell causes metabolic pathways that produce or accumulate certain cell protectants to be turned on so that higher levels of the cell protectants are produced. For example, we have observed that the key proline biosynthetic pathway enzyme, P5CS, is expressed at higher levels when the CBF-related polynucleotide is overexpressed. Then the cell protectant, such as proline, sugars or lipids, can be isolated from the plant using well known isolation and purification methods  
     [0292] The present invention can be applied to modify the biomass or increase cell protectant levels in or improve the environmental stress tolerance of a variety of plant cells or plants in particular, cells or plants monocots, dicots and gymnosperms. In particular the invention may be used for modifying the biomass or environmental stress response of agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn, potato, cotton, rice, oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherry, and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops, fruits and vegetables whose phenotype may be changed include barley, currant, avocado, citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries, nuts such as the walnut and peanut, endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish, yam, sweet potato and beans. The present invention may also be employed to modify plant biomass and increase cell protectant levels in woody plants, such pine, poplar and eucalyptus.  
     [0293] A. CBF-Related Polypeptide  
     [0294] The CBF-related protein may comprise a whole gene coding sequence or a fragment or domain of a coding sequence. A “fragment or domain”, as referred to polypeptides, may be a portion of a 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. A fragment may comprise, for example, a DNA binding domain that binds to a specific DNA promoter region (such as the AP2 domain), an activation domain or a domain for protein-protein interactions. Fragments may vary in size from as few as 6 amino acids to the length of the intact polypeptide, but are preferably at least 30 amino acids in length and more preferably at least 60 amino acids in length. For example, one can identify any number of 60 amino acid long fragments (1-60, 5-65, 10-70, 15-75, etc.) along the length of the CBF3 polypeptide shown in FIG. 13. In reference to a nucleotide sequence “a fragment” refers to any sequence of at least 18 consecutive nucleotides, preferably at least 30 nucleotides, more preferably at least 50, of any of the sequences provided herein.  
     [0295] The CBF-related polypeptides encompass naturally occurring sequences. Numerous CBF-related proteins have been previously identified and include the genes, CBF1, CBF2, and CBF3 (also known as DREB1b, DREB1c, and DREB1a, respectively), which are located in tandem on chromosome 4 in Arabidopsis (Gilmour et al. (1998) supra; Shinwari et al. (1998)  Biochem. Biophys. Res. Commun.  250: 161-170). Additional examples of CBF-related polypeptides include those described in Stockinger et al. PCT publication WO99/38977, and U.S. patent application Ser. No. 09/198,119, entitled “Plant Having Altered Environmental Stress Tolerance”, filed Nov. 23, 1998 and U.S. Provisional Patent Application No. 60/165,860 entitled “Method for Modifying the Cold Resistance of Plants”, filed Nov. 16, 1999.  
     [0296] The CBF-related polypeptides may also encompass non-naturally occurring sequences that are derivatives of the naturally-occurring CBFs described above. For example, a non-naturally occurring sequence using domains of other transcription factors described above fused in frame, but not necessarily adjacent, with functional domains derived from other sequences or sources. Additionally, the invention includes polypeptides derived from shuffling regions of transcription factors described above by methods described in Minshull and Stemmer, U.S. Pat. No. 5,837,458, entitled “Methods and Compositions for Cellular and Metabolic Engineering” and Stemmer and Crameri, U.S. Pat. No. 5,811,238, entitled “Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination”.  
     [0297] Substitutions, deletions and insertions introduced into CBF-related polypeptides are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (editor)  Meth. Enzymol.  (1993) vol. 217, Academic Press). Amino acid substitutions are typically of single residues and may be conservative (such as serine to threonine) or non-conservative (such as lysine to glutamic acid); insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, for example, a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a sequence.  
     [0298] Additionally, the CBF-related polypeptide may encompass a polypeptide sequence that is modified by chemical or enzymatic means. The homologous sequence may be a sequence modified by lipids, sugars, peptides, organic or inorganic compounds, by the use of modified amino acids or the like. Protein modification techniques are illustrated in Ausubel et al., editors, (1998)  Current Protocols in Molecular Biology,  John Wiley &amp; Sons.  
     [0299] B. Altered Expression of CBF-Related Polypeptide  
     [0300] Any of the identified sequences may be incorporated into a cassette or vector for expression in cells, in particular plant cells. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach, (1989)  Methods for Plant Molecular Biology,  Academic Press, and Gelvin et al. (1990)  Plant Molecular Biology Manual,  Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of  Agrobacterium tumefaciens,  as well as those disclosed by Herrera-Estrella et al. (1983)  Nature  303: 209, Bevan (1984)  Nucl. Acids Res.  12: 8711-8721, Klee (1985)  Bio/Technology  3: 637-642. Ti-derived plasmids can be transferred into both monocot and dicot species using Agrobacterium-mediated transformation (Ishida et al (1996)  Nat. Biotechnol.  14: 745-750; Barton et al. (1983)  Cell  32: 1033-1043).  
     [0301] Alternatively, non-Ti vectors can be used to transfer the DNA into plant cells by using free DNA delivery techniques. Such methods may involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991)  Bio/Technology  9: 957-962) and corn (Gordon-Kamm (1990)  Plant Cell  2: 603-618) can be produced. An immature embryo can also be a good target tissue for plants 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 et al. (1994)  Plant Physiol.  104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996)  Nature Biotech.  14: 745-750).  
     [0302] Typically, plant transformation vectors include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (for example, a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.  
     [0303] Examples of constitutive plant promoters which may be useful for expressing the CBF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, for example, Odell et al. (1985)  Nature  313: 810-812); the nopaline synthase promoter (An et al. (1988)  Plant Physiol.  88: 547-552); and the octopine synthase promoter (Fromm et al. (1989)  Plant Cell  1: 977-984).  
     [0304] 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 the CBFs in plants, as illustrated by seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186; fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988) supra), 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 et al. (1998)  Plant Mol. Biol.  37: 977-988), flower-specific (Kaiser et al. (1995)  Plant Mol. Biol.  28: 231-243), 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) 38: 817-825) and the like. Additional promoters are those that elicit expression in response to light (for example, the pea rbcS-3A promoter, Kuhlemeier et al. (1989)  Plant Cell  1: 471-478, and the maize rbcS promoter, Schaffner et al. (1991)  Plant Cell  3: 997-1012); wounding (for example, wunI, Siebertz et al. (1989)  Plant Cell  1: 961-968); pathogen resistance chemicals such as methyl jasmonate or salicylic acid (Gatz et al. (1997) supra). In addition, the timing of the expression can be controlled by using promoters such as those acting at late seed development (Odell et al. (1994)  Plant Physiol.  106: 447-458).  
     [0305] Plant expression vectors may also include RNA processing signals that may be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors may include additional regulatory sequences from the 3′-untranslated region of plant genes, for example, a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.  
     [0306] Finally, as noted above, plant expression vectors may also include dominant selectable marker genes to allow for the ready selection of transformants. Such genes include those encoding antibiotic resistance genes (for example, resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide resistance genes (for example, phosphinothricin acetyltransferase).  
     [0307] The polynucleotides and polypeptides of this invention may 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. For example, by ectopically expressing a gene by T-DNA activation tagging (Ichikawa et al. (1997)  Nature  390: 698-701, Kakimoto et al. (1996)  Science  274: 982-985). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated. In another example, the transcriptional machinery in a plant may be modified so as to increase transcription levels of a polynucleotide of the invention (See PCT Publications WO9606166 and WO 9853057 which describe the modification of the DNA binding specificity of zinc finger proteins by changing particular amino acids in the DNA binding motif).  
     [0308] The transgenic plant may also comprise 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.  
     [0309] In some cases, a reduction in plant biomass or the level of cryoprotectants may be desired. In such a case, a reduction of biomass or the cryoprotectant levels may be achieved by decreasing the levels of CBF expression. For example, a reduction of CBF expression in a transgenic plant to modify a plant trait may be obtained by introducing into plants antisense constructs based on the CBF cDNA. For antisense suppression, the CBF cDNA is arranged in reverse orientation relative to the promoter sequence in the expression vector. The introduced sequence need not be the full length CBF cDNA or gene, and need not be identical to the CBF cDNA or a gene found in the plant type to be transformed.  
     [0310] Vectors in which RNA encoded by the CBF cDNA (or variants thereof) is over-expressed may also be used to obtain co-suppression of the endogenous CBF gene 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 CBF cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous CBF gene. However, as with antisense suppression, the suppressive efficiency will be enhanced as (1) the introduced sequence is lengthened and (2) the sequence similarity between the introduced sequence and the endogenous CBF gene is increased.  
     [0311] Vectors expressing an untranslatable form of the CBF mRNA may also be used to suppress the expression of endogenous CBF activity to modify a trait. Methods for producing such constructs are described in U.S. Pat. No. 5,583,021 to Dougherty et al. Preferably, such constructs are made by introducing a premature stop codon into the CBF gene. Alternatively, a plant trait may be modified by gene silencing using double-strand RNA (Sharp (1999)  Genes Development  13: 139-141) or by simultaneous expression of both sense and antisense RNAs (Waterhouse et al. (1998)  Proc. Natl. Acad. Sci.  95: 13959-13964).  
     [0312] 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 CBF gene. Mutants containing a single mutation event at the desired gene may be crossed to generate homozygous plants for the mutation (Koncz et al. (1992)  Methods in Arabidopsis Research.  World Scientific).  
     [0313] A plant trait may also be modified by using the cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A plant genome may 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.  
     [0314] C. Transgenic Plants with Modified CBF Expression  
     [0315] Once an expression cassette comprising a polynucleotide encoding a CBF gene of this invention has been constructed, standard techniques may be used to introduce the polynucleotide into a plant in order to modify a trait of the plant. The plant may be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leguminosae (alfalfa ( Medicago sativa ), soybean ( Glycine max ), clover (Trifolium), etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed ( Brassica rapa  L and  Brassica napus  L), broccoli, leaf mustard ( Brassica juncea ), etc.), Curcurbitaceae (melons and cucumber), Gramineac (wheat (Triticum), corn ( Zea mays ), rice ( Oryza sativa ), barley ( Hordeum vulgare ), rye ( Secale cereale ), sorghum ( Sorghum bicolor  and  Sorghum vulgare ), millet ( Panicum miliaceum, Setaria italica,  and  Eleusine coracana ), etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al., editors, (1984)  Handbook of Plant Cell Culture—Crop Species  Macmillan Publ. Co. New York, N.Y.; Shimamoto et al. (1989)  Nature  338: 274-276; Fromm et al. (1990)  Bio/Technology  8: 833-839; and Vasil et al. (1990)  Bio/Technology  8: 429-434.  
     [0316] 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 may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and  Agrobacterium tumefaciens  mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence.  
     [0317] Successful examples of the modification of plant characteristics or traits 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.  
     [0318] 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.  
     [0319] After transformed plants are selected and grown to maturity, those plants with modified plant biomass or high levels of cell protectants are identified and used for any of the purposes 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 may 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.  
     [0320] The following examples are intended to illustrate but not limit the present invention.  
     EXAMPLES  
     Example 1  
     [0321] Arabidopsis thaliana  Plant Handling  Arabidopsis thaliana  (L.) Heynh. ecotype Ws-2 and transgenic plants in the Ws-2 background were grown in controlled environment chambers at 20° C. under constant illumination from cool-white fluorescent lights (100-150 μmol m −2  s −1 )) essentially as described (Gilmour et al. (1988)  Plant Physiol.  87:745-750) in Baccto planting mix (Michigan Peat, Houston, Tex.). Pots were sub-irrigated with deionized water as necessary. All seeds were cold-treated (5° C.) for 4 days immediately after planting to ensure uniform germination.  
     Example 2  
     [0322] RNA Hybridization and cDNA Probes  
     [0323] In the following examples, unless otherwise specified, total RNA was extracted from Arabidopsis plants as described previously (Gilmour et al. (1988)  Plant Physiol.  87: 745-750). Northern transfers were prepared and hybridized as described (Hajela et al. (1990)  Plant Physiol.  93: 1246-1252) using high stringency wash conditions (Stockinger et al. (1997) supra).  32 P-labeled probes were prepared by random priming (Feinberg and Vogelstein (1983)  Anal. Biochem.  132: 6-13). A gene-specific probe to CBF3 was made to the 3′ end of the cDNA clone by PCR as described previously (Gilmour et al. (1998) supra). Arabidopsis cDNA clones encoding Arabidopsis sucrose synthase (182C20T7), corresponding to the SUS1 gene (Martin et al. (1993)  Plant J.  4: 367-377) and Δ′-pyrroline-5-carboxylate synthase (125M17T7), corresponding to the P5CS2 gene (Strizhov, et al. (1997)  Plant J.  12: 557-569), were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Probes for P5CS2 transcripts should cross-hybridize with the highly similar P5CS1 transcripts and thus is a measure of total P5CS transcripts.  
     Example 3  
     [0324] Activation of Transcription in Yeast Containing C-Repeat/DRE Using CBF1, CBF2 and CBF3  
     [0325] This example shows that CBF1, CBF2, and CBF3 activate transcription in yeast containing CRT/DREs upstream of a reporter gene. The CBFs were expressed in yeast under control of the ADC1 promoter on a 2μ plasmid (pDB20.1; Berger et al. (1992)  Cell  70: 251-265). Constructs expressing the different CBFs were transformed into yeast reporter strains that had the indicated CRT/DRE upstream of the lacZ reporter gene. Copy number of the CRT/DREs and its orientation relative to the direction of transcription from each promoter is indicated by the direction of the arrow.  
     [0326]FIG. 15 is a graph showing transcription regulation of CRT/DRE containing reporter genes by CBF1, CBF2, and CBF3 genes in yeast. In FIG. 15, the vertical lines across the arrows of the COR15a construct represent the m3cor15a mutant CRT/DRE construct. Each CRT/DRE-lacZ construct was integrated into the URA3 locus of yeast. Error bars represent the standard deviation derived from three replicate transformation events with the same CBF activator construct into the respective reporter strain. Quantitative beta-gal assays were performed as described by Rose and Botstein (Rose et al. (1983)  Methods Enzymol.  101: 167-180).  
     Example 4  
     [0327] Isolation and Analysis of  Arabidopsis thaliana  cDNA Clone (CBF1) Encoding C-Repeat/DRE Binding Factor  
     [0328] The following example describes the isolation of an  Arabidopsis thaliana  cDNA clone that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1). Expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE (CCGAC) as an upstream activator sequence. Meanwhile, CBF1 did not activate transcription of mutant versions of the CCGAC binding element, indicating that CBF1 is a transcription factor that binds to the C-repeat/DRE. Binding of CBF1 to the C-repeat/DRE was also demonstrated in gel shift assays using recombinant CBF1 protein expressed in  Escherichia coli.  Analysis of the deduced CBF1 amino acid sequence indicated that the protein has a potential nuclear localization sequence, a possible acidic activation domain and an AP2 domain, a DNA-binding motif of about 60 amino acids that is similar to those present in Arabidopsis proteins APETALA2, AINTEGUMENTA and TINY, the tobacco ethylene response element binding proteins, and numerous other plant proteins of unknown function.  
     [0329] Cold Treatment.  
     [0330] Plants were treated by placing pots in a cold room at 2.5° C. under cool constant illumination with white florescent lamps (25 μmol m −2 s −1 ) for the indicated times.  
     [0331] Arabidopsis cDNA Expression Library.  
     [0332] The Arabidopsis pACT cDNA expression library was constructed by John Walker and colleagues (NSF/DOE/USDA Collaborative Research in Plant Biology Program grant USDA 92-37105-7675) and deposited in the Arabidopsis Biological Resource Center (stock #CD4-10).  
     [0333] Yeast Reporter Strains.  
     [0334] Oligonucleotides (Table 4) (synthesized at the MSU Macromolecular Structure Facility) encoding either wild-type or mutant versions of the C-repeat/DRE were ligated into the BglII site of the lacZ reporter vector pBgl-lacZ (Li et al. (1993)  Science  262: 1870-1874); kindly provided by Joachim Li). The resulting reported constructs were integrated into the ura3 locus of  Saccharomyces cerevisiae  strain GGY1 (MAT gal4 gal80 ura3 leu2 his3 ade2 tyr) (Li et al. (1993) supra; provided by Joachim Li) by transformation and selection for uracil prototrophy.  
     [0335] E. coli  Strains  
     [0336] Escherichia coli  strain GM2163 containing plasmid pEJS251 was deposited under the Budapest Treaty on May 17, 1996 with the American Type Culture Collection, Rockville, Md. as ATCC 98063. It is available by name and number pursuant to the provisions of the Budapest Treaty.  
               TABLE 4                          Oligonucleotides encoding wild type and mutant versions of the       C-repeat/DRE                                 C-repeat/               Oligonucleotide   DRE*   Sequence   SEQ ID NO:               MT50   COR15a   GatcATTTCATGG CCGAC CTGCTTTTT   3               MT52   M1COR15a   CACAATTTCA a G aattca CTGCTTTTTT   4               MT80   M2COR15a   GatcATTTCATGG tatgt CTGCTTTTT   5               MT125   M3COR15a   GatcATTTCATGG aatca CTGCTTTTT   6               MT68   COR15b   GatcACTTGATGG CCGAC CTCTTTTTT   7               MT66   C0R78-1   GatcAATATACTA CCGAC ATGAGTTCT   8               MT86   C0R78-2   ACTA CCGAC ATGAGTTCCAAAAAGC   9                    
     [0337] * The C-repeat/DRE sequences tested are either wild-type found in the promoters of COR15a (Baker et al. (1994)  Plant Mol. Biol.  24: 701-713), COR15b or COR78/RD29a (Horvath et al. (1993)  Plant Physiol.  103: 1047-1053; Yamaguchi-Shinozaki et al. (1994)  Plant Cell  6: 251-264) or are mutant versions of the COR15a C-repeat/DRE (M1COR15a, M2COR15a and M3COR15a).  
     [0338] # Uppercase letters designate bases in wild type C-repeat/DRE sequences. The core CCGAC sequence common to the above sequences is indicated in bold type. Lowercase letters at the beginning of a sequence indicate bases added to facilitate cloning. The lowercase letters that are underlined indicate the mutations in the C-repeat/DRE sequence of COR15a.  
     [0339] Screen of Arabidopsis cDNA Library.  
     [0340] The Arabidopsis pACT cDNA expression library was screened for clones encoding C-repeat/DRE biomass and environmental stress response regulatory elements by the following method. The cDNA library, harbored in  Escherichia coli  BNN132, was amplified by inoculating 0.5 ml of the provided glycerol stock into 1 L of M9 minimal glucose medium (Sambrook et al. (1989) supra) and shaking the bacteria for 20 h at 37° C. Plasmid DNA was isolated and purified by cesium chloride density gradient centrifugation (Sambrook et al. (1989) supra) and transformed into the yeast GGY1 reporter strains selecting for leucine prototrophy. Yeast transformants that had been grown for 2 or 3 days at 30° C. were overlaid with either a nitrocellulose membrane filter (Schleicher and Schuell, Keene, N.H.) or Whatman #50 filter paper (Hillsboro, Oreg.) and incubated overnight at 30° C. The yeast impregnated filters were then lifted from the plate and treated with X-gal (5-bromo-4-chloro-3-indolyl-D-galactosidase) to assay colonies for beta-galactosidase activity (Li et al. (1993) supra). Plasmid DNA from “positive” transformants (those forming blue colonies on the X-gal-treated filters) was recovered (Strathern et al. (1991)  Methods Enzymol.  194: 319-329), propagated in  E. coli  DH5α and transformed back into the yeast reporter strains to confirm activity.  
     [0341] Yeast Transformation and Quantitative Beta-galactosidase Assays.  
     [0342] Yeast were transformed by either electroporation (Becker et al. (1991)  Methods Enzymol.  194: 182-187) or the lithium acetate/carrier DNA method (Schiestl et al. (1989)  Current Genetics  16: 339-346). Quantitative in vitro beta-galactosidase assays were done as described (Rose et al. (1983)  Methods Enzymol.  101: 167-180).  
     [0343] Expression of CBF1 Protein in  E. coli  and Yeast.  
     [0344] CBF1 was expressed in  E. coli  using the pET-28a(+) vector (Novagen, Madison, Wis.). The BglII-BclI restriction fragment of pACT-11 encoding CBF1 was ligated into the BamHI site of the vector bringing CBF1 under control of the T7 phage promoter. The construct resulted in a “histidine tag,” a thrombin recognition sequence and a “T7 epitope tag” being fused to the amino terminus of CBF1. The construct was transformed into  E. coli  BL21 (DE3) and the recombinant CBF1 protein was expressed as recommended by the supplier (Novagen). Expression of CBF1 in yeast was accomplished by ligating restriction fragments encoding CBF1 (the BclI-BglII and BglII-BglII fragments from pACT-11) into the BglII site of pDB20.1 (Berger et al. (1992)  Cell  70: 251-265) bringing CBF1 under control of the constitutive ADC1 (alcohol dehydrogenase constitutive 1) promoter.  
     [0345] Gel Shift Assays.  
     [0346] The presence of expressed protein that binds to a C-repeat/DRE binding domain was evaluated using the following gel shift assay. Total soluble  E. coli  protein (40 ng) was 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% BSA, 1 mM DTT) plus 50 ng poly(dI-dC):poly(dI-dC) (Pharmacia, Piscataway, N.J.) with or without 100 ng competitor DNA. After 10 min, probe DNA (1 ng) that was  32 P-labeled by end-filling (Sambrook et al. supra) was added and the mixture incubated for an additional 10 min. Samples were loaded onto polyacrylamide gels (4% w/v) and fractionated by electrophoresis at 150V for 2 h (Sambrook et al. supra). Probes and competitor DNAs were prepared from oligonucleotide inserts ligated into the BamHI site of pUC118 (Vicira et al. (1987)  Methods Enzymol.  153: 3-11). The orientation and concatenation number of the inserts were determined by dideoxy DNA sequence analysis (Sambrook et al. (1989), supra). Inserts were recovered after restriction digestion with EcoRI and HindIII and fractionation on polyacrylamide gels (12% w/v) (Sambrook et al. (1989) supra).  
     [0347] Northern and Southern Analysis.  
     [0348] Northern and Southern analysis was performed as follows. Total RNA was isolated from Arabidopsis (Gilmour et al. (1988)  Plant Physiol.  87: 745-750) and the poly(A) +  fraction purified using oligo dT cellulose (Sambrook et al. (1989), supra). Northern transfers were prepared and hybridized as described (Hajela et al. (1990)  Plant Physiol.  93: 1246-1252) except that high stringency wash conditions were at 50 C in 0.1×SSPE [1.0×SSPE is 3.6 M NaCl, 20 mM EDTA, 0.2 M Na 2 —HPO 4  (pH7.7)], 0.5% SDS. Membranes were stripped in 0.1×SSPE, 0.5% SDS at 95° C. for 15 min prior to re-probing. Total Arabidopsis genomic DNA was isolated (Stockinger et al. (1996)  J. Heredity  87: 214-218) and Southern transfers prepared (Sambrook et al. supra) using nylon membranes (MSI, Westborough, Mass.). High stringency hybridization and wash conditions were as described by Walling (Walling et al. (1988)  Nucleic Acids Res.  16: 10477-10492). Low stringency hybridization was in 6×SSPE, 0.5% SDS, 0.25% low fat dried milk at 60° C. Low stringency washes were in 1×SSPE, 0.5% SDS at 50° C. Probes used for the entire CBF1 coding sequence and 3′ end of CBF1 were the BclI/BglII and EcoRV/BglII restriction fragments from pACT-11, respectively, that had been gel purified (Sambrook et al. (1989), supra). DNA probes were radiolabeled with  32 P-nucleotides by random priming (Sambrook et al. (1989), supra). Autoradiography was performed using hyperfilm-MP (Amersham, Arlington Heights Ill.). Radioactivity was quantified using a Betascope 603 blot analyzer (Betagen Corp., Waltham Mass.).  
     [0349] Screen of Arabidopsis cDNA Library for Sequence Encoding a C-Repeat/DRE Binding Domain.  
     [0350] The “one-hybrid” strategy (Li et al. (1993) supra) was used to screen for Arabidopsis cDNA clones encoding a C-repeat/DRE binding domain. In brief, yeast strains were constructed that contained a lacZ reporter gene with either wild-type or mutant C-repeat/DRE sequences in place of the normal UAS (upstream activator sequence) of the GAL1 promoter.  
     [0351]FIGS. 1A and 1B show how the yeast reporter strains were constructed. FIG. 1A is a schematic diagram showing the screening strategy. Yeast reporter strains were constructed that carried C-repeat/DRE sequences as UAS elements fused upstream of a lacZ reporter gene with a minimal GAL1 promoter. The strains were transformed with an Arabidopsis expression library that contained random cDNA inserts fused to the GAL4 activation domain (GAL4-ACT) and screened for blue colony formation on X-gal-treated filters. FIG. 1B is a chart showing activity of the “positive” cDNA clones in yeast reporter strains. The oligonucleotides (oligos) used to make the UAS elements, and their number and direction of insertion, are indicated by the arrows.  
     [0352] Yeast strains carrying these reporter constructs produced low levels of beta-galactosidase and formed white colonies on filters containing X-gal. The reporter strains carrying the wild-type C-repeat/DRE sequences were transformed with a DNA expression library that contained random Arabidopsis cDNA inserts fused to the acidic activator domain of the yeast GAL4 transcription factor, “GAL4-ACT” (FIG. 1A). The notion was that some of the clones might contain a cDNA insert encoding a C-repeat/DRE binding domain fused to GAL4-ACT and that such a hybrid protein could potentially bind upstream of the lacZ reporter genes carrying the wild type C-repeat/DRE sequence, activate transcription of the lacZ gene and result in yeast forming blue colonies on X-gal-treated filters.  
     [0353] Upon screening about 2×10 6  yeast transformants, three “positive” cDNA clones were isolated; i.e., clones that caused yeast strains carrying lacZ reporters fused to wild-type C-repeat/DRE inserts to form blue colonies on X-gal-treated filters (FIG. 1B). The three cDNA clones did not cause a yeast strain carrying a mutant C-repeat/DRE fused to LacZ to turn blue (FIG. 1B). Thus, activation of the reporter genes by the cDNA clones appeared to be dependent on the C-repeat/DRE sequence. Restriction enzyme analysis and DNA sequencing indicated that the three cDNA clones had an identical 1.8 kb insert (FIG. 2A). One of the clones, designated pACT-11, was chosen for further study.  
     [0354] Identification of 24 kDa Polypeptide with an AP2 Domain Encoded by pACT-11.  
     [0355]FIGS. 2A, 2B,  2 C and  2 D provide an analysis of the pACT-11 cDNA clone. FIG. 2A is a schematic drawing of the pACT-11 cDNA insert indicating the location and 5′ to 3′ orientation of the 24 kDa polypeptide and 25s rRNA sequences. The cDNA insert was cloned into the XhoI site of the pACT vector. FIG. 2B is a DNA and amino acid sequence of the 24 kDa polypeptide (SEQ ID NO: 1 and SEQ ID NO: 2). The AP2 domain is indicated by a double underline. The basic amino acids that potentially act as a nuclear localization signal are indicated with asterisks. The BclI site immediately upstream of the 24 kDa polypeptide used in subcloning the 24 kDa polypeptide and the EcoRV site used in subcloning the 3′ end of CBF1 are indicated by single underlines. FIG. 2C is a schematic drawing indicating the relative positions of the potential nuclear localization signal (NLS), the AP2 domain and the acidic region of the 24 kDa polypeptide. Numbers indicate amino acid residues. FIG. 2D is a chart showing comparison of the AP2 domain of the 24 kDa polypeptide (SEQ ID NO: 10) with that of the tobacco DNA binding protein EREBP2 (Okme-Takagi et al. (1995)  Plant Cell  7: 173-182; SEQ ID NO: 11). Identical amino acids are indicated with single lines; similar amino acids are indicated by double dots; amino acids that are invariant in AP2 domains are indicated with asterisks (Klucher et al. (1996)  Plant Cell  8: 137-153); and the histidine residues present in CBF1 and TINY (Wilson et al. (1996)  Plant Cell  8: 659-671) that are tyrosine residues in all other described AP2 domains are indicated with a caret. A single amino acid gap in the CBF1 sequence is indicated by a single dot.  
     [0356] Our expectation was that the cDNA insert in pACT-11 would have a C-repeat/DRE binding domain fused to the yeast GAL4-ACT sequence. However, DNA sequence analysis indicated that an open reading frame of only nine amino acids had been added to the C-terminus of GAL4-ACT. It seemed highly unlikely that such a short amino acid sequence could comprise a DNA binding domain. Also surprising was the fact that about half of the cDNA insert in pACT-11 corresponded to 25s rRNA sequences (FIG. 2A). Further analysis, however, indicated that the insert had an open reading frame, in opposite orientation to the GAL4-ACT sequence, deduced to encode a 24 kDa polypeptide (FIGS.  2 A- 2 C). The polypeptide has a basic region that could potentially serve as a nuclear localization signal (Raikhel (1992)  Plant Physiol.  100: 1627-1632) and an acidic C-terminal half (pI of 3.6) that could potentially act as an acidic transcription activator domain (Hahn (1993)  Cell  72: 481-483). A search of the nucleic acid and protein sequence databases indicated that there was no previously described homology of the 24 kDa polypeptide. However, the polypeptide did have an AP2 domain (Jofuku et al. (1994)  Plant Cell  6: 1211-1225 (FIGS. 2B, 2D), a DNA binding motif of about 60 amino acids (Ohme-Takagi et al. (1994)  Plant Cell  7: 173-182) that is present in numerous plant proteins including the APETALA2 (Jofuku et al. (1994)  Plant Cell  6: 1211-1225), AINTEGUMENTA (Klucher et al. (1996)  Plant Cell  8: 137-153; Elliot et al. (1996)  Plant Cell  8: 155-168) and TINY (Wilson et al. (1996)  Plant Cell  8: 659-671) proteins of Arabidopsis and the EREBPs (ethylene response element binding proteins) of tobacco (Ohme-Takagi et al. (1995)  Plant Cell  7: 173-182).  
     [0357] 24 kDa Polypeptide Binds to the C-Repeat/DRE and Activates Transcription in Yeast.  
     [0358] We hypothesized that the 24 kDa polypeptide was responsible for activating the lacZ reporter genes in yeast. To test this, the BclI-BglII fragment of pACT-11 containing the 24 kDa polypeptide, and the BglII-BglII fragment containing the 24 kDa polypeptide plus a small portion of the 25s rRNA sequence, was inserted into the yeast expression vector pDB20.1  
     [0359]FIG. 3 is a chart showing activation of reporter genes by the 24 kDa polypeptide. Restriction fragments of pACT-11 carrying the 24 kDa polypeptide (BclI-BglII) or the 24 kDa polypeptide plus a small amount of 25s RNA sequence (BglII-BglII) were inserted in both orientations into the yeast expression vector pDB20.1 (see FIGS. 2A and 2B for location of BclI and BglII restriction sites). These “expression constructs” were transformed into yeast strains carrying the lacZ reporter gene fused to direct repeat dimers of either the wild-type COR15a C-repeat/DRE (oligonucleotide MT50) or the mutant M2COR15a C-repeat/DRE (oligonucleotide MT80). The specific activity of beta-galactosidase (nmoles o-nitrophenol produced/min −1 ×mg protein −1 ) was determined from cultures grown in triplicate. Standard deviations are indicated. Abbreviations: pADC1, ADC1 promoter; tADC 1, ADC1 terminator.  
     [0360] Plasmids containing either insert in the same orientation as the ADC1 promoter stimulated synthesis of beta-galactosidase when transformed into yeast strains carrying the lacZ reporter gene fused to a wild-type COR15a C-repeat/DRE (FIG. 3). The plasmids did not, however, stimulate synthesis of beta-galactosidase when transformed into yeast strains carrying lacZ fused to a mutant version of the COR15a C-repeat/DRE (FIG. 3). These data indicated that the 24 kDa polypeptide could bind to the wild-type C-repeat/DRE and activate expression for the lacZ reporter gene in yeast. Additional experiments indicated that the 24 kDa polypeptide could activate expression of the lacZ reporter gene fused to either a wild-type COR78 C-repeat/DRE (dimer of MT66) or a wild-type COR15b C-repeat/DRE (dimer of MT 68) (not shown). A plasmid containing the BclI-BglII fragment (which encodes only the 24 kDa polypeptide) cloned in opposite orientation to the ADC1 promoter did not stimulate synthesis of beta-galactosidase in reporter strains carrying the wild-type COR15a C-repeat/DRE fused to lacZ (FIG.  3 ). In contrast, a plasmid carrying the BglII-BglII fragment (containing the 24 kDa polypeptide plus some 25s rRNA sequences) cloned in opposite orientation to the ADC1 promoter produced significant levels of beta-galactosidase in reporter strains carrying the wild-type COR15a C-repeat/DRE (FIG. 3). Thus, a sequence located closely upstream of the 24 kDa polypeptide was able to serve as a cryptic promoter in yeast, a result that offered an explanation for how the 24 kDa polypeptide was expressed in the original pACT-11 clone.  
     [0361] Gel Shift Analysis Indicates that the 24 kDa Polypeptide Binds to the C-Repeat/DRE.  
     [0362] Gel shift experiments were conducted to demonstrate further that the 24 kDa polypeptide bound to the C-repeat/DRE. Specifically, the open reading frame for the 24 kDa polypeptide was inserted into the pET-28a(+) bacterial expression vector and the resulting 28 kDa fusion protein was expressed at high levels in  E. coli.  (FIG. 4).  
     [0363]FIG. 4 is a photograph of an electrophoresis gel showing expression of the recombinant 24 kDa polypeptide in  E. coli.  Shown are the results of SDS-PAGE analysis of protein extracts prepared from  E. coli  harboring either the expression vector alone (vector) or the vector plus an insert encoding the 24 kDa polypeptide in sense (sense insert) or antisense (antisense insert) orientation. The 28 kDa fusion protein (see Materials and Methods) is indicated by an arrow.  
     [0364]FIG. 5 is a photograph of a gel for shift assays indicating that CBF1 binds to the C-repeat/DRE. The C-repeat/DRE probe (1 ng) used in all reactions was a  32 P-labeled dimer of the oligonucleotide MT50 (wild type C-repeat/DRE from COR15a). The protein extracts used in the first four lanes were either bovine serum albumin (BSA) or the indicated CBF1 sense, antisense and vector extracts described in FIG. 4. The eight lanes on the right side of the figure used the CBF1 sense protein extract plus the indicated competitor C-repeat/DRE sequences (100 ng). The numbers 1×, 2× and 3× indicate whether the oligonucleotides were monomers, dimers or trimers, respectively, of the indicated C-repeat/DRE sequences.  
     [0365] Protein extracts prepared from  E. coli  expressing the recombinant protein produced a gel shift when a wild-type COR15a C-repeat/DRE was used as probe (FIG. 5). No shift was detected with BSA or  E. coli  extracts prepared from strains harboring the vector alone, or the vector with an antisense insert for the 24 kDa polypeptide. Oligonucleotides encoding wild-type C-repeat/DRE sequences from COR15a or COR78 competed effectively for binding to the COR15a C-repeat/DRE probe, but mutant version of the COR15a C-repeat/DRE did not (FIG. 5). These in vitro results corroborated the in vivo yeast expression studies indicating that the 24 kDa polypeptide binds to the C-repeat/DRE sequence. The 24 kDa polypeptide was thus designated CBF1 (C-repeat/DRE binding factor 1) and the gene encoding it named CBF1.  
     [0366] CBF1 is a Unique or Low Copy Number Gene.  
     [0367]FIG. 6 is a photograph of a southern blot analysis indicating CBF1 is a unique or low copy number gene. Arabidopsis DNA (1 μg) was digested with the indicated restriction endonucleases and southern transfers were prepared and hybridized with a  32 P-labeled probe encoding the entire CBF1 polypeptide.  
     [0368] The hybridization patterns observed in southern analysis of Arabidopsis DNA using the entire CBF1 gene as probe were relatively simple indicating that CBF1 is either a unique or low copy number gene (FIG. 6). The hybridization patterns obtained were not altered if only the 3′ end of the gene was used as the probe (the EcoRV/BglII restriction fragment from pACT-11 encoding the acidic region of CBF1, but not the AP2 domain) or if hybridization was carried out at low stringency (not shown).  
     [0369] CBF1 Transcript Level Response to Low Temperature.  
     [0370]FIGS. 7A, 7B and  7 C relate to CBF1 transcripts in control and cold-treated Arabidopsis. FIG. 7A is a photograph of a membrane RNA isolated from Arabidopsis plants that were grown at 22° C. or grown at 22° C. and transferred to 2.5° C. for the indicated times. FIGS. 7B and 7C are graphs showing relative transcript levels of CBF1 and COR15a in control and cold-treated plants. The radioactivity present in the samples described in FIG. 7A were quantified using a Betascope 603 blot analyzer and plotted as relative transcript levels (the values for the 22° C. grown plants being arbitrarily set as 1) after adjusting for differences in loading using the values obtained with the pHH25 probe.  
     [0371] Based on the original data used to prepare FIGS.  7 A- 7 C, northern analysis indicated that the level of CBF1 transcripts increased about 2 to 3 fold in response to low temperature (FIG. 7B). Following this analysis, it was determined that CBF genes are induced rapidly (within 15 min of treatment) by mechanical agitation as well as cold temperatures (Gilmour et al. (1998)  Plant J.  16: 433-443), and that CBF genes are not expressed at significant levels in non-stressed plants. The detectable level of CBF1 transcripts at the “0” time point in FIGS. 7A and 7B was subsequently shown to be due to agitation, and that CBF1 transcript levels actually increased significantly more in response to low temperature than 2 to 3 fold over control plants, when agitation was eliminated as a inducer of CBF genes in all of the plants.  
     [0372] The transcript levels for COR15a increased approximately 35 fold in cold-treated plants (FIG. 7C). Only a singly hybridizing band was observed for CBF1 at either high or low stringency with probes for either the entire CBF1 coding sequence or the 3′ end of the gene (the EcoRV/BglII fragment of pACT-11) (not shown). The size of the CBF1 transcripts was about 1.0 kb.  
     [0373] The above example regarding CBF1 represents the first identification of a gene sequence that encodes a protein capable of binding to the C-repeat/DRE sequence CCGAC. The experimental results presented evidence that CBF1 binds to the C-repeat/DRE both in vitro via gel shift assays and in vivo via yeast expression assays. Further, the results demonstrate that CBF1 can activate transcription of reporter genes in yeast that contain the C-repeat/DRE.  
     [0374] The results of the southern analysis indicate that CBF1 is a unique or low copy number gene in Arabidopsis. However, the CBF1 protein contains a 60 amino acid motif, the AP2 domain that is evolutionary conserved in plants (Weigel (1995)  Plant Cell  7: 388-389). It is present in the APETALA2 (Jofuku et al. (1994)  Plant Cell  6: 1211-1225), AINTEGUMENTA (Klucher et al. (1996)  Plant Cell  8: 137-153; and Elliot et al. (1996)  Plant Cell  8: 155-168), TINY (Wilson et al.  Plant Cell  8: 659-671 (1996)) and cadmium-induced (Choi et al. (1995)  Plant Physiol.  108: 849) proteins of Arabidopsis and the EREBPs of tobacco (Ohme-Takagi et al. (1995)  Plant Cell  7: 173-182). In addition, a search of the GenBank expressed sequence tagged cDNA database indicates that there is one cDNA from  B. napus,  two from  Ricinus communis,  and more than 25 from Arabidopsis and 15 from rice, that are deduced to encode proteins with AP2 domains. The results of Ohme-Takagi and Shinshi (Ohme-Takagi et al. supra) indicate that the function of the AP2 domain is DNA-binding; this region of the putative tobacco transcription factor EREBP2 is responsible for its binding to the cis-acting ethylene response element referred to as the GCC-repeat. As discussed by Ohme-Takagi and Shinshi (Ohme-Takagi et al. supra), the DNA-binding domain of EREBP2 (the AP2 domain) contains no significant amino acid sequence similarities or obvious structural similarities with other known transcription factors or DNA binding motifs. Thus, the domain appears to be a novel DNA-binding motif that to date, has only been found in plant proteins.  
     [0375] It is generally believed that that the CCGAC core sequence is a member of family of core sequences having the common subsequence CCG, and that the binding of CBF1 to the C-repeat/DRE involves the AP2 domain. In this regard, it is germane to note that the tobacco ethylene response element, AGCCGCC, closely resembles the C-repeat/DRE sequences present in the promoters of the Arabidopsis genes COR15a, GGCCGAC, and COR78/RD29A, TACCGAC. Applicants believe that CBF1, the EREBPs and other AP2 domain proteins are members of a superfamily of DNA binding proteins that recognize a family of cis-acting regulatory elements having CCG as a common core sequence. Differences in the sequence surrounding the CCG core element could result in recruitment of different AP2 domain proteins which, in turn, could be integrated into signal transduction pathways activated by different environmental, hormonal and developmental cues. Such a scenario is akin to the situation that exists for the ACGT-family of cis-acting elements (Foster et al. (1994)  FASEB J.  8: 192-200). In this case, differences in the sequence surrounding the ACGT core element result in the recruitment of different bZIP transcription factors involved in activating transcription in response to a variety of environmental and developmental signals. Applicants believe that other C-repeat/DRE regulatory sequences exist which belong to a broader CCG family of regulatory sequences. By screening plant genomes according to the methodology taught herein using other members of the CCG family, additional regulatory sequences as well as the binding proteins which bind to these regulatory sequences can be identified. For example, plants which are known to have modified biomass or exhibit a form of environmental stress tolerance can be screened according to the blue colony assay and other screening methodologies used in the present invention with other members of the CCG family in order to identify other binding proteins and their gene sequences. Examples of other members of the CCG family include, but are not limited to, biomass or environmental stress response regulatory elements which include one of the following sequences: CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG (see U.S. Pat. No. 6,417,428).  
     [0376] The results of the yeast transformation experiments indicate that CBF1 has a domain that can serve as a transcriptional activator. The most likely candidate for this domain is the acidic C-terminal half of the polypeptide. Indeed, random acidic amino acid peptides from  E. coli  have been shown to substitute for the GAL4 acidic activator domain of GAL4 in yeast (Ma et al. (1987)  Cell  51: 113-199). Moreover, acidic activator domains have been found to function across kingdoms (Hahn (1993)  Cell  72: 481-483); the yeast GAL4 acidic activator, for instance, can activate transcription in tobacco (Ma et al. (1988)  Nature  334: 631-633). It has also been shown that certain plant transcription factors, such as Vp1 (McCarty et al. (1991)  Cell  66: 895-905), have acidic domains that function as transcriptional activators in plants. Significantly, the acidic activation domains of the yeast transcription factors VP16 and GCN4 require the “adaptor” proteins ADA2, ADA3, and GCN5 for full activity (see Guarente (1995)  Trends Biochem. Sci.  20: 517-521). These proteins form a heteromeric complex (Horiuchi et al. (1995)  Mol. Cell Biol.  15: 1203-1209) that bind to the relevant activation domains. The precise mechanism of transcriptional activation is not known, but appears to involve histone acetylation: there is a wealth of evidence showing a positive correlation between histone acetylation and the transcriptional activity of chromatin (Wolffe (1994)  Trends Biochem. Sci.  19: 240-244) and recently, the GCN5 protein has been shown to have histone acetyltransferase activity (Brownell et al. (1996)  Cell  84: 843-851). Genetic studies indicate that CBF1, like VP16 and GCN4, requires ADA2, ADA3 and GCN5 to function optimally in yeast. The fundamental question thus raised is whether plants have homologs of ADA2, ADA3 and GCN5 and whether these adaptors are required for CBF1 function (and function of other transcription factors with acidic activator regions) in Arabidopsis.  
     Example 5  
     [0377] Identification of Modified Phenotypes in Overexpression or Gene Knockout Plants  
     [0378] Experiments were performed to identify those transformants or knockouts that exhibited modified cell protectant levels. Among the biochemicals that were assayed were sugars, proline and fatty acids.  
     [0379] Proline levels in leaves were measured by preparing lyophilized leaf material; 30 mg samples of the lyophilized material were then extracted with 3 ml deionized water at 80° C. for 15 min. The samples were shaken for approximately 1 hour at room temperature and then allowed to stand overnight at 4° C. The extracts were filtered through glass wool and analyzed for proline content using the acid ninhydrin reaction (Troll and Lindsley (1955)  J. Biol. Chem.  215:655-660). Proline levels in certain samples were confirmed by amino acid analysis using an amino acid analyzer at the Macromolecular Structure Facility in the Biochemistry Department at Michigan State University.  
     [0380] Total soluble sugars (for example, sucrose, glucose, and fructose among others) were extracted from lyophilized leaf material (20 mg) in 80% ethanol (2 ml) at 80° C. for 15 min. The samples were shaken for approximately 1 hr at room temperature and allowed to stand overnight at 4° C. Extracts were filtered through glass wool and chlorophyll removed by shaking samples (0.4 ml) with water (0.4 ml) and chloroform (0.4 ml). The aqueous extract was tested for sugar content using the phenol-sulfuric acid assay (Dubois et al. (1956)  Anal. Chem.  28:350-356). Certain samples were dried down, suspended in water and the sugars analyzed by HPLC using a sugar column (Shodex, Shoko Co. Ltd., Japan) with a refractive index detector as previously described (Gao et al. (1999)  Physiol. Plant  106:1-8). Retention times were compared to those of standard glucose, fructose and sucrose, and the peaks integrated using Millennium-32 software (Waters Corp.).  
     [0381] The fatty acid composition of plant cells and tissues may be altered by transcriptional control of fatty acid biosynthesis. The presently disclosed transcription factors and variants thereof may be able to modify the expression of fatty acid biosynthetic pathways, which may, in turn, alter cell protectant levels within a plant. A number of individual fatty acids in the leaves of transgenic plants are presently of interest as cell protectants. These fatty acids of interest represent either end products or intermediates within one or more biosynthetic pathways. Modifications of the levels of intermediates generally affect the throughput and yield of an entire pathway, and thus measurements of individual fatty acid metabolites are often representative of changes to an entire biosynthetic pathway. For example, malonyl-CoA is a common intermediate in fatty acid biosynthesis of anthroquinones, cuticular waxes, flavonoids, and fatty acids. The formation of malonyl-CoA may be regulated by the action of acetyl-CoA carboxylase, which catalyzes ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. Control of malonyl-CoA synthesis at the enzymatic or transcriptional level has a profound impact on various fatty acid levels via a number of pathways (Higuchi, T. (1997)  Biochemistry and Molecular Biology of Wood,  Springer-Verlag, Berlin, pages 238-242, and Buchanan et al (2000)  Biochemistry and Molecular Biology of Plants,  American Society of Plant Physiology, Rockville, Md., pages 465-471) for example, saturated fatty acids such as 18:1, 18:2 and/or 18:3 fatty acids that may protect plants freezing stress). Thus, analysis of both total lipids and of individual fatty acids that affect entire pathways is central to understanding how fatty acid biosynthesis and composition can act to modulate a plant&#39;s cell protectant levels.  
     [0382] Total lipids from Arabidopsis leaves were measured by extraction, hydrolysis, and methylation essentially as described by Benning and Somerville ((1992)  J. Bacteriol.  174: 2352-2360). Triplicate samples of leaf material (approximately 20 mg fresh weight) were placed in TEFLON-lined glass screw cap tubes with 1 ml 1N HCl in methanol and heated at 80° C. for 40 min. Myristic acid (14:0) (5 μg) was added as an internal standard to each sample. The resulting fatty acid methyl esters were partitioned into 0.9% NaCl in hexane (1 ml), the hexane phase concentrated to a small volume and the entire sample separated by gas chromatography as detailed (Rossak et al. 1997  Arch. Biochem. Biophys.  340:219-230). The individual fatty acids were quantified using AGP_TOP software (Hewlett Packard). Experiments were performed to identify those transformants or knockouts that exhibited an improved environmental stress tolerance, including cold or freezing stress, drought stress or salt stress, as described in the following examples. For such studies, the transformants were exposed to a variety of environmental stresses. Plants were exposed to chilling stress (6 hour exposure to 4-8° C.), heat stress (6 hour exposure to 32-37° C.), high salt stress (6 hour exposure to 200 mM NaCl), drought stress (168 hours after removing water from trays), or osmotic stress (6 hour exposure to 3 M mannitol).  
     Example 6  
     [0383] Use of CBF1 to Induce Cold Regulated Gene Expression in Non-Cold Acclimated Arabidopsis Plants.  
     [0384] The following example demonstrates that increased expression of CBF1 induces COR gene expression in non-cold acclimated Arabidopsis plants. Transgenic Arabidopsis plants that overexpress CBF1 were created by placing a cDNA encoding CBF1 under the control of the strong cauliflower mosaic virus (CaMV) 35S promoter and transforming the chimeric gene into Arabidopsis ecotype RLD plants (Standard procedures were used for plasmid manipulations (Sambrook et al. (1989), supra). The CBF1-containing AseI-BglII fragment from pACT-Bgl+ (Stockinger et al. (1997)  Proc. Natl. Acad. Sci.  94: 1035) was gel-purified, BamHI linkers were ligated to both ends and the fragment was inserted into the BamHI site in pCIB710 (Rothstein et al. (1987)  Gene  53: 153-161) which contains the CaMV 35S promoter and terminator. The chimeric plasmid was linearized at the KpnI site and inserted into the KpnI site of the binary vector pCIB10g (Ciba-Geigy, Research Triangle Park, N.C.). The plasmid was transformed into  Agrobacterium tumefaciens  strain C58C1 (pMP90) by electroporation. Arabidopsis plants were transformed by the vacuum infiltration procedure (Bechtold et al. (1993)  Acad. Sci. Paris, Life Sci.  316: 1194-1199) as modified (van Hoof et al. (1996)  Plant Journal  10: 415-424). Initial screening gave rise to two transgenic lines, A6 and B16, that accumulated CBF1 transcripts at elevated levels.  
     [0385]FIG. 8 is a Northern blot showing CBF1 and COR transcript levels in RLD and transgenic Arabidopsis plants. Leaves from non-cold acclimated and three-day cold-acclimated plants ( Arabidopsis thaliana  ecotype RLD plants were grown in pots under continuous light (100 μE/m 2 /sec) at 22 C for 18-25 days as described (Gilmour et al.  Plant Physiol.  87: 735 (1988)). In some cases, plants were then cold-acclimated by placing them at 2.5° C. under continuous light (50 μE/m 2 /sec) for varying amounts of time. Leaves were harvested and total RNA prepared and analyzed for CBF1 and COR transcripts by RNA blot analysis using  32 P-radiolabeled probes (Total RNA was isolated from plant leaves and subjected to RNA blot analysis using high stringency hybridization and wash conditions as described (Stockinger et al. (1997)  Proc. Natl. Acad. Sci.  94: 1035-1040; and Gilmour et al. (1988)  Plant Physiol.  87: 745-750).  
     [0386]FIG. 9 is an immunoblot showing COR15am protein levels in RLD and transgenic Arabidopsis plants. Total soluble protein (100 μg) was prepared from leaves of the non-cold acclimated RLD (RLDw), 4-day cold-acclimated RLD (RLDc4), 7-day cold-acclimated RLD (RLDc7) and non-cold acclimated A6 and B16 plants and the levels of COR15am determined by immunoblot analysis using antiserum raised against the COR15am polypeptide (Total soluble protein was isolated from plant leaves, fractionated by tricine SDS-PAGE and transferred to 0.2 micron nitrocellulose as previously described (Artus et al. (1996)  Proc. Natl. Acad. Sci.  93: 13404). COR15am protein was detected using antiserum raised to purified COR15am and protein A conjugated alkaline phosphatase (Sigma-Aldrich, St. Louis, Mo.) (Artus et al. (1996) supra). No reacting bands were observed with pre-immune serum (not shown).  
     [0387] Southern analysis indicated that the A6 line had a single DNA insert while the B16 line had multiple inserts (not shown). Examination of fourth generation homozygous A6 and B16 plants indicated that CBF1 transcript levels were higher in non-cold acclimated A6 and B16 plants than they were in non-cold acclimated RLD plants, the levels in A6 being about three fold higher than in B16 (FIG. 8).  
     [0388] CBF1 overexpression resulted in strong induction of COR gene expression (FIG. 8). Specifically, the transcript levels of COR6.6, COR15a, COR47 and COR78 were dramatically elevated in non-cold acclimated A6 and B16 plants as compared to non-cold acclimated RLD plants. The effect was greater in the A6 line, where COR transcript levels in non-cold acclimated plants approximated those found in cold-acclimated RLD plants. The finding that COR gene expression was greater in A6 plants than in B16 plants was consistent with CBF1 transcript levels being higher in the A6 plants (FIG. 7A). Immunoblot analysis indicated that the levels of the COR15am (FIG. 9) and COR6.6 (not shown) polypeptides were also elevated in the A6 and B16 lines, the level of expression again being higher in the A6 line. Attempts to identify the CBF1 protein in either RLD or transgenic plants were unsuccessful. Overexpression of CBF1 had no effect on the transcript levels for eIF4A (eukaryotic initiation factor 4A) (Metz et al. (1992)  Gene  120: 313-314), a constitutively expressed gene that is not responsive to low temperature (FIG. 8) and had no obvious effects on plant growth and development.  
     [0389] The results from this example demonstrate that overexpression of the Arabidopsis transcriptional activator CBF1 induces expression of an Arabidopsis COR “regulon” composed of genes carrying the CRT/DRE DNA regulatory element. It appears that CBF1 binds to the CRT/DRE DNA regulatory elements present in the promoters of these genes and activates transcription that is consistent with the notion of CBF1 having a role in COR gene regulation. Significantly, there was a strong correlation between CBF1 transcript levels and the magnitude of COR gene induction in non-cold acclimated A6, B16, and RLD plants (FIG. 8). However, upon low temperature treatment the level of CBF1 transcripts remained relatively low in RLD plants, while COR gene expression was induced to about the same level as that in non-cold acclimated A6 plants (FIG. 8). Thus, it appears that CBF1 or an associated protein becomes “activated” in response to low temperature.  
     Example 7  
     [0390] CBF Overexpression Resulted in a Marked Increase in Plant Freezing Tolerance  
     [0391] A. Experiments with  Aribidopsis thaliana    
     [0392] The following example describes a comparison of the freezing tolerance of non-cold acclimated Arabidopsis plants that overexpress CBF1 to that of cold-acclimated wild-type plants. As described below, the freezing tolerance of non-cold acclimated Arabidopsis plants overexpressing CBF1 significantly exceeded that of non-cold acclimated wild-type Arabidopsis plants and approached that of cold-acclimated wild-type plants.  
     [0393] Freezing tolerance was determined using the electrolyte leakage test (Sukumaran (1972) et al.  Hort. Science  7: 467). Detached leaves were frozen to various subzero temperatures and, after thawing, cellular damage (due to freeze-induced membrane lesions) was estimated by measuring ion leakage from the tissues.  
     [0394]FIGS. 10A and 10B are graphs showing freezing tolerance of leaves from RLD and transgenic Arabidopsis plants. Leaves from non-cold acclimated RLD (RLDw) plants, cold-acclimated RLD (RLDc) plants and non-cold acclimated A6, B16 and T8 plants were frozen at the indicated temperatures and the extent of cellular damage was estimated by measuring electrolyte leakage (Electrolyte leakage tests were conducted as described (Sukumaran et al. (1972) supra; and Gilmour et al. (1988)  Plant Physiol.  87: 735) with the following modifications. Detached leaves (2-4) from non-cold acclimated or cold-acclimated plants were placed in a test tube and submerged for 1 hour in a −2° C. water-ethylene glycol bath in a completely randomized design, after which ice crystals were added to nucleate freezing. After an additional hour of incubation at −2° C., the samples were cooled in decrements of 1° C. each hour until −8° C. was reached. Samples (five replicates for each data point) were thawed overnight on ice and incubated in 3 ml distilled water with shaking at room temperature for 3 hours. Electrolyte leakage from leaves was measured with a conductivity meter. The solution was then removed, the leaves frozen at −80° C. (for at least one hour), and the solution returned to each tube and incubated for 3 hours to obtain a value for 100% electrolyte leakage. In FIG. 10A and 10B, the RLDc plants were cold-acclimated for 10 and 11 days, respectively. Error bars indicate standard deviations.  
     [0395] As can be seen from FIG. 10A and 10B, CBF1 overexpression resulted in a marked increase in plant freezing tolerance. The experiment presented in FIG. 10A indicates that the leaves from both non-cold acclimated A6 and B16 plants were more freezing tolerant than those from non-cold acclimated RLD plants. Indeed, the freezing tolerance of leaves from non-cold acclimated A6 plants approached that of leaves from cold-acclimated RLD plants. The results also indicate that the leaves from non-cold acclimated A6 plants were more freezing tolerant than those from non-cold acclimated B16 plants, a result that is consistent with the greater level of CBF1 and COR gene expression in the A6 line.  
     [0396] The results presented in FIG. 10B further demonstrate that the freezing tolerance of leaves from non-cold acclimated A6 plants was greater than that of leaves from non-cold acclimated RLD plants and that it approached the freezing tolerance of leaves from cold-acclimated RLD plants. In addition, the results indicate that overexpression of CBF1 increases freezing tolerance to a much greater extent than overexpressing COR15a alone. This conclusion comes from comparing the freezing tolerance of leaves from non-cold acclimated A6 and T8 plants (FIG. 10B). T8 plants (Artus (1996) supra) are from a transgenic line that constitutively expresses COR15a (under control of the CaMV 35S promoter) at about the same level as in A6 plants (FIG. 1). However, unlike in A6 plants, other CRT/DRE-regulated COR genes are not constitutively expressed in T8 plants (FIG. 8).  
     [0397] A comparison of EL 50  values (the freezing temperature that results in release of 50% of tissue electrolytes) of leaves from RLD, A6, B16 and T8 plants is presented in Table 5.  
     [0398] EL 50  values were calculated and compared by analysis of variance curves fitting up to third order linear polynomial trends were determined for each electrolyte leakage experiment. To insure unbiased predictions of electrolyte leakage, trends significantly improving the model fit at the 0.2 probability level were retained. EL 50  values were calculated from the fitted models. In Table 2, an unbalanced one-way analysis of variance, adjusted for the different numbers of EL 50  values for each plant type, was determined using SAS PROC GLM [SAS Institute, Inc. (1989), SAS/STAT User&#39;s Guide, Version 6, Cory, N.C.)]. EL 50  values±SE (n) are presented on the diagonal line for leaves from non-cold acclimated RLD (RLDw), cold-acclimated (7 to 10 days) RLD (RLDc) and non-cold acclimated A6, B16 and T8 plants. P values for comparisons of EL 50  values are indicated in the intersecting cells.  
               TABLE 5                          EL 50  values                                         RLDw   RLDc   A6   B16   T8                                                 RLDw   −3.9 ± 0.21 (8)   P &lt; 0.0001   P &lt; 0.0001   P = 0.0014   P = 0.7406       RLDc       −7.6 ± 0.30 (4)   P = 0.3261   P &lt; 0.0001   P &lt; 0.0001       A6           −7.2 ± 0.25 (6)   P &lt; 0.0001   P &lt; 0.0001       B16               −5.2 ± 0.27 (5)   P = 0.0044       T8                   −3.8 ± 0.35 (3)                  
 
