Patent Publication Number: US-2003233681-A1

Title: ICE1, a regulator of cold induced transcriptome and freezing tolerance in plants

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
     [0001] This application claims benefit to U.S. Provisional Application Serial No. 60/377,469, filed on May 1, 2002, and U.S. Provisional Application Serial No. 60/377,897, filed on May 2, 2002, both or which are incorporated herein by reference in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
     [0002] This work was supported by the National Science Foundation Grant No. IBN9808398 and by the U.S. Department of Agriculture USDA/CSREES Grant No. 00-35100-9426. The United States government is entitled to certain rights in the present application. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0003] 1. Field of the Invention  
       [0004] The present invention relates to proteins and nucleic acids related to regulation of cold induced transcriptome and freezing tolerance in plants.  
       [0005] 2. Discussion of the Background  
       [0006] Cold is an environmental factor that limits the geographical distribution and growing season of many plant species, and it often adversely affects crop quality and productivity (Thomashow 1999). For example, most temperate plants can acquire tolerance to freezing temperatures by a process known as cold acclimation in which tolerance arises through a prior exposure to low non-freezing temperatures (Guy 1990; Hughes and Dunn 1996; Browse and Xin 2001). However, plants of tropical and sub-tropical origins are incapable of cold acclimation and, as such, are sensitive to chilling temperatures (0-10° C.). Many studies have suggested that cold-regulated gene expression is critical in plants for both chilling tolerance (Gong et al. 2002; Hsieh et al. 2002) and cold acclimation (Thomashow 1999; Knight et al. 1999; Tahtiharju and Palva 2001). Cold-responsive genes encode a diverse array of proteins, such as enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids and antioxidants; molecular chaperones, antifreeze proteins; and others with a presumed function in tolerance to the dehydration caused by freezing (Thomashow 1999; Guy 1990; Mohapatra et al. 1989).  
       [0007] Many of the cold and dehydration responsive genes have one or several copies of the DRE/CRT cis-element in their promoters, which has the core sequence, CCGAC (Yamaguchi-Shinozaki and Shinozaki 1994; Stockinger et al. 1997). A family of transcription factors known as CBFs or DREB1s binds to this element and activates transcription of the downstream cold and dehydration-responsive genes (Stockinger et al. 1997; Liu et al. 1998). Interestingly, the CBF/DREB1 genes are themselves induced by low temperatures. This induction is transient and precedes that of the downstream genes with the DRE/CRT cis-element (Thomashow 1999). Therefore, there is a transcriptional cascade leading to the expression of the DRE/CRT class of genes under cold stress. Ectopic expression of CBFs/DREB1s in plants turns on downstream cold-responsive genes even at warm temperatures and confers improved freezing tolerance (Jagglo-Ottosen et al. 1998; Liu et al. 1998).  
       [0008] Since CBF transcripts begin accumulating within 15 min of plants&#39; exposure to cold, Gilmour et al (1998) proposed that there is a transcription factor already present in the cell at normal growth temperature that recognizes the CBF promoters and induces CBF expression upon exposure to cold stress. Gilmour et al (1998) named the unknown activator(s) as “ICE” (Inducer of CBF Expression) protein(s) and hypothesized that upon exposing a plant to cold, modification of either ICE or an associated protein would allow ICE to bind to CBF promoters and activate CBF transcription.  
       [0009] Genetic analysis in Arabidopsis plants expressing the firefly luciferase reporter gene driven by the CRT/DRE element-containing RD29A promoter (Ishitani et al. 1997) has identified several mutants with de-regulated cold-responsive gene expression. The hos1 (high expression of osmotically responsive genes) mutant shows an enhanced cold-induction of CBFs and their downstream cold responsive genes (Ishitani et al. 1998). HOSI encodes a RING finger protein that is present in the cytoplasm at normal growth temperatures but accumulates in the nucleus upon cold treatment. Since many RING-finger proteins are known to serve as ubiquitin E3 ligases, HOS1 has been proposed to function by targeting certain positive regulator(s) of CBFs for ubiquitination and degradation (Lee et al. 2001). The transcription of CBF genes is also under feedback repression by its own gene product or its downstream target gene products. This was revealed by studies on the los1 mutant that is defective in the translational elongation factor 2 gene (Guo et al. 2002). The los1 mutation blocks cold induction of genes with the CRT/DRE element but causes super-induction of the CBF genes. It was shown that protein synthesis in los1 mutant plants is disrupted specifically in the cold. Therefore, cold-induced CBF transcripts cannot be translated to activate downstream genes, and feedback repression cannot occur, leading to super-induction of CBF transcripts (Guo et al. 2002).  
       [0010] Another Arabidopsis mutation, los2, also impairs cold induction of CRT/DRE element-containing genes (Lee et al., 2002). LOS2 encodes a bi-functional enolase that can bind to the promoter of ZAT10, a zinc finger transcriptional repressor. ZAT10 expression is rapidly and transiently induced by cold in the wild type, and this induction is stronger and more sustained in the los2 mutant. Therefore, LOS2 may control the expression of delayed cold response genes via transcriptional repression of ZAT1 (Lee et al. 2002). The Arabidopsis LOS4 locus is involved in the accumulation of CBF transcripts under cold treatment (Gong et al. 2002). los4-1 mutant plants are sensitive to chilling stress, and the chilling sensitivity can be rescued by ectopic expression of CBF3 (Gong et al. 2002). LOS4 encodes a DEAD-box RNA helicase, suggesting that RNA metabolism may be involved in cold responses.  
       [0011] Since environmental factors, such as cold, limits the geographical distribution and growing season of many plant species, and often adversely affects crop quality and productivity, there remains an ongoing critical need to increase cold acclimation in plants, particularly those plants that are advantageously useful as agricultural crops.  
       SUMMARY OF THE INVENTION  
       [0012] It is an object of the present invention to provide methods and compositions for increasing cold acclimation in plants.  
       [0013] It is another object of the present invention to provide plants and plant cells, which have increased cold acclimation.  
       [0014] The objects of the present invention, and others, may be accomplished with a method of increasing cold acclimation in a plant, comprising overexpressing ICE1 in the plant.  
       [0015] The objects of the present invention may also be accomplished with a method of increasing cold acclimation in a plant cell, comprising overexpressing ICE1 in the plant cell.  
       [0016] The objects of the present invention may also be accomplished with a plant or a plant cell transformed with a nucleic acid that encodes ICE1.  
       [0017] Thus, the present invention also provides a method of producing such a plant or plant cell, by transforming a plant or plant cell with the nucleic acid that encodes ICE1.  
       [0018] The present invention also provides an isolated and purified ICE1 having the amino acid sequence of SEQ ID NO: 2.  
       [0019] The present invention also provides a method of producing the ICE1 described above, comprising culturing host cells that have been transformed with a nucleic acid encoding ICE1 under conditions in which ICE1 is expressed, and isolating ICE1.  
       [0020] In another embodiment, the present invention provides an isolated and purified enzyme having ICE1 transcriptional activator activity, wherein the amino acid sequence of the enzyme has a homology of from 70% to less than 100% to SEQ ID NO: 2.  
       [0021] The present invention also provides a method of producing the enzyme described above, comprising culturing host cells that have been transformed with a nucleic acid encoding the enzyme under conditions in which the enzyme is expressed, and isolating the enzyme.  
       [0022] The present invention also provides a method of increasing cold acclimation in a plant, comprising overexpressing an ICE1 transcriptional activator in the plant.  
       [0023] The present invention also provides a method of increasing cold acclimation in a plant by increasing the expression of one or more additional transcription factors selected from the group consisting of a CBF transcription factor and a DREB1 transcription factor and/or by increasing expression of one or more cold-responsive genes.  
       [0024] The present invention has been accomplished using a genetic screen (Chinnusamy et al. 2002) to identify cold signaling components upstream of the CBF proteins. A cold-responsive bioluminescent Arabidpsis plant was engineered by expressing the firefly luciferase (LUC) coding sequence under the control of the CBF3 promoter. Homozygous CBF3-LUC plants were chemically mutagenized and luminescence imaging isolated mutants with altered cold-induced CBF3-LUC expression. In the present specification, the Inventors report on the ice1 (for inducer of CBF expression 1) mutant, which is impaired in the cold-induction of CBF3-LUC and is defective in cold acclimation. ICE1 encodes a MYC-like basic helix-loop-helix transcriptional activator that binds to the CBF3 promoter. Thus, ICE1 plays a key role in regulating cold-responsive gene expression and cold tolerance in Arabidopsis.  
       [0025] The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention. 
     