     [0399] The data confirm that: 1) the freezing tolerance of leaves from both non-cold acclimated A6 and B16 plants is greater than that of leaves from both non-cold acclimated RLD and T8 plants; and 2) that leaves from non-cold acclimated A6 plants are more freezing tolerant than leaves from non-cold acclimated B16 plants. No significant difference was detected in EL 50  values for leaves from non-cold acclimated A6 and cold-acclimated RLD plants or from non-cold acclimated RLD and T8 plants.  
     [0400] The enhancement of freezing tolerance in the A6 line was also apparent at the whole plant level. FIG. 11 is a photograph showing freezing survival of RLD and A6 Arabidopsis plants. Non-cold acclimated (WARM) RLD and A6 plants and 5-day cold-acclimated (COLD) RLD plants were frozen at −5° C. for 2 days and then returned to a growth chamber at 22° C. (Pots (3.5 inch) containing about 40 non-cold acclimated Arabidopsis plants (20 day old) and 4 day cold-acclimated plants (25 days old) ( Arabidopsis thaliana  ecotype RLD plants were grown in pots under continuous light (100 μE/m 2 /sec) at 22° C. for 18-25 days as described (Gilmour et al. (1988)  Plant Physiol.  87: 735). In some cases, plants were then cold-acclimated by placing them at 2.5° C. under continuous light (50 μE/m 2 /sec) for varying amounts of time) were placed in a completely randomized design in a −5° C. cold chamber in the dark. After 1 hour, ice chips were added to each pot to nucleate freezing. Plants were removed after 2 days and returned to a growth chamber at 22° C.). A photograph of the plants after 7 days of regrowth is shown.  
     [0401] Although the magnitude of the difference varied from experiment to experiment, non-cold acclimated A6 plants consistently displayed greater freezing tolerance in whole plant freeze tests than did non-cold acclimated RLD plants (FIG. 11). No difference in whole plant freeze survival was detected between non-cold acclimated B16 and RLD plants or non-cold acclimated T8 and RLD plants (not shown).  
     [0402] The results of this experiment show that CBF1-induced expression of CRT/DRE-regulated COR genes result in a dramatic increase in freezing tolerance and confirms the belief that COR genes play a major role in plant cold acclimation. The increase in freezing tolerance brought about by expressing the battery of CRT/DRE-regulated COR genes was much greater than that brought about by overexpressing COR15a alone indicating that COR genes in addition to COR15a have roles in freezing tolerance.  
     [0403] Traditional plant breeding approaches have met with limited success in improving the freezing tolerance of agronomic plants (Thomashow (1990)  Adv. Genet  28: 99-131). For instance, the freezing tolerance of the best wheat varieties today is essentially the same as the most freezing-tolerance varieties developed in the early part of the 20 th  Century. Thus, in recent years there has been considerable interest that biotechnology might offer new strategies to improve the freezing tolerance of agronomic plants. By the results of the present invention, Applicants demonstrate the ability to enhance the freezing tolerance of non-cold acclimated Arabidopsis plants by increasing the expressing of the Arabidopsis regulatory gene CBF1. As described throughout this application, the ability of the present invention to modify the expression of environmental stress tolerance genes such as COR genes has wide ranging implications since the CRT/DRE DNA regulatory element is not limited to Arabidopsis (Jiang et al. (1996)  Plant Mol. Biol.  30: 679-684). Rather, CBF1 and homologous genes can be used to manipulate expression of CRT/DRE-regulated COR genes in important crop species and thereby improve their freezing tolerance. By transforming modified versions of CBF1 (or homologs) into such plants, it will extend their safe growing season, increase yield and expand areas of production.  
     [0404] B. Experiments with  Lycopersicon esculentum    
     [0405] Two CBF sequences were isolated from tomato and were transformed into Arabidopsis plants using the methods described herein (Examples 4 and 6). These tomato CBF-related polypeptides, LeCBF1 (SEQ ID NO: 331) and LeCBF2 (SEQ ID NO: 332), share amino acid sequences with SEQ ID NO:6 (SEQ ID NO: 2; see FIG. 35). Similar freezing tolerance leakage experiments to those conducted with Arabidopsis plants, as described above in Example 7a, demonstrated that CBF-induced expression of CRT/DRE-regulated COR genes from tomato are endogenously induced when wild-type tomato plants are exposed to cold-stress (FIG. 36). When tomato CBF1 was expressed as a transgene in Arabidopsis plants, expression of COR genes, including both mRNA expression and protein expression, was activated (FIG. 37). This overexpression resulted in a dramatic increase in freezing tolerance, as measured by percentage electrolyte leakage (FIG. 38).  
     [0406] The results of this experiment show that CBF1-induced expression of CRT/DRE-regulated COR genes result in a dramatic increase in freezing tolerance and confirms the belief that COR genes play a major role in plant cold acclimation. The increase in freezing tolerance brought about by expressing the battery of CRT/DRE-regulated COR genes was much greater than that brought about by overexpressing COR15a alone indicating that COR genes in addition to COR15a have roles in freezing tolerance.  
     [0407] Traditional plant breeding approaches have met with limited success in improving the freezing tolerance of agronomic plants (Thomashow (1990)  Adv. Genet  28: 99-131). For instance, the freezing tolerance of the best wheat varieties today is essentially the same as the most freezing-tolerance varieties developed in the early part of the 20 th  Century. Thus, in recent years there has been considerable interest that biotechnology might offer new strategies to improve the freezing tolerance of agronomic plants. By the results of the present invention, Applicants demonstrate the ability to enhance the freezing tolerance of non-cold acclimated Arabidopsis plants by increasing the expressing of the Arabidopsis regulatory gene CBF1. As described throughout this application, the ability of the present invention to modify the expression of environmental stress tolerance genes such as COR genes has wide ranging implications since the CRT/DRE DNA regulatory element is not limited to Arabidopsis (Jiang et al. (1996)  Plant Mol. Biol.  30: 679-684). Rather, CBF1 and homologous genes can be used to manipulate expression of CRT/DRE-regulated COR genes in important crop species and thereby improve their freezing tolerance. By transforming modified versions of CBF1 (or homologs) into such plants, it will extend their safe growing season, increase yield and expand areas of production.  
     [0408] B. Experiments with  Lycopersicon esculentum    
     [0409] CBF sequences were isolated from tomato and were transformed into Arabidopsis plants using the methods described herein (Examples 4 and 6). One tomato CBF (CBF1; SEQ ID NO: 331) shares 56 % identity (121/215 aminom acid residues) with SEQ ID NO:2 (G40). Similar freezing tolerance leakage experiments to those conducted with Arabidopsis plants, as described above in Example 7a, demonstrate that CBF-induced expression of CRT/DRE-regulated COR genes from tomato are endogenously induced when wild-type tomato plants are exposed to cold-stress. When tomato CBF1 is expressed as a transgene in Arabidopsis plants, expression of COR genes, including both mRNA expression and protein expression, is activated. This overexpression results in a dramatic increase in freezing tolerance, as measured by percentage electrolyte leakage.  
     [0410] These experiments demonstrate that CBF genes in diverse species play a major role in plant cold acclimation.  
     Example 8  
     [0411] Selection of Promoters to Control Expression of CBF1 in Plants.  
     [0412] The following examples describe the isolation of different promoters from plant genomic DNA, construction of the plasmid vectors carrying the CBF1 gene and the inducible promoters, transformation of Arabidopsis cells/plants with these constructs, and regeneration of transgenic plants that have altered biomass or increased tolerance to environmental stresses compared to non-transformed plants  
     [0413] Isolation of inducible promoters from plant genomic DNAs. Inducible promoters from different plant genomic DNAs were identified and isolated by PCR amplification using primers designed to flank the promoter region and contain suitable restriction sites for cloning into the expression vector. The following genes were used to BLAST search GenBank to find the inducible promoters: Dreb2a; P5CS; Rd22; Rd29a; Rd29b; Rab18; Cor47. Table 6 lists the accession numbers and positions of these promoters. Table 7 lists the forward and reverse primers that were used to isolate the promoters.  
                                   TABLE 6                                   Gene Name   Accession No.   Position   Length (bps)                          Dreb2a   AB010692   51901-53955   2054           P5CS   AC003000   45472-47460   1988           Rd22   D10703    17-1046   1029           Rd29a   D13044   3870-5511   1641           Rd29b   D13044    90-1785   1695           Rab18   AB013389   8070-9757   1687           Cor47   AB004872     1-1370   1370                      
 
     [0414]                           TABLE 7                                   SEQ       Promoter name   Primer name   Cloning sites   ID NO:                  Dreb2a   Dreb2a-reverse   HindIII (AAGCTT)   19                   Dreb2a-forward   BglII (AGATCT)   20               P5CS   P5CS-reverse   HindIII (AAGCTT)   21                   P5CS-forward   BglII (AGATCT)   22               Rd22   Rd22-reverse   HindIII (AAGCTT)   23                   Rd22-forward   KpnI (GGTACC)   24               Rd29a   Rd29a-reverse   HindIII (AAGCTT)   25                   Rd29a-forward   KpnI (GGTACC)   26               Rd29b   Rd29b-reverse   HindIII (AAGCTT)   27                   Rd29b-forward   KpnI (GGTACC)   28               Rab 18   Rab18-reverse   HindIII (AAGCTT)   29                   Rab18-forward   BglII (AGATCT)   30               Cor47   Cor47-reverse   HindIII (AAGCTT)   31                   Cor47-forward   BglII (AGATCT)   32                    
     [0415] (1) Dreb2a Promoter  
     [0416] A cDNA encoding DRE (C-repeat) binding protein (DREB2A) has been recently identified (Liu et al. (1998)  Plant Cell  10: 1391-1406). The transcription of the DREB2A gene is activated by dehydration and high-salt stress, but not by cold stress. The upstream untranslated region (166 bps) of dreb2a was used to BLAST-search the public database. A region containing the DREB2A promoter was identified in chromosome 5 of Arabidopsis (Accession No. AB010692) between nucleotide positions 51901-53955 (Table 6).  
     [0417] Two PCR primers designed to amplify the promoter region from  Arabidopsis thaliana  genomic DNA are as follows:  
     [0418] dreb2a-reverse: 5′-GCCC AAGCTT CAAGTTTAGTGAGCACTATGTGCTCG-3′ [SEQ ID NO: 19];  
     [0419] and dreb2a-forward: 5′-GGA AGATCT CCTTCCCAGAAACAACACAATCTAC-3′ [SEQ ID NO: 20].  
     [0420] The dre2ba-reverse primer includes a Hind III ( AAGCTT ) restriction site near the 5′-end of the primer and dreb2a-forward primer has a Bgl II ( AGATCT ) restriction site at near 5′-end of the primer. These restriction sites may be used to facilitate cloning of the fragment into an expression vector.  
     [0421] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The reaction conditions that may be used in this PCR experiment are as follows: Segment 1: 94° C., 2 minutes; Segment 2: 94° C., 30 seconds; 60° C., 1 minute; 72° C., 3 minutes, for a total of 35 cycles; Segment 3: 72° C. for 10 minutes. A PCR product of 2054 bp is expected.  
     [0422] The PCR products can be subject to electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. The DNA fragments containing the inducible promoter will be excised and purified using a QIAQUICK gel extraction kit (Qiagen, Valencia, Calif.).  
     [0423] (2) P5CS Promoter  
     [0424] A cDNA for delta 1-pyrroline-5-carboxylate synthetase (P5CS) has been isolated and characterized (Yoshiba et al. (1995)  Plant J.  7: 751-760). The cDNA encodes an enzyme involved in the biosynthesis of proline under osmotic stress (drought/high salinity). The transcription of the P5CS gene was found to be induced by dehydration, high salt, and treatment with plant hormone ABA, while it did not respond to heat or cold treatment.  
     [0425] A genomic DNA containing a promoter region of P5CS was identified by a BLAST search of GenBank using the upstream untranslated region (106 bps) of the P5CS sequence (Accession No. D32138). The sequence for the P5CS promoter is located in the region between from nucleotide positions 45472 to 47460 (Accession No. AC003000; Table 6).  
     [0426] Reverse and forward PCR primers designed to amplify this promoter region from  Arabidopsis thaliana  genomic DNA are  
     [0427] P5CS-reverse primer 5′-GCCC AAGCTT GTTTCATTTTCTCCATGAAGGAGAT-3′ [SEQ ID NO: 21]; and  
     [0428] P5CS-forward primer 5′-GGA AGATCT TATCGTCGTCGTCGTCTACCAAAACCACAC-3′ [SEQ ID NO: 22].  
     [0429] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The PCR product is expected to be 1988 bps and may be PCR amplified and gel purified following the same protocol described for the dreb2a promoter.  
     [0430] (3) rd22 Promoter  
     [0431] A cDNA clone of rd22 was isolated from Arabidopsis under dehydration conditions (Yamaguchi-Shinozaki et al. (1993)  Mol. Gen. Genet.  238: 17-25). Transcripts of rd22 were found to be induced by salt stress, water deficit and endogenous abscisic acid (ABA) but not by cold or heat stress. A promoter region was identified from GenBank by using Nucleotide Search WWW Entrez at the NCBI with the rd22 as a search word. The sequence for the rd22 promoter is located in the region between nucleotide positions 17 to 1046 (Accession No. D10703; Table 6).  
     [0432] Reverse and forward PCR primers designed to amplify this promoter region from  Arabidopsis thaliana  genomic DNA are  
     [0433] rd22-reverse primer 5′-GCTCT AAGCTT CACAAGGGGTTCGTTTGGTGC-3′ [SEQ ID NO: 23]; and  
     [0434] rd22-forward primer 5′-GG GGTACC TTTTGGGAGTTGGAATAGAAATGGGTTTGATG-3′ [SEQ ID NO: 24].  
     [0435] The rd22-reverse primer includes a Hind III ( AAGCTT ) restriction site near the 5′-end of primer and rd22-forward primer has a KpnI ( GGTACC ) restriction site at near 5′-end of primer.  
     [0436] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The PCR product is expected to be 1029 bps and may be PCR amplified and gel purified following the same protocol described for the dreb2a promoter.  
     [0437] (4) rd29a Promoter  
     [0438] The rd29a and rb29b genes were isolated and characterized by Shinozaki&#39;s group in Japan (Yamaguchi-Shinizaki et al. (1993)  Plant Physiol.  101: 1119-1120). Both rd29a and rb29b gene expressions were found to be induced by desiccation, salt stress and exogenous ABA treatment (Yamaguchi-Shinizaki et al. (1993) supra); Ishitani et al. (1998)  Plant Cell  10: 1151-1161). The rd29a gene expression was induced within 20 min after desiccation, but rd29b mRNA did not accumulate to a detectable level until 3 hours after desiccation. Expression of rd29a could also be induced by cold stress, whereas expression of rd29b could not be induced by low temperature.  
     [0439] A genomic clone carrying the rd29a promoter was identified by using Nucleotide Search WWW Entrez at the NCBI with the rd29a as a search word. The sequence for the rd29a promoter is located in the region between nucleotide positions 3870 to 5511 (Accession No. D13044, Table 6).  
     [0440] Reverse and forward primers designed to amplify this promoter region from Arabidopsis genomic DNA are:  
     [0441] rd29a-reverse primer 5′-GCCC AAGCTTA ATTTTACTCAAAATGTTTTGGTTGC-3′ [SEQ ID NO: 25]; and  
     [0442] rd29a-forward primer 5′-CC GGTACC TTTCCAAAGATTTTTTTCTTTCCAATAGAAGTAATC-3′ [SEQ ID NO: 26].  
     [0443] The rd29a-reverse primer includes a Hind III ( AAGCTT ) restriction site near the 5′-end of primer and rd29a-forward primer has a KpnI ( GGTACC ) restriction site near 5′-end of primer.  
     [0444] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The PCR product is expected to be 1641 bps and may be PCR amplified and gel purified following the same protocol described for the dreb2a promoter.  
     [0445] (5) rd29b Promoter  
     [0446] A genomic clone carrying the rd29b promoter was identified by using Nucleotide Search WWW Entrez at the NCBI with the rd29b as a search word. The sequence for the rd29a promoter was located in the region between nucleotide positions 90 to 1785 for rd29b (Accession No. D13044; Table 6).  
     [0447] Reverse and forward PCR primers designed to amplify this promoter region from  Arabidopsis thaliana  genomic DNA are:  
     [0448] rd29b-reverse primer 5′-GCGG AAGCTT CATTTTCTGCTACAGAAGTG-3′ [SEQ ID NO: 27]; and  
     [0449] rd29b-forward primer 5′-CC GGTACC TTTCCAAAGCTGTGTTTTCTCTTTTTCAAGTG-3′ [SEQ ID NO: 28].  
     [0450] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The PCR product is expected to be 1695 bps and may be PCR amplified and gel purified following the same protocol described for the dreb2a promoter.  
     [0451] (6) rab18 Promoter  
     [0452] A rab-related (responsive to ABA) gene, rab18 from Arabidopsis has been isolated. The gene encodes a hydrophilic, glycine-rich protein with the conserved serine- and lysine-rich domains. The rab18 transcripts accumulate in plants exposed to water deficit or exogenous abscisic acid (ABA) treatment. A weak induction of rab18 mRNA by low temperature was also observed (Ishitani et al. (1998)  Plant Cell  10: 1151-1161).  
     [0453] A genomic DNA containing a promoter region of rab18 was identified by a BLAST search of GenBank using the upstream untranslated region (757 bps) of the rab18 sequence (Accession No. L04173). The sequence of the rab18 promoter is located in the region between nucleotide positions 8070 to 9757 (Accession No. AB013389).  
     [0454] Reverse and forward PCR primers designed and used to amplify this promoter region from  Arabidopsis thaliana  genomic DNA are:  
     [0455] rab18-reverse primer 5′-GCCC AAGCTT CAAATTCTGAATATTCACATATCAAAAAAGTG-3′ [SEQ ID NO: 29]; and  
     [0456] rab18-forward primer 5′-GGA AGATCT GTTCTTCTTGTCTTAAGCAAACACTTTGAGC-3′ [SEQ ID NO: 30].  
     [0457] The rab18-reverse primer includes a Hind III ( AAGCTT ) restriction site near the 5′-end of the primer and rab18-forward primer has a Bgl II ( AGATCT ) restriction site near the 5′-end of the primer.  
     [0458] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The PCR product is expected to be 1687 bps and may be PCR amplified and gel purified following the same protocol described for the dreb2a promoter.  
     [0459] (7) Cor47 Promoter  
     [0460] The DNA sequence of cDNA for cold-regulated (cor47) gene of  Arabidopsis thaliana  was determined. Gilmour et al. (1992)  Plant Molec. Biol.  18: 13-21). Expression of cor47 gene was induced by cold stress, dehydration and high NaCl treatment (Ishitani et al. (1998)  Plant Cell.  10: 1151-1161). The promoter region of cor47 gene was identified in GenBank by using Nucleotide Search WWW Entrez at the NCBI with the cor47 as a search word. The sequence of the cor47 promoter is located in the region between nucleotide positions 1-1370 (Accession No. AB004872; Table 6).  
     [0461] Reverse and forward PCR primers designed to amplify this promoter region from  Arabidopsis thaliana  genomic DNA are:  
     [0462] cor47-reverse primer 5′-GCCC AAGCTT TCGTCTGTTATCATACAAGGCACAAAACGAC-3′ [SEQ ID NO: 31]; and  
     [0463] cor47-forward primer 5′-GGA AGATCT AGTTAATCTTGATTTGATTAAAAGTTTATATAG-3′ [SEQ ID NO: 32].  
     [0464] The cor47-reverse primer includes a Hind III ( AAGCTT ) restriction site near the 5′-end of the primer and cor47-forward primer has a Bgl II ( AGATCT ) restriction site near the 5′-end of the primer.  
     [0465] Total genomic DNA may be isolated from  Arabidopsis thaliana  (ecotype colombia) by using the CTAB method (Ausubel et al. (1992)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). Ten nanograms of the genomic DNA can be used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The PCR product is expected to be 1370 bps and may be PCR amplified and gel purified following the same protocol described for the dreb2a promoter.  
     [0466] Construction of the plasmids containing CBF1 and inducible promoter. The expression binary vector pMEN020 contains a kanamycin resistance gene (neomycin phosphotransferase) for antibiotic selection of the transgenic plants and a Spc/Str gene used for bacterial or agrobacterial selections. The pMEN020 plasmid is digested with restriction enzymes such as HindIII and BglII to remove the 35S promoter. The 35S promoter is then replaced with an inducible promoter.  
     [0467] (1) Cloning of the Inducible Promoter into pMEN020  
     [0468] The sequences of the inducible promoters that are PCR amplified and gel purified, as well as the plasmid pMEN020, are subject to restriction digestion with their respective restriction enzymes as listed in Table 7. Both DNA samples are purified by using the Qiaquick purification kit (Qiagen) and ligated at a ratio of 3:1 (vector to insert). Ligation reactions using T4 DNA ligase (New England Biolabs, MA) are carried out at 16° C. for 16 hours. The ligated DNAs are transformed into competent cells of the  E. coli  strain DH5α by using the heat shock method. The transformed cells are plated on LB plates containing 100 μg/ml spectinomycin (Sigma-Aldrich, St. Louis, Mo.). Individual colonies are grown overnight in five milliliters of LB broth containing 100 μg/ml spectinomycin at 37° C.  
     [0469] Plasmid DNAs from transformants are purified by using QIAQUICK Mini Prep kits (Qiagen) according to the manufacturer&#39;s instruction. The presence of the promoter insert is verified by restriction mapping with the respective restriction enzymes as listed in Table 7 to cut out the cloned insert. The plasmid DNA is also subject to double-strand DNA sequencing analysis using a vector primer  
     [0470] E9.1 primer 5′-CAAACTCAGTAGGATTCTGGTGTGT-3′ [SEQ ID NO: 33].  
     [0471] (2) Cloning of the cbf1 Gene into the Plasmids Containing the Inducible Promoters  
     [0472] To clone the CBF1 gene into the plasmids, different PCR primers with suitable restriction sites for each plasmid are used to isolate CBF1 gene from  Arabidopsis thaliana  genomic DNA. The primers that may be used are listed in Table 8.  
                               TABLE 8                                   Promoter name   Primer name   Cloning sites                                                            Dreb2a   Cbf1-reverse1   BglII   (AGATCT)               Cbf1-forward1   BamHI   (GGATCC)           P5CS   Cbf1-reverse1   BglII   (AGATCT)               Cbf1-forward1   BamHI   (GGATCC)           Rd22   Cbf1-reverse2   KpnI   (GGTACC               Cbf1-forward1   BamHI   (GGATCC)           Rd29a   Cbf1-reverse2   KpnI   (GGTACC               Cbf1-forward1   BamHI   (GGATCC)           Rd29b   Cbf1-reverse2   KpnI   (GGTACC               Cbf1-forward1   BamHI   (GGATCC)           Rab18   Cbf1-reverse1   BglII   (AGATCT)               Cbf1-forward2   XbaI   (TCTAGA           Cor47   Cbf1-reverse1   BglII   (AGATCT)               Cbf1-forward1   BamHI   (GGATCC)                      
 
     [0473] Two of the four available PCR primers (Table 8) are used for cloning the at-CBF1 gene into the expression vectors containing each inducible promoter described above. The four primers have these sequences:  
     [0474] cbf1-reverse 1 5′-GGAAGATCTTGAAACAGAGTACTCTGATCAATGAACTC-3′ [SEQ ID NO: 34],  
     [0475] cbf1-forward 1 5′-CGCGGATCCCTCGTTTCTACAACAATAAAATAAAATAAAATG-3′ [SEQ ID NO: 35] 
     [0476] cbf1-reverse 2 5′-GGGGTACCTGAAACAGAGTACTCTGATCAATGAACTC-3′ [SEQ ID NO: 36], and  
     [0477] cbf1-forward 2 5′-GCTCTAGACTCGTTTCTACAACAATAAAATAAAATAAAATG-3′ [SEQ ID NO: 37].  
     [0478] For example, for the Dreb2a, P5CS, and COR47 promoters that are ligated to a BamHI and BglII flanked insert, the cbf1-reverse 1 and cbf1-forward 1 primers [SEQ ID NO: 34 and 35, respectively] are used to isolate CBF1 gene from  Arabidopsis thaliana  genomic DNA. The cbf1-reverse primer includes a BglII ( AGATCT ) restriction site near the 5′-end of the primer and cbf1-forward primer has a BamHI ( GGATCC ) restriction site near the 5′-end of the primer. A PCR product of 764 bp is expected. The genomic DNA (10 ng) is used as a template in a PCR reaction under conditions suggested by the manufacturer (Boehringer Mannheim). The reaction conditions to be used in this PCR experiment are as follows: Segment 1: 94° C., 2 minutes; Segment 2: 94° C., 30 seconds; 55° C., 1 minute; 72° C., 1 minute, for a total of 35 cycles; Segment 3: 72° C. for 10 minutes.  
     [0479] The PCR products are subject to electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. The DNA fragment containing cbf1 is excised and purified by using a Qiaquick gel extraction kit (Qiagen). The purified fragment and the vector pMBI2001 containing the inducible promoter (Table 8) are each digested with BglII and BamHI restriction enzymes at 37° C. for 2 hours. Both DNA samples are purified by using the QIAQUICK purification kit (Qiagen) and ligated at a ratio of 3:1 (vector to insert ratio). Ligation reactions using T4 DNA ligase (New England Biolabs, MA) are carried out at 16° C. for 16 hours. The ligated DNAs are transformed into competent cells of the  E. coli  strain DH5α by using the heat shock method. The transformation are plated on LB plates containing 100 (g/ml spectinomycin (Sigma-Aldrich).  
     [0480] Individual colonies are grown overnight in five milliliters of LB broth containing 100 g/ml spectinomycin at 37° C. Plasmid DNA are purified by using QIAQUICK Mini Prep kits (Qiagen). The presence of the cbf1 insert is verified by restriction mapping with BglII and BamHI. The plasmid DNA is also subject to double-strand DNA sequencing analysis by using vector primer E9.1  
     [0481] 5′-CAAACTCAGTAGGATTCTGGTGTGT-3′ [SEQ ID NO: 33].  
     [0482] The other primers shown in Table 8 and appropriate restriction enzymes are used in a similar way to clone the CBF1 gene into plasmids containing the other inducible promoters. The resulting plasmids are listed in Table 9 and shown in FIGS.  17 A- 17 G.  
     [0483] A similar cloning strategy may be used to clone other genes, such as cbf2, cbf3, and the other full length CBF genes listed in Table 9 and shown in FIG. 18 (new CBF gene table) into plasmids containing inducible promoters.  
                               TABLE 9                                   Construct name   Promoter name   Figure name                          PMBI2008   Dreb2a               PMBI2009   P5CS               PMBI2010   Rd22               PMBI2011   Rd29a               PMBI2012   Rd29b               PMBI2013   Rab18               PMBI2014   Cor47   FIG. 17G                      
 
     Example 9  
     [0484] Transformation of Agrobacterium with Plasmids Containing CBF1 Gene and Inducible Promoters  
     [0485] After the plasmid vectors containing CBF1 gene and inducible promoters are constructed, these vectors may be used to transform  Agrobacterium tumefaciens  cells expressing the gene products. The stock of  Agrobacterium tumefaciens  cells for transformation may be made as described by Nagel et al. (1990)  FEMS Microbiol Letts  67: 325-328. Agrobacterium strain ABI may be grown in 250 ml LB medium (Sigma-Aldrich) overnight at 28° C. with shaking until an absorbance (A 600 ) of 0.5-1.0 is reached. Cells are harvested by centrifugation at 4,000×g for 15 min at 4 C. Cells are then resuspended in 250 μl chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells are centrifuged again as described above and resuspended in 125 μl chilled buffer. Cells are 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 are then distributed into 40 μl aliquots, quickly frozen in liquid nitrogen, and stored at −80° C.  
     [0486] Agrobacterium cells are transformed with plasmids formed as described above in Section 4B(2) following the protocol described by Nagel et al. (1990) supra. For each DNA construct to be transformed, 50-100 ng DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) is mixed with 40 μl of Agrobacterium cells. The DNA/cell mixture is then transferred to a chilled cuvette with a 2 mm 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 are 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 are plated onto selective medium of LB broth containing 100 μg/ml spectinomycin (Sigma-Aldrich) and incubated for 24-48 h at 28° C. Single colonies are then picked and inoculated in fresh medium. The presence of the plasmid construct are verified by PCR amplification and sequence analysis.  
     Example 10  
     [0487] Transformation of Arabidopsis Plants with  Agrobacterium tumefaciens  Carrying Expression Vector for CBF1 Protein  
     [0488] After transformation of  Agrobacterium tumefaciens  with plasmid vectors containing CBF1 gene and inducible promoters, single Agrobacterium colonies containing each of pMBI2008-pMBI2014 are identified, propagated, and used to transform Arabidopsis plants. Briefly, 500 ml cultures of LB medium containing 100 ug/ml spectinomycin are inoculated with the colonies and grown at 28 C with shaking for 2 days until an absorbance (A 600 ) of &gt;2.0 is reached. Cells are then harvested by centrifugation at 4,000×g for 10 min, and resuspended in infiltration medium (½× Murashige and Skoog salts (Sigma-Aldrich), 1× Gamborg&#39;s B-5 vitamins (Sigma-Aldrich), 5.0% (w/v) sucrose (Sigma-Aldrich), 0.044 μM benzylamino purine (Sigma-Aldrich), 200 μl/L Silwet L-77 (Lehle Seeds) until an absorbance (A 600 ) of 0.8 is reached.  
     [0489] Prior to transformation,  Arabidopsis thaliana  seeds (ecotype Columbia) are 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 are grown under continuous illumination (50-75 μE/m 2 /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 are prepared for transformation by removal of all siliques and opened flowers.  
     [0490] The pots are 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 is removed and pots are turned upright. The immersion procedure is repeated one week later, for a total of two immersions per pot. Seeds are then collected from each transformation pot and analyzed following the protocol described below.  
     Example 11  
     [0491] Identification of Arabidopsis Primary Transformants  
     [0492] Seeds collected from the transformation pots are sterilized essentially as follows. Seeds are dispersed into in a solution containing 0.1% (v/v) Triton X-100 (Sigma-Aldrich) and sterile H 2 O and washed by shaking the suspension for 20 min. The wash solution is then drained and replaced with fresh wash solution to wash the seeds for 20 min with shaking. After removal of the second wash solution, a solution containing 0.1% (v/v) Triton X-100 and 70% EtOH (Equistar) is added to the seeds and the suspension is shaken for 5 min. After removal of the ethanol/detergent solution, a solution containing 0.1% (v/v) Triton X-100 and 30% (v/v) bleach (Chlorox) is added to the seeds, and the suspension is shaken for 10 min. After removal of the bleach/detergent solution, seeds are then washed five times in sterile distilled H 2 O. The seeds are 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&#39;s B-5 vitamins, 0.9% phytagar (Life Technologies, Rockville, Md.), and 50 μg/L kanamycin). Seeds are germinated under continuous illumination (50-75 μE/m 2 /sec) at 22-23° C. After 7-10 days of growth under these conditions, kanamycin resistant primary transformants (T 1  generation) are visible and are obtained for each of constructs pMBI2008-pMBI2014. These seedlings are 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). Progeny seeds (T 2 ) are collected; kanamycin resistant seedlings selected and analyzed as described above.  
     Example 12  
     [0493] Transformation of Cereal Plants with Plasmid Vectors Containing CBF1 Gene and Inducible Promoters.  
     [0494] Cereal plants, such as corn, wheat, rice, sorghum and barley, can also be transformed with the plasmid vectors containing the CBF genes and inducible promoters to modify their biomass or increase their tolerance to environmental stresses. In these cases, the cloning vector, pMEN020, is modified to replace the NptII coding region with the BAR gene of  Streptomyces hygroscopicus  that confers resistance to phosphinothricin. The KpnI and BglII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes. After cloning of the inducible promoters into the modified plasmid by the same procedures described above, the at-cbf coding region of cbf1 gene is inserted into the plasmid following the same procedures as described above. The resulting plasmids are listed in Table 10.  
                           TABLE 10                                   Promoter name   Construct name                          Dreb2a   PMBI2015           P5CS   PMBI2016           Rd22   PMBI2017           Rd29a   PMBI2018           Rd29b   PMBI2019           Rab18   PMBI2020           Cor47   PMBI2021                      
 
     [0495] It is now routine to produce transgenic plants of most cereal crops (Vasil (1994)  Plant Molec. Biol.  25: 925-937) such as corn, wheat, rice, sorghum (Cassas et al. (1993)  Proc. Natl. Acad Sci  90: 11212-11216), and barley (Wan et al. (1994).  Plant Physiol.  104: 37-48. Other direct DNA transfer methods such as the microprojectile gun or  Agrobacterium tumefaciens -mediated transformation can be used for corn (Fromm et al. (1990)  Bio/Technology  8: 833-839; Gordon-Kamm et al. (1990)  Plant Cell  2: 603-618; Ishida (1990)  Nature Biotechnology  14: 745-750), wheat (Vasil et al. (1992)  Bio/Technology  10: 667-674; Vasil et al. (1993)  Bio/Technology  11: 1553-1558; Weeks et al. (1993)  Plant Physiol.  102: 1077-1084), rice (Christou (1991) Bio/Technology  9: 957-962; Hiei et al. (1994)  Plant J.  6: 271-282; Aldemita et al. (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) supra; Vasil (1994)  Plant Molec. Biol.  25: 925-937).  
     [0496] Plasmids 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)  Bio/Technology  8: 833-839; Gordon-Kamm et al. (1990)  Plant Cell  2: 603-618). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al. (1990) supra). Transgenic plants are regenerated by standard corn regeneration techniques (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra).  
     [0497] The plasmids prepared as described above can also be used to produce transgenic wheat and rice plants (Christou (1991) supra; Hiei et al. (1994) supra; Aldemita (1996) supra; Hiei et al. (1997) supra) by following standard transformation protocols known to those skilled in the art for rice and wheat Vasil, et al. (1992) supra; Vasil et al. (1993) supra; Weeks et al. (1993) supra), where the BAR gene is used as the selectable marker.  
     Example 13  
     [0498] Transformation of Plants with Plasmid Vectors Containing CBF1 Gene and Seed Specific Promoters.  
     [0499] The napin promoter from  Brassica campestris  (GenBank accession no. M64632) is a seed specific promoter. A fragment of the napin promoter (between nucleotides 1146 to 2148) is identified and isolated by PCR amplification using a 5′ PCR primer containing a HindIII site upstream of the promoter and a 3′ PCR primer containing a BamHI site downstream of the promoter. Deletion of the napin promoter to −211 and −152 have been shown to have reduced levels of expression (Ellerstrom et al. (1996)  Plant Mol. Biol.  32: 1019-1027; Stålberg et al. (1996)  Planta  199: 515-519; Stålberg et al. (1993)  Plant Mol. Biol.  23: 671-683). These 5′ deleted promoters are useful to have reduced levels of CBF1 expression for applications where the larger napin promoter fragment is too large.  
     [0500] Other seed-active promoters or deletions of these promoters can also be isolated from genomic DNA by using the same method described above for the napin promoter. Examples of these promoters include but are not limited to the soybean 7S seed storage protein (Chen et al. (1989)  Devel. Gen.  10: 112-122, the bean phaseolin promoter (cited in U.S. Pat. No. 5,003,045), the Arabidopsis 12S globulin (cruciferin) promoter (Pang et al. (1988)  Plant Mol. Biol.  11: 805-820, the maize globulin1 promoter (Kriz et al. (1989)  Plant Physiol.  91: 636; U.S. Pat. No. 5,773,691). These promoters maybe used for altering COR gene expression in cereals such as corn, barley, wheat, rice and rye seeds.  
     [0501] The binary constructs containing seed-specific napin promoters (pMEN1001.1-4; pMEN1002.1-4; and pMEN1003.1-4) are used to transform canola and rapeseed plants as described (Moloney et al. U.S. Pat. No. 5,750,871), except that the Bar gene selectable marker is used.  
     [0502] These constructs are also used to transform regenerable barley cells by microprojectile bombardment (Wan et al. (1994)  Plant Physiol.  104: 37-48). After bombardment the tissues are selected on phosphinothricin by standard barley regeneration techniques (Wan and Lemaux, supra).  
     Example 14  
     [0503] Identification of Homologous Sequence to CBF1 in Canola  
     [0504] This example describes the identification of homologous sequences to CBF1 in canola using PCR. Degenerate primers were designed for regions of AP2 binding domain and outside of the AP2 (carboxyl terminal domain). More specifically, the following degenerate PCR primers were used:  
     [0505] Mol 368 (reverse) 5′-CAY CCN ATH TAY MGN GGN GT-3′ 
     [0506] Mol 378 (forward) 5′-GGN ARN ARC ATN CCY TCN GCC-3′ 
     [0507] Y: C/T, N: A/C/G/T, H: A/C/T, M: A/C, R: A/G)  
     [0508] Primer Mol 368 is in the AP2 binding domain of CBF1 (amino acid sequence: H P I Y R G V) while primer Mol 378 is outside the AP2 domain (carboxyl terminal domain)(amino acid sequence: M A E G M L L P).  
     [0509] The genomic DNA isolated from  Brassica 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.  
     [0510] 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 (By Qiagen Extraction Kit). The DNA fragments were ligated into the TA clone vector from TOPO TA Cloning Kit (Invitrogen) and transformed into  E. coli  strain TOP10 (Invitrogen).  
     [0511] 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.  
     [0512]FIG. 16 shows an amino acid sequence of a homolog (CAN1; SEQ ID NO: 17) identified by this process and its alignment to the amino acid sequence of CBF1. The nucleic acid sequence for CAN1 is listed herein as SEQ ID NO: 18.  
     [0513] As illustrated in FIG. 16, the amino acid sequence alignment in four regions of BN-CBF1 shows 82% identity in the AP2 binding domain region and range from 75% to 83% with some alignment gaps due to regions of lesser homology or introns in the genomic sequence. The aligned amino acid sequences show that the polypeptide encoded by the BNCBF1 gene has 88% identity in the AP2 domain region and 85% identity outside the AP2 domain when aligned for two insertion sequences that are outside the AP2 domain. The extra amino acids in the two insertion regions are either due to the presence of introns in this region of the BNCBF1 gene, as it was derived from genomic DNA, or could be due to extra amino acids in these regions of the BNCBF1 gene. Isolation and sequencing of a cDNA of the BNCBF1 gene using the genomic DNA as a probe will resolve this.  
     Example 15  
     [0514] Identification of Homologous Sequence to CBF1 in Canola and Other Species  
     [0515] A PCR strategy similar to that described in Example 14 was used to isolate additional CBF homologs from  Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Glycine max, Raphanus sativus  and  Zea mays.  The nucleotide (for example, bjCBF1) and peptide sequences (for example, BJCBF1-PEP) of these isolated CBF homologs are shown in FIGS. 18A and 18B, respectively. Table 11 lists the sequence names and SEQ ID NOs of these isolated CBF homologs. The PCR primers are internal to the gene so partial gene sequences are initially obtained. The full length sequences of some of these genes were further isolated by inverse PCR or ligated linker PCR. One skilled in the art can use the conserved regions in these genes to design PCR primers to isolate additional CBF genes.  
                               TABLE 11                           SEQ           %       DNA SEQ Name   ID NO:   Peptide SEQ Name   SEQ ID NO:   ID*                  bjCBF1   38   BJCBF1-PEP   39   87               bjCBF2   40   BJCBF2-PEP   41   85               bjCBF3   42   BJCBF3-PEP   43   85               bjCBF4   44   BJCBF4-PEP   45   93               bnCBF1   46   BNCBF1-PEP   47   88               bnCBF2   48   BNCBF2-PEP   49   87               bnCBF3   50   BNCBF3-PEP   51   87               bnCBF4   52   BNCBF4-PEP   53   88               bnCBF5   54   BNCBF5-PEP   55   88               bnCBF6   56   BNCBF6-PEP   57   88               bnCBF7   58   BNCBF7-PEP   59   87               bnCBF8   60   BNCBF8-PEP   61   88               bnCBF9   62   BNCBF9-PEP   63   85               boCBF1   64   BOCBF1-PEP   65   88               boCBF2   66   BOCBF2-PEP   67   87               boCBF3   68   BOCBF3-PEP   69   88               boCBF4   70   BOCBF4-PEP   71   88               boCBF5   72   BOCBF5-PEP   73   87               brCBF1   74   BRCBF1-PEP   75   88               brCBF2   76   BRCBF2-PEP   77   88               brCBF3   78   BRCBF3-PEP   79   88               brCBF4   80 BRCBF4-PEP   81   88               brCBF5   82   BRCBF5-PEP   83   88               brCBF6   84   BRCBF6-PEP   85   88               brCBF7   86   BRCBF7-PEP   87   88               gmCBF1   88   GMCBF1-PEP   89   87               rsCBF1   90   RSCBF1-PEP   91   88               rsCBF2   92   RSCBF2-PEP   93   88               zmCBF1   94   ZMCBF1-PEP   95   80                          
 
     [0516]FIG. 19A shows an amino acid alignment of the AP2 domains of the CBF proteins listed in Table 11 with their consensus sequences highlighted. FIG. 19A also provides a comparison of the consensus sequence with that of the tobacco DNA binding protein EREBP2 (SEQ ID NO: 168; Okme-Takagi et al. (1995) supra). The AP2 domain sequences of these CBF proteins are: atcbf2 (SEQ ID NO:137); atcbf3 (SEQ ID NO: 138); atcbf1 (SEQ ID NO: 139); bjcbf4 (SEQ ID NO: 140); bocbf2 (SEQ ID NO: 141); rscbf2 (SEQ ID NO: 142); bjcbf2 (SEQ ID NO: 143); bncbf7 (SEQ ID NO: 144); bjcbf3 (SEQ ID NO: 145); bncbf2 (SEQ ID NO: 146); bncbf3 (SEQ ID NO: 147); bncbf1 (SEQ ID NO: 148); bncbf6 (SEQ ID NO: 149); bncbf8 (SEQ ID NO: 150); bocbf3 (SEQ ID NO: 151); brcbf2 (SEQ ID NO: 152); bncbf4 (SEQ ID NO: 153); bncbf5 (SEQ ID NO: 154); bocbf4 (SEQ ID NO: 155); brcbf1 (SEQ ID NO: 156); brcbf3 (SEQ ID NO: 157); brcbf4 (SEQ ID NO: 158); brcbf5 (SEQ ID NO: 159); brcbf6 (SEQ ID NO: 160); brcbf7 (SEQ ID NO: 161); rscbf1 (SEQ ID NO: 162); bocbf1 (SEQ ID NO: 163); bocbf5 (SEQ ID NO: 164); bncbf9 (SEQ ID NO: 165); zmcbf1 (SEQ ID NO: 166); and gmcbfl (SEQ ID NO: 167).  
     [0517] As can be seen from the consensus sequence shown in FIG. 19A, a significant portion of the AP2 domain is conserved among the different CBF proteins. In view of this data, Applicants use the conserved sequence in the AP2 domain to define a class of AP2 domain proteins comprising this conserved sequence.  
     [0518]FIG. 19B shows an amino acid alignment of the AP2 domains shown in FIG. 19A and the AP2 doamins of dreb2a (SEQ ID NO:169) and dreb2b (SEQ ID NO: 170) and a consensus sequence between the proteins highlighted. As can be seen, a very high degree of homology exists between AP2 domains shown in FIG. 19A and dreb2a and dreb2b. Applicants employ the conserved sequence in the AP2 domain shown in FIG. 19B to define a broader class of AP2 domain proteins that are capable of binding to CCG regulatory region.  
     [0519]FIG. 19C shows an amino acid alignment of the AP2 domains shown in FIG. 19B and the AP2 domain of tiny (SEQ ID NO: 171) and a consensus sequence between the proteins highlighted. As can be seen, a very high degree of homology exists between AP2 domains shown in FIG. 19A, dreb2a (SEQ ID NO: 169), dreb2b (SEQ ID NO:170), and tiny (SEQ ID NO:171). Applicants employ the conserved sequence in the AP2 domain shown in FIG. 19C to define a yet broader class of AP2 domain proteins that are capable of binding to CCG regulatory region.  
     [0520]FIG. 19D shows a consensus sequence corresponding to the difference between the consensus sequence shown in FIG. 19A and tiny. Applicants employ the highlighted portion of the conserved sequence shown in FIG. 19D to define a group of amino acid residues that may be critical to binding to a CCG regulatory region.  
     [0521]FIG. 19E shows a consensus sequence corresponding to the difference between the consensus sequence shown in FIG. 19B and tiny. Applicants employ the highlighted portion of the conserved sequence shown in FIG. 19E to define another group of amino acid residues that may be critical to binding to a CCG regulatory region.  
     [0522]FIG. 20 shows the amino acid alignment of the amino terminus of the CBF proteins with their consensus sequence highlighted. The sequences of these CBF proteins are: brcbf3 (SEQ ID NO: 172); brcbf6 (SEQ ID NO: 173); bncbf5 (SEQ ID NO: 174); atcbf2 (SEQ ID NO: 175); atcbf3 (SEQ ID NO: 176); atcbf1 (SEQ ID NO: 177); bncbf2 (SEQ ID NO: 178); bncbf6 (SEQ ID NO: 179); bocbf3 (SEQ ID NO: 180); bncbf3 (SEQ ID NO: 181); bncbf8 (SEQ ID NO: 182); bncbf9 (SEQ ID NO: 183); brcbf2 (SEQ ID NO: 184); bocbf5 (SEQ ID NO: 185); bocbf2 (SEQ ID NO: 186); rscbf2 (SEQ ID NO: 187); bncbf4 (SEQ ID NO: 188); bncbf7 (SEQ ID NO: 189); bocbf4 (SEQ ID NO: 190); brcbf7 (SEQ ID NO: 191); brcbf4 (SEQ ID NO: 192); brcbf5 (SEQ ID NO: 193); and rscbf1 (SEQ ID NO: 194).  
     [0523] As can be seen from the consensus sequence shown in FIG. 20, a significant portion of the amino terminus of CBF proteins is conserved among the different CBF proteins. In view of this data, Applicants employ the conserved sequence in the amino terminus domain to define a class of proteins comprising this conserved sequence. Of note, the conserved sequence corresponding to amino acids 31-37 of SEQ ID NO: 2, PKKPAGR; SEQ ID NO: 326), amino acids 35-40 of SEQ ID NO: 2 (AGRKKF; SEQ ID NO: 325), or amino acids 42-46 of SEQ ID NO: 2 (ETRHP; SEQ ID NO: 321), can define the class of CBF proteins. In addition, the consensus sequence corresponding to amino acids 31-37 of SEQ ID NO: 2 PKXXAGR; SEQ ID NO: 319), amino acids 35-40 of SEQ ID NO: 2 (AGRXKF; SEQ ID NO: 320), or amino acids 42-46 of SEQ ID NO: 2 (ETRHP; SEQ ID NO: 321), wherein X is any amino acid residue, also can define the class of CBF proteins.  
     [0524]FIG. 21A shows the amino acid alignment of the carboxy terminus of 24 CBF proteins with their consensus sequences highlighted. The sequences of these CBF proteins are: bncbf3 (SEQ ID NO: 195; SEQ ID NO: 219; SEQ ID NO: 243); bncbf9 (SEQ ID NO: 196; SEQ ID NO: 220; SEQ ID NO: 244); brcbf2 (SEQ ID NO: 197; SEQ ID NO: 221; SEQ ID NO: 245); bncbf1 (SEQ ID NO: 198; SEQ ID NO: 222; SEQ ID NO: 246); bncbf8 (SEQ ID NO: 199; SEQ ID NO: 223; SEQ ID NO: 247); bncbf6 (SEQ ID NO: 200; SEQ ID NO: 224; SEQ ID NO: 248); bocbf3 (SEQ ID NO: 201; SEQ ID NO: 225; SEQ ID NO: 249); bncbf2 (SEQ ID NO: 202; SEQ ID NO: 226; SEQ ID NO: 250); bocbf5 (SEQ ID NO: 203; SEQ ID NO: 227; SEQ ID NO: 251); brcbf5 (SEQ ID NO: 204; SEQ ID NO: 228; SEQ ID NO: 252); rscbf1 (SEQ ID NO: 205; SEQ ID NO: 229; SEQ ID NO: 253); bncbf4 (SEQ ID NO: 206; SEQ ID NO: 230; SEQ ID NO: 254); bocbf4 (SEQ ID NO: 207; SEQ ID NO: 231; SEQ ID NO: 255); bncbf5 (SEQ ID NO: 208; SEQ ID NO: 232; SEQ ID NO: 256); brcbf7 (SEQ ID NO: 209; SEQ ID NO: 233; SEQ ID NO: 257); brcbf6 (SEQ ID NO: 210; SEQ ID NO: 234; SEQ ID NO: 258); bocbf1 (SEQ ID NO: 211; SEQ ID NO: 235; SEQ ID NO: 259); bjcbf2 (SEQ ID NO: 212; SEQ ID NO: 236; SEQ ID NO: 260); bjcbf3 (SEQ ID NO: 213; SEQ ID NO: 237; SEQ ID NO: 261); bncbf7 (SEQ ID NO: 214; SEQ ID NO: 238; SEQ ID NO: 262); rscbf2 (SEQ ID NO: 215; SEQ ID NO: 239; SEQ ID NO: 263); atcbf1 (SEQ ID NO: 216; SEQ ID NO: 240; SEQ ID NO: 264); atcbf2 (SEQ ID NO:217; SEQ ID NO: 241; SEQ ID NO: 265); and atcbf3 (SEQ ID NO: 218; SEQ ID NO: 242; SEQ ID NO: 266).  
     [0525] As can be seen from the consensus sequence shown in FIG. 21A, a significant portion of the carboxy terminus of CBF proteins is conserved among the different CBF proteins. In view of this data, Applicants employ the conserved sequence in the carboxy terminus domain to define a class of proteins comprising this conserved sequence.  
     [0526]FIG. 21B shows the amino acid alignment of the carboxy terminus of 9 CBF proteins with their consensus sequences highlighted. The sequences of these CBF proteins are: bncbf3 (SEQ ID NO: 267; SEQ ID NO: 276; SEQ ID NO: 285); bncbf9 (SEQ ID NO: 268; SEQ ID NO: 277; SEQ ID NO: 286); brcbf2 (SEQ ID NO: 269; SEQ ID NO: 278; SEQ ID NO: 287); bncbfl (SEQ ID NO: 270; SEQ ID NO: 279; SEQ ID NO: 288); bncbf8 (SEQ ID NO: 272; SEQ ID NO: 280; SEQ ID NO: 289); bncbf6 (SEQ ID NO: 273; SEQ ID NO: 281; SEQ ID NO: 290); bocbf3 (SEQ ID NO: 274; SEQ ID NO: 282; SEQ ID NO: 291); bncbf2 (SEQ ID NO: 274; SEQ ID NO: 283; SEQ ID NO: 292); bocbf5 (SEQ ID NO: 275; SEQ ID NO: 284; SEQ ID NO: 293).  
     [0527] As can be seen from the consensus sequence shown in FIG. 21B, a greater portion of the carboxy terminus is conserved when these nine CBF proteins are used. In view of this data, Applicants employ the conserved sequence in the carboxy terminus domain to define another class of proteins comprising this conserved sequence.  
     Example 16  
     [0528] Homologous CBF Encoding Genes in other Plants.  
     [0529] This example shows that homologous sequences to CBF1 are present in other plants. The presence of these homologous sequences suggest that the same or similar cold regulated environmental stress response regulatory elements such as the C-repeat/DRE of Arabidopsis (CCGAC) exist in other plants. This example serves to indicate that genes with significant homology to CBF1, CBF2 and CBF3 exist in a wide range of plant species.  
     [0530] Total plant DNAs from  Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon pimpinellifolium, Prunus avium, Prunus cerasus, Cucumis sativus,  and  Oryza sativa  were isolated according to Stockinger al (Stockinger et al. (1996)  J. Heredity  87: 214-218). Approximately 2 to 10 μg of each DNA sample was restriction digested, transferred to nylon membrane (Micron Separations, Westboro Mass.) and hybridized according to Walling et al. (Walling et al. (1988)  Nucleic Acids Res.  16: 10477-10492). Hybridization conditions were: 42° C. in 50% formamide, 5×SSC, 20 mM phosphate buffer 1× Denhardt&#39;s, 10% dextran sulfate, and 100 μg/ml herring sperm DNA. Four low stringency washes at RT in 2×SSC, 0.05% Na sarcosyl and 0.02% Na 4  pyrophosphate were performed prior to high stringency washes at 55° C. in 0.2×SSC, 0.05% Na sarcosyl and 0.01% Na 4  pyrophosphate. High stringency washes were performed until no counts were detected in the washout. The BclI-BglII fragment of CBF1 (Stockinger et al. (1997)  Proc Natl Acad Sci  94: 1035-1040) was gel isolated (Sambrook et al. (1989), supra) and direct prime labeled (Feinberg et al. (1982)  Anal. Biochem.  132: 6-13) using the primer MT117  
     [0531] (TTGGCGGCTACGAATCCC; SEQ ID NO: 16).  
     [0532] Specific activity of the radiolabelled fragment was approximately 4×10 8  cpm/μg. Autoradiography was performed using HYPERFILM-MP (Amersham) at −80° C. with one intensifying screen for 15 hours.  
     [0533] Autoradiography of the gel showed that DNA sequences from  Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon pimpinellifolium, Prunus avium, Prunus cerasus, Cucumis sativus,  and  Oryza sativa  hybridized to the labeled BclI, BglII fragment of CBF1. These results suggest that homologous CBF encoding genes are present in a variety of other plants.  
     Example 17  
     [0534] Identification of CBF1 Homologs CBF2 and CBF3 Using CBF1  
     [0535] This example describes two homologs of CBF1 from  Arabidopsis thaliana  and named them CBF2 and CBF3.  
     [0536] CBF2 and CBF3 have been cloned and sequenced as described below. The sequences of the DNA and encoded proteins are set forth in SEQ ID NOs: 12 and 13, 14 and 15, and FIGS. 12 and 13.  
     [0537] A lambda cDNA library prepared from RNA isolated from  Arabidopsis thaliana  ecotype Columbia (Lin et al. (1992)  Plant Physiol.  99: 519-525) was screened for recombinant clones that carried inserts related to the CBF1 gene (Stockingeret al. (1997)  Proc Natl Acad Sci  94: 1035-1040). CBF1 was  32 P-radiolabeled by random priming (Sambrook et al. (1989) 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. (1989), 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 at the MSU-DOE Plant Research Laboratory sequencing facility. 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 CBF1 (SEQ ID NO:1 and SEQ ID NO:2, respectively), CBF2 (SEQ ID NO:12 and SEQ ID NO:13, respectively), and CBF3 (SEQ ID NO:14 and SEQ ID NO:15, respectively) appear in the Sequence Listing.  
     [0538] A comparison of the nucleic acid sequences of CBF1, CBF2 and CBF3 indicate that they are 83 to 85% identical as shown in Table 12. FIG. 14 shows the amino acid alignment of proteins CBF1, (SEQ ID NO:2), CBF2 (SEQ ID NO:13), and CBF3 (SEQ ID NO:15).  
                           TABLE 12                                      Percent identity a                                   DNA b     Polypeptide                                     cbf1/cbf2   85   86       cbf1/cbf3   83   84       cbf2/cbf3   84   85                                  
 
     [0539] 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 CBF1 serves as an activation domain in both yeast and Arabidopsis (not shown).  
     [0540] Residues 47 to 106 of CBF1 correspond to the AP2 domain of the protein, a DNA binding motif that, to date, has only been found in plant proteins. A comparison of the AP2 domains of CBF1, CBF2, and CBF3 indicates that there are a few differences in amino acid sequence. These differences in amino acid sequence might have an effect on DNA binding specificity.  
     Example 18  
     [0541] Activation of Transcription in Yeast Containing C-Repeat/DRE Using CBF1, CBF2, and CBF3  
     [0542] This example shows that CBF1, CBF2, and CBF3 activate transcription in yeast containing CRT/DREs upstream of a reporter gene. The CBFs were expressed in yeast under control of the ADC1 promoter on a 2μ plasmid (pDB20.1; Berger et al. (1992)  Cell  70: 251-265). Constructs expressing the different CBFs were transformed into yeast reporter strains that had the indicated CRT/DRE upstream of the lacZ reporter gene. Copy number of the CRT/DREs and its orientation relative to the direction of transcription from each promoter is indicated by the direction of the arrow.  
     [0543]FIG. 15 is a graph showing transcription regulation of CRT/DRE containing reporter genes by CBF1, CBF2, and CBF3 genes in yeast. In FIG. 15, the vertical lines across the arrows of the COR15a construct represent the m3cor15a mutant CRT/DRE construct. Each CRT/DRE-lacZ construct was integrated into the URA3 locus of yeast. Error bars represent the standard deviation derived from three replicate transformation events with the same CBF activator construct into the respective reporter strain. Quantitative B-gal assays were performed as described by Rose and Botstein (Rose et al. (1983)  Methods Enzymol.  101: 167-180).  
     Example 19  
     [0544] Identification and Isolation of Novel CBF-Related Polypeptides  
     [0545] Additionally, we identified novel CBF-related polypeptides from soybean, wheat, rice, and rye plants.  
     [0546] Soybean seeds were bought from a local supermarket (packaged by JAMECO Co, San Francisco, Calif.). DNA and mRNA were isolated using standard procedures (Ausubel et al. (1998)  Current Protocols in Molecular Biology  (Greene &amp; Wiley, New York)). A soybean seedling cDNA library was also constructed using standard procedures. Based on the sequence of the Arabidopsis CBF1 gene (SEQ ID NO: 1), degenerate primers  
     [0547] O368 (CAYCCNATHTAYMGNGGNGT (SEQ ID NO: 104));  
     [0548] O376 (GCNGCYTCNGCNGCNGCYTTYTGDAT (SEQ ID NO: 105)); and  
     [0549] O2953 (AARAARTTYMGNGARACNMGNCAY (SEQ ID NO: 106))  
     [0550] were designed. O376 and O2953 were first used in a PCR experiment using soybean genomic DNA as template. The product from this reaction was excised from the gel, purified (Ausubel et al. (1998) supra), and used as a template in a second round of PCR using primers O368 and O376. Then, the PCR product was cloned into pGEM-T and sequenced using T7 and sp6 primers (Promega Corp).  
     [0551] Based on the sequence of the cloned soybean clone, 3′ rapid amplification of cDNA ends (RACE) was performed using the MARATHON cDNA amplification kit (Clontech, Palo Alto, Calif.). Generally, the method entailed first isolating poly(A) mRNA, synthesize first and second strand cDNA 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 double-stranded cDNA. Gene-specific primers were designed to be used along with adaptor specific primers for 3′ RACE reactions. Often, nested primers were used to increase PCR specificity. In this case, the 3′ nested primers were  
     [0552] O5436 (GGAGGAACACGGATAAGTGGGTAAG (SEQ ID NO: 107)) and  
     [0553] O5437 (AGGATTTGGCTGGGGACTTTTCC (SEQ ID NO: 108)).  
     [0554] The resulting RACE fragment was cloned into the pGEM-T vector (Promega Corp) and sequenced using T7 and sp6 primers. The cloned insert was then labeled using the DIG DNA Labeling and Detection Kit following the manufacture&#39;s instructions (Boehringer Mannheim), and the labeled probe was used to screen the soybean cDNA library using standard procedures and hybridization conditions (Ausubel et al. (1998) supra). SEQ ID NO: 127 was isolated in this manner.  
     [0555] Rice seeds were obtained from the laboratory of Dr. Pam Ronald at UC Davis. Corn, wheat, and rye seeds were obtained from the USDA, ARS National Small Grains Research Facility, Aberdeen, Id. DNA and mRNA were isolated using standard procedures. Seedling cDNA libraries were also constructed using standard procedures.  
     [0556] In order to isolate CBF1 homologs from monocotyledon species, CBF1 polypeptide sequence was used to identify related sequences from public plant sequence databases. The tblastn sequence analysis program was employed. A rice homolog (Ace. No. AB023482) was identified as having a P value of 6.3e-17. Based on its sequence, primers  
     [0557] O18016 (ACGCGTCGACCCATCATCACCGAGATCGACTCGAC (SEQ ID NO: 109)) and  
     [0558] O18017 (ATAAGAATGCGGCCGCTCATTGTTCGCTCACTGGGAG (SEQ ID NO: 110)  
     [0559] were synthesized, and the rice gene was isolated from rice genomic DNA by a standard PCR procedure using those primers. The amplified fragment was cloned into the pGEM-T vector following the manufacture&#39;s protocol (Promega Corp). The clone was sequenced using O18016, O18017,  
     [0560] O18035 (GCTGACAGAACGGGTGCCGA (SEQ ID NO: 111)) and  
     [0561] O18036 (TGACCGTTTCTGGATAGGCA (SEQ ID NO: 112)).  
     [0562] Based on the rice sequence, primers  
     [0563] O18065 (GGCCGGCGGGGCGAACCAAGTTCC (SEQ ID NO: 113)) and  
     [0564] O18066 (AGGCAGAGTCGGCGAAGTTGAGGC (SEQ ID NO: 114))  
     [0565] were synthesized. These primers were used to isolate rye and wheat CBF gene fragments by PCR from their respective cDNA libraries. For some of the PCR reactions outlined above, a PCR optimization kit (Boehringer Mannheim) was used. The PCR product was cloned into the pGEM-T vector (Promega Corp). To isolate full-length rye cDNAs, the rye fragment was then labeled with  32 P dCTP using the High Prime DNA Labeling Kit (Boehringer Mannheim). Purified radiolabeled probes were used to probe a rye cDNA library using standard conditions (see Ausubel et al.  Current Protocols in Molecular Biology,  supra section 6.3). SEQ ID NO: 115, 117, 119, 121 and 123, which are rye CBF-related sequences, were isolated in this manner. SEQ ID NO: 125 is a CBF-related peptide sequence identified from wheat.  
     [0566] The percent sequence identity of the AP2 regions of polypeptide SEQ ID NO: 116, 118, 120, 122, 124 and 126, with CBF1 (SEQ ID NO: 2), are shown in Table 13. The percent sequence identity between the different polypeptides provided in the Sequence Listing varied from 53% to 96% over most of the length of the sequences. Generally, these sequences comprise an AP2 domain comprising amino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of SEQ ID NO: 2 or comprise one or more of the following peptides: PKXXAGR (SEQ ID NO:319; amino acids 31-37 of SEQ ID NO: 2), or AGRXKF (SEQ ID NO: 320; amino acids 35-40 of SEQ ID NO: 2) or ETRHP (SEQ ID NO: 321; amino acids 42-46 of SEQ ID NO: 2).  
                               TABLE 13                               Peptide       %       DNA SEQ Name   SEQ ID NO:   SEQ Name   SEQ ID NO:   ID*                                                            Rye   CBF20   115   Rye   CBF20   116   69                       PEP       Rye   CBF28   117   Rye   CBF28-   118   70                       PEP       Rye   CBF46   119   Rye   CBF46-   120   71                       PEP       Rye   CBF7   121   Rye   CBF7-   122   67                       PEP       Rye   CBF71   123   Rye   CBF71-   124   71                       PEP       Wheat   CBF   125   Wheat   CBF-   126   71                       PEP                          
 
     Example 20  
     [0567] Identification and Isolation of Additional CBF Orthologs from other Plants  
     [0568] Medicago truncatula    
     [0569] Orthologous CBF sequences from  Medicago truncatula  were identified by BLAST analysis (TBLASTN 2.1.3 [Apr. 1, 2001]).  Medicago truncatula  ESTs in the Genbank public database were queried using the conserved domain from Arabidopsis CBF1, CBF2, and CBF3. The Arabidopsis sequences included the entire AP2/EREBP conserved domain flanked by the CBF signature sequences PKK/RPAGRxKFxETRHP and DSAWR of Arabidopsis CBF1, CBF2, CBF3, and CBF4 (SEQ ID NOs: 327, 328, 329, and 330, respectively). EST sequences were considered positive hits if they had an HSP BLAST score of at least 100 bits over at least 95% of the query sequence.  
     [0570] All positive EST sequences were organized into contigs using the SEQUENCHER assembly program (SEQUENCHER version 3.1, Gene Codes Corporation, Ann Arbor Mich.). At least seven separate sequences were identified (SEQ ID NOs: 294, 295, 296, 297, 298, 299, and 300). Those that had complete predicted coding regions with a start and a stop codon were cloned directly from cDNA or genomic DNA by PCR using primers in the 5′ and 3′ flanking regions. RACE (SMART RACE, BD Clontech) was used to identify the start and stop codons of genes that did not have full-length coding sequences available in the public database. These sequences were then cloned as above. All sequences (SEQ ID NOs: 294, 295, 296, 297, 298, 299, and 300) were cloned into the transformation vector pMEN65, or a modified version with GATEWAY compatible sites (GATEWAY Technology, Invitrogen Life Technologies, Carlsbad Calif.).  
     [0571] Oryza sativa    
     [0572] Rice orthologs were identified in the GenBank public database as described above for  Medicago truncatula.  Positive hits were organized into contigs using Sequencher. Ten full length sequences (SEQ ID NOs: 301, 302, 303, 304, 305, 306, 307, 308, 309, and 310) were identified and cloned from genomic DNA by PCR using primers in the 5′ and 3′ untranslated regions. All genes were cloned into the transformation vector pMEN65, or a modified version with GATEWAY compatible sites.  
     [0573] Medicago saliva    
     [0574] No putative alfalfa orthologs were identified in GenBank public database using BLAST analysis as described above for  M. truncatula.  However, because  M. truncatula  and  M. sativa  species are so closely related, three alfalfa CBF orthologs (SEQ ID NOs: 316, 317, and 318) were cloned directly from alfalfa genomic DNA using the  M. truncatula  cloning primers. All genes were cloned into the transformation vector pMEN65.  
     [0575] Zea mays    
     [0576] Maize orthologs were identified using BLAST analysis as described for  M. truncatula  in a proprietary cDNA database. Five orthologs (SEQ ID NOs: 311, 312, 313, 314, and 315) were cloned with PCR primers in the 5′ and 3′ untranslated regions and inserted into pMEN65.  
     [0577] Alignment with Arabidopsis CBFs  
     [0578] Polynucleotides isolated from  Medicago truncatula  (SEQ ID NOs: 294-300) which encoded CBF-related peptide sequences are shown compared with Arabidopsis CBF1, CBF2, and CBF3 in FIG. 39 (SEQ ID NOs: 2, 15, 13, and 97, respectively). Polynucleotides isolated from  Oryza sativa  (SEQ ID NOs: 301-310) which encoded CBF-related peptide sequences are shown compared with Arabidopsis CBF1, CBF2, and CBF3 in FIG. 40 (SEQ ID NOs: 2, 15, 13, and 97, respectively). Polynucleotides isolated from  Zea mays  (SEQ ID NOs: 311-315) which encoded CBF-related peptide sequences are shown compared with Arabidopsis CBF1, CBF2, CBF3, and CBF4 in FIG. 41 (SEQ ID NOs: 2, 15, 13, and 97, respectively).  
     Example 21  
     [0579] Testing of CBF Orthologs in a Transient Assay  
     [0580] Preparation of Transformant Vectors  
     [0581] Seven clones from  M. truncatula,  ten clones from rice, and three clones from alfalfa ( M. sativa ) were tested for CBF activity in a transient assay. CBF activity was determined by activation of the RD29a promoter, quantitated by GUS activity. Results are shown on FIG. 42. In addition, rice CBF G3376 (SEQ ID NO:307) activated transcription from the RD29a promoter by greater than four-old when compared with the plasmid pMEN 65 vector control.  
     [0582] CBF clones were introduced into the pMEN 65 vector under the transcriptional control of the 35S promoter to create a 35S::CBF clone.  
     [0583] 35S::CBF clones transformed into Agrobacterium cells were grown up overnight in LB medium (supplemented with kanamycin 75 mg/l; spectinomycin 100 mg/l; chloramphenicol 25 mg/l) at 28° C. with shaking. The resulting culture (between 1% and 10% original volume) was then inoculated once more into LB (supplemented with kanamycin 75 mg/l; spectinomycin 100 mg/l; chloramphenicol 25 mg/l; 20 μM acetosyringone; and 10 mM MES) and grown overnight at 28° C. with shaking.  
     [0584] Plasmid P511, which contains the GUS reporter gene under the control of the RD29a promoter, was transformed and amplified as described for the 35S::CBF clone in this example. Plasmid vector pMEN 65 was similarly innoculated and amplified to provide the vector control.  
     [0585] The overnight cultures were then pelleted by low-speed centrifugation, 1500×g (3000 rpm in Sorvall RT7) for 25 minutes. Cell pellets were resuspended in ⅕ the original culture volume of freshly made Infiltration Media (IM: 10 mM MES; 10 mM MgCl 2 ; 150 μM acetosyringone) and resuspended cell pellets were kept at ambient temperature, with occasional mixing, for at least two hours before use. The resuspensed cell pellet was diluted with IM to attain an final absorption value at 600 nm of 1.0 in a 1 cm lightpath (A 600 , O.D.=1.00).  
     [0586] Equal amounts of the P511 culture and the 35S::CBF culture were mixed by inversion to a final volume of 0.8 ml per tube.  
     [0587] Transient Transformation of  Nicotiniana benthamiana  Leaf Tissue  
     [0588] The plant was watered from the top two hours prior to the infiltration procedure. Healthy symetrical leaves that had good turgor were chosen. A total of ten ˜0.5 cm-diameter circles were marked on the axial side of each leaf. A positive control was included on at least one leaf of each plant. Negative controls were included in the assay on every leaf. Control suspensions were made up for half the reporter construct mix and for the other half of the mix a binary vector without insert was used.  
     [0589] The culture mix was taken up into a 1 ml disposable syringe and approximately 0.1 ml was expeled onto each circle on the leaf. The plants were then cultivated for five days until sample collection.  
     [0590] Extraction and GUS Assay  
     [0591] Each leaf disk was excised from the plant using a cork borer and placed in 300 μl of Buffer 1 (50 mM Na+ Phosphate, pH 7.9; 16.7 mM EDTA) in a tube in a cluster tube rack (Catalogue No. 4413, Corning Inc.) which also contained a single stainless steel bead (Type 440C stainless stell balls {fraction (3/16)}″, Catalogue No. 9529K13, McMaster-Carr Supply Co.).  
     [0592] One hundred microlitres of Buffer 2 (50 mM Na+ Phosphate, pH 7.9; 50 mM β-mercaptoethanol) was then added to each tube, the tube was capped, and the rack shaken in a model MM300 shaker-mixer (F. Kurt Retch Gmbh &amp; Co. K G, Haan, Germany) for 1.5 minutes at 28 beats/minute. The rack was rotated half a turn and shaken again for 1.5 minutes. The rack was briefly centrifuged.  
     [0593] One hundred microlitres of Buffer 3 (50 mM Na+ Phosphate, pH 7.9; 0.5% lauroylsarcosine, sodium salt; 0.5% TRITON X-100) was then added to each tube and mixed by inversion. The tube was allowed to stand for two minutes at ambient temperature. The rack was centreifuged at 3000 rpm in a table top centrifuge for 15 minutes at 4° C.  
     [0594] Forty microliters of Extraction Buffer (3 parts Buffer 1 mixed with 1 part Buffer 2 with 1 part Buffer 3) were added to each well in a 96-well reaction plate (Microseal 96 Skirted V-bottom Polypropylene Microplate; Catalogue No. MSP-9601, Natural; MJ Research, Inc.) Ten microlitres of the supernatant from each tube was added to each well.  
     [0595] Ten miligrams of 4-methylumbelliferyl β-D-glucuronide (MUG; Catalogue No. M-5664, Sigma-Aldrich Chemical Co.) were disolved in 14.5 ml of Extraction Buffer to approximately 2 mM. 50 μl of this MUG solution was added to each sample in the reaction plate and the plate was incubated at 37° C. for one hour.  
     [0596] Two hundred microlitres of Stop Buffer (0.2 M Na 2 CO 3 ) were added to a black 96-well plate (Polyfiltronics 96-well, 300 μl, black, flat-bottom microplates; Catalogue No. PF030-PBX8, Phenix Research Products, Hayward Calif.), then 8 μl of each reaction transferred to each Stop Buffer plate well and mixed. The samples were then read using a Synergy HT plate reader (BIOTECK Instruments, Inc., Winoosk, Vt.).  
     Example 22  
     [0597] Overexpression of CBF1 or CBF2 Increases Arabidopsis Drought or High Salt Tolerance  
     [0598] Soil studies were done by growing seedlings for 10 days with water, and then letting the soil dry out (no further watering) until the plants were severely dehydrated. The soil was then watered and then recovery of the plants was measured. The transgenic lines were alternated with the control wild type lines, with a one-inch spacing between plants in two inches of soil. No detrimental effects were observed but the beneficial effects seen need further testing with drought inducible promoter lines.  
     [0599] Two separate root elongation assays were performed to evaluate the drought resistance phenotypes of the transgenic plants. First plants were grown on MS agar plates for two weeks, and then transferred to MS agar plates containing either 300 mM mannitol or 150 mM NaCl. Those concentrations were chosen because preliminary testing showed that wild type plants showed the most dramatic reduction in root growth in those conditions. The root lengths were then measured after seven days, and the data summarized in Table 14. The growth on sucrose (0.3% w/v), the non-inhibition control and the growth on either salt or mannitol is shown. The control line lacking a CBF gene is the 643-3 line. When the ratio of the CBF line to the 643-3 line (CBF/wild type) is significantly above 1.0, this is an indication of drought or salt tolerance.  
     [0600] From these, we concluded that the overexpression of the CBF genes did provide growth benefit under high osmotic pressure and high salt, particularly for the CBF2 lines tested.  
               TABLE 14                          Mannitol (M) and Salt (S) Root Elongation Assay (mm)                                 Ave   Std   CBF/wt                                                 CBF1#1   38.5   12.9   0.80           643-3   48.2   10.4           CBF1#1 (S)   20.0   7.1   1.30           643-3 (S)   15.4   3.3           CBF1#1 (M)   21.7   6.1   1.63           643-3 (M)   13.3   3.6           CBF1#6   43.7   16.8   0.94           643-3   46.5   14.7           CBF1#6 (S)   16.5   4.0   1.03           643-3 (S)   16.0   3.8           CBF1#6 (M)   23.2   7.7   1.25           643-3 (M)   18.5   3.6           CBF2#10   48.3   7.2   0.89           643-3   54.2   5.3           CBF2#10 (S)   15.8   3.7   1.08           643-3 (S)   14.7   2.6           CBF2#10 (M)   21.0   5.4   1.31           643-3 (M)   16.1   3.4           CBF2#14   40.5   11.3   0.89           643-3   45.3   13.0           CBF2#14 (S)   21.5   3.9   1.50           643-3 (S)   14.4   3.0           CBF2#14 (M)   24.0   5.3   1.67           643-3, (M)   14.4   2.6                      
 
     Example 23  
     [0601] Identification of Transgenic Arabidopsis Plants that Express CBF3  
     [0602] Total soluble protein was obtained essentially as described (Gilmour et al. (1996)  Plant Physiol.  111: 293-299) by grinding leaf material (about 100 mg) in 0.4 ml extraction buffer (50 mM PIPES pH 7.0, 25 mM EDTA) containing 2.5% (w/v) polyvinyl-polypyrrolidone and removing insoluble material by centrifugation (16000 g×20 min). Protein concentration in the supernatant was measured using the dye-binding method of Bradford (1976) with BSA as the standard. Protein samples (50 μg total protein) were fractionated by tricine SDS/PAGE (Schägger et al. (1987)  Anal. Biochem.  166: 368-379) and transferred to 0.2 micron nitrocellulose membranes by electroblotting. COR15am and COR6.6 were detected using the ECL kit (Amersham) with antiserum raised to recombinant COR15am and COR6.6 (Gilmour et al. (1996)  Plant Physiol.  111: 293-299).  
     [0603] Transgenic Arabidopsis plants that overexpress CBF3 at normal growth temperature were created by placing the CBF3 coding sequence under control of the cauliflower mosaic virus (CaMV) 35S promoter and transforming the construct into Arabidopsis plants using the floral dip transformation procedure. Transgenic Arabidopsis plants that overexpress CBF3 were generated by transforming the chimeric genes into Arabidopsis ecotype Ws-2 plants.  
     [0604] A 910 bp BamHI/HindIII fragment from a cDNA clone containing the whole coding region of CBF3 (Gilmour et al. (1998)  Plant J.  16: 433-442) was inserted into the BglII and HindIII sites of the binary transformation vector pGA643. PGA643 has a CaMV 35S promoter and the terminator from gene 7 of pTiA6 (An (1988) “Binary Vectors”, in Gynheung et al., editors, (1995)  Plant Molecular Biology Manual,  Kluwer Acad. Publishers). The resulting plasmid, pMPS13, which contains the CBF3 coding sequence under control of the CaMV 35S promoter, was transformed into  Agrobacterium tumefaciens  strain GV3101 by electroporation (Koncz et al. (1986)  Mol. Gen. Gen.  204: 383). Arabidopsis plants were transformed with plasmid pMPS13 or the transformation vector pGA643 using the floral dip method (Clough et al. (1998)  Plant J.  16: 735-743). Transformed plants were selected on the basis of kanamycin resistance. Homozygous T3 or T4 plants were used in all experiments.  
     [0605] Standard procedures were used for plasmid manipulations (Sambrook et al. (1989), supra). Prior to transformation,  Arabidopsis thaliana  seeds were sown at a density of ˜10 plants per 4″ pot onto Bacto planting mix (Michigan Peat Co., Houston, Tex.) covered with fiberglass mesh (18 mm×18 mm). Plants were grown under continuous illumination (100-150 μE/m 2 /sec) at 20-22° C. with 65-70% relative humidity. Plants were used when the primary inflorescences were approximately 10-12 cm high. The pots were then immersed upside down in the mixture of Agrobacterium infiltration medium as described above (Clough et al. (1998) supra) for 2-3 seconds, and placed on their sides to allow draining into a 1′×2′ flat surface covered with plastic wrap. After 24 hours, the plastic wrap is removed and the pots were turned upright. Seeds were then collected from each transformation pot and analyzed following the protocol described below  
     [0606] A construct comprising a cold-regulatable polypeptide gene coding sequence (CBF3) operably linked to a constitutive promoter was generated.  
     [0607] Twenty-two independent lines were identified in which the T2 plants segregated 3:1 for kanamycin resistance (the selectable marker carried on the transformation vector). These lines presumably carried a single active T-DNA locus. The kanamycin resistant T2 plants were then screened by western analysis for constitutive expression of COR15a, a target gene of the CBF transcriptional activators.  
     [0608] Three independent transgenic lines—A28, A30 and A40—were identified that produced the COR15am polypeptide at high levels uniformly among plants grown at normal temperatures. Northern blot analysis indicated that the transcript levels for CBF3 were about equal in non-cold acclimated and cold-treated A28, A30 and A40 plants and were much greater than those observed in either non-cold acclimated or cold-treated control plants (i.e. non-transformed plants or transgenic plants carrying the transformation vector without an insert). The transcript levels for two target COR genes, COR15a and COR6.6, were also nearly equal in non-cold acclimated and cold-treated A28, A30 and A40 plants and approximated the levels observed in cold-acclimated control plants. Western blot analysis indicated that the proteins encoded by COR15a and COR6.6 were present in both non-cold acclimated and cold-acclimated A28, A30 and A40 plants at 3 to 5 fold-higher levels than those found in cold-acclimated control plants.  
     Example 24  
     [0609] Overexpression of the CBF-Related Polypeptide in Arabidopsis Affects Physiological and Morphological Characteristics of the Plant  
     [0610] After the same number of days of vegetative growth at normal temperature, physiological and morphological characteristics of the CBF3-expressing plants were compared with those of wild type plants. The size was less than that of the control plants. Additional differences in growth characteristics were also evident. One was that the CBF3-expressing plants had a pronounced prostrate growth habit; whereas the leaves of the control plants generally had an upright stature, those of the transgenic plants laid flat to the soil. The CBF3-expressing plants also had much shorter petioles when compared to those of the control plants and the leaves had a slight bluish-green tint. Also, there was a substantial difference in time to flowering between the control and CBF3-expressing plants; i.e., control plants bolted and formed flowers well before the CBF3-expressing plants did. In one experiment, for instance, the control plants began to bolt at 17 days while A40, A30 and A28 plants took 21, 26 and 28 days, respectively, to initiate bolting (Table 15).  
               TABLE 15                          Effects of CBF3 Expression on Time to Flowering and       Rosette Leaf Number.                                 Plants   Time to Flowering (d)   Rosette Leaves Per Plant                                             Ws-2   17   4.5           B6   17   4.6           A40   21   6.0           A30   26   9.7           A28   28   12.5                      
 