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
     [0026] A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below.  
     [0027]FIG. 1. The ice1 mutation blocks the cold-induction of CBF3 and affects the expression of other cold-responsive genes. (A) Morphology (left) and CBF3-LUC luminescence images (right) of wild-type and ice1 seedlings. Luminescence images of the plants were collected after 12 h of cold (0° C.) treatment. (B) Quantitation of the luminescence intensities of wild type (solid circles) and ice1 (open circles) seedlings in response to different durations of cold treatment. (C) Transcript levels of CBFs and their downstream target genes in wild type and ice1 plants in response to cold treatment. Seedlings were either not treated (0 h) or treated with cold (0° C.) for the indicated durations (h). The tubulin gene was used as a loading control. WT, wild type.  
     [0028]FIG. 2. Morphology, and freezing and chilling sensitivity of ice1 mutant plants. (A) Wild type and ice1 seedlings in nutrient medium on agar under normal growth conditions. (B) Wild type and ice1 plants in soil under normal growth conditions. (C) ice1 plants are defective in cold acclimation. Ten-day-old seedlings grown at 22° C. were incubated for 4 days in light at 4° C. before freezing treatment at −12° C. The picture was taken 3 days after the freezing treatment. (D) Comparison of survival rates after freezing treatments at the indicated temperatures. Open circles and open triangles represent wild type and ice1 plants, respectively. (E) ice1 plants are sensitive to prolonged chilling treatment. After germination at 22° C., the plants were grown at 4° C. for 6 weeks. (F) Comparison of survival rates after 6 weeks of chilling stress.  
     [0029]FIG. 3. Confirmation of ICE1 gene cloning by expressing the dominant ice1 mutant allele in wild type plants. (A) Expression in wild type of a genomic fragment containing the ice1 mutation recapitulates the ice1 mutant phenotype. Seven-day-old seedlings of the wild type, ice1, and wild type transformed with the mutant ice1 gene grown on MS agar medium were subjected to luminescence imaging after 12 h of cold (0° C.) stress. (B) Quantitation of CBF3-LUC bioluminescence levels in wild type (WT), ice1 and WT transformed with the mutant ice1 gene after 12 h of cold (0° C.) stress.  
     [0030]FIG. 4. ICE1 encodes a bHLH protein. (A) Overall domain structure of ICE1 protein. A putative acidic domain (acidic), serine rich region (S-rich), bHLH domain, and possible zipper region (ZIP) are indicated. The arrow indicates the amino acid residue changed in the ice1 mutant. (B) Sequence alignment of the bHLH domains and ZIP regions of ICE1 and other plant and animal bHLH proteins. Identical and similar residues are shown in black and gray, respectively. A bold line indicates the basic region and open boxes connected with a loop indicate the helix-loop-helix domain. The zipper region is indicated as a dotted line. DDJB/EMBL/GenBank accession numbers and amino acid numbers (in parentheses) are: ICE1 (SEQ ID NO: 2), AY195621 (300-398); At1g12860 (SEQ ID NO: 3), NM — 101157 (638-731); At5g65640 (SEQ ID NO: 4), NM — 125962.1 (171-269); At5g10570 (SEQ ID NO: 5), NM — 121095.2 (144-242); rd22BP (SEQ ID NO: 6), AB000875 (446-544); ATR2 (SEQ ID NO: 7), NM — 124046.1 (409-507); maize R gene (SEQ ID NO: 8), M26227 (410-508); TT8 (SEQ ID NO: 9), AJ277509 (357-455); PIF3 (SEQ ID NO: 10), AF100166 (254-352); PIF4 (SEQ ID NO: 11), AJ440755 (255-353); MAX (SEQ ID NO: 12), P52161 (21-107); c-myc (SEQ ID NO: 13), 1001205A (354-435). Asterisks indicate amino acid residues of MAX that are known to interact with nucleotides (Grandori et al. 2000).  
     [0031]FIG. 5. Expression of the ICE1 gene and subcellular localization of the ICE1 protein. (A) ICE1 promoter driven GUS expression pattern in a wild type seedling. (B) ICE1 promoter-GUS expression in different plant tissues, and the corresponding ICE1 transcript levels as determined by RT-PCR analysis. The tubulin gene was used as an internal control in the RT-PCR. (C) RNA blot analysis of ICE1 expression in wild type seedlings under various abiotic stresses. Plants with the following treatments are shown: control, MS salt only; NaCl, 300 mM NaCl for 5 hr; ABA, 100 μM abscisic acid for 3 hr; Cold, 0° C. for 2 hr; Dehydration, air drying for 30 min. (D) Localization of GFP-ICE1 fusion protein in the nucleus. Panels (a)-(c) show confocal images of root cells in GFP-ICE1 transgenic plants, while panel (d) shows the location of nuclei as indicated by propidium stain.  
     [0032]FIG. 6. ICE1 protein binds to the MYC-recognition elements in the CBF3 promoter. (A) Sequences and positions of oligonucleotides within the CBF3 promoter used in the EMSA. Letters in bold indicate sequences of MYC-recognition motifs in MYC-1 (SEQ ID NO: 38), MYC-2 (SEQ ID NO: 37), MYC-3 (SEQ ID NO: 36), MYC-4 (SEQ ID NO: 35), and MYC-5 (SEQ ID NO: 34). Bold letters in the P1 (SEQ ID NO: 39) oligonucleotide are a putative MYB-recognition motif. The sequences labeled P2, MYC-2 (wt), and MYC-2 (M) correspond to (SEQ ID NO: 40), (SEQ ID NO: 41), and (SEQ ID NO: 42), respectively. (B) Interaction between ICE1 protein and  32 P-labeled MYC-1 through MYC-4 DNA fragments. (C) ICE1 binds to the MYC-2 DNA fragment more strongly than to the other DNA fragments. (D) Consensus nucleotide residues in the MYC-recognition motif are important for the interaction between ICE1 and the MYC-2 DNA fragment. (E) ice1 mutant protein also binds to the MYC-2 DNA fragment. The labeled oligonucleotides used in each experiment are indicated on the top of each panel. Triangles indicate increasing amounts of unlabeled oligonucleotides for competition in (B), (C) and (D), which correspond to 50-, 100- and 250-fold excess of each probe.  
     [0033]FIG. 7. ICE1 is a transcriptional activator and its overexpression enhances the CBF regulon in the cold and improves freezing tolerance. (A) Schematic representation of the reporter and effector plasmids used in the transient expression assay. A GAL4-responsive reporter gene was used in this experiment. Nos denotes the terminator signal of the nopaline synthase gene. Ω indicates the translational enhancer of tobacco mosaic virus. GAL4 DB is the DNA binding domain of the yeast transcription factor GAL4. (B) Relative luciferase activities after transfection with GAL4-LUC and 35S-GAL4-ICE1 or 35S-GAL4-ice1. To normalize values obtained after each transfection, a gene for luciferase from Renilla was used as an internal control. Luciferase activity is expressed in arbitrary units relative to the activity of Renilla luciferase (as described in Ohta et al. 2001). The values are averages of three bombardments, and error bars indicate standard deviations. (C) RNA blot analysis of ICE1 and cold responsive gene expression in wild type and ICE1 overexpressing transgenic (Super-ICE1 ) plants. Seedlings were either not treated (0 h) or treated with low temperature (0° C.) for 3 h or 6 h. Ethidium bromide stained rRNA bands are shown as loading control. (D) CBF3-LUC expression (indicated as luminescence intensity) in wild type and ICE1 overexpressing transgenic (Super-ICE1) plants. (E) Improved survival of ICE1 overexpressing transgenic (Super-ICE1) plants after a freezing treatment. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0034] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in biochemistry, cellular biology, and molecular biology.  
     [0035] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.  
     [0036] Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989); Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995); Arabidopsis, Meyerowitz et al, Eds., Cold Spring Harbor Laboratory Press, New York (1994) and the various references cited therein.  
     [0037] The term “plant” includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. The class of plants, which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Preferred plants include rice, corn, wheat, cotton, peanut, and soybean.  
     [0038] Thus, in one embodiment of the present invention, cold acclimation can be enhanced or increased by increasing the amount of protein available in the plant, preferably by the enhancement of the ice1 gene in the plant.  
     [0039] Thus, one embodiment of the present invention is plant cells carrying the polynucleotides of the present invention, and preferably transgenic plants carrying the isolated polynucleotides of the present invention.  
     [0040] As used herein, the term “enhancement” means increasing the intracellular activity of one or more enzymes in a plant cell and/or plant, which are encoded by the corresponding DNA. Enhancement can be achieved with the aid of various manipulations of the bacterial cell. In order to achieve enhancement, particularly over-expression, the number of copies of the corresponding gene can be increased, a strong promoter can be used, or the promoter- and regulation region or the ribosome binding site which is situated upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene may act in the same manner. In addition, it is possible to increase expression by employing inducible promoters. A gene can also be used which encodes a corresponding enzyme with a high activity. Expression can also be improved by measures for extending the life of the mRNA. Furthermore, preventing the degradation of the enzyme increases enzyme activity as a whole. Moreover, these measures can optionally be combined in any desired manner. These and other methods for altering gene activity in a plant are known as described, for example, in Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995).  
     [0041] An “expression cassette” as used herein includes a promoter, which is functional in a plant cell, operably linked to an isolated nucleic acid encoding an ICE1 protein of SEQ ID NO: 2, wherein enhanced expression of the protein in a plant cell imparts increased cold acclimation to said plant cell. In a preferred embodiment of the present invention the promoter is selected from the group consisting of a viral coat protein promoter, a tissue-specific promoter, a monocot promoter, a ubiquitin promoter, a stress inducible promoter, a CaMV 35S promoter, a CaMV 19S promoter, an actin promoter, a cab promoter, a sucrose synthase promoter, a tubulin promoter, a napin R gene complex promoter, a tomato E8 promoter, a patatin promoter, a mannopine synthase promoter, a soybean seed protein glycinin promoter, a soybean vegetative storage protein promoter, a bacteriophage SP6 promoter, a bacteriophage T3 promoter, a bacteriophage T7 promoter, a Ptac promoter, a root-cell promoter, an ABA-inducible promoter and a turgor-inducible promoter.  
     [0042] A gene can also be used which encodes a corresponding or variant enzyme with a high activity. Preferably the corresponding enzyme has a greater activity than the native form of the enzyme, more preferably at least in the range of 5, 10, 25% or 50% more activity, most preferably more than twice the activity of the native enzyme.  
     [0043] In the context of the present Application, a polynucleotide sequence is “homologous” with the sequence according to the invention if at least 70%, preferably at least 80%, most preferably at least 90% of its base composition and base sequence corresponds to the sequence according to the invention. According to the invention, a “homologous protein” is to be understood to comprise proteins which contain an amino acid sequence at least 70% of which, preferably at least 80% of which, most preferably at least 90% of which, corresponds to the amino acid sequence which is shown in SEQ ID NO: 2 or which is encoded by the ice1 gene (SEQ ID No.1), wherein corresponds is to be understood to mean that the corresponding amino acids are either identical or are mutually homologous amino acids. The expression “homologous amino acids” denotes those that have corresponding properties, particularly with regard to their charge, hydrophobic character, steric properties, etc. Thus, the protein may be from 70% up to less than 100% homologous to SEQ ID NO: 2.  
     [0044] Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.  
     [0045] The present invention also relates to polynucleotides which contain the complete gene with the polynucleotide sequence corresponding to SEQ ID NO: 1 or fragments thereof, and which can be obtained by screening by means of the hybridization of a corresponding gene bank with a probe which contains the sequence of said polynucleotide corresponding to SEQ ID NO: 1 or a fragment thereof, and isolation of said DNA sequence.  
     [0046] Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate those cDNAs or genes which exhibit a high degree of similarity to the sequence of the ice1 gene, in particular the ice1 gene of SEQ ID NO: 1.  
     [0047] Polynucleotide sequences according to the invention are also suitable as primers for polymerase chain reaction (PCR) for the production of DNA which encodes an enzyme having ICE1 transcriptional activator activity.  
     [0048] Oligonucleotides such as these, which serve as probes or primers, can contain more than 30, preferably up to 30, more preferably up to 20, most preferably at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable.  
     [0049] The term “isolated” means separated from its natural environment.  
     [0050] The term “polynucleotide” refers in general to polyribonucleotides and polydeoxyribonucleotides, and can denote an unmodified RNA or DNA or a modified RNA or DNA.  
     [0051] The term “polypeptides” is to be understood to mean peptides or proteins, which contain two or more amino acids which are bound via peptide bonds.  
     [0052] The polypeptides according to invention include polypeptides corresponding to SEQ ID NO: 2, particularly those with the biological activity of a ICE1 transcriptional activator, and also includes those, at least 70% of which, preferably at least 80% of which, are homologous with the polypeptide corresponding to SEQ ID NO: 2, and most preferably those which exhibit a homology of least 90% to 95% with the polypeptide corresponding to SEQ ID NO: 2 and which have the cited activity. Thus, the polypeptides may have a homology of from 70% up to 100% with respect to SEQ ID NO: 2.  
     [0053] The invention also relates to coding DNA sequences, which result from SEQ ID NO: 1 by degeneration of the genetic code. In the same manner, the invention further relates to DNA sequences which hybridize with SEQ ID NO: 1 or with parts of SEQ ID NO: 1. Moreover, one skilled in the art is also aware of conservative amino acid replacements such as the replacement of glycine by alanine or of aspartic acid by glutamic acid in proteins as “sense mutations” which do not result in any fundamental change in the activity of the protein, i.e. which are functionally neutral. It is also known that changes at the N- and/or C-terminus of a protein do not substantially impair the function thereof, and may even stabilize said function.  
     [0054] In the same manner, the present invention also relates to DNA sequences which hybridize with SEQ ID NO: 1 or with parts of SEQ ID NO: 1. Finally, the present invention relates to DNA sequences which are produced by polymerase chain reaction (PCR) using oligonucleotide primers which result from SEQ ID NO: 1. Oligonucleotides of this type typically have a length of at least 15 nucleotides.  
     [0055] The terms “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).  
     [0056] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.  
     [0057] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): Tm=81.5oC.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (2000).  
     [0058] Thus, with the foregoing information, the skilled artisan can identify and isolated polynucleotides that are substantially similar to the present polynucleotides. In so isolating such a polynucleotide, the polynucleotide can be used as the present polynucleotide in, for example, increasing cold acclimation of a plant.  
     [0059] One embodiment of the present invention is methods of screening for polynucleotides that have substantial homology to the polynucleotides of the present invention, preferably those polynucleotides encoding a protein having ICE1 transcriptional activator activity.  
     [0060] The polynucleotide sequences of the present invention can be carried on one or more suitable plasmid vectors, as known in the art for plants or the like.  
     [0061] In one embodiment, it may be advantageous for propagating the polynucleotide to carry it in a bacterial or fungal strain with the appropriate vector suitable for the cell type. Common methods of propagating polynucleotides and producing proteins in these cell types are known in the art and are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).  
     [0062] In another preferred embodiment the polynucleotide comprises SEQ ID NO: 1, polynucleotides which are complimentary to SEQ ID NO: 1, polynucleotides which are at least 70%, 80% and 90% identical to SEQ ID NO: 1; or those sequence which hybridize under stringent conditions to SEQ ID NO: 1, the stringent conditions comprise washing in 5×SSC at a temperature from 50 to 68° C. Thus, the polynucleotide may be from 70% up to less than 100% identical to SEQ ID NO: 1.  
     [0063] In another preferred embodiment the polynucleotides of the present invention are in a vector and/or a host cell. Preferably, the polynucleotides are in a plant cell or transgenic plant. Preferably, the plant is  Arabidopsis thaliania  or selected from the group consisting of wheat, corn, peanut cotton, oat, and soybean plant. In a preferred embodiment, the polynucleotides are operably linked to a promoter, preferably an inducible promoter.  
     [0064] In another preferred embodiment the present invention provides, a process for screening for polynucleotides which encode a protein having ICE1 transcriptional activator activity comprising hybridizing the polynucleotide of the invention to the polynucleotide to be screened; expressing the polynucleotide to produce a protein; and detecting the presence or absence of ICE1 transcriptional activator activity in the protein.  
     [0065] In another preferred embodiment, the present invention provides a method for detecting a nucleic acid with at least 70% homology to nucleotide SEQ ID NO: 1, sequences which are complimentary to SEQ ID NO: 1 and/or which encode a protein having the amino acid sequence in SEQ ID NO: 2 comprising contacting a nucleic acid sample with a probe or primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1, or at least 15 consecutive nucleotides of the complement thereof.  
     [0066] In another preferred embodiment, the present invention provides a method for producing a nucleic acid with at least 70% homology to the polynucleotides of the present invention comprising contacting a nucleic acid sample with a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1, or at least 15 consecutive nucleotides of the complement thereof.  
     [0067] In another preferred embodiment, the present invention provides a method for making ICE1 protein, comprising culturing the host cell carrying the polynucleotides of the invention for a time and under conditions suitable for expression of ICE1, and collecting the ICE1.  
     [0068] In another preferred embodiment, the present invention provides a method of making a transgenic plant comprising introducing the polynucleotides of the invention into the plant.  
     [0069] In another preferred embodiment, the present invention provides method of increasing cold acclimation of a plant in need thereof, comprising introducing the polynucleotides of the invention into said plant.  
     [0070] Methods, vectors, and compositions for transforming plants and plant cells in accordance with the invention are well-known to those skilled in the art, and are not particularly limited. For a descriptive example see Karimi et al., TRENDS in Plant Science, Vol. 7, NO: 5, May 2002, pp. 193-195, incorporated herein by reference.  
     [0071] In another preferred embodiment, the present invention provides an isolated polypeptide comprising the amino acid sequence in SEQ ID NO: 2 or those proteins that are at least 70%, preferably 80%, preferably 90% and preferably 95% identity to SEQ ID NO: 2, where the polypeptides have ICE1 transcriptional activator activity. Thus, the enzyme has a homology of from 70% to less than 100% homology to SEQ ID NO: 2.  
     [0072] In another embodiment, the present invention also provides a method of increasing cold acclimation in a plant, comprising overexpressing an ICE1 transcriptional activator in the plant.  
     [0073] The present invention also provides, in another embodiment a method of increasing cold acclimation in a plant by increasing the expression of one or more additional transcription factors selected from the group consisting of a CBF transcription factor and a DREB1 transcription factor and/or by increasing expression of one or more cold-responsive genes.  
     [0074] In the context of the present invention the term “cold responsive genes” include genes that encode a protein selected from the group consisting of an enzyme involved in respiration of carbohydrates, an enzyme involved in metabolism of carbohydrates, an enzyme involved in respiration of lipids, an enzyme involved in metabolism of lipids, an enzyme involved in respiration of phenylpropanoids, an enzyme involved in metabolism of phenylpropanoids, an enzyme involved in respiration of antioxidants, an enzyme involved in metabolism of antioxidants, a molecular chaperone, an antifreeze protein, and a protein involved in tolerance to the dehydration caused by freezing.  
     [0075] The present invention has been accomplished using a genetic screen (Chinnusamy et al. 2002) to identify cold signaling components upstream of the CBF proteins. A cold-responsive bioluminescent Arabidpsis plant was engineered by expressing the firefly luciferase (LUC) coding sequence under the control of the CBF3 promoter. Homozygous CBF3-LUC plants were chemically mutagenized and luminescence imaging isolated mutants with altered cold-induced CBF3-L UC expression. In the present specification, the Inventors report on the ice1 (for inducer of CBF expression 1) mutant, which is impaired in the cold-induction of CBF3-LUC and is defective in cold acclimation. ICE1 encodes a MYC-like basic helix-loop-helix transcriptional activator that binds to the CBF3 promoter. Thus, ICE1 plays a key role in regulating cold-responsive gene expression and cold tolerance in Arabidopsis.  
     [0076] Discussion  
     [0077] Cold temperatures trigger the transcription of the CBF family of transcription factors, which in turn activate the transcription of genes containing the DRE/CRT promoter element (Thomashow 1999). The CBF target genes presumably include some transcription factors (Fowler and Thomashow 2002). Therefore, cold signaling for freezing tolerance requires a cascade of transcriptional regulations. In the present study, we have identified ICE1, a very upstream transcription factor of this cascade. Our results show that ICE1 is a positive regulator of CBF3 and has a critical role in cold acclimation. ICE1 encodes a MYC-like bHLH transcription factor. Five putative MYC recognition sequences are present in the CBF3 promoter, while CBF1 and CBF2 promoters each contain one such element (Shinwari et al. 1998). This is consistent with the fact that CBF3 is more strongly affected by the ice1 mutation than are CBF1 or CBF2. DNA binding assays showed that ICE1 can specifically bind to the MYC recognition sequences on the CBF3 promoter but not to a putative MYB recognition sequence (FIG. 6). The ice1 mutation abolishes CBF3 expression, and reduces the expression of CBF-target genes in the cold. Consistent with its role in cold-responsive gene regulation, ICE1 is important for chilling and freezing tolerance of Arabidopsis plants.  
     [0078] The ice1 mutation also affects the cold-induction of CBF1 and CBF2; their expression is slightly reduced early in the cold, but at later time points the expression is not reduced. Instead, the expression of CBF2 is actually enhanced in the ice1 mutant after 6 and 12 hours of cold treatment. The expression of CBF genes is known to be repressed by their gene products or the products of their downstream target genes (Guo et al. 2002). The correlation between the reduced CBF3 expression and enhanced CBF2 induction suggests that CBF3 may repress CBF2 expression. When the CBF2 gene is disrupted, CBF1 and CBF3 show more sustained induction in the cold (Julio Salinas, personal communication), suggesting that CBF2 may repress the expression of CBF1 and CBF3. The potential negative regulation of each other among the CBF transcription factor genes may be important for ensuring that their expression is transient and tightly controlled.  
     [0079] The three CBF genes are generally presumed to be functionally redundant. Their individual contribution has not been examined by loss of function analysis. Even though the ice1 mutation only blocks the expression of CBF3, the downstream genes such as RD29A, COR15A and COR47 are substantially affected. This suggests that CBF3 plays a critical role in the cold regulation of these genes. In comparison, the cold regulation of KIN1 is less affected by the ice1 mutation. Therefore, it is possible that the three CBF genes may each have their own set of preferred target genes.  
     [0080] ICE1 is expressed constitutively in all tissues (FIGS. 5A and 5B), and is only slightly up-regulated by cold (FIG. 5C). Consistent with what has been speculated for “ICE” proteins. (Gilmour et al. 1998), cold induced modification of the ICE1 protein or of a transcriptional co-factor appears to be necessary for ICE1 to activate the expression of CBFs. Our evidence supports this because ICE1 is expressed constitutively and localized in the nucleus, but the CBF expression requires cold treatment; and transgenic lines constitutively overexpressing ICE1 do not show CBF3 expression at warm temperatures but have a higher level of CBF3 expression at cold temperatures. The ability of transcription factors to activate gene transcription may be regulated by protein phosphorylation and dephosphorylation in the cytoplasm or in the nucleus (reviewed by Liu et al. 1999). The ice1 mutation is very near potential serine phosphorylation residues (Ser243 and Ser245), and thus might affect the phosphorylation/dephosphorylation of ICE1.  
     [0081] It is known that MYC-related bHLH transcription factors require MYB co-transcription factors and/or WD-repeat containing factors for transcriptional activation of target genes (Spelt et al. 2000; Walker et al. 1999). The promoters of CBFs contain MYC as well as potential MYB recognition sequences (Shinwari et al. 1998), suggesting that a MYB-related transcription factor may also be involved in the cold induction of CBFs. The ice1 mutation, which substitutes Arg236 with His, may interfere with hetero-oligomer formation between ICE1 and an ICE1-like protein or a MYB-related co-factor. Alternatively, the putative dominant negative effect of ice1 could be a consequence of ice1 interference with potential ICE1 homo-oligomer formation, protein stability, nuclear localization, or cold induced post-translational modification of ICE1.  
     [0082] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.  
     EXAMPLES  
     [0083] Materials and Methods  
     [0084] Plant Materials and Mutant Isolation:  
     [0085] The CBF3 promoter, a region from 1126 to 100 bp upstream of the initiation codon, was obtained by polymerase chain reaction (PCR) using the following primer pair: 5′-TCATGGATCCACCATTTGTTAATGCATGATGG-3′(SEQ ID NO: 14) and 5′-GCTCAAGCTTTCTGTTCTAGTTCAGG-3′(SEQ ID NO: 15). This promoter was placed in front of the firefly luciferase (LUC) coding sequence in a plant transformation vector (Ishitani et al. 1997).  Arabidopsis thaliana  ecotype Columbia (with the glabrous1 mutation) was transformed with  Agrobacterium tumefaciens  containing this CBF3-LUC construct by the floral dipping method. Plants homozygous for the CBF3-LUC transgene were selected from the second generation after transformation. One such plant with a single copy of the CBF3-LUC transgene was chosen for subsequent experiments (hereafter referred to as wild type). This wild type plant did not show any bioluminescence when grown under normal growth conditions, but emitted bioluminescence when cold stress was imposed. The CBF3-LUC plant seeds were mutagenized with ethyl methanesulfonate (EMS). Seedlings of the M2 generation were used to screen for mutants defective in cold regulated CBF3-LUC expression by luminescence imaging. Seven-day-old seedlings grown on 0.6% agar plates containing 3% sucrose and 1×Murashige and Skoog (MS) salts (JRH Biosciences) were screened for de-regulated luciferase expression in response to low temperature treatment at 0° C. for 12 hours, using a low light video imaging system (Princeton Instruments). Luminescence intensities of individual seedlings were quantified with the WINVIEW software provided by the camera manufacturer (Princeton Instruments) (Chinnusamy et al. 2002).  
     [0086] Chilling and Freezing Tolerance Assays:  
     [0087] Chilling sensitivity of ice1 and wild type plants were tested by exposing the seedlings immediately after radicle emergence. After 2 days of stratification at 4° C., mutant and wild-type seeds were germinated at 22° C. on MS nutrient medium with 3% sucrose and 1.2% agar. Chilling stress was imposed by incubating the seedlings at 4±1° C. with 30±2 μmol quanta. m −2 .s −1  light. Freezing tolerance was assayed as described (Xin and Browse, 1998). Briefly, wild type and ice1 seeds were sown on agar (0.9%) plates with Gamborg basal salts and 1.5% sucrose. After 2 days of stratification at 4° C., the plates were kept at 22° C. under 50±2 μmol quanta m −2 .s −1  continuous light. Ten-day-old seedlings were cold acclimated at 4±1° C. and 30±2 μmol quanta. m −2 .s −1  light for 4 days. These plants on petri dishes were placed on ice in a freezing chamber (Percival Scientific) set to −1±0.1° C. for 16 h. Ice chips were sprinkled on these plants before the chamber was programmed to cool at 1° C. h −1 . Petri dishes of plants were removed after being frozen at desired temperatures for 2 hours unless indicated otherwise, thawed at 4° C. for 12 hours in the dark, and then transferred to 22° C. under 50±2 μmol quanta m −2 .s −1  continuous light. Survival of the seedlings was scored visually after two days.  
     [0088] Gene Expression Analysis:  
     [0089] For RNA analysis, ten-day-old seedlings of WT and ice1 plants grown on separate halves of the same MS agar plates were used. Total RNA extracted from control and stressed plants was analyzed by RNA blotting as described by Liu and Zhu (1997). The RD29A gene-specific probe was from the 3′noncoding region (Liu and Zhu 1997). COR15A and COR47 cDNAs (Gilmour et al. 1992; Lin and Thomashow 1992) were kindly provided by M. F. Thomashow (Michigan State University). The CBF2 and CBF3 gene-specific probes were generated by PCR with the following primer pairs: CBF2-forward primer, 5′-TTCGATTTTTATTTCCATTTTTGG-3′(SEQ ID NO: 16); CBF2-reverse primer, 5′-CCAAACGTCCTTGAGTCTTGAT-3′(SEQ ID NO: 17); CBF3-forward primer, 5′-TAAAACTCAGATTATTATTTCCATTT-3′(SEQ ID NO: 18); CBF3-reverse primer, 5′-GAGGAGCCACGTAGAGGGCC-3′(SEQ ID NO: 19). The probe for KINI (Kurkela and Franck, 1990) was a 0.4-kb EcoR1 fragment of the Arabidopsis EST clone YAP368T7. The β-tubulin gene was used as a loading control and was amplified by PCR with the following primer pairs: forward primer (5′-CGTGGATCACAGCAATACAGAGCC-3′(SEQ ID NO: 20)) and reverse primer (5′-CCTCCTGCACTTCCACTTCGTCTTC-3′(SEQ ID NO: 21)).  
     [0090] For Affymetrix GeneChip array analysis, 20 μg of total RNA from the wild type and ice1 seedlings with or without cold treatment (6 hours under light) were extracted using the RNeasy Plant Mini Kit (Qiagen) and used to make biotin-labeled CRNA targets. The Affymetrix Arabidopsis ATHI genome array GeneChips, which contain more than 22,500 probe sets representing approximately 24,000 genes, were used and hybridization, washing, and staining were carried out as directed in the manufacturer&#39;s manual. Microarray data were extracted from scanned GeneChip images and analyzed using Microarray Suite version 5.0.1 (Affymetrix).  
     [0091] Mapping and Cloning of the ICE1 Locus:  
     [0092] Genetic analysis of F 1  and F 2  progenies of the ice1 cross with WT showed that ice1 is a dominant mutation. Hence to clone ICE1, a homozygous ice1 plant was crossed with the  Arabidopsis Landsberg erecta  (Ler) ecotype and the F 2  progeny from self-pollinated F 1  were used to select mapping samples with the wild type phenotype. Genomic DNA extracted from these seedlings was used for PCR-based mapping with simple sequence polymorphism markers or cleaved amplified polymorphic sequence markers. New SSLP mapping markers on F16J4, MTC11, MLJ15, MDJ14, K17E12 and T32N 15BAC clones were developed based on insertion/deletions identified from the Cereon Arabidopsis polymorphism and Ler sequence collection (http://www.arabidopsis.org). Genomic DNA corresponding to candidate genes was amplified by PCR from ice1 mutant and wild type plants and sequenced to identify the ice1 mutation.  
     [0093] For ice1 mutant complementation, the MLJ15.14 gene, including 2,583 bp upstream of the initiation codon and 615 bp downstream of the stop codon, was PCR amplified by LA Taq polymerase (Takara) using ice1 mutant genomic DNA as template. The PCR primers used were as follows: forward primer: 5′-AGGGATCCGGACCACCGTCAATAACATCGTTAAGTAG-3′(SEQ ID NO: 22); reverse primer: 5′-CGAATTCTAACCGCCATTAACTATGTCTCCTCTCTATCTC-3′(SEQ ID NO: 23). The resulting 5,035-bp fragment was T-A cloned into the pCR2.1 TOPO vector (Invitrogen) and then subcloned into pCAMBIA1200 between the BamHI and EcoRI sites. This and all other constructs described here were completely sequenced to ensure that they did not contain PCR or cloning errors. The binary construct was then introduced into Agrobacterium strain GV3101 and transformed into CBF3-LUC Columbia wild type plants. Hygromycin-resistant transgenic plants were selected and their T2 progenies were tested for CBF3-LUC expression in response to cold stress.  
     [0094] Analysis of ICE1 Expression:  
     [0095] The promoter region (2,589 bp upstream from the initiation codon) of the ICE1 gene was PCR-amplified with the following primer pair: forward primer, 5′-AGGGATCCGGACCACCGTCAATAACATCGTTAAGTAG-3′(SEQ ID NO: 24); reverse primer, 5′-CGAATTCGCCAAAGTTGACACCTTTACCCCAAAG-3′(SEQ ID NO: 25). The resulting fragment was digested with BamHI and EcoRI and inserted into the pCAMBIA1391 binary vector. This ICE1 promoter-GUS construct was introduced into Agrobacterium strain GV3101 and transformed into wild type Arabidopsis. T2 transgenic lines resistant to hygromycin were analyzed for ICE1-promoter driven GUS expression. For GUS staining, T2 seedlings grown on MS agar plates were incubated with X-Gluc. for 12 h at 37° C. and then washed 5 times with 70% (v/v) ethanol at 70° C. to remove chlorophyll. ICE1 expression was also examined by quantitative RT-PCR analysis of RNA prepared from wild type roots, leaves, stems and flowers. The ICE1 cDNA was amplified by RT-PCR using the following primers: forward primer: 5′-GCGATGGGTCTTGACGGAAACAATGGTG-3′ (SEQ ID NO: 26) and reverse primer 5′-TCAGATCATACCAGCATACCCTGCTGTATCG-3′(SEQ ID NO: 27). The tubulin gene was used as an internal control in the RT-PCR analysis. Tubulin cDNA was amplified using the following primers: forward primer: 5′-GTCAAGAGGTTCTCAGCAGTA-3′ (SEQ ID NO: 28) and reverse primer 5′-TCACCTTCTTGATCCGCAGTT-3′ (SEQ ID NO: 29).  
     [0096] Overexpression of ICE1:  
     [0097] The ICE1 cDNA was amplified from Arabidopsis (ecotype Columbia) RNA by RT-PCR using the following primers: a forward primer: 5′-GCTCTAGAGCGATGGGTCTTGACGGAAACAATGGTG-3′(SEQ ID NO: 30) and a reverse primer 5′-GGGGTACCTCAGATCATACCAGCATACCCTGCTGTATCG-3′(SEQ ID NO: 31). The PCR product was digested with XbaI and KpnI, and cloned into the pBIB vector under control of the superpromoter, which consists of three copies of the octopine synthase upstream-activating sequence in front of the manopine synthase promoter (Li et al. 2001).  Agrobacterium tumefaciens  strain GV3101 containing this binary construct was used to transform Arabidopsis plants. Transformants were selected on MS medium containing hygromycin (30 mg/l).  
     [0098] Expression and Localization of GFP-ICE1 Fusion Protein:  
     [0099] The full-length ICE1 cDNA was obtained from wild type plants by RT-PCR using the following primers: forward primer, 5′-AGGAATTCGCGATGGGTCTTGACGGAAACAATGGTG-3′(SEQ ID NO: 32); reverse primer, 5′-CTGGATCCTCAGATCATACCAGCATACCCTGCTGTATCG-3′(SEQ ID NO: 33). The resulting PCR fragment was digested with EcoRI and BamHI and cloned into the binary vector pEGAD downstream from the CaMV 35S promoter. This GFP-ICE1 construct was introduced into Agrobacterium strain GV3101 and transformed into wild type Arabidopsis. T2 transgenic lines resistant to Basta (glufosinate) were selected and analyzed for GFP expression. To visualize the nucleus, root tissues were stained with propidium iodide (1 μg/mL). Green fluorescence (GFP expression) and red fluorescence (propidium iodide staining) analyses of transgenic plants were performed with a confocal laser-scanning microscope.  
     [0100] DNA Binding Assay:  
     [0101] The wild type and mutant ICE1 cDNAs were amplified by RT-PCR and inserted into NdeI and BamHI sites in the expression vector pET14b (Novagen). Wild-type and mutant His-ICE1 fusion proteins were prepared from  E. coli  cells (BL21 DE3) according to the instruction manual of His-Bind Buffer Kit (Novagen). The electrophoresis mobility shift assay (EMSA) was carried out as described (Hao et al. 1998). The following double-stranded oligonucleotides listed in FIG. 6A (MYC-1, MYC-2, MYC-3, MYC-4 and MYC-5) were used as probes and competitors in EMSAs. Nucleotide sequences P1 (−949 to −930) and P2 (−909 to −890) were also used as competitors. P1 contains a putative MYB-recognition site. P2 does not contain any typical cis-elements. DNA probes were end-labeled with [γ- 32 P]dCTP using the Klenow fragment and purified through a Sephadex G-50 column. The labeled probes (ca 0.02 pmol) were incubated for 20 min at room temperature with 2.3 μg of purified His-ICE1 fusion protein in 1×binding buffer (Hao et a.1998) supplemented with 20 pmol poly(dI-dC). The resulting DNA-protein complexes were resolved by electrophoresis on a 6% polyacrylamide gel in 0.5×TBE buffer and visualized by autoradiography. For competition experiments, unlabeled competitors were incubated with the His-ICE1 fusion protein on ice for 30 min prior to the addition of labeled probes.  
     [0102] Transient Expression Assay:  
     [0103] The wild type (ICE1) and mutant (ice1) cDNAs were amplified by RT-PCR, digested with SalI and inserted into SmaI and SalI sites of the plant expression vector 35S-GAL4 DB (Ohta et al. 2000). The plasmid DNA of the resulting effector, GAL4-ICE1, and a GAL4 responsive reporter, GAL4-LUC (Ohta et al. 2000) were delivered into Arabidopsis leaves using particle bombardment (Ohta et al. 2001).  
     Experimental Example  
     [0104] Identification of the ICE1 Locus:  