     [0611] The CBF3-expressing plants went on to form flowers and set seed, though the final plant mass and seed yield was considerably less than that obtained with control plants. The lower yield of seed was due at least in part to the CBF3-expressing plants producing fewer axillary shoots. Significantly, the delay in flowering observed in the CBF3-expressing plants did not “simply” involve a slower overall growth rate, but appeared to involve a developmental delay in flowering. In one experiment, for instance, the control plants produced an average of 4.5 and 4.6 leaves per rosette while the A40, A30, and A28 transgenic plants produced 6.0, 9.7, and 12.5 leaves per rosette, respectively (Table 15).  
     [0612] In some plants, including Arabidopsis, the transition to flowering is responsive to vernalization, a long period (weeks) of low temperature treatment. In “facultative” plants such as Arabidopsis, the effect of vernalization is to shorten the time to flowering (the magnitude of the effect varies greatly among Arabidopsis ecotypes). If CBF3 expression at warm temperature was fully mimicking exposure to low temperature, then one might think that if it had any effect on flowering, that it would decrease the transition time and cause a corresponding decrease in the number of rosette leaves per plant. Our results, however, indicate that CBF3 expression had the opposite effect; it increased the time to flowering and increased the number of rosette leaves per plant.  
     [0613] When plants expressed a CBF-related polypeptide behind an inducible promoter, the plants were late flowering and their leaf number when measured at the time of bolting (i.e. at a comparable developmental stage) was increased compared to the control as was the above ground biomass at the developmental stage as shown in FIGS. 27A through 27C. The Dreb2a::CBF1 plants grew more slowly, i.e. when plants of same age were compared the biomass of the transgenics is slightly smaller (˜30%) than that of the control plants. But as the plants flowered later they grow for a longer period of time prior to bolting and at this developmental stage they had more leaves and higher biomass than the control plant at the same developmental stage (which the control plant reached a few days earlier).  
     Example 25  
     [0614] Vegetative Growth of Canola after Transformation with Plasmids Containing CBF1, CBF2, or CBF3  
     [0615] Canola was transformed with a plasmid containing the Arabidopsis CBF1, CBF2, or CBF3 genes cloned into the vector pGA643 (An (1987)  Methods Enzymol.  253: 292). In these constructs the CBF genes were expressed constitutively under the CaMV 35S promoter. In addition, the CBF1 gene was cloned under the control of the Arabidopsis COR15 promoter in the same vector pGA643. Each construct was transformed into Agrobacterium strain GV3101. Transformed agrobacteria was grown for 2 days in Minimal AB medium containing the appropriate antibiotics.  
     [0616] Spring canola ( B. napus  cv. Westar) was transformed using the protocol of Moloney (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 uE/m 2 s light using a16 hour light/8 hour dark photoperiod. Cotyledons from 4-5 day old seedlings were collected, the petioles cut and dipped into the Agrobacterium solution. The dipped cotyledons were placed on co-cultivation medium at a density of 20 cotyledons/plate and incubated as described above for 3 days. Explants were transferred to the same media, but containing 300 mg/l timentin (GlaxoSmithKline, 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.  
     [0617] 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.  
     [0618] After the same number of days of vegetative growth at normal temperature, physiological and morphological characteristics of the CBF-expressing plants were compared with those of wild type plants. Our results indicated that CBF3 increased the time to flowering and increased the number of rosette leaves per plant. This phenotype was most obvious in transgenic canola plants overexpressing CBF1, CBF2, or CBF3 under control of the 35S promoter.  
     Example 26  
     [0619] Overexpression of CBF1, CBF2, or CBF3 Affects Stress Tolerance in Plants  
     [0620] In addition to modification to the biomass of plants that occurs due to overexpression of CBF1, CBF2, or CBF3 genes shown in the above examples, plants that have been transformed and overexpress these genes may also be made more resistant to environmental stresses. Thus, when grown under adverse conditions, the biomass of plant transformed with CBF1, CBF2, or CBF3 genes may be significantly greater than that of a plant that is more susceptible to the stress(es) and fails to thrive.  
     [0621] Overexpression of CBF1, CBF2, or CBF3 Increases Freezing Tolerance in Arabidopsis.  
     [0622] Ws-2 and A30 seedlings were grown (13 days and 20 days, respectively) on Gamborg&#39;s B-5 medium containing 0.2% sucrose under sterile conditions in Petri dishes. The plants were tested for freezing tolerance by first placing the plates at −2° C. in the dark for 24 hours followed by ice nucleation with sterile ice chips for 3 hours. The temperature of the freezer was then turned down to −6° C. and the plates were left at this temperature for an additional 24 hours. The plates were taken from the freezer and placed at 4° C. in the dark for 18 hours, followed by 2 days at 22° C. under cool white fluorescent lights (40-50 μmol m −2  s −1 ) with an 18 hour photoperiod. The plates were scored 2 days later for freezing damage.  
     [0623] Electrolyte leakage freeze tests were performed essentially as described (Uemura et al. (1995)  Plant Physiol.  109: 15-30) with minor modifications. Tubes (16×125 mm) containing 3-4 leaves were placed in a low temperature bath set at −2° C. in a randomized design. The randomization was performed with the aid of the SAS system (SAS Institute Inc, Cary N.C.). Ice chips were added to each tube after 1 hour incubation. Each tube was capped with foam plugs and incubated a further 1 hour at −2° C. The bath temperature was then lowered one degree C every 20 minutes. Tubes were removed at each temperature and incubated an additional hour at that same temperature in a separate bath. Tubes were placed on ice after removal from the bath until all tubes had been removed. The samples were then thawed overnight at 2.5° C. and electrolyte leakage was measured as described (Gilmour et al. (1988)  Plant Physiol.  87: 745-750).  
     [0624] Non-cold acclimated control plants were killed by freezing at −6° C. for 24 hours while non-cold acclimated CBF3-expressing plants were not. Results for Ws-2 and A30 plants are shown in FIG. 26A. Electrolyte leakage tests indicated that the freezing tolerance of non-cold acclimated CBF3-expressing plants was about 3 to 4° C. greater than that of the non-cold acclimated control plants. Specifically, non-cold acclimated control plants had an EL 50  (temperature that caused a 50% leakage of electrolytes) of about −4.5° C. while the three CBF3 expressing lines had EL 50  values of about −8° C. (FIG. 26B). Significantly, the freezing tolerance of cold-acclimated CBF3-expressing plants was considerably greater than that of cold-acclimated control plants. Control plants that had been cold-acclimated for 7 days had an EL 50  value of about −6° C. while 7 days cold-acclimated CBF3-expressing plants had EL 50  values of −11° C. or lower (FIGS. 26C and 26D).  
     [0625] Overexpression of CBF1, CBF2, or CBF3 Increases Salt Tolerance in Canola.  
     [0626] Canola was transformed with a plasmid containing the Arabidopsis CBF1, CBF2, or CBF3 genes cloned into the vector pGA643 (An (1987)  Methods Enzymol.  253: 292). In these constructs the CBF genes were expressed constitutively under the CaMV 35S promoter. In addition, the CBF1 gene was cloned under the control of the Arabidopsis COR15 promoter in the same vector pGA643. Each construct was transformed into Agrobacterium strain GV3101. Transformed agrobacteria was grown for 2 days in Minimal AB medium containing the appropriate antibiotics.  
     [0627] Spring canola ( B. napus  cv. Westar) was transformed using the protocol of Moloney (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 uE/m 2 s light using a16 hour light/8 hour dark photoperiod. Cotyledons from 4-5 day old seedlings were collected, the petioles cut and dipped into the Agrobacterium solution. The dipped cotyledons were placed on co-cultivation medium at a density of 20 cotyledons/plate and incubated as described above for 3 days. Explants were transferred to the same media, but containing 300 mg/L timentin (GlaxoSmithKline, 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.  
     [0628] 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.  
     [0629] 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 either CBF1 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.  
     [0630] 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.  
     Example 27  
     [0631] Overexpression of CBF3 Affects Proline Metabolism in Arabidopsis  
     [0632] Proline levels in leaf samples were analyzed by methods described in Example 5.  
     [0633] Under non-cold acclimating growth conditions, the free proline levels in the CBF3-expressing plants were about 5-fold higher than they were in the control plants, levels which were about the same as those found in cold-acclimated control plants (FIG. 22). The proline levels in the CBF3-expressing plants increased further (about 2-fold) upon cold acclimation and were 2-3 fold higher than those found in the cold-acclimated control plants (FIG. 22).  
     [0634] The proline biosynthetic enzyme Δ′-pyrroline-5-carboxylate synthase has a key role in determining proline levels in plants (Yoshiba et al. (1997)  Plant Cell Physiol.  38: 1095-1102). Because of this, and that P5CS transcript levels have been shown to increase in Arabidopsis in response to low temperature (Xin et al. (1998)  Proc. Natl. Acad. Sci.  95: 7799-7804), it was of interest to determine whether P5CS transcript levels were elevated in the CBF3-expressing plants. Northern analysis indicated that they were; P5CS transcript levels were about 4 fold higher in non-cold acclimated CBF3-expressing plants than they were in non-cold acclimated control plants and were about equal to those found in the control plants that had been cold-treated for 1 day (FIG. 23). The P5CS transcript levels in 7-day cold-acclimated CBF3-expressing plants were 2 to 3 fold higher than in cold-acclimated control plants (FIG. 23), a finding that was consistent with the 2 to 3 fold higher levels of proline found in the cold-acclimated CBF3-expressing plants (FIG. 22).  
     Example 28  
     [0635] Overexpression of CBF3 Affects Sugar Metabolism in Arabidopsis  
     [0636] Total soluble sugars (for example, sucrose, glucose, and fructose among others) were extracted from lyophilized leaf material as described in Example 5. The results showed that CBF3 expression affected the sugar levels in plants. Total soluble sugars in control and CBF3-expressing plants at both non-cold acclimating and cold acclimating temperatures were measured. The results show (FIG. 24) that the levels of total sugars in non-cold acclimated CBF3-expressing plants were about 3-fold greater than those in non-cold acclimated control plants. Upon cold acclimation, sugar levels went up in both the control and CBF3-expressing plants about 2-fold, and remained about 3-fold higher in the CBF3-expressing plants. Analysis of the sugars by HPLC indicated that CBF3 expression affected the levels of sucrose; in non-cold acclimated control plants, sucrose levels were about 0.3 μg/100 μg dry weight (DW), while in non-cold acclimated CBF3-expressing plants they were about 1.5 μg/100 μg DW.  
     Example 29  
     [0637] Overexpression of CBF3 Affects Lipid Composition  
     [0638] Total lipids were extracted from Arabidopsis leaves and measured as described in Example 5. The results indicated that CBF3 expression affected lipid composition. The representative results of Ws-2 and A28 are presented in FIG. 25. They indicate that expression of CBF3 had little or no effect on the relative amounts (mol %) of 16:0, 16:3, 18:2 or 18:3 fatty acids in non-cold acclimated plants. Significantly, no appreciable change in the relative amounts of these fatty acids occurred during cold acclimation either. Cold acclimation did result in sizable decreases (30 to 50%) in the relative amounts of 16:1, 16:2, 18:0 and 18:1 fatty acids and in three of these cases, specifically 16:1, 16:2, and 18:0 fatty acids, CBF3 expression caused similar decreases to occur in non-cold acclimated plants. In the case of 18:1 fatty acids, CBF3 expression had an opposite effect from cold acclimation; it resulted in a slight increase in the relative levels of this fatty acid in non-cold acclimated transgenic plants and about a 50% increase in cold-acclimated transgenic plants. Taken together, these results indicate that overexpression of CBF3 has an effect on fatty acid composition and that certain of the changes mimic those that occur with cold acclimation.  
     Example 30  
     [0639] Overexpression of CBF3 Increases Arabidopsis Freezing Tolerance  
     [0640] Ws-2 and A30 seedlings were grown (13 days and 20 days, respectively) on Gamborg&#39;s B-5 medium containing 0.2% sucrose under sterile conditions in Petri dishes. The plants were tested for freezing tolerance by first placing the plates at −2° C. in the dark for 24 hours followed by ice nucleation with sterile ice chips for 3 hours. The temperature of the freezer was then turned down to −6° C. and the plates were left at this temperature for an additional 24 hours. The plates were taken from the freezer and placed at 4° C. in the dark for 18 hours, followed by 2 days at 22° C. under cool white fluorescent lights (40-50 μmol m −2  s −1 ) with an 18 hour photoperiod. The plates were scored 2 days later for freezing damage.  
     [0641] Electrolyte leakage freeze tests were performed essentially as described (Uemura et al. (1995)  Plant Physiol.  109: 15-30) with minor modifications. Tubes (16×125 mm) containing 3-4 leaves were placed in a low temperature bath set at −2° C. in a randomized design. The randomization was performed with the aid of the SAS system (SAS Institute Inc, Cary N.C.). Ice chips were added to each tube after 1 hour incubation. Each tube was capped with foam plugs and incubated a further 1 hour at −2° C. The bath temperature was then lowered one degree C. every 20 minutes. Tubes were removed at each temperature and incubated an additional hour at that same temperature in a separate bath. Tubes were placed on ice after removal from the bath until all tubes had been removed. The samples were then thawed overnight at 2.5° C. and electrolyte leakage was measured as described (Gilmour et al. (1988)  Plant Physiol.  87: 745-750).  
     [0642] Non-cold acclimated control plants were killed by freezing at −6° C. for 24 hours while non-cold acclimated CBF3-expressing plants were not. Results for Ws-2 and A30 plants are shown in FIG. 26A. Electrolyte leakage tests indicated that the freezing tolerance of non-cold acclimated CBF3-expressing plants was about 3 to 4° C. greater than that of the non-cold acclimated control plants. Specifically, non-cold acclimated control plants had an EL 50  (temperature that caused a 50% leakage of electrolytes) of about −4.5° C. while the three CBF3 expressing lines had EL 50  values of about −8° C. (FIG. 26B). Significantly, the freezing tolerance of cold-acclimated CBF3-expressing plants was considerably greater than that of cold-acclimated control plants. Control plants that had been cold-acclimated for 7 days had an EL 50  value of about −6° C. while 7 days cold-acclimated CBF3-expressing plants had EL 50  values of −11° C. or lower (FIGS. 26C and 26D).  
     Example 31  
     [0643] Transformation and Expression in Arabidopsis Plants with CBF-Related Polynucleotides from other Plants  
     [0644] SEQ ID NOs: 294 through 318 were transformed into Arabidopsis plants as described in Examples 7 through 11. Transformed plants are screened for traits as described in Examples 7 through 11.  
     Example 32  
     [0645] Transformation and Expression in Alfalfa Plants with CBF Polynucleotides  
     [0646] SEQ ID NOs: 294 through 318 were transformed into  Medicago sativa  plants as described. For alfalfa transformation a well established protocol from Deborah Samac&#39;s laboratory was used which is based on a system described by Austin et al., (Austin et al. (1995)  Euphytica  85: 381-393). This system gives a regeneration rate of callus pieces producing embryos and later on transgenic plantlets of more than 75%. Transformation of the callus to the generation of transgenic plantlets took from between 9-12 weeks, and the transgenic plantlets were then propagated by cuttings. Leaf explants from the alfalfa cultivar Regen-SY are best suited for rapid production of embryos (Bingham (1991)  Crop Sci.  31: 1098).  
     [0647] For transformation, young leaves (top second to fifth node) from soil grown plants were surface sterilized by a brief rinse in 70% ethanol followed by gentle agitation in a 20% bleach solution for 90 seconds. After three rinses in sterile water, leaflet margins were trimmed away and the remaining piece was cut in half. 50 leaf pieces were generated and placed in 12 ml of liquid SH medium without hormones (Schenk and Hildebrandt (1972)  Can. J. Bot.  50: 199-204). The day before transformation a 4 ml overnight culture (YEP liquid media containing 25 mg/l rifampicin) of  A. tumefaciens  strain LBA4404 containing the binary vector of interest was inoculated. For the transformation, 3 ml of this overnight culture were added to the leaf pieces and incubated at room temperature for 10-15 minutes. After that, the leaf pieces were removed, blotted briefly on sterile filter paper, and placed on plates containing solid callus-inducing B5 media with vitamins (Brown and Atanassov (1985)  Plant Cell Tissue Organ Cult.  4:111-122): B5 salts and vitamins (Gamborg et al. (1968)  Exp. Cell Res.  50: 151-158), 30 g/l sucrose, 0.5 g/l KNO 3 , 0.25 g/l MgSO 4 -7H 2 O, 0.5 g/l proline, 798 mg/l L-glutamine, 99.6 mg/l serine, 0.48 mg/l adenine, 9.6 mg/l glutathione, 1 mg/l 2,4-D, 0.1 mg/l kinetin and 8 g/l Phytagar (Life Technologies, Inc.), pH 5.7). The plates were sealed with gas-permeable tape (#394 3M Venting Tape) and incubated for 7 days at 24° C. with a light intensity of approximately 100 μmol m −2  sect −1 .  
     [0648] After 7 days co-cultivation the leaf pieces were rinsed 3-4 times in sterile water, and transferred to fresh selection plates (B5 media with vitamins containing 25 mg/l kanamycin and 100 mg/l ticarcillin or timentin). Calli and a few embryos formed after about two weeks, and were then transferred to MMS medium (MMS: MS salts, 30 g/l sucrose, vitamins (Nitsch and Nitsch (1969)  Science  163: 85-87), 100 mg/l myo-inositol, 7 g/l phytagar plus 25 mg/l kanamycin and 100 mg/l ticarcillin or timentin) and cultured for an additional 2-3 weeks. As cotyledonary stage embryos grew out from the calli, they were cut off and transferred to fresh MMS medium with antibiotics for conversion to plantlets. Plantlets that formed a primary leaf were transferred to Magenta boxes containing fresh MMS media without kanamycin. This allowed for further root and shoot development. After 2-5 weeks plants that formed roots were removed from the boxes and placed in a test tube containing water. They were kept on the lab bench for 12-48 hours to condition the plants and which reduced leaf loss due to desiccation after transplanting. Individual plants were transplanted to a soil mix and maintained in a growth chamber under conditions to maximize leaf production: 24° C./19° C. day/night temperature, 16/8 hours of light/dark cycle with a light intensity of approximately 300 μmol m −2  sec −1 ). Plants were watered daily and were fertilized weekly with a complete fertilizer.  
     [0649] Plants from individual lines were propagated by cuttings and transferred to soil. Original transformants were maintained in Magenta boxes on selection, while the working plant material was generated by cuttings from the soil explants.  
     [0650] Up to 100 independent plants were tested by PCR for stable integration of the transgene to ensure at least 20 independent transgenic plants were generated per construct. As control for successful setup of the transformation capabilities a 35s::intGUS construct was used. An empty vector control was included with the batch of 4 CBF constructs (total of 6 constructs). The initial transformants (at least 20 per construct) were propagated by cuttings after they developed 5-8 internodes (roughly 7 weeks after transfer to soil). Each cut internode was placed in vermiculite for 1-2 weeks for root formation. 4-7 plants per transgenic line were transplanted in SC-10 Super Cell Cone-tainers (Hummert; cones: 3.8 cm diameter, 21 cm depth; 98 cones per 61×30.5 cm 2  tray) and grown under a 16/8 hours light/dark cycle either in the growth room at 24° C. or in a growth chamber at 24° C./19° C. day/night temperature (or constant at 4° C. for cold acclimation experiments) for further analysis.  
     [0651] Transformed plants are screened for traits as described in Examples 7 through 11.  
     [0652] While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims.  
    
     
       
         1 
         
           
             332  
           
           
             1  
             905  
             DNA  
             Arabidopsis thaliana  
             
               CBF1 gene  
             
           
            1 

      aaaaagaatc tacctgaaaa gaaaaaaaag agagagagat ataaatagct taccaagaca     60 
      gatatactat cttttattaa tccaaaaaga ctgagaactc tagtaactac gtactactta    120 
      aaccttatcc agtttcttga aacagagtac tctgatcaat gaactcattt tcagcttttt    180 
      ctgaaatgtt tggctccgat tacgagcctc aaggcggaga ttattgtccg acgttggcca    240 
      cgagttgtcc gaagaaaccg gcgggccgta agaagtttcg tgagactcgt cacccaattt    300 
      acagaggagt tcgtcaaaga aactccggta agtgggtttc tgaagtgaga gagccaaaca    360 
      agaaaaccag gatttggctc gggactttcc aaaccgctga gatggcagct cgtgctcacg    420 
      acgtcgctgc attagccctc cgtggccgat cagcatgtct caacttcgct gactcggctt    480 
      ggcggctacg aatcccggag tcaacatgcg ccaaggatat ccaaaaagcg gctgctgaag    540 
      cggcgttggc ttttcaagat gagacgtgtg atacgacgac cacggatcat ggcctggaca    600 
      tggaggagac gatggtggaa gctatttata caccggaaca gagcgaaggt gcgttttata    660 
      tggatgagga gacaatgttt gggatgccga ctttgttgga taatatggct gaaggcatgc    720 
      ttttaccgcc gccgtctgtt caatggaatc ataattatga cggcgaagga gatggtgacg    780 
      tgtcgctttg gagttactaa tattcgatag tcgtttccat ttttgtacta tagtttgaaa    840 
      atattctagt tcctttttta gaatggttcc ttcattttat tttattttat tgttgtagaa    900 
      acgag                                                                905 

 
           
             2  
             213  
             PRT  
             Arabidopsis thaliana  
             
               CBF1 polypeptide  
             
           
            2 

      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 Asp 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 

 
           
             3  
             27  
             DNA  
             Artificial Sequence  
             
               MT50 Description of Artificial Sequence C-repeat/DRE  
             
           
            3 

      gatcatttca tggccgacct gcttttt                                         27 

 
           
             4  
             28  
             DNA  
             Artificial Sequence  
             
               MT52 Description of Artificial Sequence C-repeat/DRE  
             
           
            4 

      cacaatttca agaattcact gctttttt                                        28 

 
           
             5  
             27  
             DNA  
             Artificial Sequence  
             
               MT80 Description of Artificial Sequence C-repeat/DRE  
             
           
            5 

      gatcatttca tggtatgtct gcttttt                                         27 

 
           
             6  
             27  
             DNA  
             Artificial Sequence  
             
               MT125 Description of Artificial Sequence C-repeat/DRE  
             
           
            6 

      gatcatttca tggaatcact gcttttt                                         27 

 
           
             7  
             27  
             DNA  
             Artificial sequence  
             
               MT68 Description of Artificial Sequence C-repeat/DRE  
             
           
            7 

      gatcacttga tggccgacct ctttttt                                         27 

 
           
             8  
             27  
             DNA  
             Artificial Sequence  
             
               MT66 Description of Artificial Sequence C-repeat/DRE  
             
           
            8 

      gatcaatata ctaccgacat gagttct                                         27 

 
           
             9  
             25  
             DNA  
             Artificial Sequence  
             
               MT86 Description of Artificial Sequence C-repeat/DRE  
             
           
            9 

      actaccgaca tgagttccaa aaagc                                           25 

 
           
             10  
             60  
             PRT  
             Arabidopsis thaliana  
             
               AP2 domain of 24 kDa polypeptide  
             
           
            10 

      Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val Ser Glu 
      1               5                   10                  15 
      Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 
                  20                  25                  30 
      Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
              35                  40                  45 
      Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             11  
             61  
             PRT  
             Nicotiana tabacum  
             
               AP2 domain of tobacco DNA binding protein EREBP2  
             
           
            11 

      His Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Phe Ala Ala Glu 
      1               5                   10                  15 
      Ile Arg Asp Pro Ala Lys Asn Gly Ala Arg Val Trp Leu Gly Thr Tyr 
                  20                  25                  30 
      Glu Thr Ala Glu Glu Ala Ala Leu Ala Tyr Asp Lys Ala Ala Tyr Arg 
              35                  40                  45 
      Met Arg Gly Ser Lys Ala Leu Leu Asn Phe Pro His Arg 
          50                  55                  60 

 
           
             12  
             651  
             DNA  
             Arabidopsis thaliana  
             
               CBF2 gene  
             
           
            12 

      atgaactcat tttctgcctt ttctgaaatg tttggctccg attacgagtc tccggtttcc     60 
      tcaggcggtg attacagtcc gaagcttgcc acgagctgcc ccaagaaacc agcgggaagg    120 
      aagaagtttc gtgagactcg tcacccaatt tacagaggag ttcgtcaaag aaactccggt    180 
      aagtgggtgt gtgagttgag agagccaaac aagaaaacga ggatttggct cgggactttc    240 
      caaaccgctg agatggcagc tcgtgctcac gacgtcgccg ccatagctct ccgtggcaga    300 
      tctgcctgtc tcaatttcgc tgactcggct tggcggctac gaatcccgga atcaacctgt    360 
      gccaaggaaa tccaaaaggc ggcggctgaa gccgcgttga attttcaaga tgagatgtgt    420 
      catatgacga cggatgctca tggtcttgac atggaggaga ccttggtgga ggctatttat    480 
      acgccggaac agagccaaga tgcgttttat atggatgaag aggcgatgtt ggggatgtct    540 
      agtttgttgg ataacatggc cgaagggatg cttttaccgt cgccgtcggt tcaatggaac    600 
      tataattttg atgtcgaggg agatgatgac gtgtccttat ggagctatta a             651 

 
           
             13  
             216  
             PRT  
             Arabidopsis thaliana  
             
               CBF2 polypeptide  
             
           
            13 

      Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 
      1               5                   10                  15 
      Ser Pro Val Ser Ser Gly Gly Asp Tyr Ser Pro Lys Leu Ala Thr 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 Gln Arg Asn Ser Gly Lys Trp Val Cys 
          50                  55                  60 
      Glu Leu 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 Ile 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 Glu Ile Gln Lys Ala Ala 
              115                 120                 125 
      Ala Glu Ala Ala Leu Asn Phe Gln Asp Glu Met Cys His Met Thr Thr 
          130                 135                 140 
      Asp Ala His Gly Leu Asp Met Glu Glu Thr Leu Val Glu Ala Ile Tyr 
      145                 150                 155                 160 
      Thr Pro Glu Gln Ser Gln Asp Ala Phe Tyr Met Asp Glu Glu Ala Met 
                      165                 170                 175 
      Leu Gly Met Ser Ser Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu 
                  180                 185                 190 
      Pro Ser Pro Ser Val Gln Trp Asn Tyr Asn Phe Asp Val Glu Gly Asp 
              195                 200                 205 
      Asp Asp Val Ser Leu Trp Ser Tyr 
          210                 215 

 
           
             14  
             651  
             DNA  
             Arabidopsis thaliana  
             
               CBF3 gene  
             
           
            14 

      atgaactcat tttctgcttt ttctgaaatg tttggctccg attacgagtc ttcggtttcc     60 
      tcaggcggtg attatattcc gacgcttgcg agcagctgcc ccaagaaacc ggcgggtcgt    120 
      aagaagtttc gtgagactcg tcacccaata tacagaggag ttcgtcggag aaactccggt    180 
      aagtgggttt gtgaggttag agaaccaaac aagaaaacaa ggatttggct cggaacattt    240 
      caaaccgctg agatggcagc tcgagctcac gacgttgccg ctttagccct tcgtggccga    300 
      tcagcctgtc tcaatttcgc tgactcggct tggagactcc gaatcccgga atcaacttgc    360 
      gctaaggaca tccaaaaggc ggcggctgaa gctgcgttgg cgtttcagga tgagatgtgt    420 
      gatgcgacga cggatcatgg cttcgacatg gaggagacgt tggtggaggc tatttacacg    480 
      gcggaacaga gcgaaaatgc gttttatatg cacgatgagg cgatgtttga gatgccgagt    540 
      ttgttggcta atatggcaga agggatgctt ttgccgcttc cgtccgtaca gtggaatcat    600 
      aatcatgaag tcgacggcga tgatgacgac gtatcgttat ggagttatta a             651 

 
           
             15  
             216  
             PRT  
             Arabidopsis thaliana  
             
               CBF3 polypeptide  
             
           
            15 

      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 

 
           
             16  
             18  
             DNA  
             Nicotiana tabacum  
             
               MT117 primer  
             
           
            16 

      ttggcggcta cgaatccc                                                   18 

 
           
             17  
             210  
             PRT  
             Brassica napus  
             
               CAN1 polypeptide  
             
           
            17 

      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 
      Leu Leu 
          210 

 
           
             18  
             632  
             DNA  
             Brassica napus  
             
               CAN1 gene  
             
           
            18 

      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 

 
           
             19  
             36  
             DNA  
             Artificial Sequence  
             
               Dreb2a-reverse PCR primer  
             
           
            19 

      gcccaagctt caagtttagt gagcactatg tgctcg                               36 

 
           
             20  
             34  
             DNA  
             Artificial Sequence  
             
               Dreb2a-forward PCR primer  
             
           
            20 

      ggaagatctc cttcccagaa acaacacaat ctac                                 34 

 
           
             21  
             35  
             DNA  
             Artificial Sequence  
             
               P5CS-reverse PCR primer  
             
           
            21 

      gcccaagctt gtttcatttt ctccatgaag gagat                                35 

 
           
             22  
             39  
             DNA  
             Artificial Sequence  
             
               P5CS-forward PCR primer  
             
           
            22 

      ggaagatctt atcgtcgtcg tcgtctacca aaaccacac                            39 

 
           
             23  
             32  
             DNA  
             Artificial Sequence  
             
               Rd22-reverse PCR primer  
             
           
            23 

      gctctaagct tcacaagggg ttcgtttggt gc                                   32 

 
           
             24  
             40  
             DNA  
             Artificial Sequence  
             
               Rd22-forward PCR primer  
             
           
            24 

      ggggtacctt ttgggagttg gaatagaaat gggtttgatg                           40 

 
           
             25  
             36  
             DNA  
             Artificial Sequence  
             
               Rd29a-reverse PCR primer  
             
           
            25 

      gcccaagctt aattttactc aaaatgtttt ggttgc                               36 

 
           
             26  
             44  
             DNA  
             Artificial Sequence  
             
               Rd29a-forward PCR primer  
             
           
            26 

      ccggtacctt tccaaagatt tttttctttc caatagaagt aatc                      44 

 
           
             27  
             30  
             DNA  
             Artificial Sequence  
             
               Rd29b-reverse PCR primer  
             
           
            27 

      gcggaagctt cattttctgc tacagaagtg                                      30 

 
           
             28  
             40  
             DNA  
             Artificial Sequence  
             
               Rd29b-forward PCR primer  
             
           
            28 

      ccggtacctt tccaaagctg tgttttctct ttttcaagtg                           40 

 
           
             29  
             42  
             DNA  
             Artificial Sequence  
             
               Rab18-reverse PCR primer  
             
           
            29 

      gcccaagctt caaattctga atattcacat atcaaaaaag tg                        42 

 
           
             30  
             40  
             DNA  
             Artificial Sequence  
             
               Rab18-forward PCR primer  
             
           
            30 

      ggaagatctg ttcttcttgt cttaagcaaa cactttgagc                           40 

 
           
             31  
             41  
             DNA  
             Artificial Sequence  
             
               Cor47-reverse PCR primer  
             
           
            31 

      gcccaagctt tcgtctgtta tcatacaagg cacaaaacga c                         41 

 
           
             32  
             42  
             DNA  
             Artificial Sequence  
             
               Cor47-forward PCR primer  
             
           
            32 

      ggaagatcta gttaatcttg atttgattaa aagtttatat ag                        42 

 
           
             33  
             25  
             DNA  
             Artificial Sequence  
             
               E9.1 primer PCR primer  
             
           
            33 

      caaactcagt aggattctgg tgtgt                                           25 

 
           
             34  
             38  
             DNA  
             Artificial Sequence  
             
               cbf1-reverse 1 PCR primer  
             
           
            34 

      ggaagatctt gaaacagagt actctgatca atgaactc                             38 

 
           
             35  
             42  
             DNA  
             Artificial Sequence  
             
               cbf1-forward 1 PCR primer  
             
           
            35 

      cgcggatccc tcgtttctac aacaataaaa taaaataaaa tg                        42 

 
           
             36  
             37  
             DNA  
             Artificial Sequence  
             
               cbf1-reverse 2 PCR primer  
             
           
            36 

      ggggtacctg aaacagagta ctctgatcaa tgaactc                              37 

 
           
             37  
             41  
             DNA  
             Artificial Sequence  
             
               cbf1-forward 2 PCR primer  
             
           
            37 

      gctctagact cgtttctaca acaataaaat aaaataaaat g                         41 

 
           
             38  
             577  
             DNA  
             Brassica juncea  
             
               bjCBF1 gene  
             
           
            38 

      tttcacccta tctaccgggg agttcgcctg agaaagtcag gtaagtgggt gtgtgaagtg     60 
      agggagccaa acaagaaatc taggatttgg cttggaactt tcaaaaccgc agagatcgct    120 
      gctcgtgctc acgacgttgc cgccttagcc ctccgtggaa gagcggcctg tctcaacttc    180 
      gccgactcgg cttggcggct ccgtatcccg gagacaactt gcgccaagga tatccagaag    240 
      gctgctgctg aagctgcgtt ggcttttggg gccgaaaaga gtgataccac gacgaatgat    300 
      caaggcatga acatggagga gatgacggtg gtggcttctc aggctgaggt gagcgacacg    360 
      acgacatatc atggcctgga catggaggag actatggtgg aggctgtttt tgctgaggaa    420 
      cagagagaag ggttttactt ggcggaggag acgacggtgg agggtgttgt tacggaggaa    480 
      cagagcaaag ggttttatat gtacgaggag tggacgttcg ggatgcagtc ctttttggcc    540 
      gatatggctg aaggcatgct cttttcaaag ggcgaat                             577 

 
           
             39  
             130  
             PRT  
             Brassica juncea  
             
               bjCBF1 polypeptide  
             
           
            39 

      Leu Pro Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 
      1               5                   10                  15 
      Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys Thr 
                  20                  25                  30 
      Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg 
              35                  40                  45 
      Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg 
          50                  55                  60 
      Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 
      65                  70                  75                  80 
      Ala Ala Leu Ala Phe Gly Ala Glu Lys Ser Asp Thr Thr Thr Asn Asp 
                      85                  90                  95 
      Gln Gly Met Asn Met Glu Glu Met Thr Ala Val Ala Ser Gln Ala Glu 
                  100                 105                 110 
      Val Ser Asp Thr Thr Thr Tyr His Gly Leu Asp Met Glu Glu Thr Met 
              115                 120                 125 
      Val Asp 
          130 

 
           
             40  
             431  
             DNA  
             Brassica juncea  
             
               bjCBF2 gene  
             
           
            40 

      catccgatct acaggggagt tcgtctgaga aaatcaggta agtgggtgtg tgaagtgagg     60 
      gaaccaaaca agagatctag gatctggctc ggtactttcc taaccgccga gatcgcagct    120 
      cgcgctcacg acgtcgccgc catagccctc cgtggcaaat ccgcatgtct caatttcgct    180 
      gactcggctt ggcggctccg tatctcggag acaacatgcc ctaaggagat tcagaaggct    240 
      gctgctgaag ccgcggtggc ttttcaggct gagctaaatg atacgacggc cgatcatggc    300 
      cttgacgtgg aggagacgat cgtggaggct attttcacgg aggaaagcag cgaagggttt    360 
      tatatggacg aggagttcat gttcgggatg ccgaccttgt gggctagtat ggcagaaggg    420 
      atgcttcttc c                                                         431 

 
           
             41  
             143  
             PRT  
             Brassica juncea  
             
               bjCBF2 polypeptide  
             
           
            41 

      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 Arg Ser Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 
          50                  55                  60 
      Arg Leu Arg Ile Ser Glu Thr Thr Cys Pro Lys Glu Ile Gln Lys Ala 
      65                  70                  75                  80 
      Ala Ala Glu Ala Ala Val Ala Phe Gln Ala Glu Leu Asn Asp Thr Thr 
                      85                  90                  95 
      Ala Asp His Gly Leu Asp Val Glu Glu Thr Ile Val Glu Ala Ile Phe 
                  100                 105                 110 
      Thr Glu Glu Ser Ser Glu Gly Phe Tyr Met Asp Glu Glu Phe Met Phe 
              115                 120                 125 
      Gly Met Pro Thr Leu Trp Ala Ser Met Ala Glu Gly Met Leu Leu 
          130                 135                 140 

 
           
             42  
             431  
             DNA  
             Brassica juncea  
             
               bjCBF3 gene  
             
           
            42 

      catccaattt accgtggagt tcgtctgaga aaatcaggta agtgggtgtg tgaagtgagg     60 
      gagccaaaca agaaatctag gatctggccc ggtactttcc taaccgccga gatcgcagct    120 
      cgcgctcacg acgtcgccgc catagccctc cgtggcaaat ccgcatgtct caatttcgct    180 
      gactcggctt ggcggctccg tatcccggag acaacatgcc ctaaggagat tcagaaggct    240 
      gctgctgaag ccgcggtggc ttttcaggct gagctaaatg atacgacggc cgatcatggc    300 
      cttgacgtgg aggagacgat cgtggaggct attttcacgg aggaaagcag cgaagggttt    360 
      tatatggacg aggagttcat gttcgggatg ccgaccttgt gggctagtat ggcggagggc    420 
      atgctccttc c                                                         431 

 
           
             43  
             143  
             PRT  
             Brassica juncea  
             
               bjCBF3- polypeptide  
             
           
            43 

      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 Pro Gly Thr 
                  20                  25                  30 
      Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 
          50                  55                  60 
      Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln Lys Ala 
      65                  70                  75                  80 
      Ala Ala Glu Ala Ala Val Ala Phe Gln Ala Glu Leu Asn Asp Thr Thr 
                      85                  90                  95 
      Ala Asp His Gly Leu Asp Val Glu Glu Thr Ile Val Glu Ala Ile Phe 
                  100                 105                 110 
      Thr Glu Glu Ser Ser Glu Gly Phe Tyr Met Ala Glu Glu Phe Met Phe 
              115                 120                 125 
      Gly Met Pro Thr Leu Trp Ala Ser Val Ala Glu Gly Met Leu Leu 
          130                 135                 140 

 
           
             44  
             425  
             DNA  
             Brassica juncea  
             
               bjCBF4 gene  
             
           
            44 

      catccaatct accggggtgt tcgacagaga aactcaggga aatgggtttg tgaagttagg     60 
      gagcctaata agaaatctag gatctggtta gggacttttc cgaccgtcga aatggccgct    120 
      cgtgctcacg acgtcgccgc tttagccctt cgtggccgct ccgcttgtct taatttcgcc    180 
      gactcggcgt ggtgtctacg gattcccgag tctacttgtc ctaaagagat tcagaaagct    240 
      gcggccgaag ctgcaatggc gtttcagaac gagacggcta cgactgagac gactatggtt    300 
      gagggagtca taccggcgga ggagacggtg gggcagacgc gtgtggagac agcagaggag    360 
      aacggtgtgt tttatatgga cgatccaagg tttcttgaga atatggcaga gggcatgttc    420 
      ctacc                                                                425 

 
           
             45  
             142  
             PRT  
             Brassica juncea  
             
               bjCBF4- polypeptide  
             
           
            45 

      His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn 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 Pro Thr Val Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 
          50                  55                  60 
      Cys Leu Arg Ile Pro Glu Ser Thr Cys Pro Lys Glu Ile Gln Lys Ala 
      65                  70                  75                  80 
      Ala Ala Glu Ala Ala Met Ala Phe Gln Asn Glu Glu Thr Ala Thr Thr 
                      85                  90                  95 
      Glu Thr Thr Met Val Glu Gly Val Ile Pro Ala Glu Glu Thr Val Gly 
                  100                 105                 110 
      Gln Thr Arg Val Glu Thr Ala Glu Glu Asn Gly Val Glu Tyr Met Asp 
              115                 120                 125 
      Asp Pro Arg Phe Leu Glu Asn Met Ala Glu Gly Met Leu Phe 
          130                 135                 140 

 
           
             46  
             632  
             DNA  
             Brassica napus  
             
               bnCBF1 gene  
             
           
            46 

      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 

 
           
             47  
             210  
             PRT  
             Brassica napus  
             
               bnCBF1- polypeptide  
             
           
            47 

      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 
      Leu Leu 
          210 

 
           
             48  
             876  
             DNA  
             Brassica napus  
             
               bnCBF2 gene  
             
           
            48 

      accgctcgag caacaatgaa cacattccct gcttccactg aaatggttgg ctccgagaac     60 
      gagtctccgg ttactacggt agtaggaggt gattattatc ccatgttggc ggcaagctgt    120 
      ccgaagaagc cagcgggtag gaagaagttt caggagacac gtcaccccat ttaccgagga    180 
      gttcgtctga gaaagtcagg taagtgggtg tgtgaagtga gggaaccaaa caagaaatct    240 
      agaatttggc ccggaacttt caaaacagct gagatggcag ctcgtgctca cgacgtcgct    300 
      gccctagccc tccgtggaag aggcgcctgc ctcaattatg cggactcggc ttggcggctc    360 
      cgcatcccgg aaacaacctg ccacaaggat atccagaagg ctgctgctga agccgcattg    420 
      gcttttgagg ctgagaaaag tgatgtgacg atgcaaaatg gcctgaacat ggaggagacg    480 
      acggcggtgg cttctcaggc tgaagtgaat gacacgacga cagaacatgg catgaacatg    540 
      gaggaggcaa cagcggtggc ttctcaggct gaggtgaatg acacgacgac agatcatggc    600 
      gtagacatgg aggagacgat ggtggaggct gtttttacgg aggaacaaag tgaagggttc    660 
      aacatggcgg aggagtcgac ggtggaggct gctgttgtta cggatgaact gagcaaagga    720 
      ttttacatgg acgaggagtg gacgtacgag atgccgacct tgttggctga tatggcggca    780 
      gggatgcttt tgccgccacc atctgtacaa tggggacata atgatgactt ggaaggagat    840 
      gcggacatga acctctggag ttattaagga tccgcg                              876 

 
           
             49  
             283  
             PRT  
             Brassica napus  
             
               bnCBF2- polypeptide  
             
           
            49 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala Ser 
      145                 150                 155                 160 
      Gln Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu 
                      165                 170                 175 
      Glu Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 
                  180                 185                 190 
      Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 
              195                 200                 205 
      Glu Glu Gln Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu 
          210                 215                 220 
      Ala Ala Val Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu 
      225                 230                 235                 240 
      Glu Trp Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 
                      245                 250                 255 
      Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Leu 
                  260                 265                 270 
      Glu Gly Asp Ala Asp Met Asn Leu Trp Ser Tyr 
              275                 280 

 
           
             50  
             884  
             DNA  
             Brassica napus  
             
               bnCBF3 gene  
             
           
            50 

      actacactca gccttatcca gtttttttca aaagattttt caacaatgaa cacattccct     60 
      gcgtccactg aaatggttgg ctccgagaac gagtctccgg ttactacggt agcaggaggt    120 
      gattattatc ccatgttggc ggcaagctgt ccgaagaagc cagcaggtag gaagaagttt    180 
      caggagacac gtcaccccat ttaccgagga gttcgtctga gaaagtcagg taagtgggtg    240 
      tgtgaagtga gggaaccaaa caagaaatct agaatttggc ccggaacttt caaaacagct    300 
      gagatggcag ctcgtgctca cgacgtcgct gccctagccc tccgtggaag aggcgcctgc    360 
      ctcaattatg cggactcggc ttggcggctc cgcatcccgg agacaacctg ccacaaggat    420 
      atccagaagg ctgctgctga agccgcattg gcttttgagg ctgagaaaag tgatgtgacg    480 
      atgcaaaatg gcctgaacat ggaggagacg acggcggtgg cttctcaggc tgaagtgaat    540 
      gacacgacga cagaacatgg catgaacatg gaggaggcaa cggcagtggc ttctcaggct    600 
      gaggtgaatg acacgacgac ggatcatggc gtagacatgg aggagacaat ggtggaggct    660 
      gtttttactg gggaacaaag tgaagggttt aacatggcga aggagtcgac ggtggaggct    720 
      gctgttgtta cggaggaacc gagcaaagga tcttacatgg acgaggagtg gatgctcgag    780 
      atgccgacct tgttggctga tatggcggaa gggatgcttt tgccgccgcc gtccgtacaa    840 
      tggggacaga atgatgactt cgaaggagat gctgacatga acct                     884 

 
           
             51  
             279  
             PRT  
             Brassica napus  
             
               bnCBF3- polypeptide  
             
           
            51 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala Ser 
      145                 150                 155                 160 
      Gln Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu 
                      165                 170                 175 
      Glu Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 
                  180                 185                 190 
      Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 
              195                 200                 205 
      Gly Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu 
          210                 215                 220 
      Ala Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu 
      225                 230                 235                 240 
      Glu Trp Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly 
                      245                 250                 255 
      Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly Gln Asn Asp Asp Phe 
                  260                 265                 270 
      Glu Gly Asp Ala Asp Met Asn 
              275 

 
           
             52  
             874  
             DNA  
             Brassica napus  
             
               bnCBF4 gene  
             
           
            52 

      gtaattcgat taccgctcga gtacttacta tactacactc agccttatcc agtttttcaa     60 
      aagaagtttt caactatgaa ctcagtctct actttttctg aacttcttgg ctctgagaac    120 
      gagtctccgg taggtggtga ttactgtccc atgttggcgg cgagctgtcc gaagaagccg    180 
      gcgggtagga agaagtttcg ggagacacgt caccccattt accgaggagt tcgccttaga    240 
      aaatcaggta agtgggtgtg tgaagtgagg gaaccaaaca aaaaatctag gatttggctc    300 
      ggaactttca aaacagctga gatcgcagct cgtgctcacg acgtcgccgc cttagctctc    360 
      cgtggaagag gcgcctgcct caacttcgcc gactcggctt ggcggctccg tatcccggag    420 
      acaacctgcg ccaaggatat ccagaaggct gctgctgaag ccgcattggc ttttgaggcc    480 
      gagaagagtg ataccacgac gaatgatcat ggcatgaaca tggcttctca ggccgaggtt    540 
      aatgacacaa cggatcatgg cctggacatg gaggagacga tggtggaggc tgtttttact    600 
      gaggagcaga gagacgggtt ttacatggcg gaggagacga cggtggaggg tgttgttccg    660 
      gaggaacaga tgagcaaagg gttttacatg gacgaggagt ggatgttcgg gatgccgacc    720 
      ttgttggctg atatggcggc agggatgctc ttaccgccgc cgtccgtaca atggggacat    780 
      aatgatgact tcgaaggaga tgttgacatg aacctctgga attattagta ctcatatttt    840 
      tttaaattat tttttgaacg aataatattt tatt                                874 

 
           
             53  
             250  
             PRT  
             Brassica napus  
             
               bnCBF4- polypeptide  
             
           
            53 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile 
              35                  40                  45 
      Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 
          50                  55                  60 
      Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys Thr 
      65                  70                  75                  80 
      Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg 
                      85                  90                  95 
      Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg 
                  100                 105                 110 
      Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 
              115                 120                 125 
      Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn Asp 
          130                 135                 140 
      His Gly Met Asn Met Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Asp 
      145                 150                 155                 160 
      His Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
                      165                 170                 175 
      Glu Gln Arg Asp Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly 
                  180                 185                 190 
      Val Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu 
              195                 200                 205 
      Trp Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met 
          210                 215                 220 
      Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Phe Glu 
      225                 230                 235                 240 
      Gly Asp Val Asp Met Asn Leu Trp Asn Tyr 
                      245                 250 

 
           
             54  
             898  
             DNA  
             Brassica napus  
             
               bnCBF5 gene  
             
           
            54 

      aataaatatc ttatcaaacc agtcagaaca gagatcttgt tacttactat actacactca     60 
      gccttatcca gttttcaaaa aaagtattca acgatgaact cagtctctac tttttctgaa    120 
      ctgctccgct ccgagaacga gtctccggtt aatacggaag gtggtgatta cattttggcg    180 
      gcgagctgtc ccaagaaacc tgctggtagg aagaagtttc aggagacacg ccaccccatt    240 
      tacagaggag ttcgtctgag gaagtcaggt aagtgggtgt gtgaagtgag ggaaccaaac    300 
      aagaaatcta gaatttggct cggaactttc aaaacagctg agatcgcagc tcgtgctcac    360 
      gacgttgccg ccttagctct ccgtggaaga ggcgcctgcc tcaacttcgc cgactcggct    420 
      tggcggctcc gtatcccgga gacgacctgc gccaaggata tccagaaggc tgctgctgaa    480 
      gccgcattgg cttttgaggc cgagaagagt gataccacga cgaatgatca tggcatgaac    540 
      atggcttctc aggttgaggt taatgacacg acggatcatg acctggacat ggaggagacg    600 
      atagtggagg ctgtttttag ggaggaacag agagaagggt tttacatggc ggaggagacg    660 
      acggttgtgg gtgttgttcc ggaggaacag atgagcaaag ggttttacat ggacgaggag    720 
      tggatgttcg ggatgccgac cttgttggct gatatggcgg cagggatgct cttaccgctg    780 
      ccgtccgtac aatggggaca taatgatgac ttcgaaggag atgctgacat gaacctctgg    840 
      aattattagt actcatattt ttttaaatta ttttttgaac gaataatatt ttattgaa      898 

 
           