     [0105] Using the genetic screen noted above, Arabidopsis plants containing the CBF3-LUC transgene emitted bioluminescence in response to cold stress (FIGS. 1A and 1B). The homozygous CBF3-LUC plants (herein referred to as wild type) were mutagenized by ethylmethane sulfonate, and the resulting M2 population was screened for mutants with aberrant bioluminescence responses under cold stress using a low light imaging system (Chinnusamy et al. 2002). Several mutants showing abnormal cold regulation of CBF3-LUC expression were recovered. One of these mutant lines, designated as ice1, is virtually blocked in CBF3-LUC expression in the cold (FIGS. 1A and 1B). In response to treatment at 0° C., wild type plants showed strong luminescence, while the ice1 mutant showed very little induction of luminescence throughout the duration of cold treatment (FIGS. 1A and 1B). After 12 hours of cold treatment, ice1 plants showed nearly 10 times less luminescence than that of wild type plants, and are obviously defective in the cold regulation of CBF3-L UC expression (FIG. 1B).  
     [0106] The ice1 mutant plant was crossed with CBF3-LUC wild type plants and the resulting F1 plants were examined for CBF3-LUC expression after 12 hours of cold treatment at 0° C. As determined by luminescence imaging, all F1 plants showed reduced cold-induced CBF3-LUC expression similar to that of ice1. An F2 population from the selfed F1 segregated in an approximately 3 to 1 ratio between mutant and wild type. These results show that ice1 is a dominant mutation in a single nuclear gene.  
     [0107] ice1 Mutant Plants are Defective in Cold-Regulated Gene Expression:  
     [0108] RNA blot analysis was carried out to analyze the effect of ice1 mutation on the transcript levels of endogenous CBFs and their target cold stress-responsive genes. Consistent with the imaging results, cold induction of the endogenous CBF3 gene was greatly impaired (almost abolished) in ice1 mutant plants (FIG. 1C). Wild type plants showed CBF3 induction after 1 hour of cold stress and the expression peaked at 6 hour. In contrast, CBF3 induction was almost abolished in ice1 plants (FIG. 1C). While the CBF1 induction level was lower in the ice1 mutant was lower than that of wild type at 1 and 3 hours of cold stress, its induction level at 6 and 12 hours was similar to that in the wild type. The CBF2 induction level was slightly lower in ice1 at 1 hour of cold treatment, whereas at 6 and 12 hours, the induction level was higher in the mutant (FIG. 1C). We also examined the cold induction of the downstream target genes of CBFs. The expression levels of RD29A, COR15A and COR47A under cold stress were lower in ice1 than in the wild type, while the induction of KIN1 was lower in ice1 only after 48 hours of cold stress (FIG. 1C).  
     [0109] Consistent with these RNA blot results, microarray analysis using Affymetrix near full genome genechips showed that out of 306 genes induced 3-fold or more in the wild type by a 6-hour cold treatment, 217 are either not induced in the ice1 mutant or their induction is 50% or less of that in the wild type (Table 1A). Thirty-two of these encode putative transcription factors, suggesting that ICE1 may control many cold-responsive regulons. For 87 of the 306 cold induced genes, their induction levels in the wild type and ice1 differ by less than 2-fold (Table 1B). Interestingly, 2 genes show higher levels of cold induction in the ice1 mutant (Table 1C).  
     [0110] Table 1. Cold-Responsive Gene Expression in the Wild Type and ice1.  
     [0111] For cold treatment, the wild-type and ice1 seedlings were placed at 0±1° C. under light for 6 hours. Affymetrix GeneChip analysis was carried out as described in materials and methods. Gene expression changes were analyzed by comparing values for a cold-treated sample to those for a control sample in each genotype. ‘Fold Change’ value of +1 or −1 indicates no change in gene expression. Up-regulation or down-regulation is expressed by either + or − in ‘Fold Change’ values, respectively. Cold-responsive genes were determined in the wild type by the following standards; 1) signal intensities from cold treated sample were greater than background (i.e. genes with ‘Present’ calls, determined by Affymetrix Microarray Suite Program, in a cold treated sample); 2) ‘Change’ calls, made by Affymetrix Microarray Suite, in pair-comparison were ‘I’(for ‘increase’); 3) the ‘Fold Change’ in pair-comparison was 3-fold or higher. The expression of the resulting 306 genes was further analyzed and compared with that in ice1 mutant. A two-fold difference between changes in the wild type and ice1 was used as a threshold to categorize genes. Transcription factors are shown in gray blocks. Genes used for RNA hybridization analysis are in bold. The fold change values for 22 genes in cold-treated ice1 were not determined (ND) because their signal intensities were similar to the background value (i.e. genes with ‘Absent’ calls in cold-treated ice1). These 22 genes were all cold-induced in the wild type. Therefore, they were included in the category of cold-responsive genes with lower induction in ice1 than in the wild type.  
               TABLE 1A                          Cold-responsive genes with lower induction in ice1                         Fold Change                                 Probe Set   AGI ID   Gene Title   WT   ice1                                         254074_at   At4g25490   CBF1/DREB1B   445.7   64.0       254066_at   At4g25480   CBF3/DREB1A   78.8   29.9       258325_at   At3g22830   putative heat shock transcription factor1   42.2   5.7       246432_at   At5g17490   RGA-like protein   34.3   ND       261648_at   At1g27730   salt-tolerance zinc finger protein   24.3   6.5       247655_at   At5g59820   zinc finger protein Zat12   19.7   7.0       248160_at   At5g54470   CONSTANS B-box zinc finger family protein   19.7   9.8       250781_at   At5g05410   DRE binding protein (DREB2A)   14.9   4.9       251745_at   At3g55980   Zn finger transcription factor (PE11)   13.9   3.0       258139_at   At3g24520   heat shock transcription factor HSF1, putative   13.9   3.7       245711_at   At5g04340   putative c2h2 zinc finger transcription factor   11.3   5.3       245250_at   At4g17490   ethylene-responsive element binding factor 6 (AtERF6)   8.6   4.3       252214_at   At3g50260   EREBP-3 homolog   8.6   2.1       261613_at   At1g49720   abscisic acid responsive elements-binding factor   7.0   3.5       245078_at   At2g23340   putative AP2 domain transcription factor   5.7   1.4       263379_at   At2g40140   putative CCCH-type zinc finger protein   5.7   2.6       253405_at   At4g32800   transcription factor TINY, putative   5.3   ND       245807_at   At1g46768   AP2 domain protein RAP2.1   4.9   1.9       259432_at   At1g01520   myb family transcription factor   4.9   2.3       252278_at   At3g49530   NAC2-like protein   4.6   2.0       253485_at   At4g31800   WRKY family transcription factor   4.6   −1.2       251272_at   At3g61890   homeobox-leucine zipper protein ATHB-12   4.3   1.3       261470_at   At1g28370   ethylene-responsive element binding factor 11 (AtERF11)   4.3   1.7       261892_at   At1g80840   WRKY family transcription factor   4.3   1.2       263783_at   At2g46400   WRKY family transcription factor   4.3   1.4       257022_at   At3g19580   zinc finger protein, putative   3.7   1.4       267252_at   At2g23100   CHP-rich zinc finger protein, putative   3.7   ND       249746_at   At5g24590   NAC2-like protein   3.5   1.6       256093_at   At1g20823   putative RING zinc finger protein   3.5   1.4       252009_at   At3g52800   zinc finger-like protein   3.2   1.3       256185_at   At1g51700   Dof zinc finger protein   3.2   1.6       260763_at   At1g49220   RING-H2 finger protein RHA3a, putative   3.2   ND       245749_at   At1g51090   proline-rich protein, putative   73.5   7.5       264217_at   At1g60190   hypothetical protein   68.6   26.0       246467_at   At5g17040   UDP glucose:flavonoid 3-o-glucosyltransferase-like protein   29.9   ND       251793_at   At3g55580   regulator of chromosome condensation-like protein   27.9   6.1       262452_at   At1g11210   expressed protein   27.9   7.0       264661_at   At1g09950   hypothetical protein   27.9   ND       258947_at   At3g01830   expressed protein   26.0   2.5       246178_s_at   At5g28430   putative protein   19.7   7.0       253104_at   At4g36010   thaumatin-like protein   19.7   2.5       257391_at   At2g32050   hypothetical protein   19.7   ND       245627_at   At1g56600   water stress-induced protein, putative   18.4   ND       247208_at   At5g64870   nodulin-like   18.4   1.2       256114_at   At1g16850   expressed protein   18.4   2.6       256356_s_at   At1g66500   hypothetical protein   18.4   3.0       250098_at   At5g17350   putative protein   17.1   3.2       264758_at   At1g61340   late embryogenesis abundant protein, putative   17.1   5.3       246099_at   At5g20230   blue copper binding protein   16.0   1.3       257280_at   At3g14440   9-cis-epoxycarotenoid dioxygenase (neoxanthin cleavage   16.0   1.0               enzyme) (NC1) (NCED1), putative       251336_at   At3g61190   putative protein   13.9   3.0       260264_at   At1g68500   hypothetical protein   13.9   3.0       263497_at   At2g42540   COR15a   13.9   3.5       248337_at   At5g52310   RD29A/COR78/LTI78   13.0   4.6       248959_at   At5g45630   putative protein   13.0   2.0       259977_at   At1g76590   expressed protein   13.0   2.5       260399_at   At1g72520   putative lipoxygenase   13.0   1.5       259879_at   At1g76650   putative calmodulin   12.1   2.8       265290_at   At2g22590   putative anthocyanidin-3-glucoside rhamnosyltransferase   12.1   ND       267411_at   At2g34930   disease resistance protein family   12.1   1.1       250648_at   At5g06760   late embryogenesis abundant protein LEA like   11.3   1.9       257876_at   At3g17130   hypothetical protein   11.3   2.3       260727_at   At1g48100   polygalacturonase, putative   11.3   2.3       246125_at   At5g19875   Expressed protein   10.6   2.0       251603_at   At3g57760   putative protein   10.6   1.1       256017_at   At1g19180   expressed protein   10.6   1.5       264617_at   At2g17660   unknown protein   10.6   ND       264787_at   At2g17840   putative senescence-associated protein 12   10.6   3.5       245757_at   At1g35140   phosphate-induced (phi-1) protein, putative   9.8   1.6       252346_at   At3g48650   hypothetical protein   9.8   2.0       253643_at   At4g29780   expressed protein   9.8   3.2       254667_at   At4g18280   glycine-rich cell wall protein-like   9.8   1.2       264389_at   At1g11960   unknown protein   9.8   1.7       266545_at   At2g35290   hypothetical protein   9.8   1.4       266720_s_at   At2g46790   expressed protein   9.8   4.9       245251_at   At4g17615   calcineurin B-like protein 1   9.2   3.0       247431_at   At5g62520   putative protein   9.2   1.5       248964_at   At5g45340   cytochrome p450 family   9.2   3.0       252368_at   At3g48520   cytochrome p450, putative   9.2   1.3       262164_at   At1g78070   expressed protein   9.2   4.3       252102_at   At3g50970   dehydrin Xero2   8.6   4.0       262359_at   At1g73070   leucine rich repeat protein family   8.6   ND       262731_at   At1g16420   hypothetical protein common family   8.6   2.8       245677_at   At1g56660   hypothetical protein   8.0   2.8       245734_at   At1g73480   lysophospholipase homolog, putative   8.0   2.6       247177_at   At5g65300   expressed protein   8.0   3.7       250053_at   At5g17850   potassium-dependent sodium-calcium exchanger-like protein   8.0   2.0       254120_at   At4g24570   mitochondrial carrier protein family   8.0   3.0       254926_at   At4g11280   ACC synthase (AtACS-6)   8.0   2.3       263789_at   At2g24560   putative GDSL-motif lipase/hydrolase   8.0   ND       245346_at   At4g17090   glycosyl hydrolase family 14 (beta-amylase)   7.5   2.6       253425_at   At4g32190   putative protein   7.5   3.2       254085_at   At4g24960   abscisic acid-induced-like protein   7.5   2.3       259076_at   At3g02140   expressed protein   7.5   1.1       260227_at   At1g74450   expressed protein   7.5   2.0       260915_at   At1g02660   expressed protein   7.5   1.7       262677_at   At1g75860   unknown protein   7.5   2.6       266532_at   At2g16890   putative glucosyltransferase   7.5   3.0       247925_at   At5g57560   xyloglucan endotransglycosylase (TCH4)   7.0   2.1       252563_at   At3g45970   putative protein   7.0   1.2       254850_at   At4g12000   putative protein   7.0   2.3       260744_at   At1g15010   expressed protein   7.0   1.7       263931_at   At2g36220   expressed protein   7.0   3.0       245306_at   At4g14690   Expressed protein   6.5   2.6       246495_at   At5g16200   putative protein   6.5   1.7       248870_at   At5g46710   putative protein   6.5   2.6       253292_at   At4g33985   Expressed protein   6.5   2.0       253872_at   At4g27410   putative protein   6.5   1.5       258792_at   At3g04640   expressed protein   6.5   3.0       259516_at   At1g20450   expressed protein   6.5   3.2       262050_at   At1g80130   expressed protein   6.5   1.2       245427_at   At4g17550   putative protein   6.1   1.2       253859_at   At4g27657   Expressed protein   6.1   ND       261187_at   At1g32860   glycosyl hydrolase family 17   6.1   1.4       262448_at   At1g49450   En/Spm-like transposon protein, putative   6.1   ND       266757_at   At2g46940   unknown protein   6.1   1.3       252131_at   At3g50930   BCS1 protein-like protein   5.7   1.6       255795_at   At2g33380   RD20 protein   5.7   −1.3       258321_at   At3g22840   early light-induced protein   5.7   2.3       262496_at   At1g21790   expressed protein   5.7   2.0       265119_at   At1g62570   flavin-containing monooxygenase, putative   5.7   2.0       246018_at   At5g10695   Expressed protein   5.3   1.7       248820_at   At5g47060   putative protein   5.3   1.7       249918_at   At5g19240   putative protein   5.3   1.9       253830_at   At4g27652   Expressed protein   5.3   1.7       246490_at   At5g15950   S-adenosylmethionine decarboxylase (adoMetDC2)   4.9   2.1       253284_at   At4g34150   putative protein   4.9   1.9       253323_at   At4g33920   putative protein   4.9   2.5       253614_at   At4g30350   putative protein   4.9   1.5       264655_at   At1g09070   expressed protein   4.9   1.9       245533_at   At4g15130   putative phosphocholine cytidylyltransferase   4.6   2.1       246831_at   At5g26340   hexose transporter-like protein   4.6   2.0       247137_at   At5g66210   calcium-dependent protein kinase   4.6   1.4       247226_at   At5g65100   putative protein   4.6   ND       250467_at   At5g10100   trehalose-6-phosphate phosphatase-like protein   4.6   ND       252414_at   At3g47420   putative protein   4.6   1.9       252997_at   At4g38400   putative pollen allergen   4.6   1.1       253595_at   At4g30830   putative protein   4.6   ND       253832_at   At4g27654   Expressed protein   4.6   1.3       258188_at   At3g17800   expressed protein   4.6   1.4       259479_at   At1g19020   Expressed protein   4.6   1.7       261405_at   At1g18740   expressed protein   4.6   2.0       262881_at   At1g64890   expressed protein   4.6   2.1       264000_at   At2g22500   mitochondrial carrier protein family   4.6   2.0       265668_at   At2g32020   putative alanine acetyl transferase   4.6   2.3       265797_at   At2g35715   Expressed protein   4.6   ND       248686_at   At5g48540   33 kDa secretory protein-like   4.3   1.6       250676_at   At5g06320   harpin-induced protein-like   4.3   1.6       251259_at   At3g62260   protein phosphatase 2C (PP2C)   4.3   2.1       254300_at   At4g22780   Translation factor EF-1 alpha-like protein   4.3   −1.1       261356_at   At1g79660   unknown protein   4.3   1.6       264636_at   At1g65490   expressed protein   4.3   1.4       246468_at   At5g17050   UDP glucose:flavonoid 3-o-glucosyltransferase-like protein   4.0   2.0       248607_at   At5g49480   NaCl-inducible Ca2+-binding protein-like; calmodulin-like   4.0   1.5       250279_at   At5g13200   ABA-responsive protein-like   4.0   1.2       252053_at   At3g52400   syntaxin SYP122   4.0   1.9       256633_at   At3g28340   unknown protein   4.0   2.0       258207_at   At3g14050   putative GTP pyrophosphokinase   4.0   1.7       258805_at   At3g04010   glycosyl hydrolase family 17   4.0   1.3       261912_s_at   At1g66000   hypothetical protein   4.0   ND       264989_at   At1g27200   expressed protein   4.0   1.6       265276_at   At2g28400   hypothetical protein   4.0   −1.1       267261_at   At2g23120   expressed protein   4.0   1.7       247693_at   At5g59730   putative protein   3.7   1.9       253113_at   At4g35985   putative protein   3.7   1.4       253165_at   At4g35320   putative protein   3.7   1.9       253879_s_at   At4g27570   UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase-like protein   3.7   1.2       253915_at   At4g27280   putative protein   3.7   1.9       259426_at   At1g01470   hypothetical protein   3.7   1.6       259445_at   At1g02400   dioxygenase, putative   3.7   1.9       260410_at   At1g69870   putative peptide transporter   3.7   1.1       261581_at   At1g01140   serine threonine kinase, putative   3.7   1.5       262113_at   At1g02820   late embryogenis abundant protein, putative   3.7   1.1       262382_at   At1g72920   disease resistance protein (TIR-NBS class), putative   3.7   1.9       265665_at   At2g27420   cysteine proteinase   3.7   1.0       267069_at   At2g41010   unknown protein   3.7   1.2       245450_at   At4g16880   disease resistance RPP5 like protein (fragment)   3.5   ND       246289_at   At3g56880   putative protein   3.5   1.3       249204_at   At5g42570   expressed protein   3.5   1.5       249622_at   At5g37550   putative protein   3.5   1.4       250335_at   At5g11650   lysophospholipase-like protein   3.5   1.7       251372_at   At3g60520   putative protein   3.5   1.5       254707_at   At4g18010   putative protein   3.5   1.1       257154_at   At3g27210   expressed protein   3.5   1.5       259705_at   At1g77450   GRAB1-like protein   3.5   1.3       261037_at   At1g17420   lipoxygenase   3.5   1.2       261937_at   At1g22570   peptide transporter, putative   3.5   1.6       264024_at   At2g21180   expressed protein   3.5   1.1       264458_at   At1g10410   unknown protein   3.5   1.2       266799_at   At2g22860   unknown protein   3.5   1.4       247280_at   At5g64260   phi-1-like protein   3.2   1.6       251356_at   At3g61060   putative protein   3.2   1.5       252316_at   At3g48700   putative protein   3.2   1.3       253824_at   At4g27940   putative protein   3.2   1.1       256526_at   At1g66090   disease resistance protein (TIR-NBS class), putative   3.2   1.4       256595_x_at   At3g28530   hypothetical protein   3.2   1.1       265648_at   At2g27500   glycosyl hydrolase family 17   3.2   1.1       266097_at   At2g37970   expressed protein   3.2   1.6       267335_s_at   At2g19440   glycosyl hydrolase family 17   3.2   1.6       245699_at   At5g04250   putative protein   3.0   1.2       247467_at   At5g62130   putative protein   3.0   1.5       249583_at   At5g37770   CALMODULIN-RELATED PROTEIN 2, TOUCH-INDUCED (TCH2)   3.0   1.1       249626_at   At5g37540   putative protein   3.0   1.2       252474_at   At3g46620   putative protein   3.0   1.3       253628_at   At4g30280   xyloglucan endotransglycosylase, putative   3.0   1.4       253835_at   At4g27820   glycosyl hydrolase family 1   3.0   1.2       254158_at   At4g24380   putative protein   3.0   1.4       254188_at   At4g23920   UDPglucose 4-epimerase like protein   3.0   1.2       254634_at   At4g18650   putative protein   3.0   ND       254973_at   At4g10460   putative retrotransposon   3.0   ND       256763_at   At3g16860   unknown protein   3.0   1.0       257519_at   At3g01210   RRM-containing protein   3.0   −1.1       258894_at   At3g05650   disease resistance protein family   3.0   1.4       265841_at   At2g35710   putative glycogenin   3.0   1.5       266271_at   At2g29440   glutathione transferase, putative   3.0   1.2       266316_at   At2g27080   expressed protein   3.0   1.1       267631_at   At2g42150   hypothetical protein   3.0   1.1                  
 