             55  
             251  
             PRT  
             Brassica napus  
             
               bnCBF5- polypeptide  
             
           
            55 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Arg Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg His Pro 
              35                  40                  45 
      Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu 
          50                  55                  60 
      Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys 
      65                  70                  75                  80 
      Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
                      85                  90                  95 
      Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu 
                  100                 105                 110 
      Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala 
              115                 120                 125 
      Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn 
          130                 135                 140 
      Asp His Gly Met Asn Met Ala Ser Gln Val Glu Val Asn Asp Thr Thr 
      145                 150                 155                 160 
      Asp His Asp Leu Asp Met Glu Glu Thr Ile Val Glu Ala Val Phe Arg 
                      165                 170                 175 
      Glu Glu Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Val 
                  180                 185                 190 
      Gly Val Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu 
              195                 200                 205 
      Glu Trp Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 
          210                 215                 220 
      Met Leu Leu Pro Leu Pro Ser Val Gln Trp Gly His Asn Asp Asp Phe 
      225                 230                 235                 240 
      Glu Gly Asp Ala Asp Met Asn Leu Trp Asn Tyr 
                      245                 250 

 
           
             56  
             1132  
             DNA  
             Brassica napus  
             
               bnCBF6 gene  
             
           
            56 

      gattaccgct cgagtactta ctatactaca ctcagcctta tccagttttt ctcaaaagat     60 
      ttttcaacaa tgaacacatt ccctgcttcc actgaaatgg ttggctccga gaacgagtct    120 
      ccggttacta cggtagtagg aggtgattat tatcccatgt tggcggcaag ctgtccgaag    180 
      aagccagcgg gtaggaagaa gtttcaggag acacgtcacc ccatttaccg aggagttcgt    240 
      ctgagaaagt caggtaagtg ggtgtgtgaa gtgagggaac caaacaagaa atctagaatt    300 
      tggcttggaa ctttcaaaac agctgagatg gcagctcgtg ctcacgacgt ggctgcccta    360 
      gccctccgtg gaagaggcgc ctgcctcaat tatgcggact cggcttcgcg gctccgcatc    420 
      ccggagacaa cctgccacaa ggatatccag aaggctgctg ctgaagccgc attggctttt    480 
      gaggctgaga aaagtgatgt gacgatggag gagacgatgg cggtggcttc tcaggctgaa    540 
      gtgaatgaca cgacgacaga tcatggcatg aacatggagg aggcaacagc ggtggcttct    600 
      caggctgagg tgaatgacac gacgacagat catggcgtag acatggagga gacgatggtg    660 
      gaggctgttt ttacggagga acaaagtgaa gggttcaaca tggcggagga gtcgacggtg    720 
      gaggctgctg ttgttacgga tgaactgagc aaaggatttt acatggacga ggagtggacg    780 
      tacgagatgc cgaccttgtt ggctgatatg gcggcaggga tgcttttgcc gccaccatct    840 
      gtacaatggg gacataatga tgacttggaa ggagatgctg acatgaacct ctggaattat    900 
      taatactcgt gttttaaaaa ttatacattg tgcaataata ttttatcgaa tttctaattc    960 
      tgcctttaac ttttaatggg gatctttatt agtgtaggaa acgagtgtaa atgttccgcc   1020 
      gtggtgttgt caaaatgctg attatttttt gtgtgcagca taatcacgtt tggtttcctt   1080 
      tacactccaa atttagttga aatacaaata gaatagaaaa gtgaaaaaat gt           1132 

 
           
             57  
             277  
             PRT  
             Brassica napus  
             
               bnCBF6- polypeptide  
             
           
            57 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln Ala Glu Val Asn Asp 
      145                 150                 155                 160 
      Thr Thr Thr Asp His Gly Met Asn Met Glu Glu Ala Thr Ala Val Ala 
                      165                 170                 175 
      Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met 
                  180                 185                 190 
      Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu Glu Gln Ser Glu Gly 
              195                 200                 205 
      Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val Val Thr Asp 
          210                 215                 220 
      Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr Tyr Glu Met 
      225                 230                 235                 240 
      Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu Pro Pro Pro 
                      245                 250                 255 
      Ser Val Gln Trp Gly His Asn Asp Asp Leu Glu Gly Asp Ala Asp Met 
                  260                 265                 270 
      Asn Leu Trp Asn Tyr 
              275 

 
           
             58  
             768  
             DNA  
             Brassica napus  
             
               bnCBF7 gene  
             
           
            58 

      agtgatgttt ttcaaaagaa gttttcaact atgaactcag tctctacttt ttctgaactt     60 
      cttggctctg agaacgagtc tccggtaggt ggtgattact gtcccatgtt ggcggcgagc    120 
      tgtccgaaga agccggcggg taggaagaag tttcgggaga cacgtcaccc catttaccga    180 
      ggagttcgcc ttagaaaatc aggtaagtgg gtgtgtgaag tgagggagcc aaacaagaaa    240 
      tctaggattt ggctcggtac tttcctaaca gccgagatcg cagcccgtgc tcacgacgtc    300 
      gccgccatag ccctccgcgg caaatcagct tgtctcaatt ttgccgactc cgcttggcgg    360 
      ctccgtatcc cggagacaac atgccccaag gagattcaga aggcggctgc tgaagccgcg    420 
      gtggctttta aggctgagat aaataatacg acggcggatc atggcattga cgtggaggag    480 
      acgatcgttg aggctatttt cacggaggaa aacaacgatg gtttttatat ggacgaggag    540 
      gagtccatgt tcgggatgcc ggccttgttg gctagtatgg ctgaaggaat gcttttgccg    600 
      cctccgtccg tacaattcgg acatacctat gactttgacg gagatgctga cgtgtccctt    660 
      tggagttatt agtacaaaga ttttttattt ccatttttgg tataatactt ctttttgatt    720 
      ttcggattct acctttttat gggtatcatt ttttttttag gaaacggg                 768 

 
           
             59  
             213  
             PRT  
             Brassica napus  
             
               bnCBF7 polypeptide  
             
           
            59 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile 
              35                  40                  45 
      Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 
          50                  55                  60 
      Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Leu Thr 
      65                  70                  75                  80 
      Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg 
                      85                  90                  95 
      Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg 
                  100                 105                 110 
      Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln Lys Ala Ala Ala Glu 
              115                 120                 125 
      Ala Ala Val Ala Phe Lys Ala Glu Ile Asn Asn Thr Thr Ala Asp His 
          130                 135                 140 
      Gly Ile Asp Val Glu Glu Thr Ile Val Glu Ala Ile Phe Thr Glu Glu 
      145                 150                 155                 160 
      Asn Asn Asp Gly Phe Tyr Met Asp Glu Glu Glu Ser Met Phe Gly Met 
                      165                 170                 175 
      Pro Ala Leu Leu Ala Ser Met Ala Glu Gly Met Leu Leu Pro Pro Pro 
                  180                 185                 190 
      Ser Val Gln Phe Gly His Thr Tyr Asp Phe Asp Gly Asp Ala Asp Val 
              195                 200                 205 
      Ser Leu Trp Ser Tyr 
          210 

 
           
             60  
             953  
             DNA  
             Brassica napus  
             
               bnCBF8 gene  
             
           
            60 

      accgctcgag caacaatgaa cacattccct gcttccactg aaatggttgg ctccgagaac     60 
      gagtctccgg ttactacggt agcaggaggt gattattatc ccatgttggc ggcaagctgt    120 
      ccgaagaagc cagcgggtag gaagaagttt caggagacac gtcaccccat ttaccgagga    180 
      gttcgtctga gaaagtcagg taagtgggtg tgtgaagtga gggaaccaaa caagaaatct    240 
      agaatttggc ttggaacttt caaaacagct gagatggcag ctcgtgctca cgacgtggct    300 
      gccctagccc tccgtggaag aggcgcctgc ctcaattatg cggactcggc ttcgcggctc    360 
      cgcatcccgg agacaacctg ccacaaggat atccagaagg ctgctgctga agccgcattg    420 
      gcttttgagg ctgagaaaag tgatgtgacg atggaggaga cgatggcggt ggcttctcag    480 
      gctgaagtga atgacacgac gacagatcat ggcatgaaca tggaggaggc aacggcagtg    540 
      gcttctcagg ctgaggtgaa tgacacgacg acggatcatg gcgtagacat ggaggagaca    600 
      atggtggagg ctgtttttac tggggaacaa agtgaagggt ttaacatggc gaaggagtcg    660 
      acggtggagg ctgctgttgt tacggaggaa ccgagcaaag gatcttacat ggacgaggag    720 
      tggatgctcg agatgccgac cttgttggct gatatggcgg aagggatgct tttgccgccg    780 
      ccgtccgtac aatggggaca gaatgatgac ttcgaaggag atgcggacat gaacctctgg    840 
      agttattaat actcgtattt ttaaaattat ttattgtgca ataatttttt atcgaatttc    900 
      gaattctgcc tttaattttt aatggggatc tttatttgcc aaaaaaaaaa aaa           953 

 
           
             61  
             277  
             PRT  
             Brassica napus  
             
               bnCBF8 polypeptide  
             
           
            61 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln Ala Glu Val Asn Asp 
      145                 150                 155                 160 
      Thr Thr Thr Asp His Gly Met Asn Met Glu Glu Ala Thr Ala Val Ala 
                      165                 170                 175 
      Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met 
                  180                 185                 190 
      Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly Glu Gln Ser Glu Gly 
              195                 200                 205 
      Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val Val Thr Glu 
          210                 215                 220 
      Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met Leu Glu Met 
      225                 230                 235                 240 
      Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu Pro Pro Pro 
                      245                 250                 255 
      Ser Val Gln Trp Gly Gln Asn Asp Asp Phe Glu Gly Asp Ala Asp Met 
                  260                 265                 270 
      Asn Leu Trp Ser Tyr 
              275 

 
           
             62  
             889  
             DNA  
             Brassica napus  
             
               bnCBF9 gene  
             
           
            62 

      ctagtgatta ccgctcgagc aacaatgaac acattccctg cttccactga aatggttggc     60 
      tccgagaacg agtctccggt tactacggta gcaggaggtg attattatcc catgttggcg    120 
      gcaagctgtc cgaagaagcc agcgggtagg aagaagtttc aggagacacg tcaccccatt    180 
      taccgaggag ttcgtctgag aaagtcaggt aagtgggtgt gtgaagtgag ggaaccaaac    240 
      aagaaatcta gaatttggcc cggaactttc aaaacagctg agatggcagc tcgtgctcac    300 
      gacgtcgctg ccctagccct ccgtggaaga ggcgcccgcc tcaattatgc ggactcagct    360 
      tggcggctcc gcatcccgga gacaacctgc cacaaggata tccagaaggc tgctgctgaa    420 
      gccgcattgg cttttgaggc tgagaaaagt gatgtgacga tgcaaaatgg cctgaacatg    480 
      gaggagacga cggcggtggc ttctcaggct gaagtgaatg acacgacgac agaacatggc    540 
      atgaacatgg aggaggcaac ggcagtggct tctcaggctg aggtgaatga cacgacgacg    600 
      gatcatggcg tagacatgga ggagacaatg gtggaggctg tttttactgg ggaacaaagt    660 
      gaagggttta acatggcgaa ggagtcgacg gtggaggctg ctgttgttac ggaggaaccg    720 
      agcaaaggat cttacatgga cgaggagtgg atgctcgaga tgccgacctt gttggctgat    780 
      atggcggaag ggatgctttt gccgccgccg tccgtacaat ggggacagaa tgatgacttc    840 
      gaaggagatg cgcacatgaa cctctggagt tattaaggat ccgcgaatc                889 

 
           
             63  
             283  
             PRT  
             Brassica napus  
             
               bnCBF9 polypeptide  
             
           
            63 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Pro Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Arg Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala Ser 
      145                 150                 155                 160 
      Gln Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu 
                      165                 170                 175 
      Glu Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 
                  180                 185                 190 
      Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 
              195                 200                 205 
      Gly Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu 
          210                 215                 220 
      Ala Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu 
      225                 230                 235                 240 
      Glu Trp Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly 
                      245                 250                 255 
      Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly Gln Asn Asp Asp Phe 
                  260                 265                 270 
      Glu Gly Asp Ala His Met Asn Leu Trp Ser Tyr 
              275                 280 

 
           
             64  
             563  
             DNA  
             Brassica oleracea  
             
               boCBF1 gene  
             
           
            64 

      caccctatct accggggagt tcgcctgaga aagtcaggta agtgggtgtg tgaagtgagg     60 
      gagccaaaca agaaatctag gatttggctt ggaactttca aaaccgcaga gatcgctgct    120 
      cgtgctcacg acgttgccgc cttagccctc cgtggaagag cggcctgtct caacttcgcc    180 
      gactcggctt ggcggctccg tatcccggag acaacttgcg ccaaggatat ccagaaggct    240 
      gctgctgaag ctgcgttggc ttttggggcc gaaaagagtg ataccacgac gaatgatcaa    300 
      ggcatgaaca tggaggagat gacggtggtg gcttctcagg ctgaggtgag cgacacgacg    360 
      acatatcatg gcctggacat ggaggagact atggtggagg ctgtttttgc tgaggaacag    420 
      agagaagggt tttacttggc ggaggagacg acggtggagg gtgttgttac ggaggaacag    480 
      agcaaagggt tttatatgga cgaggagtgg acgttcggga tgcagtcctt tttggccgat    540 
      atggctgaag gcatgctctt tcc                                            563 

 
           
             65  
             188  
             PRT  
             Brassica oleracea  
             
               boCBF1 polypeptide  
             
           
            65 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 
          50                  55                  60 
      Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala 
      65                  70                  75                  80 
      Ala Ala Glu Ala Ala Leu Ala Phe Gly Ala Glu Lys Ser Asp Thr Thr 
                      85                  90                  95 
      Thr Asn Asp Gln Gly Met Asn Met Glu Glu Met Thr Val Val Ala Ser 
                  100                 105                 110 
      Gln Ala Glu Val Ser Asp Thr Thr Thr Tyr His Gly Leu Asp Met Glu 
              115                 120                 125 
      Glu Thr Met Val Glu Ala Val Phe Ala Glu Glu Gln Arg Glu Gly Phe 
          130                 135                 140 
      Tyr Leu Ala Glu Glu Thr Thr Val Glu Gly Val Val Thr Glu Glu Gln 
      145                 150                 155                 160 
      Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr Phe Gly Met Gln Ser 
                      165                 170                 175 
      Phe Leu Ala Asp Met Ala Glu Gly Met Leu Phe Pro 
                  180                 185 

 
           
             66  
             533  
             DNA  
             Brassica oleracea  
             
               boCBF2 gene  
             
           
            66 

      gaaacataga tctttgtact tactatactt caccttatcc agttttattt ttttatttat     60 
      aaagagtttt caacaatgac ctcattttct accttttctg aactgttggg ctccgagcat    120 
      gagtctccgg ttacattagg cgaagagtat tgtccgaagc tggccgcaag ctgtccgaag    180 
      aaaccagccg gccggaagaa gtttcgagag acgcgtcacc cagtttacag aggagttcgt    240 
      ctgagaaact caggtaagtg ggtgtgtgaa gtgagggagc caaacaagaa atctaggatt    300 
      tggctcggta ctttcctaac agccgagatc gcagcccgtg ctcacgacgt cgccgccata    360 
      gccctccgcg gcaaatcagc ttgtctcaat tttgccgact ccgcttggcg gctccgtatc    420 
      ccggagacaa catgccccaa ggagattcag aaggcggctg ctgaagccgc ggtggctttt    480 
      aaggctgaga taaataatac gacggcggat cacggcctcg acatggaaga gac           533 

 
           
             67  
             152  
             PRT  
             Brassica oleracea  
             
               boCBF2 polypeptide  
             
           
            67 

      Met Thr Ser Phe Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu His Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Leu Gly Glu Glu Tyr Cys Pro Lys Leu Ala Ala Ser 
                  20                  25                  30 
      Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His 
              35                  40                  45 
      Pro Val Tyr Arg Gly Val Arg Leu Arg Asn Ser Gly Lys Trp Val Cys 
          50                  55                  60 
      Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe 
      65                  70                  75                  80 
      Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala 
                      85                  90                  95 
      Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 
                  100                 105                 110 
      Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln Lys Ala Ala 
              115                 120                 125 
      Ala Glu Ala Ala Val Ala Phe Lys Ala Glu Ile Asn Asn Thr Thr Ala 
          130                 135                 140 
      Asp His Gly Leu Asp Met Glu Glu 
      145                 150 

 
           
             68  
             887  
             DNA  
             Brassica oleracea  
             
               boCBF3 gene  
             
           
            68 

      actcagcctt atccagtttt tctcaaaaga tttttcaaca atgaacacat tccctgcttc     60 
      cactgaaatg gttggctccg agaacgagtc tccggttact acggtagtag gaggtgatta    120 
      ttatcccatg ttggcggcaa gctgtccgaa gaagccagcg ggtaggaaga agtttcagga    180 
      gacacgtcac cccatttacc gaggagttcg tctgagaaag tcaggtaagt gggtgtgtga    240 
      agtgagggaa ccaaacaaga aatctagaat ttggcttgga actttcaaaa cagctgagat    300 
      ggcagctcgt gctcacgacg tggctgccct agccctccgt ggaagaggcg cctgcctcaa    360 
      ttatgcggac tcggcttggc ggctccgcat cccggagaca acctgccaca aggatatcca    420 
      gaaggctgct gctgaagccg cattggcttt tgaggctgag aaaagtgatg tgacgatgga    480 
      ggagacgatg gcggtggctt ctcaggctga agtgaatgac acgacgacag atcatggcat    540 
      gaacatggag gaggcaacag cggtggcttc tcaggctgag gtgaatgaca cgacgacaga    600 
      tcatggcgta gacatggagg agacgatggt ggaggctgtt tttacggagg aacaaagtga    660 
      agggttcaac atggcggagg agtcgacggt ggaggctgct gttgttacgg atgaactgag    720 
      caaaggattt tacatggacg aggagtggac gtacgagatg ccgaccttgt tggctgatat    780 
      ggcggcaggg atgcttttgc cgccaccatc tgtacaatgg ggacataatg atgacttgga    840 
      aggagatgcg gacatgaacc tctggagtta ttaatactcg tattttt                  887 

 
           
             69  
             277  
             PRT  
             Brassica oleracea  
             
               boCBF3 polypeptide  
             
           
            69 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln Ala Glu Val Asn Asp 
      145                 150                 155                 160 
      Thr Thr Thr Asp His Gly Met Asn Met Glu Glu Ala Thr Ala Val Ala 
                      165                 170                 175 
      Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met 
                  180                 185                 190 
      Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu Glu Gln Ser Glu Gly 
              195                 200                 205 
      Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val Val Thr Asp 
          210                 215                 220 
      Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr Tyr Glu Met 
      225                 230                 235                 240 
      Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu Pro Pro Pro 
                      245                 250                 255 
      Ser Val Gln Trp Gly His Asn Asp Asp Leu Glu Gly Asp Ala Asp Met 
                  260                 265                 270 
      Asn Leu Trp Ser Tyr 
              275 

 
           
             70  
             950  
             DNA  
             Brassica oleracea  
             
               boCBF4 gene  
             
           
            70 

      ctgaaaagaa gataaaagag agagaaataa atatcttatc aaaccagaca gaacagagat     60 
      cttgttactt actatactac actcagcctt atccagtttt tcaaaagaag ttttcaacta    120 
      tgaactcagt ctctactttt tctgaacttc ttggctctga gaacgagtct ccggtaggtg    180 
      gtgattactg tcccatgttg gcggcgagct gtccgaagaa gccggcgggt aggaagaagt    240 
      ttcgggagac acgtcacccc atttaccgag gagttcgcct tagaaaatca ggtaagtggg    300 
      tgtgtgaagt gagggaacca aacaaaaaat ctaggatttg gctcggaact ttcaaaacag    360 
      ctgagatcgc agctcgtgct cacgacgtcg ccgccttagc tctccgtgga agaggcgcct    420 
      gcctcaactt cgccgactcg gcttggcggc tccgtatccc ggagacaacc tgcgccaagg    480 
      atatccagaa ggctgctgct gaagccgcat tggcttttga ggccgagaag agtgatacca    540 
      cgacgaatga tcatggcatg aacatggctt ctcaggctga ggttaatgac acgacggatc    600 
      atggcctgga catggaggag acgatggtgg aggctgtttt tactgaggag cagagagacg    660 
      ggttttacat ggcggaggag acgacggtgg agggtgttgt tccggaggaa cagatgagca    720 
      aagggtttta catggacgag gagtggatgt tcgggatgcc gaccttgttg gctgatatgg    780 
      cggcagggat gctcttaccg ccgccgtccg tacaatgggg acataatgat gacttcgaag    840 
      gagatgctga catgaacctc tggaattatt agtactcgta tttttttaaa ttattttttg    900 
      aacgaataat attttattga attcggattc tacctgtttt tttaatggat               950 

 
           
             71  
             250  
             PRT  
             Brassica oleracea  
             
               boCBF4 polypeptide  
             
           
            71 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile 
              35                  40                  45 
      Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 
          50                  55                  60 
      Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys Thr 
      65                  70                  75                  80 
      Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg 
                      85                  90                  95 
      Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg 
                  100                 105                 110 
      Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 
              115                 120                 125 
      Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn Asp 
          130                 135                 140 
      His Gly Met Asn Met Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Asp 
      145                 150                 155                 160 
      His Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
                      165                 170                 175 
      Glu Gln Arg Asp Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly 
                  180                 185                 190 
      Val Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu 
              195                 200                 205 
      Trp Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met 
          210                 215                 220 
      Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Phe Glu 
      225                 230                 235                 240 
      Gly Asp Ala Asp Met Asn Leu Trp Asn Tyr 
                      245                 250 

 
           
             72  
             877  
             DNA  
             Brassica oleracea  
             
               boCBF5 gene  
             
           
            72 

      accgctcgag caacaatgaa cacattccct gcttccactg aaatggttag ctccgagaac     60 
      gagtctccgg ttactacggt agtaggaggt gattattatc ccatgttggc ggcaagctgt    120 
      ccgaagaagc cagcgggtag gaagaagttt caggagacac gtcaccccat ttaccgagga    180 
      gttcgtctga gaaagtcagg taagtgggtg tgtgaagtga gggaactaaa caagaaatct    240 
      agaatttggc ttggaacttt caaaacagct gagatggcag ctcgtgctca cgacgtggct    300 
      gccctagccc tccgtggaag aggcgcctgc ctcaattatg cggactcggc ttggcggctc    360 
      cgcatcccgg agacaacctg ccacaaggat atccagaagg ctgctgctga agccgcattg    420 
      gcttttgagg ctgagaagag tgatgcgacg atgcaaaatg gcctgaacat ggaggagacg    480 
      acggcggcgg cttctcagac tgaagtgagt gacacgacga cagatcatgg catgaacatg    540 
      gaggagacaa cggcggtggc ttctcaggct gaggtgaatg acacgacgac agatcatggc    600 
      gtagacatgg aggagacgat ggtggaggct gtttttactg aggaacaaag tgaagggttc    660 
      aacatggcga aggagtcgac ggcggaggct gctgttgtta cggaggaact gagcaaagga    720 
      gtttacatgg acgaggagtg gacgtacgag atgccgacct tgttggctga tatggcggca    780 
      gggatgcttt tgccgccacc atctgtacaa tggggacata atgatgactt ggaaggagat    840 
      gcggacatga acctactgga gttattaagg atccgcg                             877 

 
           
             73  
             287  
             PRT  
             Brassica oleracea  
             
               boCBF5 polypeptide  
             
           
            73 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Ser Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Leu Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Ala 
          130                 135                 140 
      Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Ala Ala Ser 
      145                 150                 155                 160 
      Gln Thr Glu Val Ser Asp Thr Thr Thr Asp His Gly Met Asn Met Glu 
                      165                 170                 175 
      Glu Thr Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 
                  180                 185                 190 
      Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 
              195                 200                 205 
      Glu Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Ala Glu 
          210                 215                 220 
      Ala Ala Val Val Thr Glu Glu Leu Ser Lys Gly Val Tyr Met Asp Glu 
      225                 230                 235                 240 
      Glu Trp Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 
                      245                 250                 255 
      Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Leu 
                  260                 265                 270 
      Glu Gly Asp Ala Asp Met Asn Leu Leu Glu Leu Leu Arg Ile Arg 
              275                 280                 285 

 
           
             74  
             374  
             DNA  
             Brassica rapa  
             
               brCBF1 gene  
             
           
            74 

      catcccattt acaggggggt tcgtttaaga aagtcaggta agtgggtgtg tgaagtgagg     60 
      gaaccaaaca agaaatctag gatttggctc ggaactttca aaaccgctga gatcgctgct    120 
      cgtgctcacg acgttgctgc cttagccctc cgcgggagag gcgcctgcct caacttcgcc    180 
      gactcggctt ggcggctccg tatcccggag acaacctgcg ccaaggacat ccagaaggcg    240 
      gctgctgaag ctgcattggc ttttgaggcc gagaagagtg atcatggcat gaacatcaag    300 
      aatactacgg cggtggtttc tcaggttgag gtgaatgaca cgacgacgga ccacggcttg    360 
      gacatggagg agac                                                      374 

 
           
             75  
             124  
             PRT  
             Brassica rapa  
             
               brCBF1 polypeptide  
             
           
            75 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp 
          50                  55                  60 
      Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala 
      65                  70                  75                  80 
      Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp His Gly 
                      85                  90                  95 
      Met Asn Ile Lys Asn Thr Thr Ala Val Val Ser Gln Val Glu Val Asn 
                  100                 105                 110 
      Asp Thr Thr Thr Asp His Gly Leu Asp Met Glu Glu 
              115                 120 

 
           
             76  
             884  
             DNA  
             Brassica rapa  
             
               brCBF2 gene  
             
           
            76 

      tacactcagc cttatccagt ttttttcaaa agacttttca acaatgaaca cattccctgc     60 
      gtccactgaa atggttggct ccgagaacga gtctccggtt actacggtag caggaggtga    120 
      ttattatccc atgttggcgg caagctgtcc gaagaagcca gcgggtagga agaagtttca    180 
      ggagacacgt caccccattt accgaggagt tcgtctgaga aagtcaggta agtgggtgtg    240 
      tgaagtgagg gaaccaaaca agaaatctag aatttggctt ggaactttca aaacagctga    300 
      gatggcagct cgtgctcacg acgtcgctgc cctagccctc cgtggaagag gcgcctgcct    360 
      caattatgcg gactcggctt ggcggctccg catcccggag acaacctgcc acaaggatat    420 
      ccagaaggct gctgctgaag ccgcattggc ttttgaggct gagaaaagtg atgtgacgat    480 
      gcaaaatggc ctgaacatgg aggagatgac ggcggtggct tctcaggctg aagtgaatga    540 
      cacgacgaca gaacatggca tgaacatgga ggaggcaacg gcagtggctt ctcaggctga    600 
      ggtgaatgac acgacgacgg atcatggcgt agacatggag gagacaatgg tggaggctgt    660 
      ttttactgag gaacaaagtg aagggtttaa catggcgaag gagtcgacgg tggaggctgc    720 
      tgttgttacg gaggaaccga gcaaaggatc ttacatggac gaggagtgga tgctcgagat    780 
      gccgaccttg ttggctgata tggcggaagg gatgcttttg ccgccgccgt ccgtacaatg    840 
      gggacagaat gatgacttcg aaggagatgc tgacatgaac ctct                     884 

 
           
             77  
             280  
             PRT  
             Brassica rapa  
             
               brCBF2 polypeptide  
             
           
            77 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Tyr Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Val 
          130                 135                 140 
      Thr Met Gln Asn Gly Leu Asn Met Glu Glu Met Thr Ala Val Ala Ser 
      145                 150                 155                 160 
      Gln Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu 
                      165                 170                 175 
      Glu Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr 
                  180                 185                 190 
      Asp His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr 
              195                 200                 205 
      Glu Glu Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu 
          210                 215                 220 
      Ala Ala Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu 
      225                 230                 235                 240 
      Glu Trp Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly 
                      245                 250                 255 
      Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly Gln Asn Asp Asp Phe 
                  260                 265                 270 
      Glu Gly Asp Ala Asp Met Asn Leu 
              275                 280 

 
           
             78  
             806  
             DNA  
             Brassica rapa  
             
               brCBF3 gene  
             
           
            78 

      acactcagcc ttatccagtt ttcaaaaaaa gtattcaacg atgaactcag tctctacttt     60 
      ttctgaactg ctctgctccg agaacgagtc tccggttaat acggaaggtg gtgattacat    120 
      tttggcggcg agctgtccca agaaacctgc tggtaggaag aagtttcagg agacacgcca    180 
      ccccatttac agaggagttc gtctgaggaa gtcaggtaag tgggtgtgtg aagtgaggga    240 
      accaaacaag aaatctagaa tttggctcgg aactttcaaa acagctgaga tcgcagctcg    300 
      tgctcacgac gttgccgcct tagctctccg tggaagaggc gcctgcctca acttcgccga    360 
      ctcggcttgg cggctccgta tcccggagac gacctgcgcc aaggatatcc agaaggctgc    420 
      tgctgaagcc gcattggctt ttgaggccga gaagagtgat accacgacga atgatcgtgg    480 
      catgaacatg gaggagacgt cggcggtggc ttctccggct gagttgaatg atacgacggc    540 
      ggatcatggc ctggacatgg aggagacgat ggtggaggct gtttttaggg aggaacagag    600 
      agaagggttt tacatggcgg aggagacgac ggtggagggt gttgttccgg agtaacagat    660 
      gagcaaaggg ttttacatgg acgaggagtg gacgttcgag atgccgaggt tgttggctga    720 
      tatggcggaa gggatgcttt tgccgccccc gtccgtacaa tggggacata acgatgactt    780 
      cgaaggagat gctgacatga acctct                                         806 

 
           
             79  
             204  
             PRT  
             Brassica rapa  
             
               brCBF3 polypeptide  
             
           
            79 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Cys Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg His Pro 
              35                  40                  45 
      Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu 
          50                  55                  60 
      Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys 
      65                  70                  75                  80 
      Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
                      85                  90                  95 
      Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu 
                  100                 105                 110 
      Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala 
              115                 120                 125 
      Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn 
          130                 135                 140 
      Asp Arg Gly Met Asn Met Glu Glu Thr Ser Ala Val Ala Ser Pro Ala 
      145                 150                 155                 160 
      Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Met Glu Glu Thr 
                      165                 170                 175 
      Met Val Glu Ala Val Phe Arg Glu Glu Gln Arg Glu Gly Phe Tyr Met 
                  180                 185                 190 
      Ala Glu Glu Thr Thr Val Glu Gly Val Val Pro Glu 
              195                 200 

 
           
             80  
             755  
             DNA  
             Brassica rapa  
             
               brCBF4 gene  
             
           
            80 

      accgctcgag tacttactat actacactca gccttatcca gtttttcttc caacgatgga     60 
      ctcaatctct acttttcctg aactgcttgg ctcagagaac gagtctccgg ttactacggt    120 
      agtaggaggt gattattgtc ccaggttggc ggcaagctgt ccgaagaagc cagcgggtag    180 
      gaagaagttt caggagacac gtcaccccat ttaccgtgga gttcgtttaa gaaagtccgg    240 
      taagtgggtg tgtgaagtga gggaaccaaa caagaaatct aggatttggc tcggaacttt    300 
      caaaaccgct gagatcgctg ctcgtgctca cgacgttgct gccttagccc tccgcggaag    360 
      aggcgcctgc ctcaacttcg ccgactcggc ttgacggctc cgtatcccgg agacaacctg    420 
      cgccaaggat atccagaagg ctgctgctga agctgcattg gcttttgagg ccgagaagag    480 
      tgatcatggc atgaacatga agaatactac ggcggtggct tctcaggttg aggtgaatga    540 
      tacgacgacg gaccatggcg tggacatgga ggagacgagg gtggagggtg ttgttacgga    600 
      ggaacagaac aattggtttt acatggacga ggagtggatg tttgggatgc cgacgttgtt    660 
      ggttgatatg gcggaaggga tgcttatacc gcggcagtcc gtacaatcgg gacactacga    720 
      tgacttcgaa ggagatgctg acatgaacct ctgga                               755 

 
           
             81  
             112  
             PRT  
             Brassica rapa  
             
               brCBF4 polypeptide  
             
           
            81 

      Met Asp Ser Ile Ser Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala 
                  100                 105                 110 

 
           
             82  
             832  
             DNA  
             Brassica rapa  
             
               brCBF5 gene  
             
           
            82 

      accgctcgag tacttactat actacactca gccttatcca gtttttcttc caacgatgga     60 
      ctcaatctct acttttcctg aactgcttgg ctcagagaac gagtctccgg ttactacggt    120 
      agtaggaggt gattattgtc ccaggttggc ggcaagctgt ccgaagaagc cagcgggtag    180 
      gaagaagttt caggagacac gtcaccccat ttaccgtgga gttcgtttaa gaaagtccgg    240 
      taagtgggtg tgtgaagtga gggaaccaaa caagaaatct aggatttggc tcggaacttt    300 
      caaaaccgct gagatcgctg ctcgtgctca cgacgttgct gccttagccc tccgcggaag    360 
      aggcgcctgc ctcaacttcg ccgactcggc ttggcggctc cgtatcccgg agacaacctg    420 
      cgccaaggat atccagaagg ctgctgctga agctgctttg gcttttgagg ccgagaagag    480 
      tgatcatggc atgaacatga agaatactac ggcggtggct tctcaggttg aggtgaatga    540 
      tacgacgacg gaccatggcg tggacatgga ggagacgttg gtggaggctg tttttacgga    600 
      ggaacagaga gaagggtttt acatgacgga ggagacgagg gtggagggtg ttgttacgga    660 
      ggaacagaac aattggtttt acatggacga ggagtggatg tttgggatgc cgacgttgtt    720 
      ggttgatatg gcggaaggga tgcttatacc gcggcagtcc gtacaatcgg gacactacga    780 
      tgacttcgaa ggagatgctg acatgaacct ctggaattat tagggatccg cg            832 

 
           
             83  
             255  
             PRT  
             Brassica rapa  
             
               brCBF5 polypeptide  
             
           
            83 

      Met Asp Ser Ile Ser Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp His 
          130                 135                 140 
      Gly Met Asn Met Lys Asn Thr Thr Ala Val Ala Ser Gln Val Glu Val 
      145                 150                 155                 160 
      Asn Asp Thr Thr Thr Asp His Gly Val Asp Met Glu Glu Thr Leu Val 
                      165                 170                 175 
      Glu Ala Val Phe Thr Glu Glu Gln Arg Glu Gly Phe Tyr Met Thr Glu 
                  180                 185                 190 
      Glu Thr Arg Val Glu Gly Val Val Thr Glu Glu Gln Asn Asn Trp Phe 
              195                 200                 205 
      Tyr Met Asp Glu Glu Trp Met Phe Gly Met Pro Thr Leu Leu Val Asp 
          210                 215                 220 
      Met Ala Glu Gly Met Leu Ile Pro Arg Gln Ser Val Gln Ser Gly His 
      225                 230                 235                 240 
      Tyr Asp Asp Phe Glu Gly Asp Ala Asp Met Asn Leu Trp Asn Tyr 
                      245                 250                 255 

 
           
             84  
             830  
             DNA  
             Brassica rapa  
             
               brCBF6 gene  
             
           
            84 

      tactacactc agccttatcc agttttcaaa aaaagtattc aactatgaac tcagtctcta     60 
      ctttttctga actgctctgc tccgagaaca agtctccggt taatacggaa ggtggtgatt    120 
      acattttggc ggcgagctgt cccaagaaac ctgctggtag gaagaagttt caggagacac    180 
      gccaccccat ttacagagga gttcgcctaa gaaagtcagg taagtgggtg tgtgaagtga    240 
      gggaaccaaa caagaaatct agaatttggc tcggaacttt caaaacagct gagatagcag    300 
      ctcgtgctca cgacgtcgcc gccttagctc tccgtggaag aggcgcctgc ctcaacttcg    360 
      ccgactcggc ttggcggctc cgtatcccag agacaacctg cgccaaggat atccagaagg    420 
      ctgctgctga agccgcattg gcttttgagg ccgagaagag tgataccacg acgaatgatc    480 
      gtggcatgaa catggaggag acgtccgcgg tggcttctcc ggctgagttg aatgatacga    540 
      cggcggatca tggcctggac atggaggaga cgatggtgga ggctgttttt agggacgaac    600 
      agagagaagg gttttacatg gcggaggaga cgacggtgga gggtgttgtt ccggaggaac    660 
      agatgagcaa agggttttac atggacgagg agtggacgtt cgagatgccg aggttgttgg    720 
      ctgatatggc ggaagggatg cttctgcctc ccccgtccgt acaatgggga cataacgatg    780 
      acttcgaagg agatgctgac atgaacctct ggaattatta gggatccgcg               830 

 
           
             85  
             258  
             PRT  
             Brassica rapa  
             
               brCBF6- polypeptide  
             
           
            85 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Cys Ser Glu Asn Lys 
      1               5                   10                  15 
      Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg His Pro 
              35                  40                  45 
      Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu 
          50                  55                  60 
      Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys 
      65                  70                  75                  80 
      Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
                      85                  90                  95 
      Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu 
                  100                 105                 110 
      Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala 
              115                 120                 125 
      Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Asn 
          130                 135                 140 
      Asp Arg Gly Met Asn Met Glu Glu Thr Ser Ala Val Ala Ser Pro Ala 
      145                 150                 155                 160 
      Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Met Glu Glu Thr 
                      165                 170                 175 
      Met Val Glu Ala Val Phe Arg Asp Glu Gln Arg Glu Gly Phe Tyr Met 
                  180                 185                 190 
      Ala Glu Glu Thr Thr Val Glu Gly Val Val Pro Glu Glu Gln Met Ser 
              195                 200                 205 
      Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr Phe Glu Met Pro Arg Leu 
          210                 215                 220 
      Leu Ala Asp Met Ala Glu Gly Met Leu Leu Pro Pro Pro Ser Val Gln 
      225                 230                 235                 240 
      Trp Gly His Asn Asp Asp Phe Glu Gly Asp Ala Asp Met Asn Leu Trp 
                      245                 250                 255 
      Asn Tyr 

 
           
             86  
             854  
             DNA  
             Brassica rapa  
             
               brCBF7 gene  
             
           
            86 

      ctatactaca cacagcctta tccagccgct cgagtactta ctatactaca ctcagccttt     60 
      tccagttttt caaaagaagt tttcaacgat gaactcagtc tctactcttt ctgaagttct    120 
      tggctcccag aacgagtctc ccgtaggtgg tgattactgt cccatgttgg cggcgagctg    180 
      tccgaagaag ccggcgggta ggaagaagtt tcgggagaca cgtcacccca tttacagagg    240 
      agttcgtctt agaaagtcag gtaagtgggt gtgtgaagtg agggaaccaa acaagaaatc    300 
      taggatttgg ctcggaactt tcaaaacagc tgagatcgca gctcgtgctc acgacgttgc    360 
      cgccttagct ctccgtggaa gaggcgcctg cctcaacttc gccgactcgg cttggcggct    420 
      ccgtatcccg gagacaacct gcgccaagga tatccagaag gctgctgctg aagccgcatt    480 
      ggcttttgag gcggagaaga gtgataccac gacgacgaat gatcatggca tgaacatggc    540 
      ttctcaggtt gaggttaatg acacgacgga tcatgacctg gacatggagg agacgatggt    600 
      ggaggctgtt tttagggagg aacagagaga agggttttac atggcggagg agacgacggt    660 
      ggagggtatt gttccggagg aacagatgag caaagggttt tacatggacg aggagtggat    720 
      gttcgggatg ccgaccttgt tggctgatat ggcggcaggg atgctcttac cgccgccgtc    780 
      cgtacaatgg ggacataatg atgacttcga aggagatgct gacatgaacc tctggaatta    840 
      ttaagggatc cgcg                                                      854 

 
           
             87  
             251  
             PRT  
             Brassica rapa  
             
               brCBF7 polypeptide  
             
           
            87 

      Met Asn Ser Val Ser Thr Leu Ser Glu Val Leu Gly Ser Gln Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile 
              35                  40                  45 
      Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp Val Cys Glu Val 
          50                  55                  60 
      Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Lys Thr 
      65                  70                  75                  80 
      Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg 
                      85                  90                  95 
      Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg 
                  100                 105                 110 
      Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 
              115                 120                 125 
      Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp Thr Thr Thr Thr Asn 
          130                 135                 140 
      Asp His Gly Met Asn Met Ala Ser Gln Val Glu Val Asn Asp Thr Thr 
      145                 150                 155                 160 
      Asp His Asp Leu Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Arg 
                      165                 170                 175 
      Glu Glu Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu 
                  180                 185                 190 
      Gly Ile Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu 
              195                 200                 205 
      Glu Trp Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly 
          210                 215                 220 
      Met Leu Leu Pro Pro Pro Ser Val Gln Trp Gly His Asn Asp Asp Phe 
      225                 230                 235                 240 
      Glu Gly Asp Ala Asp Met Asn Leu Trp Asn Tyr 
                      245                 250 

 
           
             88  
             738  
             DNA  
             Glycine max  
             
               gmCBF1 gene  
             
           
            88 

      catccgattt atagtggcgt gaggaggagg aacacggata agtgggtaag tgaggtgagg     60 
      gagcccaaca aaaagaccag gatttggctg gggacttttc ccacgccgga gatggcggca    120 
      cgggcccacg acgtggcggc aatggccctg aggggccggt atgcctgtct caacttcgct    180 
      gactcgacgt ggcggttacc aattcccgcc actgctaacg caaaggatat acagaaagca    240 
      gcagcagagg ctgccgaggc tttcagacca agtcagacct tagaaaatac gaatacaaag    300 
      caagagtgtg taaaagtggt gacgacaaca acgatcacag aacaaaaacg aggaatgttt    360 
      tatacggagg aagaagagca agtgttagat atgcctgagt tgcttaggaa tatggtgctt    420 
      atgtccccaa cacattgcat agggtatgag tatgaagatg ctgacttgga tgctcaagat    480 
      gctgaggtgt ccctatggag tttctcaatt taataacgtg cttttggttt ggttttttat    540 
      gttagttttg gagtgtgact gtctgtactg gttttttatt agtagtacgg atactagcta    600 
      taggtggcag attgaaaggg accaaaagga attttctttt gaaacccttt ttgtcaaagt    660 
      aatcaatcgc gtatcatcaa gtgaatccct tgatcaagtt tatgtatgaa ttaaataaaa    720 
      gaagaatcta gttttggt                                                  738 

 
           
             89  
             170  
             PRT  
             Glycine max  
             
               gmCBF1 polypeptide  
             
           
            89 

      His Pro Ile Tyr Ser Gly Val Arg Arg Arg Asn Thr Asp Lys Trp Val 
      1               5                   10                  15 
      Ser Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Pro Thr Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Met 
              35                  40                  45 
      Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe Ala Asp Ser Thr Trp 
          50                  55                  60 
      Arg Leu Pro Ile Pro Ala Thr Ala Asn Ala Lys Asp Ile Gln Lys Ala 
      65                  70                  75                  80 
      Ala Ala Glu Ala Ala Glu Ala Phe Arg Pro Ser Gln Thr Leu Glu Asn 
                      85                  90                  95 
      Thr Asn Thr Lys Gln Glu Cys Val Lys Val Val Thr Thr Thr Thr Ile 
                  100                 105                 110 
      Thr Glu Gln Lys Arg Gly Met Phe Tyr Thr Glu Glu Glu Glu Gln Val 
              115                 120                 125 
      Leu Asp Met Pro Glu Leu Leu Arg Asn Met Val Leu Met Ser Pro Thr 
          130                 135                 140 
      His Cys Ile Gly Tyr Glu Tyr Glu Asp Ala Asp Leu Asp Ala Gln Asp 
      145                 150                 155                 160 
      Ala Glu Val Ser Leu Trp Ser Phe Ser Ile 
                      165                 170 

 
           
             90  
             793  
             DNA  
             Raphanus sativus  
             
               rsCBF1 gene  
             
           
            90 

      actacactca gccttatcca gtttttcttc caacgatgga ctcaatctct actttttctg     60 
      aactgcttgg ctccgagaac gagtctccgg ttactacggt agtaggaggt gattattttc    120 
      ccaggttggc ggcaagctgt ccgaagaagc cagcgggtag gaagaagttt caggagacac    180 
      gtcaccccat ttaccgcgga gttcgtttaa gaaagtcagg taagtgggtg tgtgaagtga    240 
      gggaaccaaa caagaaatct aggatttggc tcggaacttt caaaaccgct gagatcgctg    300 
      ctcgtgctca cgacgttgct gccttagccc tccgcggaag aggcgcctgc ctcaacttcg    360 
      ccgactcggc ttggcggctc cgtatcccgg agacaacctg cgccaaggat atccagaagg    420 
      ctgctgctga agctgcattg gcttttgagg ccgagaagag tgatcatggc atgaacatga    480 
      agaatactac ggcggtggct tctcaggttg aggtgaatga cacgacgacg gaccatggcg    540 
      tggacatgga ggagacgttg gtggaggctg tttttacgga ggaacagaga gaagggtttt    600 
      acatgacgga ggagacgagg gtggagggtg ttgttacgga ggaacagaac aattggtttt    660 
      acatggacga ggagtggatg tttgggatgc cgacgttgtt ggttgatatg gcggaaggga    720 
      tgcttttacc gcggccgtcc gtacaatcgg gacactacga tgacttcgaa ggagatgctg    780 
      acatgaacct ctg                                                       793 

 
           
             91  
             252  
             PRT  
             Raphanus sativus  
             
               rsCBF1 polypeptide  
             
           
            91 

      Met Asp Ser Ile Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Phe Pro Arg Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg His Pro Ile Tyr Arg Gly Val Arg Leu Arg Lys Ser Gly Lys Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Lys Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Leu Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala 
                  100                 105                 110 
      Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln Lys 
              115                 120                 125 
      Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp His 
          130                 135                 140 
      Gly Met Asn Met Lys Asn Thr Thr Ala Val Ala Ser Gln Val Glu Val 
      145                 150                 155                 160 
      Asn Asp Thr Thr Thr Asp His Gly Val Asp Met Glu Glu Thr Leu Val 
                      165                 170                 175 
      Glu Ala Val Phe Thr Glu Glu Gln Arg Glu Gly Phe Tyr Met Thr Glu 
                  180                 185                 190 
      Glu Thr Arg Val Glu Gly Val Val Thr Glu Glu Gln Asn Asn Trp Phe 
              195                 200                 205 
      Tyr Met Asp Glu Glu Trp Met Phe Gly Met Pro Thr Leu Leu Val Asp 
          210                 215                 220 
      Met Ala Glu Gly Met Leu Leu Pro Arg Pro Ser Val Gln Ser Gly His 
      225                 230                 235                 240 
      Tyr Asp Asp Phe Glu Gly Asp Ala Asp Met Asn Leu 
                      245                 250 

 
           
             92  
             682  
             DNA  
             Raphanus sativus  
             
               rsCBF2 gene  
             
           
            92 

      acacctaaac cttatccagg tttaactttt tttttcataa agagttttca acaatgacca     60 
      cattttctac cttttccgaa atgttgggct ccgagtacga gtctccggtt acattaggcg    120 
      gagagtattg tccgaagctg gccgcgagct gtccgaagaa accagctggt cgtaagaagt    180 
      ttcgagagac gcgccaccca atatacagag gagttcgtct gagaaactca ggtaagtggg    240 
      tgtgtgaagt gagggagcca aacaagaaat ctaggatttg gctcggtact ttcctaaccg    300 
      ccgagatcgc agcgcgtgcc cacgacgtcg ccgccatagc cctccgcggc aaatccgcat    360 
      gtctcaattt cgctgactcg gcttggcggc tccgtatccc ggagacaaca tgccccaagg    420 
      atatacagaa ggcggctgct gaagccgcgg tggcttttca ggctgagata aatgatacga    480 
      cgacggatca tggcctggac ttggaggaga cgatcgtgga ggctattttt acggaggtaa    540 
      acaacgatga gttttatatg gacgaggagt ccatgttcgg gatgccgtct ttgttggcta    600 
      gtatggcgga agggatgctt ttgccgctgc cgtccgtaca atctgaacat aactgtgact    660 
      tcgacggaga tgctgacatg aa                                             682 

 
           
             93  
             209  
             PRT  
             Raphanus sativus  
             
               rsCBF2- polypeptide  
             
           
            93 

      Met Thr Thr Phe Ser Thr Phe Ser Glu Met Leu Gly Ser Glu Tyr Glu 
      1               5                   10                  15 
      Ser Pro Val Thr Leu Gly Gly Glu Tyr Cys Pro Lys Leu Ala Ala 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 Leu Arg Asn Ser Gly Lys Trp Val Cys 
          50                  55                  60 
      Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe 
      65                  70                  75                  80 
      Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala 
                      85                  90                  95 
      Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 
                  100                 105                 110 
      Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Asp Ile Gln Lys Ala Ala 
              115                 120                 125 
      Ala Glu Ala Ala Val Ala Phe Gln Ala Glu Ile Asn Asp Thr Thr Thr 
          130                 135                 140 
      Asp His Gly Leu Asp Leu Glu Glu Thr Ile Val Glu Ala Ile Phe Thr 
      145                 150                 155                 160 
      Glu Val Asn Asn Asp Glu Phe Tyr Met Asp Glu Glu Ser Met Phe Gly 
                      165                 170                 175 
      Met Pro Ser Leu Leu Ala Ser Met Ala Glu Gly Met Leu Leu Pro Leu 
                  180                 185                 190 
      Pro Ser Val Gln Ser Glu His Asn Cys Asp Phe Asp Gly Asp Ala Asp 
              195                 200                 205 
      Met 

 
           
             94  
             349  
             DNA  
             Zea mays  
             
               zmCBF1 gene  
             
           
            94 

      cggagtccgc ggacggcggc ggcggcggcg acgacgagta cgcgacggtg ctgtcggcgc     60 
      cacccaagcg gccggcgggg cggaccaagt tccgggagac gcggcacccc gtgtaccgcg    120 
      gcgtgcggcg gcgcgggccc gcggggcgct gggtgtgcga ggtccgcgag cccaacaaga    180 
      agtcgcgcat ctggctcggc accttcgcca cccccgaggc cgccgcgcgc gcgcacgacg    240 
      tggccgcgct ggccctgcgg ggccgcgccg cgtgcctcaa cttcgccgac tcggcgcgcc    300 
      tgctccaagt cgaccccgcc acgctcgcca cccccgacga catccgccg                349 

 
           
             95  
             115  
             PRT  
             Zea mays  
             
               zmCBF1- polypeptide  
             
           
            95 

      Glu Ser Ala Asp Gly Gly Gly Gly Gly Asp Asp Glu Tyr Ala Thr Val 
      1               5                   10                  15 
      Leu Ser Ala Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu 
                  20                  25                  30 
      Thr Arg His Pro Val Tyr Arg Gly Val Arg Arg Arg Gly Pro Ala Gly 
              35                  40                  45 
      Arg Trp Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp 
          50                  55                  60 
      Leu Gly Thr Phe Ala Thr Pro Glu Ala Ala Ala Arg Ala His Asp Val 
      65                  70                  75                  80 
      Ala Ala Leu Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp 
                      85                  90                  95 
      Ser Ala Arg Leu Leu Gln Val Asp Pro Ala Thr Leu Ala Thr Pro Asp 
                  100                 105                 110 
      Asp Ile Arg 
              115 

 
           
             96  
             675  
             DNA  
             Arabidopsis thaliana  
             
               G912 gene  
             
           
            96 

      ttactcgtca aaactccaga gtgacacgtc acccactccg tcaaagtcgt tatgattcca     60 
      gccaacttcc ggcggcggca aaagcatccc ctccgccata ttctcaaaaa agttgggcat    120 
      ccccaaaagc gcctcatcat ccatataaaa cacaccacca ttctgctcct ccgccctcct    180 
      ctccccctcc ctcaccccct cccctgccgc ctcctctgcc tccgccgcag ttttagatcc    240 
      ctccgtcgta gtctcattct gaaacgccat tgcagcttca gacgcagctt tctgaatctc    300 
      cttaggacaa gtagtctcag gaatacgaag ccgccaagca gaatcagcga aattgagaca    360 
      agcagagcga ccacgaagag ctaaagcagc aacatcatga gcacgagcag ccatttcaac    420 
      cgtcggaaaa gtacctaacc aaatcctaga tttcttatta ggctctctaa cttcacaaac    480 
      ccatttacca gaattcctct gacgaactcc tctgtaaatc ggatgacgtg tctcacgaaa    540 
      cttcttcctc ccagctcgtt tctttggaca acttgaagct aactttggtg aacactcact    600 
      actgtctgaa accggagatc tatgatcgga gattgagaga aacgagtctg ggaatgtaga    660 
      gtaaaatgga ttcat                                                     675 

 
           
             97  
             224  
             PRT  
             Arabidopsis thaliana  
             
               G912 polypeptide  
             
           
            97 

      Met Asn Pro Phe Tyr Ser Thr Phe Pro Asp Ser Phe Leu Ser Ile Ser 
      1               5                   10                  15 
      Asp His Arg Ser Pro Val Ser Asp Ser Ser Glu Cys Ser Pro Lys Leu 
                  20                  25                  30 
      Ala Ser Ser Cys Pro Lys Lys Arg Ala Gly Arg Lys Lys Phe Arg Glu 
              35                  40                  45 
      Thr Arg His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys 
          50                  55                  60 
      Trp Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu 
      65                  70                  75                  80 
      Gly Thr Phe Pro Thr Val Glu Met Ala Ala Arg Ala His Asp Val Ala 
                      85                  90                  95 
      Ala Leu Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 
                  100                 105                 110 
      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln 
              115                 120                 125 
      Lys Ala Ala Ser Glu Ala Ala Met Ala Phe Gln Asn Glu Thr Thr Thr 
          130                 135                 140 
      Glu Gly Ser Lys Thr Ala Ala Glu Ala Glu Glu Ala Ala Gly Glu Gly 
      145                 150                 155                 160 
      Val Arg Glu Gly Glu Arg Arg Ala Glu Glu Gln Asn Gly Gly Val Phe 
                      165                 170                 175 
      Tyr Met Asp Asp Glu Ala Leu Leu Gly Met Pro Asn Phe Phe Glu Asn 
                  180                 185                 190 
      Met Ala Glu Gly Met Leu Leu Pro Pro Pro Glu Val Gly Trp Asn His 
              195                 200                 205 
      Asn Asp Phe Asp Gly Val Gly Asp Val Ser Leu Trp Ser Phe Asp Glu 
          210                 215                 220 

 
           
             98  
             630  
             DNA  
             Arabidopsis thaliana  
             
               G2513 gene  
             
           
            98 

      atgaataatg atgatattat tctggcggag atgaggccta agaagcgtgc gggaaggaga     60 
      gtgtttaagg agacacgtca cccagtttac agaggcataa ggcggaggaa cggtgacaaa    120 
      tgggtctgcg aagtcagaga accgacgcac caacgccgca tttggctcgg gacttatccc    180 
      acagcagata tggcagcgcg tgcacacgac gtggcggttt tagctctgcg tgggagatcc    240 
      gcatgtttga atttcgccga ctccgcttgg cggcttccgg tgccggaatc caatgatccg    300 
      gatgtgataa gaagagttgc ggcggaagct gcggagatgt ttaggccggt ggatttagaa    360 
      agtggaatta cggttttgcc ttgtgcggga gatgatgtgg atttgggttt tggttcgggt    420 
      tccggctctg gttcgggatc ggaggagagg aattcttctt cgtatggatt tggagactac    480 
      gaagaagtct caacgacgat gatgagactc gcggaggggc cactaatgtc gccgccgcga    540 
      tcgtatatgg aagacatgac tcctactaat gtttacacgg aagaagagat gtgttatgaa    600 
      gatatgtcat tgtggagtta cagatattaa                                     630 

 
           
             99  
             209  
             PRT  
             Arabidopsis thaliana  
             
               G2513 polypeptide  
             
           
            99 

      Met Asn Asn Asp Asp Ile Ile Leu Ala Glu Met Arg Pro Lys Lys Arg 
      1               5                   10                  15 
      Ala Gly Arg Arg Val Phe Lys Glu Thr Arg His Pro Val Tyr Arg Gly 
                  20                  25                  30 
      Ile Arg Arg Arg Asn Gly Asp Lys Trp Val Cys Glu Val Arg Glu Pro 
              35                  40                  45 
      Thr His Gln Arg Arg Ile Trp Leu Gly Thr Tyr Pro Thr Ala Asp Met 
          50                  55                  60 
      Ala Ala Arg Ala His Asp Val Ala Val Leu Ala Leu Arg Gly Arg Ser 
      65                  70                  75                  80 
      Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Pro Val Pro Glu 
                      85                  90                  95 
      Ser Asn Asp Pro Asp Val Ile Arg Arg Val Ala Ala Glu Ala Ala Glu 
                  100                 105                 110 
      Met Phe Arg Pro Val Asp Leu Glu Ser Gly Ile Thr Val Leu Pro Cys 
              115                 120                 125 
      Ala Gly Asp Asp Val Asp Leu Gly Phe Gly Ser Gly Ser Gly Ser Gly 
          130                 135                 140 
      Ser Gly Ser Glu Glu Arg Asn Ser Ser Ser Tyr Gly Phe Gly Asp Tyr 
      145                 150                 155                 160 
      Glu Glu Val Ser Thr Thr Met Met Arg Leu Ala Glu Gly Pro Leu Met 
                      165                 170                 175 
      Ser Pro Pro Arg Ser Tyr Met Glu Asp Met Thr Pro Thr Asn Val Tyr 
                  180                 185                 190 
      Thr Glu Glu Glu Met Cys Tyr Glu Asp Met Ser Leu Trp Ser Tyr Arg 
              195                 200                 205 
      Tyr 

 
           
             100  
             546  
             DNA  
             Arabidopsis thaliana  
             
               G2107 gene  
             
           
            100 

      ttagtaactc caaagtgaca aatcttcgta acacatttct tcgtccacgt acacactcgt     60 
      attcatatca atgtacgatc ttggcggcga catcaacggc tcctccgcga gcctcatcat    120 
      cattccagcg actccttcat ccgacgtgtc aaactcactg gctgagggta aaaccgtaat    180 
      tcctgtacta aactccggcg gcctgaacat ctccgctgct tcggccgccg tgcgcctgat    240 
      cgtgtccgga tcagtggatg ccggcaccgg caacctccaa gcagaatcgg agaaattcaa    300 
      acacgcggat ctcccgcgca gagcaagaac cgccacgtcg tgagcacgtg cggccatatc    360 
      tgccgtcgga taagttccga gccagactcg acgctgatga atcggttcac ggacttcgca    420 
      tacccatttg tcgccgtccc tacgccgcac gcctctgtag attgggtgac gtgtctcctt    480 
      gaaaatcctc cgtccagcac gcttctttgg cttcatctcc gccacggtga tatcgtcgtt    540 
      ttccat                                                               546 

 
           
             101  
             181  
             PRT  
             Arabidopsis thaliana  
             
               G2107 polypeptide  
             
           
            101 

      Met Glu Asn Asp Asp Ile Thr Val Ala Glu Met Lys Pro Lys Lys Arg 
      1               5                   10                  15 
      Ala Gly Arg Arg Ile Phe Lys Glu Thr Arg His Pro Ile Tyr Arg Gly 
                  20                  25                  30 
      Val Arg Arg Arg Asp Gly Asp Lys Trp Val Cys Glu Val Arg Glu Pro 
              35                  40                  45 
      Ile His Gln Arg Arg Val Trp Leu Gly Thr Tyr Pro Thr Ala Asp Met 
          50                  55                  60 
      Ala Ala Arg Ala His Asp Val Ala Val Leu Ala Leu Arg Gly Arg Ser 
      65                  70                  75                  80 
      Ala Cys Leu Asn Phe Ser Asp Ser Ala Trp Arg Leu Pro Val Pro Ala 
                      85                  90                  95 
      Ser Thr Asp Pro Asp Thr Ile Arg Arg Thr Ala Ala Glu Ala Ala Glu 
                  100                 105                 110 
      Met Phe Arg Pro Pro Glu Phe Ser Thr Gly Ile Thr Val Leu Pro Ser 
              115                 120                 125 
      Ala Ser Glu Phe Asp Thr Ser Asp Glu Gly Val Ala Gly Met Met Met 
          130                 135                 140 
      Arg Leu Ala Glu Glu Pro Leu Met Ser Pro Pro Arg Ser Tyr Ile Asp 
      145                 150                 155                 160 
      Met Asn Thr Ser Val Tyr Val Asp Glu Glu Met Cys Tyr Glu Asp Leu 
                      165                 170                 175 
      Ser Leu Trp Ser Tyr 
                  180 

 
           
             102  
             888  
             DNA  
             Arabidopsis thaliana  
             
               G21  
             
           
            102 

      tcattcagat agaaaaaacg gctcttcaag ccgaaaccca gcatcggctc cacaaagctg     60 
      ccacgtggac gagtagtagc aaaacgcatc gtttcgtatc atcatctcat tctcatcggt    120 
      aaacaaatcc ggcaaatcaa acagcttctc ttcctcactg tctttgtccg tacacgccga    180 
      agtcgaagca cacgaagctt ccgaatactc ttgactctga gtcgtcgtcg tcgtgcttgt    240 
      gtccgaagaa aacaactgag ccaccacggc tcgactcggc tcggcttcaa ctatttcagc    300 
      cacttcagaa ttactcacat cgttgaccga atcttgccag ttaacggccg ctaaagaggc    360 
      ggcggcttga atgtctttag gagaatttgt gactggacga ggaagctcgc cggctaactt    420 
      gggaaaattg aggtaagccg ttgtaccttt aatggctaaa gccgctacgt catgagctcg    480 
      agctgccatc tcagccgttg gataagtccc gagccagatt cttgatttct ttctcggctc    540 
      tctaatctcc gacacccatt ttccccaact cctcatcctc actcctctat acgtcggatg    600 
      tttatctcca ccgttggtct ttcgccgttt cccacctcca ccgttttgat catcatcgtc    660 
      ttcaacggag actaacgatg acaatgatct tcgagatgct tttcttttag agttgtcttc    720 
      ctcaatgaag ttagaatttg ttgatgacga agaagaggaa gatgtagtag gtgaagagga    780 
      caatgaggcg gaagcggtga tggaggagga ggaagatacg gcggggatgg cggaggagat    840 
      aaaggtaact tgagaaacac tactctctat gttgatttgt cttgccat                 888 

 
           
             103  
             295  
             PRT  
             Arabidopsis thaliana  
             
               G21 polypeptide  
             
           
            103 

      Met Ala Arg Gln Ile Asn Ile Glu Ser Ser Val Ser Gln Val Thr Phe 
      1               5                   10                  15 
      Ile Ser Ser Ala Ile Pro Ala Val Ser Ser Ser Ser Ser Ile Thr Ala 
                  20                  25                  30 
      Ser Ala Ser Leu Ser Ser Ser Pro Thr Thr Ser Ser Ser Ser Ser Ser 
              35                  40                  45 
      Ser Thr Asn Ser Asn Phe Ile Glu Glu Asp Asn Ser Lys Arg Lys Ala 
          50                  55                  60 
      Ser Arg Arg Ser Leu Ser Ser Leu Val Ser Val Glu Asp Asp Asp Asp 
      65                  70                  75                  80 
      Gln Asn Gly Gly Gly Gly Lys Arg Arg Lys Thr Asn Gly Gly Asp Lys 
                      85                  90                  95 
      His Pro Thr Tyr Arg Gly Val Arg Met Arg Ser Trp Gly Lys Trp Val 
                  100                 105                 110 
      Ser Glu Ile Arg Glu Pro Arg Lys Lys Ser Arg Ile Trp Leu Gly Thr 
              115                 120                 125 
      Tyr Pro Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 
          130                 135                 140 
      Ala Ile Lys Gly Thr Thr Ala Tyr Leu Asn Phe Pro Lys Leu Ala Gly 
      145                 150                 155                 160 
      Glu Leu Pro Arg Pro Val Thr Asn Ser Pro Lys Asp Ile Gln Ala Ala 
                      165                 170                 175 
      Ala Ser Leu Ala Ala Val Asn Trp Gln Asp Ser Val Asn Asp Val Ser 
                  180                 185                 190 
      Asn Ser Glu Val Ala Glu Ile Val Glu Ala Glu Pro Ser Arg Ala Val 
              195                 200                 205 
      Val Ala Gln Leu Phe Ser Ser Asp Thr Ser Thr Thr Thr Thr Thr Gln 
          210                 215                 220 
      Ser Gln Glu Tyr Ser Glu Ala Ser Cys Ala Ser Thr Ser Ala Cys Thr 
      225                 230                 235                 240 
      Asp Lys Asp Ser Glu Glu Glu Lys Leu Phe Asp Leu Pro Asp Leu Phe 
                      245                 250                 255 
      Thr Asp Glu Asn Glu Met Met Ile Arg Asn Asp Ala Phe Cys Tyr Tyr 
                  260                 265                 270 
      Ser Ser Thr Trp Gln Leu Cys Gly Ala Asp Ala Gly Phe Arg Leu Glu 
              275                 280                 285 
      Glu Pro Phe Phe Leu Ser Glu 
          290                 295 

 
           
             104  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer O368  
             
           
            104 

      cayccnatht aymgnggngt                                                 20 

 
           
             105  
             26  
             DNA  
             Artificial Sequence  
             
               PCR primer O376  
             
           
            105 

      gcngcytcng cngcngcytt ytgdat                                          26 

 
           
             106  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer O2953  
             
           
            106 

      aaraarttym gngaracnmg ncay                                            24 

 
           
             107  
             25  
             DNA  
             Artificial Sequence  
             
               PCR primer O5436  
             
           
            107 

      ggaggaacac ggataagtgg gtaag                                           25 

 
           
             108  
             23  
             DNA  
             Artificial Sequence  
             
               APCR primer O5437  
             
           
            108 

      aggatttggc tggggacttt tcc                                             23 

 
           
             109  
             35  
             DNA  
             Artificial Sequence  
             
               PCR primer O18016  
             
           
            109 

      acgcgtcgac ccatcatcac cgagatcgac tcgac                                35 

 
           
             110  
             37  
             DNA  
             Artificial Sequence  
             
               PCR primer O18017  
             
           
            110 

      ataagaatgc ggccgctcat tgttcgctca ctgggag                              37 

 
           
             111  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer O18035  
             
           
            111 

      gctgacagaa cgggtgccga                                                 20 

 
           
             112  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer O18036  
             
           
            112 

      tgaccgtttc tggataggca                                                 20 

 
           
             113  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer O18065  
             
           
            113 

      ggccggcggg gcgaaccaag ttcc                                            24 

 
           
             114  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer O18066  
             
           
            114 

      aggcagagtc ggcgaagttg aggc                                            24 

 
           
             115  
             670  
             DNA  
             Secale cereale  
             
               Rye CBF20 gene  
             
           
            115 

      cgtcgaccca cgcgtccgga tccatcgatc aaacctctca acacagctgc tgattcttcc     60 
      agcactccac acttacaagc agcctcgatc tccgctagct ctagacctag atgccgtctg    120 
      gtcaggaggg gcaacggcac aggacggtga ggtcggagcc gccgggcggt gggtctgcga    180 
      ggtgcgcgtg ctcgggatga ggggctccag gctctggctt ggcaccttcg tcaccgcgga    240 
      gatggcggcg cgcgcccacg acgccgccgt gctcgcgctc tccggccgca aggcctgcct    300 
      caacttcgcc gactccgcct ggcggatgct gcccgtgctc gcggctggct ccttcggctt    360 
      cggcagcgcg cgggagatca agaccgccgt cgccgtcgcc gtcctcgcgt tccagcggca    420 
      gcagatcatt cttccagtag cccgcccggc ggaggagccg gccgacgtcc cgagcggcgc    480 
      gctgttctcc atgtcatcag gcgacttgct ggagctcgac gaggagcagt ggtttggcgg    540 
      catggttgcc gggtcctact acgagagctt ggcgcagggg atgctcgtcg agccgccgga    600 
      cgccggagcg tggcgagagg acagcgagca cagcggcgtg gcggagacgc agacgccgtt    660 
      gtggagctaa                                                           670 

 
           
             116  
             222  
             PRT  
             Secale cereale  
             
               Rye CBF20 polypeptide  
             
           
            116 

      Val Asp Pro Arg Val Arg Ile His Arg Ser Asn Leu Ser Thr Gln Leu 
      1               5                   10                  15 
      Leu Ile Leu Pro Ala Leu His Thr Tyr Lys Gln Pro Arg Ser Pro Leu 
                  20                  25                  30 
      Ala Leu Asp Leu Asp Ala Val Trp Ser Gly Gly Ala Thr Ala Gln Asp 
              35                  40                  45 
      Gly Glu Val Gly Ala Ala Gly Arg Trp Val Cys Glu Val Arg Val Leu 
          50                  55                  60 
      Gly Met Arg Gly Ser Arg Leu Trp Leu Gly Thr Phe Val Thr Ala Glu 
      65                  70                  75                  80 
      Met Ala Ala Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser Gly Arg 
                      85                  90                  95 
      Lys Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Met Leu Pro Val 
                  100                 105                 110 
      Leu Ala Ala Gly Ser Phe Gly Phe Gly Ser Ala Arg Glu Ile Lys Thr 
              115                 120                 125 
      Ala Val Ala Val Ala Val Leu Ala Phe Gln Arg Gln Gln Ile Ile Leu 
          130                 135                 140 
      Pro Val Ala Arg Pro Ala Glu Glu Pro Ala Asp Val Pro Ser Gly Ala 
      145                 150                 155                 160 
      Leu Phe Ser Met Ser Ser Gly Asp Leu Leu Glu Leu Asp Glu Glu Gln 
                      165                 170                 175 
      Trp Phe Gly Gly Met Val Ala Gly Ser Tyr Tyr Glu Ser Leu Ala Gln 
                  180                 185                 190 
      Gly Met Leu Val Glu Pro Pro Asp Ala Gly Ala Trp Arg Glu Asp Ser 
              195                 200                 205 
      Glu His Ser Gly Val Ala Glu Thr Gln Thr Pro Leu Trp Ser 
          210                 215                 220 

 
           
             117  
             813  
             DNA  
             Secale cereale  
             
               Rye CBF28 gene  
             
           
            117 

      atggacgtcg ccgacatcgc ctccccgtct ggccagcagg agcaggggca ccggacggtg     60 
      tcgtcggagc cgccgaagcg ccccgcgggg cggaccaagt tccacgagac gcgccacccg    120 
      ctgtaccgcg gcgtgcggcg ccgtggccgc gtcgggcagt gggtgtgcga ggtgcgcgtg    180 
      cccgggatca agggctccag gctctggctc ggcaccttca acacggccga gatggcggcg    240 
      cgcgcccacg acgcagccgt gctcgcgctc tcctgccgcg ccgcctgcct caacttcgcc    300 
      gactccgcct ggcggatgct gcccgtgctc gcggccgggt cgttcgggtt cggcagcccg    360 
      cgggagatca aggcagccgt cgccgtcgcc gtcatcgcgt tccagcggaa gcagattatt    420 
      ccggtcgccg tcgccgtcgt ggcgctccag cagcagcagg ttccagtcgc cgtcgccgtc    480 
      gttgcgctaa agcagaagca ggttccggtc gctgtggccg tcgtggcgct ccagcagctg    540 
      catgttccgg tagccgtcgc cgtcgtggcg ctccagcagc agcagattat tcttccagtc    600 
      gcgtgcctgg cgcccgagtt ttacatgtct tccggcgacc tgctggagct cgacgaggag    660 
      cactggtttg gcggcatgga cgccgggtcg tactacgcga gcttggcgca ggggatgctc    720 
      gtggctccgc cggacgaaag agcgaggccg gagaacggcg agcaagagcg gcgtccagac    780 
      gccgctatgg agctgtttgt tcgactaatt tag                                 813 

 
           
             118  
             270  
             PRT  
             Secale cereale  
             
               Rye CBF28 polypeptide  
             
           
            118 

      Met Asp Val Ala Asp Ile Ala Ser Pro Ser Gly Gln Gln Glu Gln Gly 
      1               5                   10                  15 
      His Arg Thr Val Ser Ser Glu Pro Pro Lys Arg Pro Ala Gly Arg Thr 
                  20                  25                  30 
      Lys Phe His Glu Thr Arg His Pro Leu Tyr Arg Gly Val Arg Arg Arg 
              35                  40                  45 
      Gly Arg Val Gly Gln Trp Val Cys Glu Val Arg Val Pro Gly Ile Lys 
          50                  55                  60 
      Gly Ser Arg Leu Trp Leu Gly Thr Phe Asn Thr Ala Glu Met Ala Ala 
      65                  70                  75                  80 
      Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser Cys Arg Ala Ala Cys 
                      85                  90                  95 
      Leu Asn Phe Ala Asp Ser Ala Trp Arg Met Leu Pro Val Leu Ala Ala 
                  100                 105                 110 
      Gly Ser Phe Gly Phe Gly Ser Pro Arg Glu Ile Lys Ala Ala Val Ala 
              115                 120                 125 
      Val Ala Val Ile Ala Phe Gln Arg Lys Gln Ile Ile Pro Val Ala Val 
          130                 135                 140 
      Ala Val Val Ala Leu Gln Gln Gln Gln Val Pro Val Ala Val Ala Val 
      145                 150                 155                 160 
      Val Ala Leu Lys Gln Lys Gln Val Pro Val Ala Val Ala Val Val Ala 
                      165                 170                 175 
      Leu Gln Gln Leu His Val Pro Val Ala Val Ala Val Val Ala Leu Gln 
                  180                 185                 190 
      Gln Gln Gln Ile Ile Leu Pro Val Ala Cys Leu Ala Pro Glu Phe Tyr 
              195                 200                 205 
      Met Ser Ser Gly Asp Leu Leu Glu Leu Asp Glu Glu His Trp Phe Gly 
          210                 215                 220 
      Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Met Leu 
      225                 230                 235                 240 
      Val Ala Pro Pro Asp Glu Arg Ala Arg Pro Glu Asn Gly Glu Gln Glu 
                      245                 250                 255 
      Arg Arg Pro Asp Ala Ala Met Glu Leu Phe Val Arg Leu Ile 
                  260                 265                 270 

 
           
             119  
             807  
             DNA  
             Secale cereale  
             
               Rye CBF46 gene  
             
           
            119 

      atggacgtcg ccgacatcgc ctcccggtct ggccagcagc agcaggggca ccggaccgtg     60 
      tcgtcggagc cgccgaagcg ccccgcgggg aggaccaagt tccacgagac gcgccacccg    120 
      ctgtaccgcg gcgtgcggcg ccgtggccgc gtcgggcagt gggtgtgcga ggtgcgcgtt    180 
      cccgggatca agggctccag gctctggctc ggcaccttca acacggccga gatggcggcg    240 
      cgcgcgcacg acgccgccgt gctcgcgctc tccggccgca aagcctgcct caacttcgcc    300 
      gactccgcct ggcggatgct gcccgtgctc gcggccggct ccttcggctt tgatagcgcg    360 
      cgggaggtca aggccgccgt cgccgtcgcc gtcgtcgcgt tccagcggaa acagattatt    420 
      ccagtcgccg tcgctgtcgt tgctctccag aagcagcagg ttccggtcgc cgtggccatc    480 
      gtggcgctcc agcagaggca ggttccggtc gccgtcgccg tcgtggcgct ccagaagctg    540 
      caggttccgg tcgccgtcgc cgtcgtagcg ctccagaaga agcagattat tcttccagcc    600 
      gcgtgcctgg cgccggagtt ttacatgtct tccggcgacc tgttggagct cgacgaggag    660 
      cagtggtttg gcggcatgga cgccgggtcg tactacgcca gcttggcgca ggggatgctc    720 
      gtggcgccgc cggacgacag agcgaggccg gagaacggcg agcagagcgg cgtccagact    780 
      ccgctatgga gctgcttgtt cgactaa                                        807 

 
           
             120  
             268  
             PRT  
             Secale cereale  
             
               Rye CBF46 polypeptide  
             
           
            120 

      Met Asp Val Ala Asp Ile Ala Ser Arg Ser Gly Gln Gln Gln Gln Gly 
      1               5                   10                  15 
      His Arg Thr Val Ser Ser Glu Pro Pro Lys Arg Pro Ala Gly Arg Thr 
                  20                  25                  30 
      Lys Phe His Glu Thr Arg His Pro Leu Tyr Arg Gly Val Arg Arg Arg 
              35                  40                  45 
      Gly Arg Val Gly Gln Trp Val Cys Glu Val Arg Val Pro Gly Ile Lys 
          50                  55                  60 
      Gly Ser Arg Leu Trp Leu Gly Thr Phe Asn Thr Ala Glu Met Ala Ala 
      65                  70                  75                  80 
      Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser Gly Arg Lys Ala Cys 
                      85                  90                  95 
      Leu Asn Phe Ala Asp Ser Ala Trp Arg Met Leu Pro Val Leu Ala Ala 
                  100                 105                 110 
      Gly Ser Phe Gly Phe Asp Ser Ala Arg Glu Val Lys Ala Ala Val Ala 
              115                 120                 125 
      Val Ala Val Val Ala Phe Gln Arg Lys Gln Ile Ile Pro Val Ala Val 
          130                 135                 140 
      Ala Val Val Ala Leu Gln Lys Gln Gln Val Pro Val Ala Val Ala Ile 
      145                 150                 155                 160 
      Val Ala Leu Gln Gln Arg Gln Val Pro Val Ala Val Ala Val Val Ala 
                      165                 170                 175 
      Leu Gln Lys Leu Gln Val Pro Val Ala Val Ala Val Val Ala Leu Gln 
                  180                 185                 190 
      Lys Lys Gln Ile Ile Leu Pro Ala Ala Cys Leu Ala Pro Glu Phe Tyr 
              195                 200                 205 
      Met Ser Ser Gly Asp Leu Leu Glu Leu Asp Glu Glu Gln Trp Phe Gly 
          210                 215                 220 
      Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Met Leu 
      225                 230                 235                 240 
      Val Ala Pro Pro Asp Asp Arg Ala Arg Pro Glu Asn Gly Glu Gln Ser 
                      245                 250                 255 
      Gly Val Gln Thr Pro Leu Trp Ser Cys Leu Phe Asp 
                  260                 265 

 
           
             121  
             639  
             DNA  
             Secale cereale  
             
               Rye CBF7 gene  
             
           
            121 

      atggacgccg ccgacgccgg ctccccccgt tttgggcaca ggacggtgtg ctcggagccg     60 
      cccaagaggc cggcagggcg gaccaagttt aaggagaccc gccacccgct gtaccgcggc    120 
      gtgcggcggc ggggtcggct cgggcagtgg gtgtgcgagg tgcgcgtgcg cggcgcgcaa    180 
      gggtacaggc tctggctcgg cacattcacc accgccgaga tggcggcgcg cgcgcacgac    240 
      tccgccgtgc tcgcgctcct cgaccgcgcc gcttgcctca acttcgccga ctccgcctgg    300 
      cggatgctgc ccgtcctcgc ggcaggctcg tcccgcttca gcagcgcgcg ggaaatcaag    360 
      gacgccgtcg ccgtcgccgt cgtggagttc cagcggcagc gccccttcgt gtccacgtcg    420 
      gagacggccg acggcgagaa ggacgtccaa ggctcgccga ggccgagcga gctgtccacg    480 
      tccagcgact tgttggacga gcactggttt agcggcatgg acgccggctc ttactacgcg    540 
      agcttggcgc aggggatgct catggagccg ccggccgcca gagcgtggag cgaggatggc    600 
      ggcgaataca gcggcgtcca cacgccgctt tggaactag                           639 

 
           
             122  
             212  
             PRT  
             Secale cereale  
             
               Rye CBF7 polypeptide  
             
           
            122 

      Met Asp Ala Ala Asp Ala Gly Ser Pro Arg Phe Gly His Arg Thr Val 
      1               5                   10                  15 
      Cys Ser Glu Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Lys Glu 
                  20                  25                  30 
      Thr Arg His Pro Leu Tyr Arg Gly Val Arg Arg Arg Gly Arg Leu Gly 
              35                  40                  45 
      Gln Trp Val Cys Glu Val Arg Val Arg Gly Ala Gln Gly Tyr Arg Leu 
          50                  55                  60 
      Trp Leu Gly Thr Phe Thr Thr Ala Glu Met Ala Ala Arg Ala His Asp 
      65                  70                  75                  80 
      Ser Ala Val Leu Ala Leu Leu Asp Arg Ala Ala Cys Leu Asn Phe Ala 
                      85                  90                  95 
      Asp Ser Ala Trp Arg Met Leu Pro Val Leu Ala Ala Gly Ser Ser Arg 
                  100                 105                 110 
      Phe Ser Ser Ala Arg Glu Ile Lys Asp Ala Val Ala Val Ala Val Val 
              115                 120                 125 
      Glu Phe Gln Arg Gln Arg Pro Phe Val Ser Thr Ser Glu Thr Ala Asp 
          130                 135                 140 
      Gly Glu Lys Asp Val Gln Gly Ser Pro Arg Pro Ser Glu Leu Ser Thr 
      145                 150                 155                 160 
      Ser Ser Asp Leu Leu Asp Glu His Trp Phe Ser Gly Met Asp Ala Gly 
                      165                 170                 175 
      Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Met Leu Met Glu Pro Pro Ala 
                  180                 185                 190 
      Ala Arg Ala Trp Ser Glu Asp Gly Gly Glu Tyr Ser Gly Val His Thr 
              195                 200                 205 
      Pro Leu Trp Asn 
          210 

 
           
             123  
             807  
             DNA  
             Secale cereale  
             
               Rye CBF71  
             
           
            123 

      atggacgtcg ccgacatcgc ctcccggtct ggccagcagc agcaggggca ccggaccgtg     60 
      tcgtcggagc cgccgaagcg ccccgcgggg aggaccaagt tccacgagac gcgccacccg    120 
      ctgtaccgcg gcgtgcggcg ccgtggccgc gtcgggcagt gggtgtgcga ggtgcgcgtg    180 
      cccgggatca agggctccag gctctggctc ggcaccttca acacggccga gatggcggcg    240 
      cgcgcgcacg acgctgccgt gctcgcgctc tccggccgcg ccgcctgcct caacttcgcc    300 
      gactccgcct ggcggatgct gcccgtgctc gcggccggct ccttcggctt tgatagcgcg    360 
      cgggaggtca aggccgccgt cgccgtcgcc gtcgtcgcgt tccagcggaa acagattatt    420 
      ccagtcgccg tcgctgtcgt tgctctccag aagcagcagg ttccggtcgc cgtggccgtc    480 
      gtggcgctcc agcagaggca ggttccggtc accgtcgccg tcgtggcgct ccagaagctg    540 
      caggttccgg tcgccgtcgc cgtcgtggcg ctccagaaga agcagattat tcttccagcc    600 
      gcgtgtctgg cgccggagtt ttacatgtct tccggcgacc tgttggagct cgacgaggag    660 
      cagtggtttg gcggcatgga cgccgggtcg tactacgcca gcttggcgca ggggatgctc    720 
      gtggcgccgc cggacgacag agcgaggccg gagaacggcg agcagagcgg cgtccagact    780 
      ccgctatgga gctgcttgtt cgactaa                                        807 

 
           
             124  
             268  
             PRT  
             Secale cereale  
             
               Rye CBF71 polypeptide  
             
           
            124 

      Met Asp Val Ala Asp Ile Ala Ser Arg Ser Gly Gln Gln Gln Gln Gly 
      1               5                   10                  15 
      His Arg Thr Val Ser Ser Glu Pro Pro Lys Arg Pro Ala Gly Arg Thr 
                  20                  25                  30 
      Lys Phe His Glu Thr Arg His Pro Leu Tyr Arg Gly Val Arg Arg Arg 
              35                  40                  45 
      Gly Arg Val Gly Gln Trp Val Cys Glu Val Arg Val Pro Gly Ile Lys 
          50                  55                  60 
      Gly Ser Arg Leu Trp Leu Gly Thr Phe Asn Thr Ala Glu Met Ala Ala 
      65                  70                  75                  80 
      Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser Gly Arg Ala Ala Cys 
                      85                  90                  95 
      Leu Asn Phe Ala Asp Ser Ala Trp Arg Met Leu Pro Val Leu Ala Ala 
                  100                 105                 110 
      Gly Ser Phe Gly Phe Asp Ser Ala Arg Glu Val Lys Ala Ala Val Ala 
              115                 120                 125 
      Val Ala Val Val Ala Phe Gln Arg Lys Gln Ile Ile Pro Val Ala Val 
          130                 135                 140 
      Ala Val Val Ala Leu Gln Lys Gln Gln Val Pro Val Ala Val Ala Val 
      145                 150                 155                 160 
      Val Ala Leu Gln Gln Arg Gln Val Pro Val Thr Val Ala Val Val Ala 
                      165                 170                 175 
      Leu Gln Lys Leu Gln Val Pro Val Ala Val Ala Val Val Ala Leu Gln 
                  180                 185                 190 
      Lys Lys Gln Ile Ile Leu Pro Ala Ala Cys Leu Ala Pro Glu Phe Tyr 
              195                 200                 205 
      Met Ser Ser Gly Asp Leu Leu Glu Leu Asp Glu Glu Gln Trp Phe Gly 
          210                 215                 220 
      Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Met Leu 
      225                 230                 235                 240 
      Val Ala Pro Pro Asp Asp Arg Ala Arg Pro Glu Asn Gly Glu Gln Ser 
                      245                 250                 255 
      Gly Val Gln Thr Pro Leu Trp Ser Cys Leu Phe Asp 
                  260                 265 

 
           
             125  
             240  
             DNA  
             Triticum aestivum  
             
               Wheat CBF gene  
             
           
            125 

      agtgattggc cggcggggcg aaccaagttc cgtgagacac gacacccgct gtaccgcggc     60 
      gtgcggcgcc gtggccgggt cgggcagtgg gtgtgcgagg tgcgcgtgcc aggagtgaag    120 
      ggctccaggc tctggctcgg caccttcacc accgccgaga tggcggcgcg cgcgcacgac    180 
      gccgcggtgc tcgcgctctc cggccgcgcc gcctgcctca acttcgccga ctctgcctaa    240 

 
           
             126  
             76  
             PRT  
             Triticum aestivum  
             
               Wheat CBF polypeptide  
             
           
            126 

      Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro Leu Tyr Arg 
      1               5                   10                  15 
      Gly Val Arg Arg Arg Gly Arg Val Gly Gln Trp Val Cys Glu Val Arg 
                  20                  25                  30 
      Val Pro Gly Val Lys Gly Ser Arg Leu Trp Leu Gly Thr Phe Thr Thr 
              35                  40                  45 
      Ala Glu Met Ala Ala Arg Ala His Asp Ala Ala Val Leu Ala Leu Ser 
          50                  55                  60 
      Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser Ala 
      65                  70                  75 

 
           
             127  
             705  
             DNA  
             Glycine max  
             
               Soy CBF gene  
             
           
            127 

      atgtttacct tgaatcattc ttctgatttg taccatgttt cccctgagct ctcatcttcc     60 
      ttggacacat cctcgccggc ttcggagggc tctcgtggcg tggcattttc cgacgaggag    120 
      gtgcggctgg cggtgaggca cccgaagaag cgggcaggtc ggaagaagtt ccgggagacg    180 
      cgccacccgg tgtaccgggg ggtgaggagg aggaactcgg ataagtgggt gtgtgaggtg    240 
      agggagccca acaagaagac caggatttgg ctggggactt tccccacgcc ggagatggcg    300 
      gctcgggcgc acgacgtggc ggcaatggcc ctgaggggcc ggtatgcctg tctaaacttt    360 
      gctgactcgg cctggcggtt acctgttccc gccacggccg aggcaaagga tatacagaag    420 
      gcagcagcag aagctgccca ggctttcaga ccagatcaaa ccttaaaaaa tgctaataca    480 
      aggcaggagt gtgtggaggc ggtggcggtg gcggtggcgg agacaacaac ggcgacggca    540 
      caaggggtgt tttatatgga ggaagaagag caggtgttgg atatgcctga gttgcttagg    600 
      aatatggtgc tcatgtcccc aacacattgc ttagggtatg agtatgaaga tgctgacttg    660 
      gatgcccaag atgctgaggt gtcactatgg aatttctcaa tttaa                    705 

 
           
             128  
             234  
             PRT  
             Glycine max  
             
               Soy CBF polypeptide  
             
           
            128 

      Met Phe Thr Leu Asn His Ser Ser Asp Leu Tyr His Val Ser Pro Glu 
      1               5                   10                  15 
      Leu Ser Ser Ser Leu Asp Thr Ser Ser Pro Ala Ser Glu Gly Ser Arg 
                  20                  25                  30 
      Gly Val Ala Phe Ser Asp Glu Glu Val Arg Leu Ala Val Arg His Pro 
              35                  40                  45 
      Lys Lys Arg Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val 
          50                  55                  60 
      Tyr Arg Gly Val Arg Arg Arg Asn Ser Asp Lys Trp Val Cys Glu Val 
      65                  70                  75                  80 
      Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Pro Thr 
                      85                  90                  95 
      Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Met Ala Leu Arg 
                  100                 105                 110 
      Gly Arg Tyr Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Pro 
              115                 120                 125 
      Val Pro Ala Thr Ala Glu Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu 
          130                 135                 140 
      Ala Ala Gln Ala Phe Arg Pro Asp Gln Thr Leu Lys Asn Ala Asn Thr 
      145                 150                 155                 160 
      Arg Gln Glu Cys Val Glu Ala Val Ala Val Ala Val Ala Glu Thr Thr 
                      165                 170                 175 
      Thr Ala Thr Ala Gln Gly Val Phe Tyr Met Glu Glu Glu Glu Gln Val 
                  180                 185                 190 
      Leu Asp Met Pro Glu Leu Leu Arg Asn Met Val Leu Met Ser Pro Thr 
              195                 200                 205 
      His Cys Leu Gly Tyr Glu Tyr Glu Asp Ala Asp Leu Asp Ala Gln Asp 
          210                 215                 220 
      Ala Glu Val Ser Leu Trp Asn Phe Ser Ile 
      225                 230 

 
           
             129  
             2396  
             DNA  
             Arabidopsis thaliana  
             
               Artificial fusion between rab18 promoter and CBF1 gene  
             
           
            129 

      aagcttcaaa ttctgaatat tcacatatca aaaaagtgat gcgagggaga gaagggtatt     60 
      tcatcttact tgcagcgata gaactccttg tggtagaagt gacatataac tttttcactc    120 
      cttgtgactt ctcccaagaa gtcgccttcg ctaacttctc ggtattcacc atgtccttgt    180 
      cttttgaatg cttctctctt ttccacttct ctctgtccag acacatcttg ggattagatc    240 
      agtccatact aatgaagaca aaagtgttaa ccaaatatag ataatgagag aatgtggggg    300 
      gtgaagttga accctgagtg ctgcaatcct atctgcgtgc aacttttcta gctctggatc    360 
      ctacagaaca agagcaagtc agtctcccat agagacagaa gagaaacata acatttcatt    420 
      gtacagatta taaaagaaca attcaaagag agcccccatt gaacttacat ccatcaattc    480 
      gtcaagatca acttcctcgt tgacaggtct tgatccttgt gccttttcat ttgcaagaac    540 
      ttcctgcaca acaaaaatac ggaaatgttt ttgatccaag aacatcaaac tctaattgca    600 
      attgtaacca acaaaaaaac acttaaaatt cgctatacac tatcaaattt cctgacacgt    660 
      agacctcatg tcagcacaaa tagaatctag cctatctctg ttaattgggt taccaaacaa    720 
      catgagattg attatgtgga aaaccaagca cattatttac cctttgaaaa tacgcgaaat    780 
      caagatccaa gaagaattta tggagaacag cttcgaagta cgaaataaac aatgaagatt    840 
      aaattacctt tttataatct ctagcagctg ccgccaatac attcccgaat gccagattcg    900 
      agagggtcga cttcaccgta tccggatcca tctctttacc aaccaactaa tccaactcag    960 
      aaaattttaa aatctcaatc aaaaatccct ctaagatagc cagagaagag attgtaaaca   1020 
      aggatttgaa atctggtgca gagaggagaa actccccgac aatgaacacc aacgatctaa   1080 
      acgcggcgtt tggtaaaagt tgagtaaatt ttgttagggc ttagttttag tccatgggct   1140 
      aattagtaag tgatttacgg cccacacatg agcccaaatg tttcagaccc agccaagttt   1200 
      cttcaaattc acccaatcaa cgacgatgta cgtgtgtatg aaaatcatta acacgacgca   1260 
      tcgctttcga ggaggagcat tacgtgtcct gttagctacg ataatgttag taccgccaca   1320 
      aagaaaagga tagatatttt gctttccagc accctgtcat gggattgata tgaacacgta   1380 
      cttggtatcg acatgaaagc tcaaaaataa attcaatccg attcctttag tgatatcaga   1440 
      agttcatttt aaatacgaac acgtatggcg aaacaccacg ccgacatttt ctgctgctgc   1500 
      cacgcgtcac tttccaaata ttgattcatt aaactaatag ttgatccata tccgaaaccg   1560 
      gactataaaa ctatcttcaa tgcgttaacg aatcttcatc gatcaaactc atcaaagtct   1620 
      aatatcacaa agaaagagtt tttttaacta gcttagctca aagtgtttgc ttaagacaag   1680 
      aagaacgaat tcaatgaact cattttcagc tttttctgaa atgtttggct ccgattacga   1740 
      gcctcaaggc ggagattatt gtccgacgtt ggccacgagt tgtccgaaga aaccggcggg   1800 
      ccgtaagaag tttcgtgaga ctcgtcaccc aatttacaga ggagttcgtc aaagaaactc   1860 
      cggtaagtgg gtttctgaag tgagagagcc aaacaagaaa accaggattt ggctcgggac   1920 
      tttccaaacc gctgagatgg cagctcgtgc tcacgacgtc gctgcattag ccctccgtgg   1980 
      ccgatcagca tgtctcaact tcgctgactc ggcttggcgg ctacgaatcc cggagtcaac   2040 
      atgcgccaag gatatccaaa aagcggctgc tgaagcggcg ttggcttttc aagatgagac   2100 
      gtgtgatacg acgaccacgg atcatggcct ggacatggag gagacgatgg tggaagctat   2160 
      ttatacaccg gaacagagcg aaggtgcgtt ttatatggat gaggagacaa tgtttgggat   2220 
      gccgactttg ttggataata tggctgaagg catgctttta ccgccgccgt ctgttcaatg   2280 
      gaatcataat tatgacggcg aaggagatgg tgacgtgtcg ctttggagtt actaatattc   2340 
      gatagtcgtt tccatttttg tactatagtt tgaaaatatt ctagttccgc ggccgc       2396 