     [0112]               TABLE 1B                          Cold-responsive genes with similar induction in the wild type and ice1                         Fold Change                                 Probe Set   AGI ID   Gene Title   WT   ice1                                         254075_at   At4g25470   CBF2/DREB1C   104.0   137.2       261263_at   At1g26790   Dof zinc finger protein   68.6   55.7       257262_at   At3g21890   CONSTANS B-box zinc finger family protein   7.5   5.3       259834_at   At1g69570   Dof zinc finger protein   7.0   5.7       256430_at   At3g11020   DREB2B   6.1   4.9       248389_at   At5g51990   DRE binding protein   5.3   4.0       257053_at   At3g15210   AtERF4   5.3   2.8       248744_at   At5g48250   CONSTANS B-box zinc finger family protein   4.9   3.0       249606_at   At5g37260   CCA1, putative   4.9   9.2       267028_at   At2g38470   WRKY family transcription factor   4.9   3.0       246523_at   At5g15850   CONSTANS-LIKE 1   4.0   3.2       248799_at   At5g47230   AtERF5   4.0   3.5       247452_at   At5g62430   Dof zinc finger protein   3.7   3.7       251190_at   At3g62690   RING-H2 zinc finger protein ATL5   3.7   2.1       253722_at   At4g29190   Zn finger protein, putative   3.7   4.6       259992_at   At1g67970   putative heat shock transcription factor   3.7   2.3       263252_at   At2g31380   CONSTANS-like B-box zinc finger protein   3.7   3.7       263739_at   At2g21320   CONSTANS B-box zinc finger family protein   3.7   2.3       252429_at   At3g47500   Dof zinc finger protein   3.5   4.3       253140_at   At4g35480   RING-H2 finger protein RHA3b   3.5   2.0       258742_at   At3g05800   bHLH protein   3.5   6.5       265939_at   At2g19650   CHP-rich zinc finger protein, putative   3.5   2.8       249415_at   At5g39660   Dof zinc finger protein   3.2   3.7       259364_at   At1g13260   DNA-binding protein (RAV1)   3.2   2.1       262590_at   At1g15100   putative RING-H2 zinc finger protein   3.2   2.0       263823_s_at   At2g40350   AP2 domain transcription factor   3.0   5.7       264511_at   At1g09350   putative galactinol synthase   17.1   12.1       264314_at   At1g70420   expressed protein   13.0   9.8       247478_at   At5g62360   DC1.2 homologue-like protein   11.3   11.3       253322_at   At4g33980   putative protein   11.3   8.0       249741_at   At5g24470   putative protein   8.0   6.5       247047_at   At5g66650   putative protein   7.0   4.3       263495_at   At2g42530   COR15b   6.5   9.8       265536_at   At2g15880   unknown protein   6.5   5.3       249174_at   At5g42900   putative protein   6.1   3.5       249191_at   At5g42760   putative protein   6.1   4.3       264153_at   At1g65390   disease resistance protein (TIR class), putative   6.1   4.9       250099_at   At5g17300   expressed protein   5.7   7.5       265725_at   At2g32030   putative alanine acetyl transferase   5.7   3.0       246922_at   At5g25110   serine/threonine protein kinase-like protein   4.9   4.9       251494_at   At3g59350   protein kinase-like protein   4.9   2.8       246821_at   At5g26920   calmodulin-binding protein   4.6   2.6       255733_at   At1g25400   expressed protein   4.6   3.0       257650_at   At3g16800   protein phosphatase 20 (PP2C)   4.6   2.5       266832_at   At2g30040   putative protein kinase   4.6   2.8       267357_at   At2g40000   putative nematode-resistance protein   4.6   3.7       249411_at   At5g40390   glycosyl hydrolase family 36   4.3   3.5       256266_at   At3g12320   expressed protein   4.3   4.0       252956_at   At4g38580   copper chaperone (CCH)-related   4.0   2.3       253455_at   At4g32020   putative protein   4.0   2.6       259570_at   At1g20440   hypothetical protein   4.0   2.3       262383_at   At1g72940   disease resistance protein (TIR-NBS class), putative   4.0   2.1       247393_at   At5g63130   unknown protein   3.7   3.2       252661_at   At3g44450   putative protein   3.7   2.5       259990_s_at   At1g68050   F-box protein FKF1/ADO3, AtFBX2a   3.7   2.3       264213_at   At1g65400   hypothetical protein   3.7   2.0       245777_at   At1g73540   unknown protein   3.5   2.5       248745_at   At5g48260   unknown protein   3.5   2.5       248846_at   At5g46500   putative protein   3.5   2.6       249063_at   At5g44110   ABC transporter family protein   3.5   2.1       257654_at   At3g13310   DnaJ protein, putative   3.5   2.1       257925_at   At3g23170   expressed protein   3.5   1.9       261048_at   At1g01420   flavonol 3-o-glucosyltransferase, putative   3.5   2.0       263216_s_at   At1g30720   FAD-linked oxidoreductase family   3.5   2.3       265184_at   At1g23710   expressed protein   3.5   2.3       245558_at   At4g15430   hypothetical protein   3.2   3.5       248164_at   At5g54490   putative protein   3.2   2.5       248502_at   At5g50450   putative protein   3.2   4.3       252010_at   At3g52740   expressed protein   3.2   2.5       253679_at   At4g29610   cytidine deaminase 6 (CDA6)   3.2   2.0       256548_at   At3g14770   expressed protein   3.2   1.9       256577_at   At3g28220   unknown protein   3.2   2.3       257083_s_at   At3g20590   non-race specific disease resistance protein, putative   3.2   2.3       260046_at   At1g73800   Expressed protein   3.2   2.0       261958_at   At1g64500   peptide transporter, putative   3.2   2.6       263352_at   At2g22080   En/Spm-like transposon protein   3.2   1.9       263452_at   At2g22190   putative trehalose-6-phosphate phosphatase   3.2   2.3       265093_at   At1g03905   ABC transporter family protein   3.2   1.7       267293_at   At2g23810   hypothetical protein   3.2   1.7       245119_at   At2g41640   expressed protein   3.0   2.6       246270_at   At4g36500   putative protein   3.0   3.0       247793_at   At5g58650   putative protein   3.0   1.7       256442_at   At3g10930   expressed protein   3.0   1.9       256487_at   At1g31540   disease resistance protein (TIR-NBS-LRR class), putative   3.0   2.1       259428_at   At1g01560   MAP kinase, putative   3.0   1.7       266834_s_at   At2g30020   protein phosphatase 2C (PP2C)   3.0   2.6       267364_at   At2g40080   expressed protein   3.0   2.3                    
     [0113]               TABLE 1C                          Cold responsive genes with higher induction in ice1                         Fold Change                                 Probe Set   AGI ID   Gene Title   WT   ice1                                         261248_at   At1g20030   calreticulin, putative   4.6   13.9       258383_at   At3g15440   hypothetical protein   4.3   9.2                    
     [0114] The ice1 Mutation Impairs Chilling and Freezing Tolerance  
     [0115] At normal growth temperatures, ice1 and wild type seedlings were similar in size (FIG. 2A). Although adult ice1 plants were smaller, they were not very different from the wild type in flowering time and fertility (FIG. 2B). Ten-day-old seedlings of ice1 and wild type grown on separate halves of the same agar plates were cold acclimated at 4° C. for four days and then subjected to a freezing tolerance assay. The ice1 mutant was less freezing-tolerant than the wild type at all freezing temperatures (FIGS. 2C and 2D). Freezing at −10° C. for 2 hours killed about 50% of ice1 mutant plants whereas less than 20% of wild type plants were killed at this temperature (FIG. 2D). When newly germinated (at 22° C.) ice1 and wild type seedlings were transferred to 4° C. (with 30±2 μmol quanta. m −2 .s −1  light), chilling injury became apparent in the mutant after 4 weeks of cold treatment (FIG. 2E). After 6 weeks of chilling stress, 100% of wild type but only 20% of ice1 mutant plants survived (FIG. 2F).  
     [0116] Positional Cloning of ICE1  
     [0117] To map the ice1 mutation, a homozygous ice1 mutant in the CBF3-LUC Columbia background was crossed to wild type plants of the Ler ecotype. F1 plants from the cross were selfed to produce F2 seeds. Since the ice1 mutation is dominant, we selected from the segregating F2 population seedlings with the wild type phenotype (based on plant size and morphology) for mapping. A total of 662 wild type plants were selected and used for mapping with simple sequence length polymorphism and cleaved amplified polymorphic sequence markers (see Materials and Methods section for details), which initially placed ice1 on the middle of chromosome 3, then narrowed its location to a 58 kb region on the MLJ15 and MDJ14 BAC clones. Candidate genes in this region were amplified from homozygous ice1 mutant plants and sequenced. The sequences were compared with the published sequence of Arabidopsis ecotype Columbia and a single G to A mutation in the hypothetical MLJ15.14 gene was found.  
     [0118] To confirm that MLJ15.14 is the ICE1 gene, the MLJ15.14 gene including 2,583 bp upstream of the initiation codon and 615 bp downstream of the stop codon was cloned from ice1 mutant plants. This fragment was inserted into a binary vector and introduced into CBF3-LUC Columbia wild type plants by Agrobacterium-mediated transformation. Transgenic plants were selected based on their hygromycin resistance, and cold-induced bioluminescence in the T2 lines was compared with that of the wild type. The MLJ 15.14 gene from ice1 suppressed cold-induced luminescence from the wild type plants (FIGS. 3A and 3B) and reduced the plant height to that of ice1 mutant, thus confirming that MLJ 15.14 is ICE1.  
     [0119] ICE1 Encodes a Constitutively Expressed and Nuclear Localized MYC-Like Basic Helix-Loop-Helix Transcription Factor  
     [0120] The open reading frame of ICE1 (SEQ ID NO: 1)was determined by sequencing cDNAs obtained by RT-PCR. The open reading frame was determined to be:  
                              1   atcaaaaaaa aagtttcaat ttttgaaagc tctgagaaat gaatctatca ttctctctct                   61   ctatctctat cttccttttc agatttcgct tcttcaattc atgaaatcct cgtgattcta               121   ctttaatgct tctctttttt tacttttcca agtctctgaa tattcaaagt atatatcttt               181   tgttttcaaa cttttgcaga attgtcttca agcttccaaa tttcagttaa aggtctcaac               241   tttgcagaat tttcctctaa aggttcagac tttggggtaa aggtgtcaac tttggcgatg               301   ggtcttgacg gaaacaatgg tggaggggtt tggttaaacg gtggtggtgg agaaagggaa               361   gagaacgagg aaggttcatg gggaaggaat caagaagatg gttcttctca gtttaagcct               421   atgcttgaag gtgattggtt tagtagtaac caaccacatc cacaagatct tcagatgtta               481   cagaatcagc cagatttcag atactttggt ggttttcctt ttaaccctaa tgataatctt               541   cttcttcaac actctattga ttcttcttct tcttgttctc cttctcaagc ttttagtctt               601   gacccttctc agcaaaatca gttcttgtca actaacaaca acaagggttg tcttctcaat               661   gttccttctt ctgcaaaccc ttttgataat gcttttgagt ttggctctga atctggtttt               721   cttaaccaaa tccatgctcc tatttcgatg gggtttggtt ctttgacaca attggggaac               781   agggatttga gttctgttcc tgatttcttg tctgctcggt cacttcttgc gccggaaagc               841   aacaacaaca acacaatgtt gtgtggtggt ttcacagctc cgttggagtt ggaaggtttt               901   ggtagtcctg ctaatggtgg ttttgttggg aacagagcga aagttctgaa gcctttagag               961   gtgttagcat cgtctggtgc acagcctact ctgttccaga aacgtgcagc tatgcgtcag               1021   agctctggaa gcaaaatggg aaattcggag agttcgggaa tgaggaggtt tagtgatgat               1081   ggagatatgg atgagactgg gattgaggtt tctgggttga actatgagtc tgatgagata               1141   aatgagagcg gtaaagcggc tgagagtgtt cagattggag gaggaggaaa gggtaagaag               1201   aaaggtatgc ctgctaagaa tctgatggct gagaggagaa ggaggaagaa gcttaatgat               1261   aggctttata tgcttagatc agttgtcccc aagatcagca aaatggatag agcatcaata               1321   cttggagatg caattgatta tctgaaggaa cttctacaaa ggatcaatga tcttcacaat               1381   gaacttgagt caactcctcc tggatctttg cctccaactt catcaagctt ccatccgttg               1441   acacctacac cgcaaactct ttcttgtcgt gtcaaggaag agttgtgtcc ctcttcttta               1501   ccaagtccta aaggccagca agctagagtt gaggttagat taagggaagg aagagcagtg               1561   aacattcata tgttctgtgg tcgtagaccg ggtctgttgc tcgctaccat gaaagctttg               1621   gataatcttg gattggatgt tcagcaagct gtgatcagct gttttaatgg gtttgccttg               1681   gatgttttcc gcgctgagca atgccaagaa ggacaagaga tactgcctga tcaaatcaaa               1741   gcagtgcttt tcgatacagc agggtatgct ggtatgatct gatctgatcc tgacttcgag               1801   tccattaagc atctgttgaa gcagagctag aagaactaag tccctttaaa tctgcaattt               1861   tcttctcaac tttttttctt atgtcataac ttcaatctaa gcatgtaatg caattgcaaa               1921   tgagagttgt ttttaaatta agcttttgag aacttgaggt tgttgttgtt ggatacataa               1981   cttcaacctt ttattagcaa tgttaacttc catttatgtc t          
 