 
           
             130  
             2655  
             DNA  
             Arabidopsis thaliana  
             
               Artificial fusion between DREB2a promoter and CBF1 gene  
             
           
            130 

      aagcttgtaa tcgataacct aaatcattgt aatgaatgcg ttcctcccta tcgatcctag     60 
      gccttagaca atgctgaatg attcatagcc acgcgaataa cctattcata ctaatatgga    120 
      aagaaagaag ccaaacttac agagctcttc tcacggtcgt cgaagaaaag agagtctaca    180 
      gtcagcaaca aaattagtgt tgccatcatg gcatcatctt gcagcttttt ccgcacaaat    240 
      aatttatcat ccaaatgttg ttcactaaac caaaaacaaa caagcattac agcgaagaaa    300 
      ccaactcgta gatacagatt tccaaattca tgccttattt agaccaataa aaactgaaat    360 
      ttctcttcag cgaaaaaaaa aacaaacaaa ctgaaatttc atatagatcc agaagataga    420 
      aacttgtagg ctcaatcgac tagactagaa gatgctcacc cgatcgtgct tgagatagcg    480 
      agatagtagc aacaccgacg gtagaaataa aatggacgac acccatccaa tgggctaatt    540 
      tagattaacg ggctttaagg gtttgataat ggatgttaat taactgaggc acatgggatt    600 
      gtatcacgta ggcaatgggt ttgataatgg atgttaagta actaaggccc atgaggttga    660 
      agtacgtagg caattgcgtg agcttacgtt agcgatgccg ttagagacac gtagtggatg    720 
      aagtggcttt ggttagcaaa ggacacatga ggcacatgca aaggctataa atgactgctg    780 
      ctttgctaca acttgcgatt cccaaatttt ataaggtaat ggaccctatc acctctgctc    840 
      gaagctaagc cacccaagtt tgagcttcac catttgacac gtctctagct aatacttaat    900 
      gcttaaactt taaactaata cttcatgttt aagacatttc tggctgacac atttatgaat    960 
      tcgctctatg tcgtacgtac accggaaacc tttaatttta caatattaac cgtgatcttt   1020 
      ttaaaaatat ctatataaac gataagtatc tcatcaaaac aaaattgaat aatgtgcacg   1080 
      tttgtaaaaa ctagaaaaac aggcaataaa catcatcatc caattacaca tctagtaagt   1140 
      gtgatgcagt ggcaaactgg caataaacca agaaaaatcg agaaagagca gatgagacag   1200 
      tgttgtgttg tcagggttaa taaaaaaaaa atgaagatat tttaaaattt cataatattt   1260 
      aaataatgaa gtagttttat gatcttatcc ataaatcaat tttaaaaagg tttaaacttt   1320 
      acttttccgt atcaacagcg tgtttcgaga agattcggga ggacactcgt cgaacggaaa   1380 
      agtcgtctaa gcctttatgt tcgaatcaaa aactgacacg taaccttgct ctcaaacaga   1440 
      aaaataaaat aatgttagaa aaatctagag aaggctataa atactccgta gatactttgt   1500 
      cttccttaga tattttgatt tctgctaaag ctgtctgata aaaagaagag gaaaactcga   1560 
      aaaagctaca cacaagaaga agaagaaaag atacgagcaa gaagactaaa cacgaaagcg   1620 
      atttatcaac tcgaaggaag agactttgat tttcaaattt cgtcccctat aggtacgttg   1680 
      atttctgatt tcttacaaat tttcaattaa tttctctgat taggtctcgt caattggtga   1740 
      ttctagggtt tatcttcgtc tttaggtttt cgattatttc cttttaggta tagctgtgaa   1800 
      attgggaaat tttaggtgtt cctaatttga taacaaataa gtaaatcgat ctgattttga   1860 
      accaagttta agttctcttg ccttatgttt cagcttgtgt tctgatattg aagtgttgtt   1920 
      gtattgtaga ttgtgttgtt tctgggaagg ccatggactc attttcagct ttttctgaaa   1980 
      tgtttggctc cgattacgag cctcaaggcg gagattattg tccgacgttg gccacgagtt   2040 
      gtccgaagaa accggcgggc cgtaagaagt ttcgtgagac tcgtcaccca atttacagag   2100 
      gagttcgtca aagaaactcc ggtaagtggg tttctgaagt gagagagcca aacaagaaaa   2160 
      ccaggatttg gctcgggact ttccaaaccg ctgagatggc agctcgtgct cacgacgtcg   2220 
      ctgcattagc cctccgtggc cgatcagcat gtctcaactt cgctgactcg gcttggcggc   2280 
      tacgaatccc ggagtcaaca tgcgccaagg atatccaaaa agcggctgct gaagcggcgt   2340 
      tggcttttca agatgagacg tgtgatacga cgaccacgga tcatggcctg gacatggagg   2400 
      agacgatggt ggaagctatt tatacaccgg aacagagcga aggtgcgttt tatatggatg   2460 
      aggagacaat gtttgggatg ccgactttgt tggataatat ggctgaaggc atgcttttac   2520 
      cgccgccgtc tgttcaatgg aatcataatt atgacggcga aggagatggt gacgtgtcgc   2580 
      tttggagtta ctaatattcg atagtcgttt ccatttttgt actatagttt gaaaatattc   2640 
      tagttccgcg gccgc                                                    2655 

 
           
             131  
             21  
             DNA  
             Artificial sequence  
             
               O6792 Synthetic oligonucleotide primer  
             
           
            131 

      gcttgtccat tatcctagaa t                                               21 

 
           
             132  
             28  
             DNA  
             Artificial sequence  
             
               O6793 Synthetic oligonucleotide primer  
             
           
            132 

      cggaattcgt tcttcttgtc ttaagcaa                                        28 

 
           
             133  
             35  
             DNA  
             Artificial sequence  
             
               O6805 Synthetic oligonucleotide primer  
             
           
            133 

      cggaattcaa tgaactcatt ttcagctttt tctga                                35 

 
           
             134  
             43  
             DNA  
             Artificial sequence  
             
               O6806 Synthetic oligonucleotide primer  
             
           
            134 

      ataagaatgc ggccgcggaa ctagaatatt ttcaaactat agt                       43 

 
           
             135  
             32  
             DNA  
             Artificial sequence  
             
               O6798  Synthetic oligonucleotide primer  
             
           
            135 

      gcccaagctt gtaatcgata acctaaatca tt                                   32 

 
           
             136  
             31  
             DNA  
             Artificial sequence  
             
               O6799 Synthetic oligonucleotide primer  
             
           
            136 

      aactgccatg gccttcccag aaacaacaca a                                    31 

 
           
             137  
             62  
             PRT  
             Arabidopsis thaliana  
           
            137 

      His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val 
       1               5                  10                  15 
      Cys Glu Leu Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             138  
             62  
             PRT  
             Arabidopsis thaliana  
           
            138 

      His Pro Ile Tyr Arg Gly Val Arg Arg Arg Asn Ser Gly Lys Trp Val 
       1               5                  10                  15 
      Cys Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             139  
             62  
             PRT  
             Arabidopsis thaliana  
           
            139 

      His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val 
       1               5                  10                  15 
      Ser Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             140  
             62  
             PRT  
             Brassica juncea  
           
            140 

      His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn 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 Pro Thr Val Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             141  
             62  
             PRT  
             Brassica oleracea  
           
            141 

      His Pro Val Tyr Arg Gly Val Arg Leu Arg Asn 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 Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             142  
             62  
             PRT  
             Raphanus sativus  
           
            142 

      His Pro Ile Tyr Arg Gly Val Arg Leu Arg Asn 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 Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             143  
             62  
             PRT  
             Brassica juncea  
           
            143 

      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 Arg Ser Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             144  
             62  
             PRT  
             Brassica napus  
           
            144 

      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 Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             145  
             62  
             PRT  
             Brassica juncea  
           
            145 

      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 Pro Gly Thr 
                  20                  25                  30 
      Phe Leu Thr Ala Glu Ile Ala Ala Arg Ala His Asp Val Ala Ala Ile 
              35                  40                  45 
      Ala Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             146  
             62  
             PRT  
             Brassica napus  
           
            146 

      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 Pro 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 
          50                  55                  60 

 
           
             147  
             62  
             PRT  
             Brassica napus  
           
            147 

      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 Pro 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 
          50                  55                  60 

 
           
             148  
             62  
             PRT  
             Brassica napus  
           
            148 

      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 
          50                  55                  60 

 
           
             149  
             62  
             PRT  
             Brassica napus  
           
            149 

      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 
          50                  55                  60 

 
           
             150  
             62  
             PRT  
             Brassica napus  
           
            150 

      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 
          50                  55                  60 

 
           
             151  
             62  
             PRT  
             Brassica oleracea  
           
            151 

      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 
          50                  55                  60 

 
           
             152  
             62  
             PRT  
             Brassica rapa  
           
            152 

      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 
          50                  55                  60 

 
           
             153  
             62  
             PRT  
             Brassica napus  
           
            153 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             154  
             62  
             PRT  
             Brassica napus  
           
            154 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             155  
             62  
             PRT  
             Brassica oleracea  
           
            155 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             156  
             62  
             PRT  
             Brassica rapa  
           
            156 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             157  
             62  
             PRT  
             Brassica rapa  
           
            157 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             158  
             62  
             PRT  
             Brassica rapa  
           
            158 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             159  
             62  
             PRT  
             Brassica rapa  
           
            159 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             160  
             62  
             PRT  
             Brassica rapa  
           
            160 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             161  
             62  
             PRT  
             Brassica rapa  
           
            161 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             162  
             62  
             PRT  
             Raphanus sativus  
           
            162 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             163  
             62  
             PRT  
             Brassica oleracea  
           
            163 

      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 Ile Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             164  
             62  
             PRT  
             Brassica oleracea  
           
            164 

      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 Leu 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 
          50                  55                  60 

 
           
             165  
             62  
             PRT  
             Brassica napus  
           
            165 

      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 Pro 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 Arg Leu Asn Tyr Ala Asp Ser 
          50                  55                  60 

 
           
             166  
             63  
             PRT  
             Zea mays  
           
            166 

      His Pro Val Tyr Arg Gly Val Arg Arg Arg Gly Pro Ala Gly Arg Trp 
       1               5                  10                  15 
      Val Cys Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly 
                  20                  25                  30 
      Thr Phe Ala Thr Pro Glu Ala Ala Ala Arg Ala His Asp Val Ala Ala 
              35                  40                  45 
      Leu Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             167  
             62  
             PRT  
             Glycine max  
           
            167 

      His Pro Ile Tyr Ser Gly Val Arg Arg Arg Asn Thr Asp Lys Trp Val 
       1               5                  10                  15 
      Ser Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Pro Thr Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Met 
              35                  40                  45 
      Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe Ala Asp Ser 
          50                  55                  60 

 
           
             168  
             63  
             PRT  
             Nicotiana tabacum  
           
            168 

      Gly Arg His Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Phe Ala 
       1               5                  10                  15 
      Ala Glu Ile Arg Asp Pro Ala Lys Asn Gly Ala Arg Val Trp Leu Gly 
                  20                  25                  30 
      Thr Tyr Glu Thr Ala Glu Glu Ala Ala Leu Ala Tyr Asp Lys Ala Ala 
              35                  40                  45 
      Tyr Arg Met Arg Gly Ser Lys Ala Leu Leu Asn Phe Pro His Arg 
          50                  55                  60 

 
           
             169  
             62  
             PRT  
             Arabidopsis thaliana  
           
            169 

      Arg Cys Ser Phe Arg Gly Val Arg Gln Arg Ile Trp Gly Lys Trp Val 
       1               5                  10                  15 
      Ala Glu Ile Arg Glu Pro Asn Arg Gly Ser Arg Leu Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Pro Thr Ala Gln Glu Ala Ala Ser Ala Tyr Asp Glu Ala Ala Lys 
              35                  40                  45 
      Ala Met Tyr Gly Pro Leu Ala Arg Leu Asn Phe Pro Arg Ser 
          50                  55                  60 

 
           
             170  
             62  
             PRT  
             Arabidopsis thaliana  
           
            170 

      His Cys Ser Phe Arg Gly Val Arg Gln Arg Ile Trp Gly Lys Trp Val 
       1               5                  10                  15 
      Ala Glu Ile Arg Glu Pro Lys Ile Gly Thr Arg Leu Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Pro Thr Ala Glu Lys Ala Ala Ser Ala Tyr Asp Glu Ala Ala Thr 
              35                  40                  45 
      Ala Met Tyr Gly Ser Leu Ala Arg Leu Asn Phe Pro Gln Ser 
          50                  55                  60 

 
           
             171  
             62  
             PRT  
             Arabidopsis thaliana  
           
            171 

      His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn Trp Gly Lys Trp Val 
       1               5                  10                  15 
      Ser Glu Ile Arg Glu Pro Arg Lys Lys Ser Arg Ile Trp Leu Gly Thr 
                  20                  25                  30 
      Phe Pro Ser Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu 
              35                  40                  45 
      Ser Ile Lys Gly Ala Ser Ala Ile Leu Asn Phe Pro Asp Leu 
          50                  55                  60 

 
           
             172  
             46  
             PRT  
             Brassica rapa  
           
            172 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Cys Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg 
              35                  40                  45 

 
           
             173  
             46  
             PRT  
             Brassica rapa  
           
            173 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Cys Ser Glu Asn Lys 
       1               5                  10                  15 
      Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg 
              35                  40                  45 

 
           
             174  
             46  
             PRT  
             Brassica napus  
           
            174 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Arg Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Asn Thr Glu Gly Gly Asp Tyr Ile Leu Ala Ala Ser Cys 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr Arg 
              35                  40                  45 

 
           
             175  
             47  
             PRT  
             Arabidopsis thaliana  
           
            175 

      Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 
       1               5                  10                  15 
      Ser Pro Val Ser Ser Gly Gly Asp Tyr Ser Pro Lys Leu Ala Thr Ser 
                  20                  25                  30 
      Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             176  
             47  
             PRT  
             Arabidopsis thaliana  
           
            176 

      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 
              35                  40                  45 

 
           
             177  
             44  
             PRT  
             Arabidopsis thaliana  
           
            177 

      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 
              35                  40 

 
           
             178  
             49  
             PRT  
             Brassica napus  
           
            178 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             179  
             49  
             PRT  
             Brassica napus  
           
            179 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             180  
             49  
             PRT  
             Brassica oleracea  
           
            180 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             181  
             49  
             PRT  
             Brassica napus  
           
            181 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             182  
             49  
             PRT  
             Brassica napus  
           
            182 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             183  
             49  
             PRT  
             Brassica napus  
           
            183 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             184  
             49  
             PRT  
             Brassica rapa  
           
            184 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Ala Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             185  
             49  
             PRT  
             Brassica oleracea  
           
            185 

      Met Asn Thr Phe Pro Ala Ser Thr Glu Met Val Ser Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Tyr Pro Met Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             186  
             47  
             PRT  
             Brassica oleracea  
           
            186 

      Met Thr Ser Phe Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu His Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Leu Gly Glu Glu Tyr Cys Pro Lys Leu Ala Ala Ser 
                  20                  25                  30 
      Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             187  
             47  
             PRT  
             Raphanus sativus  
           
            187 

      Met Thr Thr Phe Ser Thr Phe Ser Glu Met Leu Gly Ser Glu Tyr Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Leu Gly Gly Glu Tyr Cys Pro Lys Leu Ala Ala Ser 
                  20                  25                  30 
      Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             188  
             45  
             PRT  
             Brassica napus  
           
            188 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             189  
             45  
             PRT  
             Brassica napus  
           
            189 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             190  
             45  
             PRT  
             Brassica oleracea  
           
            190 

      Met Asn Ser Val Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             191  
             45  
             PRT  
             Brassica rapa  
           
            191 

      Met Asn Ser Val Ser Thr Leu Ser Glu Val Leu Gly Ser Gln Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Gly Gly Asp Tyr Cys Pro Met Leu Ala Ala Ser Cys Pro 
                  20                  25                  30 
      Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg 
              35                  40                  45 

 
           
             192  
             49  
             PRT  
             Brassica rapa  
           
            192 

      Met Asp Ser Ile Ser Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             193  
             49  
             PRT  
             Brassica rapa  
           
            193 

      Met Asp Ser Ile Ser Thr Phe Pro Glu Leu Leu Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Cys Pro Arg Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             194  
             49  
             PRT  
             Raphanus sativus  
           
            194 

      Met Asp Ser Ile Ser Thr Phe Ser Glu Leu Leu Gly Ser Glu Asn Glu 
       1               5                  10                  15 
      Ser Pro Val Thr Thr Val Val Gly Gly Asp Tyr Phe Pro Arg Leu Ala 
                  20                  25                  30 
      Ala Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Gln Glu Thr 
              35                  40                  45 
      Arg 

 
           
             195  
             50  
             PRT  
             Brassica napus  
           
            195 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             196  
             50  
             PRT  
             Brassica napus  
           
            196 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             197  
             50  
             PRT  
             Brassica rapa  
           
            197 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Met Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             198  
             50  
             PRT  
             Brassica napus  
           
            198 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Gln Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             199  
             44  
             PRT  
             Brassica napus  
           
            199 

      Ala Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 
              35                  40 

 
           
             200  
             44  
             PRT  
             Brassica napus  
           
            200 

      Ala Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 
              35                  40 

 
           
             201  
             44  
             PRT  
             Brassica oleracea  
           
            201 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 
              35                  40 

 
           
             202  
             50  
             PRT  
             Brassica napus  
           
            202 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             203  
             50  
             PRT  
             Brassica oleracea  
           
            203 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Ala Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Ala Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             204  
             31  
             PRT  
             Brassica rapa  
           
            204 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser 
                  20                  25                  30 

 
           
             205  
             31  
             PRT  
             Raphanus sativus  
           
            205 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser 
                  20                  25                  30 

 
           
             206  
             33  
             PRT  
             Brassica napus  
           
            206 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Thr 

 
           
             207  
             33  
             PRT  
             Brassica oleracea  
           
            207 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Thr 

 
           
             208  
             33  
             PRT  
             Brassica napus  
           
            208 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Thr 

 
           
             209  
             34  
             PRT  
             Brassica rapa  
           
            209 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Thr Thr 

 
           
             210  
             33  
             PRT  
             Brassica rapa  
           
            210 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Thr 

 
           
             211  
             33  
             PRT  
             Brassica oleracea  
           
            211 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Gly Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Thr 

 
           
             212  
             26  
             PRT  
             Brassica juncea  
           
            212 

      Ala Trp Arg Leu Arg Ile Ser Glu Thr Thr Cys Pro Lys Glu Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Val Ala Phe 
                  20                  25 

 
           
             213  
             26  
             PRT  
             Brassica juncea  
           
            213 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Val Ala Phe 
                  20                  25 

 
           
             214  
             26  
             PRT  
             Brassica napus  
           
            214 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Glu Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Val Ala Phe 
                  20                  25 

 
           
             215  
             26  
             PRT  
             Raphanus sativus  
           
            215 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys Pro Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Val Ala Phe 
                  20                  25 

 
           
             216  
             26  
             PRT  
             Arabidopsis thaliana  
           
            216 

      Ala Trp Arg Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe 
                  20                  25 

 
           
             217  
             26  
             PRT  
             Arabidopsis thaliana  
           
            217 

      Ala Trp Arg Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys Glu Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Asn Phe 
                  20                  25 

 
           
             218  
             26  
             PRT  
             Arabidopsis thaliana  
           
            218 

      Ala Trp Arg Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe 
                  20                  25 

 
           
             219  
             49  
             PRT  
             Brassica napus  
           
            219 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu 

 
           
             220  
             49  
             PRT  
             Brassica napus  
           
            220 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu 

 
           
             221  
             49  
             PRT  
             Brassica rapa  
           
            221 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu 

 
           
             222  
             49  
             PRT  
             Brassica napus  
           
            222 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu 

 
           
             223  
             49  
             PRT  
             Brassica napus  
           
            223 

      Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu 

 
           
             224  
             49  
             PRT  
             Brassica napus  
           
            224 

      Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu 

 
           
             225  
             49  
             PRT  
             Brassica oleracea  
           
            225 

      Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu 

 
           
             226  
             49  
             PRT  
             Brassica napus  
           
            226 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu 

 
           
             227  
             49  
             PRT  
             Brassica oleracea  
             
               VARIANT  
               (1)...(49)  
               Xaa = Any Amino Acid  
             
           
            227 

      Thr Glu Val Ser Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Thr Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Xaa Xaa Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu 

 
           
             228  
             41  
             PRT  
             Brassica rapa  
           
            228 

      Asp His Gly Met Asn Met Lys Asn Thr Thr Ala Val Ala Ser Gln Val 
       1               5                  10                  15 
      Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met Glu Glu Thr 
                  20                  25                  30 
      Leu Val Glu Ala Val Phe Thr Glu Glu 
              35                  40 

 
           
             229  
             41  
             PRT  
             Raphanus sativus  
           
            229 

      Asp His Gly Met Asn Met Lys Asn Thr Thr Ala Val Ala Ser Gln Val 
       1               5                  10                  15 
      Glu Val Asn Asp Thr Thr Thr Asp His Gly Val Asp Met Glu Glu Thr 
                  20                  25                  30 
      Leu Val Glu Ala Val Phe Thr Glu Glu 
              35                  40 

 
           
             230  
             37  
             PRT  
             Brassica napus  
           
            230 

      Thr Thr Asn Asp His Gly Met Asn Met Ala Ser Gln Ala Glu Val Asn 
       1               5                  10                  15 
      Asp Thr Thr Asp His Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala 
                  20                  25                  30 
      Val Phe Thr Glu Glu 
              35 

 
           
             231  
             37  
             PRT  
             Brassica oleracea  
           
            231 

      Thr Thr Asn Asp His Gly Met Asn Met Ala Ser Gln Ala Glu Val Asn 
       1               5                  10                  15 
      Asp Thr Thr Asp His Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala 
                  20                  25                  30 
      Val Phe Thr Glu Glu 
              35 

 
           
             232  
             37  
             PRT  
             Brassica napus  
           
            232 

      Thr Thr Asn Asp His Gly Met Asn Met Ala Ser Gln Val Glu Val Asn 
       1               5                  10                  15 
      Asp Thr Thr Asp His Asp Leu Asp Met Glu Glu Thr Ile Val Glu Ala 
                  20                  25                  30 
      Val Phe Arg Glu Glu 
              35 

 
           
             233  
             37  
             PRT  
             Brassica rapa  
           
            233 

      Thr Thr Asn Asp His Gly Met Asn Met Ala Ser Gln Val Glu Val Asn 
       1               5                  10                  15 
      Asp Thr Thr Asp His Asp Leu Asp Met Glu Glu Thr Met Val Glu Ala 
                  20                  25                  30 
      Val Phe Arg Glu Glu 
              35 

 
           
             234  
             44  
             PRT  
             Brassica rapa  
           
            234 

      Thr Thr Asn Asp Arg Gly Met Asn Met Glu Glu Thr Ser Ala Val Ala 
       1               5                  10                  15 
      Ser Pro Ala Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Met 
                  20                  25                  30 
      Glu Glu Thr Met Val Glu Ala Val Phe Arg Asp Glu 
              35                  40 

 
           
             235  
             44  
             PRT  
             Brassica oleracea  
           
            235 

      Thr Thr Asn Asp Gln Gly Met Asn Met Glu Glu Met Thr Val Val Ala 
       1               5                  10                  15 
      Ser Gln Ala Glu Val Ser Asp Thr Thr Thr Tyr His Gly Leu Asp Met 
                  20                  25                  30 
      Glu Glu Thr Met Val Glu Ala Val Phe Ala Glu Glu 
              35                  40 

 
           
             236  
             26  
             PRT  
             Brassica juncea  
           
            236 

      Gln Ala Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Val Glu 
       1               5                  10                  15 
      Glu Thr Ile Val Glu Ala Ile Phe Thr Glu 
                  20                  25 

 
           
             237  
             26  
             PRT  
             Brassica juncea  
           
            237 

      Gln Ala Glu Leu Asn Asp Thr Thr Ala Asp His Gly Leu Asp Val Glu 
       1               5                  10                  15 
      Glu Thr Ile Val Glu Ala Ile Phe Thr Glu 
                  20                  25 

 
           
             238  
             26  
             PRT  
             Brassica napus  
           
            238 

      Lys Ala Glu Ile Asn Asn Thr Thr Ala Asp His Gly Ile Asp Val Glu 
       1               5                  10                  15 
      Glu Thr Ile Val Glu Ala Ile Phe Thr Glu 
                  20                  25 

 
           
             239  
             26  
             PRT  
             Raphanus sativus  
           
            239 

      Gln Ala Glu Ile Asn Asp Thr Thr Thr Asp His Gly Leu Asp Ile Glu 
       1               5                  10                  15 
      Glu Thr Ile Val Glu Ala Ile Phe Thr Glu 
                  20                  25 

 
           
             240  
             28  
             PRT  
             Arabidopsis thaliana  
           
            240 

      Gln Asp Glu Thr Cys Asp Thr Thr Thr Thr Asp His Gly Leu Asp Met 
       1               5                  10                  15 
      Glu Glu Thr Met Val Glu Ala Ile Tyr Thr Pro Glu 
                  20                  25 

 
           
             241  
             28  
             PRT  
             Arabidopsis thaliana  
           
            241 

      Gln Asp Glu Met Cys His Met Thr Thr Asp Ala His Gly Leu Asp Met 
       1               5                  10                  15 
      Glu Glu Thr Leu Val Glu Ala Ile Tyr Thr Pro Glu 
                  20                  25 

 
           
             242  
             27  
             PRT  
             Arabidopsis thaliana  
           
            242 

      Gln Asp Glu Met Cys Asp Ala Thr Thr Asp His Gly Phe Asp Met Glu 
       1               5                  10                  15 
      Glu Thr Leu Val Glu Ala Ile Tyr Thr Ala Glu 
                  20                  25 

 
           
             243  
             49  
             PRT  
             Brassica napus  
           
            243 

      Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             244  
             49  
             PRT  
             Brassica napus  
           
            244 

      Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             245  
             49  
             PRT  
             Brassica rapa  
           
            245 

      Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             246  
             49  
             PRT  
             Brassica napus  
           
            246 

      Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             247  
             49  
             PRT  
             Brassica napus  
           
            247 

      Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             248  
             49  
             PRT  
             Brassica napus  
           
            248 

      Gln Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             249  
             49  
             PRT  
             Brassica oleracea  
           
            249 

      Gln Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             250  
             49  
             PRT  
             Brassica napus  
           
            250 

      Gln Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             251  
             49  
             PRT  
             Brassica oleracea  
           
            251 

      Gln Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Ala Glu Ala Ala 
       1               5                  10                  15 
      Val Val Thr Glu Glu Leu Ser Lys Gly Val Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Thr Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             252  
             48  
             PRT  
             Brassica rapa  
           
            252 

      Gln Arg Glu Gly Phe Tyr Met Thr Glu Glu Thr Arg Val Glu Gly Val 
       1               5                  10                  15 
      Val Thr Glu Glu Gln Asn Asn Trp Phe Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Phe Gly Met Pro Thr Leu Leu Val Asp Met Ala Glu Gly Met Leu Ile 
              35                  40                  45 

 
           
             253  
             48  
             PRT  
             Raphanus sativus  
           
            253 

      Gln Arg Glu Gly Phe Tyr Met Thr Glu Glu Thr Arg Val Glu Gly Val 
       1               5                  10                  15 
      Val Thr Glu Glu Gln Asn Asn Trp Phe Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Phe Gly Met Pro Thr Leu Leu Val Asp Met Ala Glu Gly Met Leu Leu 
              35                  40                  45 

 
           
             254  
             49  
             PRT  
             Brassica napus  
           
            254 

      Gln Arg Asp Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly Val 
       1               5                  10                  15 
      Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             255  
             49  
             PRT  
             Brassica oleracea  
           
            255 

      Gln Arg Asp Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly Val 
       1               5                  10                  15 
      Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             256  
             49  
             PRT  
             Brassica napus  
           
            256 

      Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Val Gly Val 
       1               5                  10                  15 
      Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             257  
             49  
             PRT  
             Brassica rapa  
           
            257 

      Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly Ile 
       1               5                  10                  15 
      Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Met Phe Gly Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             258  
             49  
             PRT  
             Brassica rapa  
           
            258 

      Gln Arg Glu Gly Phe Tyr Met Ala Glu Glu Thr Thr Val Glu Gly Val 
       1               5                  10                  15 
      Val Pro Glu Glu Gln Met Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp 
                  20                  25                  30 
      Thr Phe Glu Met Pro Arg Leu Leu Ala Asp Met Ala Glu Gly Met Leu 
              35                  40                  45 
      Leu 

 
           
             259  
             48  
             PRT  
             Brassica oleracea  
           
            259 

      Gln Arg Glu Gly Phe Tyr Leu Ala Glu Glu Thr Thr Val Glu Gly Val 
       1               5                  10                  15 
      Val Thr Glu Glu Gln Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr 
                  20                  25                  30 
      Phe Gly Met Gln Ser Phe Leu Ala Asp Met Ala Glu Gly Met Leu Phe 
              35                  40                  45 

 
           
             260  
             29  
             PRT  
             Brassica juncea  
           
            260 

      Glu Ser Ser Glu Gly Phe Tyr Met Asp Glu Glu Phe Met Phe Gly Met 
       1               5                  10                  15 
      Pro Thr Leu Trp Ala Ser Met Ala Glu Gly Met Leu Leu 
                  20                  25 

 
           
             261  
             29  
             PRT  
             Brassica juncea  
           
            261 

      Glu Ser Ser Glu Gly Phe Tyr Met Ala Glu Glu Phe Met Phe Gly Met 
       1               5                  10                  15 
      Pro Thr Leu Trp Ala Ser Val Ala Glu Gly Met Leu Leu 
                  20                  25 

 
           
             262  
             30  
             PRT  
             Brassica napus  
           
            262 

      Glu Asn Asn Asp Gly Phe Tyr Met Asp Glu Glu Glu Ser Met Phe Gly 
       1               5                  10                  15 
      Met Pro Ala Leu Leu Ala Ser Met Ala Glu Gly Met Leu Leu 
                  20                  25                  30 

 
           
             263  
             29  
             PRT  
             Raphanus sativus  
           
            263 

      Val Asn Asn Asp Glu Phe Tyr Met Asp Glu Glu Ser Met Phe Gly Met 
       1               5                  10                  15 
      Pro Ser Leu Leu Ala Ser Met Ala Glu Gly Met Leu Leu 
                  20                  25 

 
           
             264  
             29  
             PRT  
             Arabidopsis thaliana  
           
            264 

      Gln Ser Glu Gly Ala Phe Tyr Met Asp Glu Glu Thr Met Phe Gly Met 
       1               5                  10                  15 
      Pro Thr Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu 
                  20                  25 

 
           
             265  
             29  
             PRT  
             Arabidopsis thaliana  
           
            265 

      Gln Ser Gln Asp Ala Phe Tyr Met Asp Glu Glu Ala Met Leu Gly Met 
       1               5                  10                  15 
      Ser Ser Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu 
                  20                  25 

 
           
             266  
             29  
             PRT  
             Arabidopsis thaliana  
           
            266 

      Gln Ser Glu Asn Ala Phe Tyr Met His Asp Glu Ala Met Phe Glu Met 
       1               5                  10                  15 
      Pro Ser Leu Leu Ala Asn Met Ala Glu Gly Met Leu Leu 
                  20                  25 

 
           
             267  
             50  
             PRT  
             Brassica napus  
           
            267 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             268  
             50  
             PRT  
             Brassica napus  
           
            268 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             269  
             50  
             PRT  
             Brassica rapa  
           
            269 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Met Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             270  
             50  
             PRT  
             Brassica napus  
           
            270 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Gln Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             271  
             44  
             PRT  
             Brassica napus  
           
            271 

      Ala Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 
              35                  40 

 
           
             272  
             44  
             PRT  
             Brassica napus  
           
            272 

      Ala Ser Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 
              35                  40 

 
           
             273  
             44  
             PRT  
             Brassica oleracea  
           
            273 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Glu Glu Thr Met Ala Val Ala Ser Gln 
              35                  40 

 
           
             274  
             50  
             PRT  
             Brassica napus  
           
            274 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Val Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Val Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             275  
             50  
             PRT  
             Brassica oleracea  
           
            275 

      Ala Trp Arg Leu Arg Ile Pro Glu Thr Thr Cys His Lys Asp Ile Gln 
       1               5                  10                  15 
      Lys Ala Ala Ala Glu Ala Ala Leu Ala Phe Glu Ala Glu Lys Ser Asp 
                  20                  25                  30 
      Ala Thr Met Gln Asn Gly Leu Asn Met Glu Glu Thr Thr Ala Ala Ala 
              35                  40                  45 
      Ser Gln 
          50 

 
           
             276  
             50  
             PRT  
             Brassica napus  
           
            276 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             277  
             50  
             PRT  
             Brassica napus  
           
            277 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             278  
             50  
             PRT  
             Brassica rapa  
           
            278 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             279  
             50  
             PRT  
             Brassica napus  
           
            279 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             280  
             50  
             PRT  
             Brassica napus  
           
            280 

      Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Gly 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             281  
             50  
             PRT  
             Brassica napus  
           
            281 

      Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             282  
             50  
             PRT  
             Brassica oleracea  
           
            282 

      Ala Glu Val Asn Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             283  
             50  
             PRT  
             Brassica napus  
           
            283 

      Ala Glu Val Asn Asp Thr Thr Thr Glu His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Ala Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             284  
             50  
             PRT  
             Brassica oleracea  
           
            284 

      Thr Glu Val Ser Asp Thr Thr Thr Asp His Gly Met Asn Met Glu Glu 
       1               5                  10                  15 
      Thr Thr Ala Val Ala Ser Gln Ala Glu Val Asn Asp Thr Thr Thr Asp 
                  20                  25                  30 
      His Gly Val Asp Met Glu Glu Thr Met Val Glu Ala Val Phe Thr Glu 
              35                  40                  45 
      Glu Gln 
          50 

 
           
             285  
             50  
             PRT  
             Brassica napus  
           
            285 

      Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             286  
             50  
             PRT  
             Brassica rapa  
           
            286 

      Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             287  
             50  
             PRT  
             Brassica napus  
           
            287 

      Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             288  
             48  
             PRT  
             Brassica napus  
           
            288 

      Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 
              35                  40                  45 

 
           
             289  
             50  
             PRT  
             Brassica napus  
           
            289 

      Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Glu Glu Pro Ser Lys Gly Ser Tyr Met Asp Glu Glu Trp Met 
                  20                  25                  30 
      Leu Glu Met Pro Thr Leu Leu Ala Asp Met Ala Glu Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             290  
             50  
             PRT  
             Brassica napus  
           
            290 

      Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr 
                  20                  25                  30 
      Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             291  
             50  
             PRT  
             Brassica oleracea  
           
            291 

      Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr 
                  20                  25                  30 
      Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             292  
             50  
             PRT  
             Brassica napus  
           
            292 

      Ser Glu Gly Phe Asn Met Ala Glu Glu Ser Thr Val Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Asp Glu Leu Ser Lys Gly Phe Tyr Met Asp Glu Glu Trp Thr 
                  20                  25                  30 
      Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             293  
             50  
             PRT  
             Brassica oleracea  
           
            293 

      Ser Glu Gly Phe Asn Met Ala Lys Glu Ser Thr Ala Glu Ala Ala Val 
       1               5                  10                  15 
      Val Thr Glu Glu Leu Ser Lys Gly Val Tyr Met Asp Glu Glu Trp Thr 
                  20                  25                  30 
      Tyr Glu Met Pro Thr Leu Leu Ala Asp Met Ala Ala Gly Met Leu Leu 
              35                  40                  45 
      Pro Pro 
          50 

 
           
             294  
             205  
             PRT  
             Medicago truncatula  
             
               G3362 polypeptide  
             
           
            294 

      Met Phe Thr Met Asn Gln Phe Ser Glu Ser His Asp Pro Cys Ser Ser 
      1               5                   10                  15 
      Ser Ser Glu Arg Phe Leu Ala Glu Thr Met Pro Lys Lys Arg Ala Gly 
                  20                  25                  30 
      Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg 
              35                  40                  45 
      Lys Arg Asp Ser Gly Lys Trp Val Cys Glu Val Arg Glu Pro Asn Lys 
          50                  55                  60 
      Lys Thr Arg Ile Trp Leu Gly Thr Phe Pro Thr Pro Glu Met Ala Ala 
      65                  70                  75                  80 
      Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly Arg Ser Ala Cys 
                      85                  90                  95 
      Leu Asn Phe Ala Asp Ser Ala Trp Lys Leu Pro Val Pro Ala Thr Ser 
                  100                 105                 110 
      Glu Ala Arg Asp Ile Gln Lys Ala Ala Ala Glu Ala Ala Glu Ala Phe 
              115                 120                 125 
      Arg Pro Glu Ser Val Phe Glu Asn Ser Glu Glu Arg Lys Asp Ser Glu 
          130                 135                 140 
      Pro Ser Ser Thr Val Ala Val Ser Glu Thr Val Met Glu Gln Arg Glu 
      145                 150                 155                 160 
      Glu Glu Glu Asp Thr Val Pro Glu Tyr Leu Arg Asn Met Val Leu Met 
                      165                 170                 175 
      Ser Pro Ala His Tyr Trp Gly Ser Asp Cys Gly Val Ala Asp Val Glu 
                  180                 185                 190 
      Phe Asp Glu Thr Glu Val Ser Leu Trp Ser Tyr Ser Phe 
              195                 200                 205 

 
           
             295  
             215  
             PRT  
             Medicago truncatula  
             
               G3364 polypeptide  
             
           
            295 

      Met Phe Thr Thr Asn Asn Ser Ser Tyr Ser His Ser Ile Ser Ser Glu 
      1               5                   10                  15 
      Ala Ser Ser Ser Tyr Tyr Asn Ser Leu Pro Glu Ser Glu Ile Arg Leu 
                  20                  25                  30 
      Ala Ala Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys Ile Phe Lys Glu 
              35                  40                  45 
      Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn Leu Asp Lys 
          50                  55                  60 
      Trp Val Cys Glu Met Arg Glu Pro Asn Thr Lys Thr Arg Ile Trp Leu 
      65                  70                  75                  80 
      Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala 
                      85                  90                  95 
      Ala Met Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe Ala Asp Ser 
                  100                 105                 110 
      Val Trp Arg Leu Pro Ile Pro Ala Thr Ser Ser Ile Lys Asp Ile Gln 
              115                 120                 125 
      Lys Ala Ala Thr Lys Ala Ala Glu Ala Phe Arg Pro Asp Asn Thr Ile 
          130                 135                 140 
      Met Ile Thr Asn Ile Glu Thr Val Val Ala Val Val Ala Thr Lys Glu 
      145                 150                 155                 160 
      Leu Asn Met Phe Cys Val Glu Glu Glu Glu Glu Met Leu Asn Met Pro 
                      165                 170                 175 
      Glu Phe Trp Arg Asn Met Ala Leu Met Ser Pro Thr His Ser Phe Glu 
                  180                 185                 190 
      Tyr His Asp Gln Tyr Glu Asp Phe His Phe Gln Asp Phe Gln Asp Asp 
              195                 200                 205 
      Glu Val Ser Leu Trp Asn Phe 
          210                 215 

 
           
             296  
             220  
             PRT  
             Medicago truncatula  
             
               G3365 polypeptide  
             
           
            296 

      Met Lys Ser Ser Leu Asp Glu Ser Ser Tyr Val Glu Asn Asn Ser Ser 
      1               5                   10                  15 
      Ser Ser Glu Ile Leu Leu Ala Ser Glu Gln Pro Lys Lys Arg Ala Gly 
                  20                  25                  30 
      Arg Arg Lys Phe Lys Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg 
              35                  40                  45 
      Arg Arg Asn Asn Asn Asn Asn Lys Trp Val Cys Glu Val Arg Val Pro 
          50                  55                  60 
      Asn Asp Lys Ser Thr Arg Ile Trp Leu Gly Thr Tyr Pro Thr Pro Glu 
      65                  70                  75                  80 
      Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg Gly Lys 
                      85                  90                  95 
      Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Ala Leu Pro 
                  100                 105                 110 
      Ala Thr Asn Asn Ala Lys Glu Ile Arg Lys Met Ala Ala Glu Ala Ala 
              115                 120                 125 
      Leu Ala Phe Ala Val Val Ala Asp Ser Lys Glu Gln Thr Met Ile Ser 
          130                 135                 140 
      Asn Cys Asp Val Asn Ser Val Gly Val Met Glu Val Asp Asn Lys Pro 
      145                 150                 155                 160 
      Leu Gln Gly Leu Cys Val Glu Val Pro Glu Glu Glu Met Leu His Asp 
                      165                 170                 175 
      Trp Phe Arg Ser Met Ala Asp Glu Pro Leu Arg Ser Pro Ile Thr Pro 
                  180                 185                 190 
      Phe Ile Arg His Gly Arg Asp Gln Trp Asn Asn Val Asp Ile Asp Gln 
              195                 200                 205 
      Val Asp Ala Glu Val Ser Leu Trp Asn Phe Thr Ile 
          210                 215                 220 

 
           
             297  
             227  
             PRT  
             Medicago truncatula  
             
               G3366 polypeptide  
             
           
            297 

      Met Tyr Pro Thr Thr Asn Ser Val Ser Ser Ser Ser Ser Asp Met Ser 
      1               5                   10                  15 
      Leu Pro Asn Ser Glu Gly Ser His Trp Met Ser Ile Cys Asn Glu Glu 
                  20                  25                  30 
      Met Arg Leu Ala Ala Thr Thr Pro Lys Lys Arg Ala Gly Arg Lys Lys 
              35                  40                  45 
      Phe Lys Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn 
          50                  55                  60 
      Leu Asp Lys Trp Val Cys Glu Met Arg Glu Pro Asn Lys Lys Thr Lys 
      65                  70                  75                  80 
      Ile Trp Leu Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Arg Ala His 
                      85                  90                  95 
      Asp Val Ala Ala Met Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe 
                  100                 105                 110 
      Ala Asp Ser Ala Trp Arg Leu Pro Lys Pro Ala Thr Thr Gln Ala Lys 
              115                 120                 125 
      Asp Ile Gln Lys Ala Ala Thr Glu Ala Ala Glu Ala Phe Arg Pro Asp 
          130                 135                 140 
      Lys Thr Leu Leu Thr Asn His Asn Asp Asn Asp Asn Asp Asn Asp Lys 
      145                 150                 155                 160 
      Glu Asn Asp Met Ala Val Val Ala Thr Ala Thr Glu Glu Gln Ser Met 
                      165                 170                 175 
      Ile Cys Met Glu Glu Lys Glu Glu Gly Val Met Asn Met Gln Glu Met 
                  180                 185                 190 
      Trp Ser Asn Met Ala Leu Met Ser Pro Thr His Ser Leu Gly Tyr Tyr 
              195                 200                 205 
      Glu Tyr Gln Tyr Ile Asn Glu Asp Phe Gln Asp Glu Glu Val Ser Leu 
          210                 215                 220 
      Trp Ser Phe 
      225 

 
           
             298  
             248  
             PRT  
             Medicago truncatula  
             
               G3367 polypeptide  
             
           
            298 

      Met Ile Ser Thr Asn Asn Ser Ser Tyr Ser His Ser Ile Ser Ser Lys 
      1               5                   10                  15 
      Asp Phe Ser Pro Phe Asp Ala Ser Ser Pro Asp Ser Glu Val Arg Leu 
                  20                  25                  30 
      Ala Ser Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys Ile Phe Lys Glu 
              35                  40                  45 
      Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn Leu Asn Lys 
          50                  55                  60 
      Trp Val Cys Glu Met Arg Glu Pro Asn Thr Lys Asn Arg Ile Trp Leu 
      65                  70                  75                  80 
      Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala 
                      85                  90                  95 
      Ala Ile Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe Ala Asp Ser 
                  100                 105                 110 
      Val Trp Arg Leu Pro Ile Pro Ala Thr Ser Ala Ile Lys Asp Ile Gln 
              115                 120                 125 
      Lys Ala Ala Thr Lys Ala Ala Glu Ala Phe Arg Pro Asp Asn Thr Leu 
          130                 135                 140 
      Met Thr Ser Asp Ile Asp Thr Val Val Ala Val Val Ala Thr Gln Glu 
      145                 150                 155                 160 
      Leu Asn Met Phe Arg Val Glu Val Glu Glu Glu Glu Val Leu Asn Met 
                      165                 170                 175 
      Pro Glu Leu Trp Arg Asn Met Ala Leu Met Ser Pro Thr His Ser Phe 
                  180                 185                 190 
      Gly Tyr His Asp Gln Tyr Glu Asp Ile His Ile Gln Asp Phe Gln Asp 
              195                 200                 205 
      Asp Glu Asp Phe Lys Lys Arg Ser Val Thr Thr Ile Trp Ala Val Thr 
          210                 215                 220 
      Ser Ile Gly Val His Ser Leu His Phe Thr Val Ile Ser Arg Ile Val 
      225                 230                 235                 240 
      Met Arg Thr Leu Leu Leu Cys Val 
                      245 

 
           
             299  
             229  
             PRT  
             Medicago truncatula  
             
               G3368 polypeptide  
             
           
            299 

      Met Asp Phe Phe Met Ser Ser Phe Ser Asp Tyr Ser Asp Thr Ser Ser 
      1               5                   10                  15 
      Ser Glu Thr Ala Ser Ser Asn Arg Thr Ser Ser Ser Glu Val Ile Leu 
                  20                  25                  30 
      Ala Pro Ala Arg Pro Lys Lys Arg Ala Gly Arg Arg Val Phe Lys Glu 
              35                  40                  45 
      Thr Arg His Pro Val Tyr Arg Gly Val Arg Arg Arg Lys Asn Asn Lys 
          50                  55                  60 
      Trp Val Cys Glu Met Arg Val Pro Asn Asn Ile Val Asn Lys Asn Asn 
      65                  70                  75                  80 
      Lys Ser Arg Ile Trp Leu Gly Thr Tyr Pro Thr Pro Glu Met Ala Ala 
                      85                  90                  95 
      Arg Ala His Asp Val Ala Ala Leu Thr Leu Lys Gly Lys Ser Ala Cys 
                  100                 105                 110 
      Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg Leu Pro Glu Ser Asn 
              115                 120                 125 
      Asp Ala Thr Glu Ile Arg Arg Ala Ala Met Glu Ala Ala Gln Leu Phe 
          130                 135                 140 
      Ala Val Glu Asp Lys Gln Cys Cys Val Thr Val Glu Asp Gly Val Phe 
      145                 150                 155                 160 
      Met Asp Met Glu Asp Ser Lys Asn Met Leu Glu Ala Gln Val Pro Val 
                      165                 170                 175 
      Val Ser Ser Glu Phe Glu Asp Met His His Leu Leu Leu Ser Ile Ala 
                  180                 185                 190 
      Asn Glu Pro Leu Arg Ser Ala Pro Pro Ser Pro Thr Asn Tyr Gly Ser 
              195                 200                 205 
      Tyr Asn Trp Gly Asp Met Glu Ile Phe Asp Thr Gln Leu Val Ser Leu 
          210                 215                 220 
      Trp Asn Phe Ser Ile 
      225 

 
           