     [0121] ICE1 is predicted to encode a protein of 494 amino acids, with an estimated molecular mass of 53.5 kDa as follows (SEQ ID NO: 2):  
                          MGLDGNNGGGVWLNGGGGEREENEEGSWGRNQEDGSSQFKPMLEGDWFSSNQPHPQDLQMLQNQP                   DFRYFGGFPFNPNDNLLLQHSIDSSSSCSPSQAFSLDPSQQNQFLSTNNNKGCLLNVPSSANPFDNAFEF               GSESGFLNQIHAPISMGFGSLTQLGNRDLSSVPDFLSARSLLAPESNNNNTMLCGGFTAPLELEGFGSPA               NGGFVGNRAKVLKPLEVLASSGAQPTLFQKRAAMRQSSGSKMGNSESSGMRRFSDDGDMDETGIEVS               GLNYESDEINESGKAAESVQIGGGGKGKKKGMPAKNLMAERRRRKKLNDRLYMLRSVVPKISKMDR               ASILGDAIDYLKELLQRINDLHNELESTPPGSLPPTSSSFHPLTPTPQTLSCRVKEELCPSSLPSPKGQQAR               VEVRLREGRAVNIHMFCGRRPGLLLATMKALDNLGLDVQQAVISCFNGFALDVFRAEQCQEGQEILPD               QIKAVLFDTAGYAGMI          
 
     [0122] Database searches revealed that ICE1 contains a MYC-like basic helix-loop-helix (bHLH) domain at its C-terminal half (FIGS. 4A and 4B). Over the entire length of the protein, ICE1 shows amino acid sequence similarity to an unknown protein of Arabidopsis (At1g12860). The ice1 mutation changes Arg236, conserved in these two Arabidopsis proteins, to His. The bHLH domain of ICE1 shows high amino acid similarity to that of known MYC-related bHLH transcription factors (FIG. 4B). All MYC binding promoter elements contain the CA nucleotides that are contacted by a conserved glutamic acid in the bHLH zipper domain (Grandori et al., 2000). This glutamic acid residue (Glu312) is also conserved in the basic DNA binding domain of ICE1 (FIG. 4B). An acidic domain near the amino terminus characterizes the bHLH family of transcription factors and a conserved bHLH DNA binding and dimerization domain near the carboxyl terminus (Purugganan and Wessler 1994). All these features are present in ICE1 protein (FIG. 4A).  
     [0123] To analyze the expression pattern of ICE1 in different tissues, T2 lines of transgenic Arabidopsis plants expressing an ICE1 promoter-GUS transgene were analyzed. GUS expression was detected in roots, leaves, stem and floral parts. Semi-quantitative RT-PCR analysis also showed that ICE1 was expressed constitutively and the expression was stronger in leaves and stems than in other tissues (FIGS. 5A and 5B). RNA blot analysis showed that the ICE1 transcript was slightly up-regulated by cold, NaCl and ABA but not by dehydration (FIG. 5C).  
     [0124] To examine the subcellular localization of the ICE1 protein, ICE1 was fused in-frame to the C-terminal side of the green fluorescent protein (GFP) and expressed under control of the CaMV 35S promoter. Confocal imaging of GFP fluorescence in T2 transgenic plants showed that the GFP-ICE1 fusion protein is present in the nucleus under either warm (FIG. 5D) or cold temperatures.  
     [0125] ICE1 Binds to MYC Recognition Sites in the CBF3 Promoter  
     [0126] ICE1 has a basic helix-loop-helix (bHLH) domain and its amino acid sequence in the basic region is highly conserved with other bHLH proteins (FIG. 4B), and therefore may recognize promoter elements similar to the DNA-binding sites for known bHLH proteins. These proteins recognize DNA with the consensus sequence CANNTG (Meshi and Iwabuchi 1995). In the promoter region of CBF3, there are five potential MYC-recognition elements within a 1 kb region upstream of the transcription initiation site (Shinwari et al. 1998). These possible MYC-recognition sites, designated MYC-1 through MYC-5, fall into four groups because MYC-3 and MYC-5 share the same consensus sequence, CATTTG (FIG. 6A). Thus, MYC-3 was used to represent both MYC-3 and MYC-5. To determine whether ICE1 binds to these MYC-recognition sites in the CBF3 promoter, we expressed and purified His-ICE1 fusion protein from  E. coli.  Four DNA fragments encompassing each possible MYC-recognition site were used for interaction with His-ICE1 in an electrophoresis mobility shift assay (EMSA).  
     [0127] Several complexes were observed when ICE1 was incubated with any of the four DNA fragments (MYC-1 through MYC-4), indicating that ICE1 is able to bind to these sequences (FIG. 6B). The MYC-2 fragment formed one major complex with ICE1, while the other DNA fragments formed several complexes with ICE1. These complexes were abolished by the addition of increasing amounts of cold competitors with the same sequences, but not by P1 or P2, which contains a putative MYB- recognition site and a non-related sequence, respectively (FIG. 6B). This specificity of competition strengthens the hypothesis that the interaction between DNA and ICE1 requires the MYC-recognition sequences. When the MYC-2 fragment was used as a probe, the complex was most efficiently competed off by the cold MYC-2 competitor, suggesting that ICE1 has a higher affinity for the MYC-2 site than for the other sites (FIG. 6C). The complex formed by ICE1 and the MYC-2 fragment was less affected by a mutated competitor than by the wild type competitor (FIG. 6D). Together, these results show that ICE1 interacts specifically with the MYC-recognition sites in the CBF3 promoter. The ice1 mutation does not appear to affect ICE1 interaction with the CBF3 promoter, because the Arg236 to His mutant form of ICE1 was also able to bind to the MYC-2 probe (FIG. 6E).  
     [0128] ICE1 is a Transcriptional Activator that Positively Regulates CBF Expression  
     [0129] Transient expression assays were carried out to determine whether ICE1 acts as a transcriptional activator or repressor. An effector plasmid was constructed by fusing ICE1 with the DNA binding domain of the yeast GAL4 transcriptional activator (GAL4-ICE1, FIG. 7A). When the wild type GAL4-ICE1 and a GAL4-responsive reporter gene, GAL4-LUC, were delivered into Arabidopsis leaves by particle bombardment, the luciferase activity increased 20 fold relative to the control with or without an effector plasmid containing only the GAL4 DNA binding domain (FIG. 7B). The Arg236 to His mutant form of GAL4-ICE1 also activated the GAL4-responsive transcription (FIG. 7B). These results suggest that ICE1 is a transcriptional activator, and that the ice1 mutation does not affect the function of the transcriptional activation domain.  
     [0130] A null allele of ice1 created by T-DNA insertion does not show any phenotypes of the dominant ice1 mutant, suggesting that there is functional redundancy in the ICE1 gene family. We overexpressed ICE1 in wild type Arabidopsis plants by using the strong constitutive super promoter. None of the overexpression lines showed any ice1 mutant phenotypes. RNA blot analysis showed that ICE1 -overexpression did not activate CBF3 expression at warm temperatures. However, ICE1 -overexpression enhanced the expression of the endogenous CBF3 gene as well as the CBF3-LUC reporter gene in the cold (FIGS. 7C and 7D). Cold-induction of CBF2, RD29A and COR15A was also enhanced in the Super-25 ICE1 transgenic plants (FIG. 7C). When the Super-ICE1transgenic plants and wild type control plants in the same agar plates were cold acclimated at 4° C. for 5 days and then subjected to freezing treatment at −8° C. for 4 hours, the ICE1 overexpression transgenic seedlings showed a higher survival rate (75.9±6.5%) than that of control plants (37.2±12.6%) (FIG. 7E). The ICE1 overexpression transgenic plants did not exhibit obvious growth or developmental abnormalities. These results suggest that ICE1 is a positive regulator of CBF3, and that the dominant nature of ice1 is likely caused by a dominant negative effect of the mutation.  
     [0131] Numerous modifications and variations on the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.  
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     [0146] Jaglo-Ottosen, K. R., Gilmour, S. J., Zarka, D. G., Schabenberger, O., and Thomashow, M. F. 1998. Arabidopsis CBF1 overexpression induces cor genes and enhances freezing tolerance.  Science  280: 104-106.  
     [0147] Knight, H., Veale, E. L., Warren, G. J., and Knight, M. R. 1999. The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif.  Plant Cell  11: 875-886.  
     [0148] Kurkela, S., and Franck, M. 1990. Cloning and characterization of a cold- and ABA-inducible Arabidopsis gene.  Plant Mol. Biol.  15: 137-144.  
     [0149] Lee, H., Guo, Y., Ohta, M., Xiong, L., Stevenson, B., and Zhu, J.-K. 2002. LOS2, a genetic locus required for cold-responsive gene transcription encodes a bifunctional enolase.  EMBO. J.  21: 2692-2702.  
     [0150] Lee, H., Xiong, L., Gong, Z. , Ishitani, M., Stevenson, B., and Zhu, J. K. 2001. The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning.  Genes Dev.  15: 912-924.  
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     [0159] Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H., and Ohme-Takagi, M. 2001. Repression Domains of Class II ERF transcriptional repressors share an essential motif for active repression.  Plant Cell  13: 1959-1968.  
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     [0162] Spelt, C., Quattrocchio, F., Mol, J. N. M., and Koes, R. 2000. Anthocyanin1 of petunia encodes a basic-helix-loop-helix protein that directly activates transcription of structural anthocyanin genes.  Plant Cell  12: 1619-1631.  
     [0163] Stockinger, E. J. Gilmour, S. J., and Thomashow, M. F. 1997 . Arabidopsis thaliana  CBF1 encodes an AP2 domain-containing transcription activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit.  Proc. Natl. Acad. Sci. USA  94: 1035-1040.  
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     [0167] Xin, Z., and J. Browse 1998. eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant.  Proc. Natl. Acad. Sci. USA  95: 7799-7804.  
     [0168] Yamaguchi-Shinozaki, K., and Shinozaki, K. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress.  Plant Cell  6: 251-264.  
    