             300  
             260  
             PRT  
             Medicago truncatula  
             
               G3369 polypeptide  
             
           
            300 

      Met Asp Met Phe Thr Asn Asn Asn Ser Tyr Ser His Pro Phe Ser Pro 
      1               5                   10                  15 
      Thr Cys Ser Glu Ser Ser Phe Pro Asn Ser Glu Gly Ser Gln Gly Met 
                  20                  25                  30 
      Ser Ile Ser Asn Glu Glu Val Arg Leu Ala Ala Thr Thr Pro Lys Lys 
              35                  40                  45 
      Arg Ala Gly Arg Lys Lys Phe Lys Glu Thr Arg His Pro Val Tyr Arg 
          50                  55                  60 
      Gly Val Arg Lys Arg Asn Leu Asp Lys Trp Val Cys Glu Met Arg Glu 
      65                  70                  75                  80 
      Pro Asn Lys Lys Thr Lys Ile Trp Leu Gly Thr Phe Pro Thr Ala Glu 
                      85                  90                  95 
      Met Ala Ala Arg Ala His Asp Val Ala Ala Met Ala Leu Arg Gly Arg 
                  100                 105                 110 
      Tyr Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Pro Ile Pro 
              115                 120                 125 
      Ala Thr Thr Gln Ala Lys Asp Ile Gln Lys Ala Ala Ala Gln Ala Ala 
          130                 135                 140 
      Glu Ala Phe Arg Pro Asp Lys Thr Ser Ile Thr Asn Asp Ile Asp Thr 
      145                 150                 155                 160 
      Ala Ile Ser Thr Ser Ala Thr Ala Glu Gln Ser Arg Thr Phe Met Glu 
                      165                 170                 175 
      Glu Glu Glu Glu Gly Val Met Asn Met Pro Glu Leu Leu Arg Asn Met 
                  180                 185                 190 
      Ala Leu Met Ser Pro Thr His Ser Ser Gly Tyr Asn Glu Tyr Glu Asn 
              195                 200                 205 
      Ile His Val Gln Asp Phe Gln Asp Leu Gln Asp Phe Gln Asp Glu Glu 
          210                 215                 220 
      Val Leu Ile Lys His Lys Val Leu Leu Ile Pro Ser Ile Ser Ile Tyr 
      225                 230                 235                 240 
      Glu Arg Arg Ile Glu Val Trp Tyr Val Lys Ile Ser Val Asn Phe Ile 
                      245                 250                 255 
      Ser Tyr Leu Asn 
                  260 

 
           
             301  
             218  
             PRT  
             Oryza sativa  
             
               G3370 polypeptide  
             
           
            301 

      Met Glu Val Glu Glu Ala Ala Tyr Arg Thr Val Trp Ser Glu Pro Pro 
      1               5                   10                  15 
      Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro Val 
                  20                  25                  30 
      Tyr Arg Gly Val Arg Arg Arg Gly Gly Arg Pro Gly Ala Ala Gly Arg 
              35                  40                  45 
      Trp Val Cys Glu Val Arg Val Pro Gly Ala Arg Gly Ser Arg Leu Trp 
          50                  55                  60 
      Leu Gly Thr Phe Ala Thr Ala Glu Ala Ala Ala Arg Ala His Asp Ala 
      65                  70                  75                  80 
      Ala Ala Leu Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp 
                      85                  90                  95 
      Ser Ala Trp Arg Met Pro Pro Val Pro Ala Ser Ala Ala Leu Ala Gly 
                  100                 105                 110 
      Ala Arg Gly Val Arg Asp Ala Val Ala Val Ala Val Glu Ala Phe Gln 
              115                 120                 125 
      Arg Gln Ser Ala Ala Pro Ser Ser Pro Ala Glu Thr Phe Ala Asp Asp 
          130                 135                 140 
      Gly Asp Glu Glu Glu Asp Asn Lys Asp Val Leu Pro Val Ala Ala Ala 
      145                 150                 155                 160 
      Glu Val Phe Asp Ala Gly Ala Phe Glu Leu Asp Asp Gly Phe Arg Phe 
                      165                 170                 175 
      Gly Gly Met Asp Ala Gly Ser Tyr Tyr Ala Ser Leu Ala Gln Gly Leu 
                  180                 185                 190 
      Leu Val Glu Pro Pro Ala Ala Gly Ala Trp Trp Glu Asp Gly Glu Leu 
              195                 200                 205 
      Ala Gly Ser Asp Met Pro Leu Trp Ser Tyr 
          210                 215 

 
           
             302  
             253  
             PRT  
             Oryza sativa  
             
               G3371 polypeptide  
             
           
            302 

      Met Glu Lys Asn Thr Ala Ala Ser Gly Gln Leu Met Thr Ser Ser Ala 
      1               5                   10                  15 
      Glu Ala Thr Pro Ser Ser Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe 
                  20                  25                  30 
      Gln Glu Thr Arg His Leu Val Phe Arg Gly Val Arg Trp Arg Gly Cys 
              35                  40                  45 
      Ala Gly Arg Trp Val Cys Lys Val Arg Val Pro Gly Ser Arg Gly Asp 
          50                  55                  60 
      Arg Phe Trp Ile Gly Thr Ser Asp Thr Ala Glu Glu Thr Ala Arg Thr 
      65                  70                  75                  80 
      His Asp Ala Ala Met Leu Ala Leu Cys Gly Ala Ser Ala Ser Leu Asn 
                      85                  90                  95 
      Phe Ala Asp Ser Ala Trp Leu Leu His Val Pro Arg Ala Pro Val Val 
                  100                 105                 110 
      Ser Gly Leu Arg Pro Pro Ala Ala Arg Cys Ala Thr Arg Cys Leu Gln 
              115                 120                 125 
      Gly His Arg Arg Val Pro Ala Pro Gly Arg Gly Ser Asn Ala Thr Ala 
          130                 135                 140 
      Thr Ala Thr Ser Gly Asp Ala Ala Ser Thr Ala Pro Pro Ser Ala Pro 
      145                 150                 155                 160 
      Val Leu Ser Ala Lys Gln Cys Glu Phe Ile Phe Leu Ser Ser Leu Asp 
                      165                 170                 175 
      Cys Trp Met Leu Met Ser Lys Leu Ile Ser Ser Ser Arg Ala Lys Gly 
                  180                 185                 190 
      Ser Leu Cys Leu Arg Lys Asn Pro Ile Ser Phe Cys Met Val Thr Asn 
              195                 200                 205 
      Ser Tyr Thr Ala Leu Leu Leu Glu Tyr Ile Ile Leu Gln Met Asn Ser 
          210                 215                 220 
      Met Ile Val Leu Ile His Glu Leu Ser Lys Tyr Gln Val Phe Leu Leu 
      225                 230                 235                 240 
      Leu Thr Met Ile Thr His His Leu Phe Gln Trp Arg Arg 
                      245                 250 

 
           
             303  
             214  
             PRT  
             Oryza sativa  
             
               G3372 polypeptide  
             
           
            303 

      Met Glu Tyr Tyr Glu Gln Glu Glu Tyr Ala Thr Val Thr Ser Ala Pro 
      1               5                   10                  15 
      Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro 
                  20                  25                  30 
      Val Tyr Arg Gly Val Arg Arg Arg Gly Pro Ala Gly Arg Trp Val Cys 
              35                  40                  45 
      Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe 
          50                  55                  60 
      Ala Thr Ala Glu Ala Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 
      65                  70                  75                  80 
      Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Arg Leu 
                      85                  90                  95 
      Leu Arg Val Asp Pro Ala Thr Leu Ala Thr Pro Asp Asp Ile Arg Arg 
                  100                 105                 110 
      Ala Ala Ile Glu Leu Ala Glu Ser Cys Pro His Asp Ala Ala Ala Ala 
              115                 120                 125 
      Ala Ala Ser Ser Ser Ala Ala Ala Val Glu Ala Ser Ala Ala Ala Ala 
          130                 135                 140 
      Pro Ala Met Met Met Gln Tyr Gln Asp Asp Met Ala Ala Thr Pro Ser 
      145                 150                 155                 160 
      Ser Tyr Asp Tyr Ala Tyr Tyr Gly Asn Met Asp Phe Asp Gln Pro Ser 
                      165                 170                 175 
      Tyr Tyr Tyr Asp Gly Met Gly Gly Gly Gly Glu Tyr Gln Ser Trp Gln 
                  180                 185                 190 
      Met Asp Gly Asp Asp Asp Gly Gly Ala Gly Gly Tyr Gly Gly Gly Asp 
              195                 200                 205 
      Val Thr Leu Trp Ser Tyr 
          210 

 
           
             304  
             219  
             PRT  
             Oryza sativa  
             
               G3373 polypeptide  
             
           
            304 

      Met Asp Thr Glu Asp Thr Ser Ser Ala Ser Ser Ser Ser Val Ser Pro 
      1               5                   10                  15 
      Pro Ser Ser Pro Gly Gly Gly His His His Arg Leu Pro Pro Lys Arg 
                  20                  25                  30 
      Arg Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg 
              35                  40                  45 
      Gly Val Arg Ala Arg Ala Gly Gly Ser Arg Trp Val Cys Glu Val Arg 
          50                  55                  60 
      Glu Pro Gln Ala Gln Ala Arg Ile Trp Leu Gly Thr Tyr Pro Thr Pro 
      65                  70                  75                  80 
      Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly 
                      85                  90                  95 
      Glu Arg Gly Ala Glu Leu Asn Phe Pro Asp Ser Pro Ser Thr Leu Pro 
                  100                 105                 110 
      Arg Ala Arg Thr Ala Ser Pro Glu Asp Ile Arg Leu Ala Ala Ala Gln 
              115                 120                 125 
      Ala Ala Glu Leu Tyr Arg Arg Pro Pro Pro Pro Leu Ala Leu Pro Glu 
          130                 135                 140 
      Asp Pro Gln Glu Gly Thr Ser Gly Gly Gly Ala Thr Ala Thr Ser Gly 
      145                 150                 155                 160 
      Arg Pro Ala Ala Val Phe Val Asp Glu Asp Ala Ile Phe Asp Met Pro 
                      165                 170                 175 
      Gly Leu Ile Asp Asp Met Ala Arg Gly Met Met Leu Thr Pro Pro Ala 
                  180                 185                 190 
      Ile Gly Arg Ser Leu Asp Asp Trp Ala Ala Ile Asp Asp Asp Asp Asp 
              195                 200                 205 
      His Tyr His Met Asp Tyr Lys Leu Trp Met Asp 
          210                 215 

 
           
             305  
             251  
             PRT  
             Oryza sativa  
             
               G3374 polypeptide  
             
           
            305 

      Met Cys Thr Ser Lys Leu Glu Glu Ile Thr Gly Glu Trp Pro Pro Pro 
      1               5                   10                  15 
      Ala Leu Gln Ala Ala Ser Thr Thr Ser Ser Ser Glu Pro Cys Arg Arg 
                  20                  25                  30 
      Leu Ser Pro Pro Ser Ser Lys Arg Pro Ala Gly Arg Thr Lys Phe His 
              35                  40                  45 
      Glu Thr Arg His Pro Val Phe Arg Gly Val Arg Arg Arg Gly Arg Ala 
          50                  55                  60 
      Gly Arg Trp Val Cys Glu Val Arg Val Pro Gly Arg Arg Gly Cys Arg 
      65                  70                  75                  80 
      Leu Trp Leu Gly Thr Phe Asp Ala Ala Asp Ala Ala Ala Arg Ala His 
                      85                  90                  95 
      Asp Ala Ala Met Leu Ala Leu Arg Gly Arg Ala Ala Ala Cys Leu Asn 
                  100                 105                 110 
      Phe Ala Asp Ser Ala Trp Leu Leu Ala Val Pro Pro Pro Ala Thr Leu 
              115                 120                 125 
      Arg Cys Ala Ala Asp Val Gln Arg Ala Val Ala Arg Ala Leu Glu Asp 
          130                 135                 140 
      Phe Glu Gln Arg Glu Ser Ser Ser Ser Val Phe Pro Leu Ala Ile Asp 
      145                 150                 155                 160 
      Val Val Ala Glu Asp Ala Met Ser Ala Thr Ser Glu Pro Ser Ala Ala 
                      165                 170                 175 
      Ser Asp Asp Asp Ala Val Thr Ser Ser Ser Ser Thr Thr Asp Ala Asp 
                  180                 185                 190 
      Glu Glu Ala Ser Pro Phe Glu Leu Asp Val Val Ser Asp Met Gly Trp 
              195                 200                 205 
      Ser Leu Tyr Tyr Ala Ser Leu Ala Glu Gly Leu Leu Met Glu Pro Pro 
          210                 215                 220 
      Ala Ser Gly Ala Ser Ser Asp Asp Asp Asp Asp Ala Ile Val Asp Ser 
      225                 230                 235                 240 
      Gly Asp Ile Ala Asp Val Ser Leu Trp Ser Tyr 
                      245                 250 

 
           
             306  
             219  
             PRT  
             Oryza sativa  
             
               G3375 polypeptide  
             
           
            306 

      Met Glu Trp Ala Tyr Tyr Gly Ser Gly Tyr Ser Ser Ser Gly Thr Pro 
      1               5                   10                  15 
      Ser Pro Val Gly Gly Asp Gly Asp Glu Asp Ser Tyr Met Thr Val Ser 
                  20                  25                  30 
      Ser Ala Pro Pro Lys Arg Arg Ala Gly Arg Thr Lys Phe Lys Glu Thr 
              35                  40                  45 
      Arg His Pro Val Tyr Lys Gly Val Arg Ser Arg Asn Pro Gly Arg Trp 
          50                  55                  60 
      Val Cys Glu Val Arg Glu Pro His Gly Lys Gln Arg Ile Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Glu Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala 
                      85                  90                  95 
      Met Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp Ser Pro 
                  100                 105                 110 
      Arg Arg Leu Arg Val Pro Pro Leu Gly Ala Gly His Glu Glu Ile Arg 
              115                 120                 125 
      Arg Ala Ala Val Glu Ala Ala Glu Leu Phe Arg Pro Ala Pro Gly Gln 
          130                 135                 140 
      His Asn Ala Ala Ala Glu Ala Ala Ala Ala Val Ala Ala Gln Ala Thr 
      145                 150                 155                 160 
      Ala Ala Ser Ala Glu Leu Phe Ala Asp Phe Pro Cys Tyr Pro Met Asp 
                      165                 170                 175 
      Gly Leu Glu Phe Glu Met Gln Gly Tyr Leu Asp Met Ala Gln Gly Met 
                  180                 185                 190 
      Leu Ile Glu Pro Pro Pro Leu Ala Gly Gln Ser Thr Trp Ala Glu Glu 
              195                 200                 205 
      Asp Tyr Asp Cys Glu Val Asn Leu Trp Ser Tyr 
          210                 215 

 
           
             307  
             224  
             PRT  
             Oryza sativa  
             
               G3376 polypeptide  
             
           
            307 

      Met Asp Val Ser Ala Ala Leu Ser Ser Asp Tyr Ser Ser Gly Thr Pro 
      1               5                   10                  15 
      Ser Pro Val Ala Ala Asp Ala Asp Asp Gly Ser Ser Ala Tyr Met Thr 
                  20                  25                  30 
      Val Ser Ser Ala Pro Pro Lys Arg Arg Ala Gly Arg Thr Lys Phe Lys 
              35                  40                  45 
      Glu Thr Arg His Pro Val Phe Lys Gly Val Arg Arg Arg Asn Pro Gly 
          50                  55                  60 
      Arg Trp Val Cys Glu Val Arg Glu Pro His Gly Lys Gln Arg Ile Trp 
      65                  70                  75                  80 
      Leu Gly Thr Phe Glu Thr Ala Glu Met Ala Ala Arg Ala His Asp Val 
                      85                  90                  95 
      Ala Ala Leu Ala Leu Arg Gly Arg Ala Ala Cys Leu Asn Phe Ala Asp 
                  100                 105                 110 
      Ser Pro Arg Arg Leu Arg Val Pro Pro Ile Gly Ala Ser His Asp Asp 
              115                 120                 125 
      Ile Arg Arg Ala Ala Ala Glu Ala Ala Glu Ala Phe Arg Pro Pro Pro 
          130                 135                 140 
      Asp Glu Ser Asn Ala Ala Thr Glu Val Ala Ala Ala Ala Ser Gly Ala 
      145                 150                 155                 160 
      Thr Asn Ser Asn Ala Glu Gln Phe Ala Ser His Pro Tyr Tyr Glu Val 
                      165                 170                 175 
      Met Asp Asp Gly Leu Asp Leu Gly Met Gln Gly Tyr Leu Asp Met Ala 
                  180                 185                 190 
      Gln Gly Met Leu Ile Asp Pro Pro Pro Met Ala Cys Asp Pro Ala Val 
              195                 200                 205 
      Gly Gly Gly Glu Asp Asp Asn Asp Gly Glu Val Gln Leu Trp Ser Tyr 
          210                 215                 220 

 
           
             308  
             236  
             PRT  
             Oryza sativa  
             
               G3377 polypeptide  
             
           
            308 

      Met Glu Lys Asn Thr Thr Ala Met Gly Gln Leu Met Ser Ser Ser Ala 
      1               5                   10                  15 
      Thr Thr Ala Ala Thr Ala Thr Gly Pro Ala Ser Pro Lys Arg Pro Ala 
                  20                  25                  30 
      Gly Arg Thr Lys Phe Gln Glu Thr Arg His Pro Val Phe Arg Gly Val 
              35                  40                  45 
      Arg Arg Arg Gly Arg Ala Gly Arg Trp Val Cys Glu Val Arg Val Pro 
          50                  55                  60 
      Gly Ser Arg Gly Asp Arg Leu Trp Val Gly Thr Phe Asp Thr Ala Glu 
      65                  70                  75                  80 
      Glu Ala Ala Arg Ala His Asp Ala Ala Met Leu Ala Leu Cys Gly Ala 
                      85                  90                  95 
      Ser Ala Ser Leu Asn Phe Ala Asp Ser Ala Trp Leu Leu His Val Pro 
                  100                 105                 110 
      Arg Ala Pro Val Ala Ser Gly His Asp Gln Leu Pro Asp Val Gln Arg 
              115                 120                 125 
      Ala Ala Ser Glu Ala Val Ala Glu Phe Gln Arg Arg Gly Ser Thr Ala 
          130                 135                 140 
      Ala Thr Ala Thr Ala Thr Ser Gly Asp Ala Ala Ser Thr Ala Pro Pro 
      145                 150                 155                 160 
      Ser Ser Ser Pro Val Leu Ser Pro Asn Asp Asp Asn Ala Ser Ser Ala 
                      165                 170                 175 
      Ser Thr Pro Ala Val Ala Ala Ala Leu Asp His Gly Asp Met Phe Gly 
                  180                 185                 190 
      Gly Met Arg Thr Asp Leu Tyr Phe Ala Ser Leu Ala Gln Gly Leu Leu 
              195                 200                 205 
      Ile Glu Pro Pro Pro Pro Pro Thr Thr Ala Glu Gly Phe Cys Asp Asp 
          210                 215                 220 
      Glu Gly Cys Gly Gly Ala Glu Met Glu Leu Trp Ser 
      225                 230                 235 

 
           
             309  
             286  
             PRT  
             Oryza sativa  
             
               G3378 polypeptide  
             
           
            309 

      Met His Thr Tyr Ile Tyr Thr Pro Arg Ala Ala Glu Leu Glu His Ser 
      1               5                   10                  15 
      His Ser Ala Ser Ala Thr Arg Ser His Ser Leu Gly Gln Ala Pro Pro 
                  20                  25                  30 
      Ser Leu Asp Arg Ser Arg Ala Ala Met Asp Met Ala Gly His Glu Val 
              35                  40                  45 
      Asn Ser Ser Ser Ser Ser Ser Gly Ala Glu Ser Ser Ser Ser Ser Ser 
          50                  55                  60 
      Gly Arg Gln Gln Tyr Lys Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg 
      65                  70                  75                  80 
      Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg Arg Arg Gly Gly Ala 
                      85                  90                  95 
      Gly Arg Trp Val Cys Glu Val Arg Val Pro Gly Lys Arg Gly Ala Arg 
                  100                 105                 110 
      Leu Trp Leu Gly Thr Tyr Val Thr Ala Glu Ala Ala Ala Arg Ala His 
              115                 120                 125 
      Asp Ala Ala Met Ile Ala Leu Arg Gly Gly Ala Gly Gly Gly Gly Ala 
          130                 135                 140 
      Ala Cys Leu Asn Phe Gln Asp Ser Ala Trp Leu Leu Ala Val Pro Pro 
      145                 150                 155                 160 
      Ala Ala Pro Ser Asp Leu Ala Gly Val Arg Arg Ala Ala Thr Glu Ala 
                      165                 170                 175 
      Val Ala Gly Phe Leu Gln Arg Asn Lys Thr Thr Asn Gly Ala Ser Val 
                  180                 185                 190 
      Ala Glu Ala Ile Asp Glu Ala Thr Ser Gly Val Ser Lys Pro Pro Pro 
              195                 200                 205 
      Leu Ala Asn Asn Ala Asp Ser Ser Glu Thr Pro Gly Pro Ser Ser Ile 
          210                 215                 220 
      Asp Gly Thr Ala Asp Thr Ala Ala Gly Ala Ala Leu Asp Met Phe Glu 
      225                 230                 235                 240 
      Leu Asp Phe Phe Gly Glu Met Asp Tyr Asp Thr Tyr Tyr Ala Ser Leu 
                      245                 250                 255 
      Ala Glu Gly Leu Leu Met Glu Pro Pro Pro Ala Ala Thr Ala Leu Trp 
                  260                 265                 270 
      Asp Asn Gly Asp Glu Gly Ala Asp Ile Ala Leu Trp Ser Tyr 
              275                 280                 285 

 
           
             310  
             238  
             PRT  
             Oryza sativa  
             
               G3379 polypeptide  
             
           
            310 

      Met Cys Gly Ile Lys Gln Glu Met Ser Gly Glu Ser Ser Gly Ser Pro 
      1               5                   10                  15 
      Cys Ser Ser Ala Ser Ala Glu Arg Gln His Gln Thr Val Trp Thr Ala 
                  20                  25                  30 
      Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His 
              35                  40                  45 
      Pro Val Phe Arg Gly Val Arg Arg Arg Gly Asn Ala Gly Arg Trp Val 
          50                  55                  60 
      Cys Glu Val Arg Val Pro Gly Arg Arg Gly Cys Arg Leu Trp Leu Gly 
      65                  70                  75                  80 
      Thr Phe Asp Thr Ala Glu Gly Ala Ala Arg Ala His Asp Ala Ala Met 
                      85                  90                  95 
      Leu Ala Ile Asn Ala Gly Gly Gly Gly Gly Gly Gly Ala Cys Cys Leu 
                  100                 105                 110 
      Asn Phe Ala Asp Ser Ala Trp Leu Leu Ala Val Pro Arg Ser Tyr Arg 
              115                 120                 125 
      Thr Leu Ala Asp Val Arg His Ala Val Ala Glu Ala Val Glu Asp Phe 
          130                 135                 140 
      Phe Arg Arg Arg Leu Ala Asp Asp Ala Leu Ser Ala Thr Ser Ser Ser 
      145                 150                 155                 160 
      Ser Thr Thr Pro Ser Thr Pro Arg Thr Asp Asp Asp Glu Glu Ser Ala 
                      165                 170                 175 
      Ala Thr Asp Gly Asp Glu Ser Ser Ser Pro Ala Ser Asp Leu Ala Phe 
                  180                 185                 190 
      Glu Leu Asp Val Leu Ser Asp Met Gly Trp Asp Leu Tyr Tyr Ala Ser 
              195                 200                 205 
      Leu Ala Gln Gly Met Leu Met Glu Pro Pro Ser Ala Ala Leu Gly Asp 
          210                 215                 220 
      Asp Gly Asp Ala Ile Leu Ala Asp Val Pro Leu Trp Ser Tyr 
      225                 230                 235 

 
           
             311  
             246  
             PRT  
             Zea mays  
             
               G3438 polypeptide  
             
           
            311 

      Met Asp Met Gly Arg His Gln Leu Gln Leu Gln His Ala Ala Ser Ser 
      1               5                   10                  15 
      Ser Ser Thr Ser Ala Ser Ser Ser Ser Glu Gln Asp Lys Pro Leu Cys 
                  20                  25                  30 
      Cys Ser Gly Pro Lys Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu 
              35                  40                  45 
      Thr Arg His Pro Val Phe Arg Gly Val Arg Arg Arg Gly Ala Ala Gly 
          50                  55                  60 
      Arg Trp Val Cys Glu Val Arg Val Pro Gly Arg Arg Gly Ala Arg Leu 
      65                  70                  75                  80 
      Trp Leu Gly Thr Tyr Leu Gly Ala Glu Ala Ala Ala Arg Ala His Asp 
                      85                  90                  95 
      Ala Ala Met Leu Ala Leu Gly Arg Gly Ala Ala Cys Leu Asn Phe Pro 
                  100                 105                 110 
      Asp Ser Ala Trp Leu Leu Ala Val Pro Pro Pro Pro Ala Leu Ser Gly 
              115                 120                 125 
      Gly Leu Asp Gly Ala Arg Arg Ala Ala Leu Glu Ala Val Ala Glu Phe 
          130                 135                 140 
      Gln Arg Arg Arg Phe Gly Ala Ala Ala Ala Asp Glu Ala Thr Ser Gly 
      145                 150                 155                 160 
      Thr Ser Pro Pro Ser Ser Ser Ser Ser Ala Thr Lys Pro Ala Pro Ala 
                      165                 170                 175 
      Ile Glu Arg Val Pro Val Glu Ala Ser Glu Thr Val Ala Leu Asp Gly 
                  180                 185                 190 
      Ala Val Phe Glu Pro Asp Trp Phe Gly Asp Met Asp Leu Asp Leu Tyr 
              195                 200                 205 
      Tyr Ala Ser Leu Ala Glu Gly Leu Leu Val Glu Pro Pro Pro Pro Pro 
          210                 215                 220 
      Pro Pro Ala Ala Trp Asp His Gly Asp Cys Cys Asp Ser Gly Ala Asp 
      225                 230                 235                 240 
      Val Ala Leu Trp Ser Tyr 
                      245 

 
           
             312  
             256  
             PRT  
             Zea mays  
             
               G3439 polypeptide  
             
           
            312 

      Met Asp Met Gly Arg Leu Gln Leu Gln Leu Gln His Ala Ala Ser Ser 
      1               5                   10                  15 
      Ser Ser Thr Ser Ala Ser Ser Ser Ser Ser Ser Glu Gln Asn Lys Leu 
                  20                  25                  30 
      Ala Trp Ser Pro Ser Ser Pro Gln Pro Pro Lys Lys Arg Pro Ala Gly 
              35                  40                  45 
      Arg Thr Lys Phe Arg Glu Thr Arg His Pro Val Phe Arg Gly Val Arg 
          50                  55                  60 
      Arg Arg Gly Ala Ala Gly Arg Trp Val Cys Glu Val Arg Val Pro Gly 
      65                  70                  75                  80 
      Arg Arg Gly Ala Arg Leu Trp Leu Gly Thr Tyr Leu Gly Ala Glu Ala 
                      85                  90                  95 
      Ala Ala Arg Ala His Asp Ala Ala Met Leu Ala Leu Gly Arg Gly Ala 
                  100                 105                 110 
      Ala Cys Leu Asn Phe Pro Asp Ser Ala Trp Leu Leu Ala Val Pro Pro 
              115                 120                 125 
      Pro Pro Ala Leu Ser Gly Gly Leu Asp Gly Ala Arg Arg Ala Ala Leu 
          130                 135                 140 
      Glu Ala Val Ala Glu Phe Gln Arg Arg Arg Phe Gly Ala Val Ala Ala 
      145                 150                 155                 160 
      Asp Glu Ala Thr Ser Gly Thr Ser Pro Pro Ser Ser Ser Ser Ser Pro 
                      165                 170                 175 
      Ser Gly Thr Tyr Val Ser Gln Ala Pro Ala Pro Ala Ile Glu Arg Val 
                  180                 185                 190 
      Pro Val Glu Ala Ser Glu Thr Ala Ala Leu Asp Gly Ala Val Phe Glu 
              195                 200                 205 
      Pro Asp Trp Phe Arg Asp Met Asp Leu Asp Leu Tyr Tyr Ala Ser Leu 
          210                 215                 220 
      Ala Glu Gly Leu Leu Val Glu Pro Pro Pro Pro Pro Ala Ala Trp Asp 
      225                 230                 235                 240 
      His Gly Asp Cys Ser His Ser Gly Ala Asp Val Ala Leu Trp Ser Tyr 
                      245                 250                 255 

 
           
             313  
             231  
             PRT  
             Zea mays  
             
               G3440 polypeptide  
             
           
            313 

      Met Cys Pro Thr Lys Lys Gly Met Thr Gly Glu Pro Ser Ser Pro Cys 
      1               5                   10                  15 
      Ser Ser Ala Ser Ala Ser Thr Leu Pro Glu His His Gln Thr Val Trp 
                  20                  25                  30 
      Thr Ser Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr 
              35                  40                  45 
      Arg His Pro Val Phe Arg Gly Val Arg Arg Arg Gly Ser Ala Gly Arg 
          50                  55                  60 
      Trp Val Cys Glu Val Arg Val Pro Gly Arg Arg Gly Cys Arg Leu Trp 
      65                  70                  75                  80 
      Leu Gly Thr Phe Asp Thr Ala Glu Ala Ala Ala Arg Ala His Asp Ala 
                      85                  90                  95 
      Ala Met Leu Ala Leu Ala Gly Ala Gly Ala Cys Cys Leu Asn Phe Ala 
                  100                 105                 110 
      Asp Ser Ala Trp Leu Leu Ala Val Pro Ala Ser Cys Ala Ser Leu Ala 
              115                 120                 125 
      Glu Val Arg His Ala Val Ala Asp Ala Val Asp Asp Phe Leu Arg His 
          130                 135                 140 
      Gln Leu Val Pro Glu Asp Asp Ala Leu Ala Ala Thr Pro Ser Ser Pro 
      145                 150                 155                 160 
      Ser Ser Glu Asp Gly Asn Thr Ser Asp Gly Gly Glu Ser Ser Ser Asp 
                      165                 170                 175 
      Ser Ser Pro Pro Thr Gly Ala Ser Pro Phe Glu Phe Asp Val Phe Asn 
                  180                 185                 190 
      Asp Met Ser Trp Asp Leu His Tyr Ala Ser Leu Ala Gln Gly Leu Leu 
              195                 200                 205 
      Val Glu Pro Pro Ser Ala Val Thr Ala Phe Met Asp Glu Gly Phe Ala 
          210                 215                 220 
      Asp Val Pro Leu Trp Ser Tyr 
      225                 230 

 
           
             314  
             231  
             PRT  
             Zea mays  
             
               G3441 polypeptide  
             
           
            314 

      Met Glu Tyr Ala Ala Val Gly Tyr Gly Tyr Gly Tyr Gly Tyr Asp Glu 
      1               5                   10                  15 
      Arg Gln Glu Pro Ala Glu Ser Ala Asp Gly Gly Gly Gly Gly Asp Asp 
                  20                  25                  30 
      Glu Tyr Ala Thr Val Leu Ser Ala Pro Pro Lys Arg Pro Ala Gly Arg 
              35                  40                  45 
      Thr Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg Arg 
          50                  55                  60 
      Arg Gly Pro Ala Gly Arg Trp Val Cys Glu Val Arg Glu Pro Asn Lys 
      65                  70                  75                  80 
      Lys Ser Arg Ile Trp Leu Gly Thr Phe Ala Thr Pro Glu Ala Ala Ala 
                      85                  90                  95 
      Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg Gly Arg Ala Ala Cys 
                  100                 105                 110 
      Leu Asn Phe Ala Asp Ser Ala Arg Leu Leu Gln Val Asp Pro Ala Thr 
              115                 120                 125 
      Leu Ala Thr Pro Asp Asp Ile Arg Arg Ala Ala Ile Gln Leu Ala Asp 
          130                 135                 140 
      Ala Ala Ser Gln Gln Asp Glu Thr Ala Ala Val Ala Ala Asp Val Val 
      145                 150                 155                 160 
      Ala Pro Ser Gln Ala Asp Asp Val Ala Ala Ala Ala Ala Ala Ala Ala 
                      165                 170                 175 
      Ala Ala Ala Met Tyr Gly Gly Gly Met Glu Phe Asp His Ser Tyr Cys 
                  180                 185                 190 
      Tyr Asp Asp Gly Met Val Ser Gly Ser Ser Asp Cys Trp Gln Ser Gly 
              195                 200                 205 
      Gly Gly Gly Trp His Ser Ser Val Asp Gly Asp Asp Asp Gly Ala Gly 
          210                 215                 220 
      Asp Met Thr Leu Trp Ser Tyr 
      225                 230 

 
           
             315  
             267  
             PRT  
             Zea mays  
             
               G3442 polypeptide  
             
           
            315 

      Met Asp Thr Ala Gly Leu Val Gln His Ala Thr Ser Ser Ser Ser Thr 
      1               5                   10                  15 
      Ser Thr Ser Ala Ser Ser Ser Ser Ser Glu Gln Gln Ser Arg Lys Ala 
                  20                  25                  30 
      Ala Trp Pro Pro Ser Thr Ala Ser Ser Pro Gln Gln Pro Pro Lys Lys 
              35                  40                  45 
      Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro Val Phe 
          50                  55                  60 
      Arg Gly Val Arg Arg Arg Gly Ala Ala Gly Arg Trp Val Cys Glu Val 
      65                  70                  75                  80 
      Arg Val Pro Gly Arg Arg Gly Ala Arg Leu Trp Leu Gly Thr Tyr Leu 
                      85                  90                  95 
      Ala Ala Glu Ala Ala Ala Arg Ala His Asp Ala Ala Ile Leu Ala Leu 
                  100                 105                 110 
      Gln Gly Arg Gly Ala Gly Arg Leu Asn Phe Pro Asp Ser Ala Arg Leu 
              115                 120                 125 
      Leu Ala Val Pro Pro Pro Ser Ala Leu Pro Gly Leu Asp Asp Ala Arg 
          130                 135                 140 
      Arg Ala Ala Leu Glu Ala Val Ala Glu Phe Gln Arg Arg Ser Gly Ser 
      145                 150                 155                 160 
      Gly Ser Gly Ala Ala Asp Glu Ala Thr Ser Gly Ala Ser Pro Pro Ser 
                      165                 170                 175 
      Ser Ser Pro Ser Leu Pro Asp Val Ser Ala Ala Gly Ser Pro Ala Ala 
                  180                 185                 190 
      Ala Leu Glu His Val Pro Val Lys Ala Asp Glu Ala Val Ala Leu Asp 
              195                 200                 205 
      Leu Asp Gly Asp Val Phe Gly Pro Asp Trp Phe Gly Asp Met Gly Leu 
          210                 215                 220 
      Glu Leu Asp Ala Tyr Tyr Ala Ser Leu Ala Glu Gly Leu Leu Val Glu 
      225                 230                 235                 240 
      Pro Pro Pro Pro Pro Ala Ala Trp Asp His Gly Asp Cys Cys Asp Ser 
                      245                 250                 255 
      Gly Ala Ala Asp Val Ala Leu Trp Ser Tyr Tyr 
                  260                 265 

 
           
             316  
             215  
             PRT  
             Medicago sativa  
             
               G3497 polypeptide  
             
           
            316 

      Met Leu Thr Thr Asn Asn Ser Ser Asn Ser Gln Ser Ile Ser Ser Thr 
      1               5                   10                  15 
      Ala Ser Ser Ser Tyr Asp Met Ser Thr Pro Asn Leu Glu Val Arg Leu 
                  20                  25                  30 
      Ala Ala Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys Ile Phe Lys Glu 
              35                  40                  45 
      Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn Leu Asp Lys 
          50                  55                  60 
      Trp Val Cys Glu Met Arg Glu Pro Asn Thr Lys Thr Arg Ile Trp Leu 
      65                  70                  75                  80 
      Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Gln Ala His Asp Val Ala 
                      85                  90                  95 
      Ala Met Ala Leu Arg Gly Arg Tyr Ala Cys Val Asn Phe Ala Asp Ser 
                  100                 105                 110 
      Val Trp Arg Leu Pro Ile Pro Ala Thr Ser Lys Ile Lys Asp Ile Gln 
              115                 120                 125 
      Lys Ala Ala Ala Glu Ala Ala Glu Ala Phe Arg Pro Asp Lys Thr Leu 
          130                 135                 140 
      Met Thr Asn Asp Ile Asp Thr Val Val Ala Val Val Ala Thr Lys Glu 
      145                 150                 155                 160 
      Leu Asn Met Phe Cys Val Glu Val Glu Asp Asp Val Leu Asn Met Pro 
                      165                 170                 175 
      Glu Leu Trp Arg Asn Met Ala Leu Met Ser Arg Thr His Ser Phe Gly 
                  180                 185                 190 
      Tyr Asp Asp Gln Tyr Glu Asp Ile His Val Gln Asp Phe Gln Asp Asp 
              195                 200                 205 
      Glu Val Ser Leu Trp Asn Phe 
          210                 215 

 
           
             317  
             214  
             PRT  
             Medicago sativa  
             
               G3498 polypeptide  
             
           
            317 

      Met Phe Thr Thr Asn Asn Ser Ser Tyr Ser His Ser Ile Ser Ser Lys 
      1               5                   10                  15 
      Ala Ser Ser Ser Tyr Tyr Asn Ser Leu Pro Asp Ser Glu Ile Arg Leu 
                  20                  25                  30 
      Ala Ala Ser Asn Pro Lys Lys Arg Ala Gly Arg Lys Ile Phe Lys Glu 
              35                  40                  45 
      Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys Arg Asn Leu Asp Lys 
          50                  55                  60 
      Trp Val Cys Glu Met Arg Glu Pro Asn Met Lys Thr Arg Ile Trp Leu 
      65                  70                  75                  80 
      Gly Thr Phe Pro Thr Ala Asp Met Ala Ala Arg Ala His Asp Val Ala 
                      85                  90                  95 
      Ala Lys Ala Leu Arg Gly Arg Tyr Ala Cys Leu Asn Phe Ala Tyr Ser 
                  100                 105                 110 
      Val Trp Arg Leu Pro Ile Pro Ala Thr Ser Ser Ile Lys Asp Ile Gln 
              115                 120                 125 
      Lys Ala Ala Thr Lys Ala Ala Glu Ala Phe Arg Pro Asp His Thr Ile 
          130                 135                 140 
      Met Ile Thr Asp Ile Glu Thr Val Val Ala Val Val Ala Thr Lys Asp 
      145                 150                 155                 160 
      Leu Asn Ile Phe Cys Gly Glu Glu Glu His Glu Met Leu Asp Met Ser 
                      165                 170                 175 
      Glu Leu Trp Arg Asn Met Ala Leu Met Ser Pro Thr His Ser Phe Ser 
                  180                 185                 190 
      Asn Asp His Tyr Glu Asp Ile Gln Ala Gln Asp Phe Gln Asp Asp Glu 
              195                 200                 205 
      Val Ser Leu Trp Asn Tyr 
          210 

 
           
             318  
             215  
             PRT  
             Medicago sativa  
             
               G3499 polypeptide  
             
           
            318 

      Met Lys Ser Ser Leu Asp Glu Ser Ser Tyr Val Glu Asn Asn Ser Ser 
      1               5                   10                  15 
      Ser Ser Glu Thr Ser Cys Tyr Glu Glu Ile Leu Leu Ala Ser Glu Arg 
                  20                  25                  30 
      Pro Lys Lys Pro Ala Gly Arg Arg Lys Phe Lys Glu Thr Arg His Pro 
              35                  40                  45 
      Val Tyr Arg Gly Val Arg Arg Arg Asn Asn Asn Lys Trp Val Cys Glu 
          50                  55                  60 
      Val Arg Val Pro Asn Asp Lys Ser Thr Arg Ile Trp Leu Gly Thr Tyr 
      65                  70                  75                  80 
      Pro Thr Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 
                      85                  90                  95 
      Leu Arg Gly Lys Ser Ala Cys Leu Asn Phe Ala Asn Ser Ala Trp Arg 
                  100                 105                 110 
      Leu Ala Leu Pro Glu Thr Asn Asn Ala Lys Glu Ile Arg Lys Met Ala 
              115                 120                 125 
      Ala Glu Ala Ala Leu Ala Phe Ala Val Glu Asp Ser Lys Glu Gln Ile 
          130                 135                 140 
      Met Ile Ser Asn Cys Asp Val Cys Ser Ser Asp Asn Val Met Glu Val 
      145                 150                 155                 160 
      Asp Asn Lys Pro Leu Gln Gly Leu Cys Val Glu Val Pro Glu Gln Glu 
                      165                 170                 175 
      Met Leu His Asp Trp Phe Arg Ser Met Ala Asp Glu Pro Leu Arg Ser 
                  180                 185                 190 
      Pro Met Thr Pro Phe Ile Arg Tyr Gly Ile Gly Arg Asp His Ser Asn 
              195                 200                 205 
      Asn Val Asp Val Asp Pro Cys 
          210                 215 

 
           
             319  
             7  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            319 

      Pro Lys Xaa Xaa Ala Gly Arg 
      1               5 

 
           
             320  
             6  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            320 

      Ala Gly Arg Xaa Lys Phe 
      1               5 

 
           
             321  
             5  
             PRT  
             oligopeptide  
             
               oligopeptide  
             
           
            321 

      Glu Thr Arg His Pro 
      1               5 

 
           
             322  
             7  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            322 

      Pro Lys Lys Xaa Ala Gly Arg 
      1               5 

 
           
             323  
             7  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            323 

      Pro Lys Lys Arg Ala Gly Arg 
      1               5 

 
           
             324  
             6  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            324 

      Ala Gly Arg Lys Xaa Phe 
      1               5 

 
           
             325  
             6  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            325 

      Ala Gly Arg Lys Lys Phe 
      1               5 

 
           
             326  
             7  
             PRT  
             Artificial sequence  
             
               oligopeptide  
             
           
            326 

      Pro Lys Lys Pro Ala Gly Arg 
      1               5 

 
           
             327  
             79  
             PRT  
             Arabidopsis thaliana  
             
               CBF1 AP2 domain and flanking signature sequences  
             
           
            327 

      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro 
      1               5                   10                  15 
      Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val Ser Glu 
                  20                  25                  30 
      Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 
              35                  40                  45 
      Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
          50                  55                  60 
      Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 
      65                  70                  75 

 
           
             328  
             79  
             PRT  
             Arabidopsis thaliana  
             
               CBF2 AP2 domain and flanking signature sequences  
             
           
            328 

      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro 
      1               5                   10                  15 
      Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val Cys Glu 
                  20                  25                  30 
      Leu Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 
              35                  40                  45 
      Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu 
          50                  55                  60 
      Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 
      65                  70                  75 

 
           
             329  
             79  
             PRT  
             Arabidopsis thaliana  
             
               CBF3 AP2 domain and flanking signature sequences  
             
           
            329 

      Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro 
      1               5                   10                  15 
      Ile Tyr Arg Gly Val Arg Arg Arg Asn Ser Gly Lys Trp Val Cys Glu 
                  20                  25                  30 
      Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln 
              35                  40                  45 
      Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
          50                  55                  60 
      Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 
      65                  70                  75 

 
           
             330  
             79  
             PRT  
             Arabidopsis thaliana  
             
               G912 AP2 domain and flanking signature sequences  
             
           
            330 

      Pro Lys Lys Arg Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro 
      1               5                   10                  15 
      Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly Lys Trp Val Cys Glu 
                  20                  25                  30 
      Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Pro 
              35                  40                  45 
      Thr Val Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu 
          50                  55                  60 
      Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 
      65                  70                  75 

 
           
             331  
             210  
             PRT  
             Lycopersicon esculentum  
             
               Lycopersicon esculentum CBF1 polypeptide  
             
           
            331 

      Met Asn Ile Phe Glu Thr Tyr Tyr Ser Asp Ser Leu Ile Leu Thr Glu 
      1               5                   10                  15 
      Ser Ser Ser Ser Ser Ser Ser Ser Ser Phe Ser Glu Glu Glu Val Ile 
                  20                  25                  30 
      Leu Ala Ser Asn Asn Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg 
              35                  40                  45 
      Glu Thr Arg His Pro Ile Tyr Arg Gly Ile Arg Lys Arg Asn Ser Gly 
          50                  55                  60 
      Lys Trp Val Cys Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp 
      65                  70                  75                  80 
      Leu Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Arg Ala His Asp Val 
                      85                  90                  95 
      Ala Ala Leu Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ser Asp 
                  100                 105                 110 
      Ser Ala Trp Arg Leu Pro Ile Pro Ala Ser Ser Asn Ser Lys Asp Ile 
              115                 120                 125 
      Gln Lys Ala Ala Ala Gln Ala Val Glu Ile Phe Arg Ser Glu Glu Val 
          130                 135                 140 
      Ser Gly Glu Ser Pro Glu Thr Ser Glu Asn Val Gln Glu Ser Ser Asp 
      145                 150                 155                 160 
      Phe Val Asp Glu Glu Ala Ile Phe Phe Met Pro Gly Leu Leu Ala Asn 
                      165                 170                 175 
      Met Ala Glu Gly Leu Met Leu Pro Pro Pro Gln Cys Ala Glu Met Gly 
                  180                 185                 190 
      Asp His Cys Val Glu Thr Asp Ala Tyr Met Ile Thr Leu Trp Asn Tyr 
              195                 200                 205 
      Ser Ile 
          210 

 
           
             332  
             220  
             PRT  
             Lycopersicon esculentum  
             
               Lycopersicon esculentum CBF2 polypeptide  
             
           
            332 

      Met Asp Ile Phe Glu Ser Tyr Tyr Ser Asn Ser Phe Val Glu Ser Leu 
      1               5                   10                  15 
      Leu Ser Ser Ser Leu Ser Ile Ser Asp Thr Asn Asn Leu Asn His Tyr 
                  20                  25                  30 
      Ser Pro Asn Glu Glu Val Ile Ile Leu Ala Ser Asn Asn Pro Lys Lys 
              35                  40                  45 
      Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg 
          50                  55                  60 
      Gly Ile Arg Lys Arg Asn Ser Gly Lys Trp Val Cys Glu Val Arg Glu 
      65                  70                  75                  80 
      Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe Pro Thr Ala Glu 
                      85                  90                  95 
      Met Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly Arg 
                  100                 105                 110 
      Ser Ala Cys Leu Asn Phe Ala Asp Ser Tyr Trp Arg Leu Pro Ile Pro 
              115                 120                 125 
      Ala Ser Ser Asn Ser Lys Asp Ile Gln Lys Ala Ala Ala Glu Ala Ala 
          130                 135                 140 
      Glu Ile Phe Arg Ser Glu Glu Val Ser Gly Glu Ser Pro Glu Thr Ser 
      145                 150                 155                 160 
      Glu Asn Val Gln Glu Ser Ser Asp Phe Val Asp Glu Glu Ala Leu Phe 
                      165                 170                 175 
      Ser Met Pro Gly Leu Leu Ala Asn Met Ala Glu Gly Leu Met Leu Pro 
                  180                 185                 190 
      Pro Pro Gln Cys Leu Glu Ile Gly Asp His Tyr Val Glu Leu Ala Asp 
              195                 200                 205 
      Val His Ala Tyr Met Pro Leu Trp Asn Tyr Ser Ile 
          210                 215                 220