     
       
         1 
         
           
             42  
           
           
             1  
             2021  
             DNA  
             Arabidopsis thaliania  
           
            1 

atcaaaaaaa aagtttcaat ttttgaaagc tctgagaaat gaatctatca ttctctctct     60 

ctatctctat cttccttttc agatttcgct tcttcaattc atgaaatcct cgtgattcta    120 

ctttaatgct tctctttttt tacttttcca agtctctgaa tattcaaagt atatatcttt    180 

tgttttcaaa cttttgcaga attgtcttca agcttccaaa tttcagttaa aggtctcaac    240 

tttgcagaat tttcctctaa aggttcagac tttggggtaa aggtgtcaac tttggcgatg    300 

ggtcttgacg gaaacaatgg tggaggggtt tggttaaacg gtggtggtgg agaaagggaa    360 

gagaacgagg aaggttcatg gggaaggaat caagaagatg gttcttctca gtttaagcct    420 

atgcttgaag gtgattggtt tagtagtaac caaccacatc cacaagatct tcagatgtta    480 

cagaatcagc cagatttcag atactttggt ggttttcctt ttaaccctaa tgataatctt    540 

cttcttcaac actctattga ttcttcttct tcttgttctc cttctcaagc ttttagtctt    600 

gacccttctc agcaaaatca gttcttgtca actaacaaca acaagggttg tcttctcaat    660 

gttccttctt ctgcaaaccc ttttgataat gcttttgagt ttggctctga atctggtttt    720 

cttaaccaaa tccatgctcc tatttcgatg gggtttggtt ctttgacaca attggggaac    780 

agggatttga gttctgttcc tgatttcttg tctgctcggt cacttcttgc gccggaaagc    840 

aacaacaaca acacaatgtt gtgtggtggt ttcacagctc cgttggagtt ggaaggtttt    900 

ggtagtcctg ctaatggtgg ttttgttggg aacagagcga aagttctgaa gcctttagag    960 

gtgttagcat cgtctggtgc acagcctact ctgttccaga aacgtgcagc tatgcgtcag   1020 

agctctggaa gcaaaatggg aaattcggag agttcgggaa tgaggaggtt tagtgatgat   1080 

ggagatatgg atgagactgg gattgaggtt tctgggttga actatgagtc tgatgagata   1140 

aatgagagcg gtaaagcggc tgagagtgtt cagattggag gaggaggaaa gggtaagaag   1200 

aaaggtatgc ctgctaagaa tctgatggct gagaggagaa ggaggaagaa gcttaatgat   1260 

aggctttata tgcttagatc agttgtcccc aagatcagca aaatggatag agcatcaata   1320 

cttggagatg caattgatta tctgaaggaa cttctacaaa ggatcaatga tcttcacaat   1380 

gaacttgagt caactcctcc tggatctttg cctccaactt catcaagctt ccatccgttg   1440 

acacctacac cgcaaactct ttcttgtcgt gtcaaggaag agttgtgtcc ctcttcttta   1500 

ccaagtccta aaggccagca agctagagtt gaggttagat taagggaagg aagagcagtg   1560 

aacattcata tgttctgtgg tcgtagaccg ggtctgttgc tcgctaccat gaaagctttg   1620 

gataatcttg gattggatgt tcagcaagct gtgatcagct gttttaatgg gtttgccttg   1680 

gatgttttcc gcgctgagca atgccaagaa ggacaagaga tactgcctga tcaaatcaaa   1740 

gcagtgcttt tcgatacagc agggtatgct ggtatgatct gatctgatcc tgacttcgag   1800 

tccattaagc atctgttgaa gcagagctag aagaactaag tccctttaaa tctgcaattt   1860 

tcttctcaac tttttttctt atgtcataac ttcaatctaa gcatgtaatg caattgcaaa   1920 

tgagagttgt ttttaaatta agcttttgag aacttgaggt tgttgttgtt ggatacataa   1980 

cttcaacctt ttattagcaa tgttaacttc catttatgtc t                       2021 

 
           
             2  
             494  
             PRT  
             Arabidopsis thaliania  
           
            2 

Met Gly Leu Asp Gly Asn Asn Gly Gly Gly Val Trp Leu Asn Gly Gly 
1               5                   10                  15 

Gly Gly Glu Arg Glu Glu Asn Glu Glu Gly Ser Trp Gly Arg Asn Gln 
            20                  25                  30 

Glu Asp Gly Ser Ser Gln Phe Lys Pro Met Leu Glu Gly Asp Trp Phe 
        35                  40                  45 

Ser Ser Asn Gln Pro His Pro Gln Asp Leu Gln Met Leu Gln Asn Gln 
    50                  55                  60 

Pro Asp Phe Arg Tyr Phe Gly Gly Phe Pro Phe Asn Pro Asn Asp Asn 
65                  70                  75                  80 

Leu Leu Leu Gln His Ser Ile Asp Ser Ser Ser Ser Cys Ser Pro Ser 
                85                  90                  95 

Gln Ala Phe Ser Leu Asp Pro Ser Gln Gln Asn Gln Phe Leu Ser Thr 
            100                 105                 110 

Asn Asn Asn Lys Gly Cys Leu Leu Asn Val Pro Ser Ser Ala Asn Pro 
        115                 120                 125 

Phe Asp Asn Ala Phe Glu Phe Gly Ser Glu Ser Gly Phe Leu Asn Gln 
    130                 135                 140 

Ile His Ala Pro Ile Ser Met Gly Phe Gly Ser Leu Thr Gln Leu Gly 
145                 150                 155                 160 

Asn Arg Asp Leu Ser Ser Val Pro Asp Phe Leu Ser Ala Arg Ser Leu 
                165                 170                 175 

Leu Ala Pro Glu Ser Asn Asn Asn Asn Thr Met Leu Cys Gly Gly Phe 
            180                 185                 190 

Thr Ala Pro Leu Glu Leu Glu Gly Phe Gly Ser Pro Ala Asn Gly Gly 
        195                 200                 205 

Phe Val Gly Asn Arg Ala Lys Val Leu Lys Pro Leu Glu Val Leu Ala 
    210                 215                 220 

Ser Ser Gly Ala Gln Pro Thr Leu Phe Gln Lys Arg Ala Ala Met Arg 
225                 230                 235                 240 

Gln Ser Ser Gly Ser Lys Met Gly Asn Ser Glu Ser Ser Gly Met Arg 
                245                 250                 255 

Arg Phe Ser Asp Asp Gly Asp Met Asp Glu Thr Gly Ile Glu Val Ser 
            260                 265                 270 

Gly Leu Asn Tyr Glu Ser Asp Glu Ile Asn Glu Ser Gly Lys Ala Ala 
        275                 280                 285 

Glu Ser Val Gln Ile Gly Gly Gly Gly Lys Gly Lys Lys Lys Gly Met 
    290                 295                 300 

Pro Ala Lys Asn Leu Met Ala Glu Arg Arg Arg Arg Lys Lys Leu Asn 
305                 310                 315                 320 

Asp Arg Leu Tyr Met Leu Arg Ser Val Val Pro Lys Ile Ser Lys Met 
                325                 330                 335 

Asp Arg Ala Ser Ile Leu Gly Asp Ala Ile Asp Tyr Leu Lys Glu Leu 
            340                 345                 350 

Leu Gln Arg Ile Asn Asp Leu His Asn Glu Leu Glu Ser Thr Pro Pro 
        355                 360                 365 

Gly Ser Leu Pro Pro Thr Ser Ser Ser Phe His Pro Leu Thr Pro Thr 
    370                 375                 380 

Pro Gln Thr Leu Ser Cys Arg Val Lys Glu Glu Leu Cys Pro Ser Ser 
385                 390                 395                 400 

Leu Pro Ser Pro Lys Gly Gln Gln Ala Arg Val Glu Val Arg Leu Arg 
                405                 410                 415 

Glu Gly Arg Ala Val Asn Ile His Met Phe Cys Gly Arg Arg Pro Gly 
            420                 425                 430 

Leu Leu Leu Ala Thr Met Lys Ala Leu Asp Asn Leu Gly Leu Asp Val 
        435                 440                 445 

Gln Gln Ala Val Ile Ser Cys Phe Asn Gly Phe Ala Leu Asp Val Phe 
    450                 455                 460 

Arg Ala Glu Gln Cys Gln Glu Gly Gln Glu Ile Leu Pro Asp Gln Ile 
465                 470                 475                 480 

Lys Ala Val Leu Phe Asp Thr Ala Gly Tyr Ala Gly Met Ile 
                485                 490 

 
           
             3  
             828  
             PRT  
             Arabidopsis thaliania  
           
            3 

Met Glu Ser Arg Glu Asp Ser Phe Ile Ser Lys Glu Lys Lys Ser Thr 
1               5                   10                  15 

Met Lys Lys Glu Lys Gln Ala Ile Ala Ser Gln Arg Asn Arg Arg Arg 
            20                  25                  30 

Val Ile Lys Asn Arg Gly Asn Gly Lys Arg Leu Ile Ala Ser Leu Ser 
        35                  40                  45 

Gln Arg Lys Arg Arg Arg Ile Pro Arg Gly Arg Gly Asn Glu Lys Ala 
    50                  55                  60 

Val Phe Ala Pro Ser Ser Leu Pro Asn Asp Val Val Glu Glu Ile Phe 
65                  70                  75                  80 

Leu Arg Leu Pro Val Lys Ala Ile Ile Gln Leu Lys Ser Leu Ser Lys 
                85                  90                  95 

Gln Trp Arg Ser Thr Ile Glu Ser Arg Ser Phe Glu Glu Arg His Leu 
            100                 105                 110 

Lys Ile Val Glu Arg Ser Arg Val Asp Phe Pro Gln Val Met Val Met 
        115                 120                 125 

Ser Glu Glu Tyr Ser Leu Lys Gly Ser Lys Gly Asn Gln Pro Arg Pro 
    130                 135                 140 

Asp Thr Asp Ile Gly Phe Ser Thr Ile Cys Leu Glu Ser Ala Ser Ile 
145                 150                 155                 160 

Leu Ser Ser Thr Leu Ile Thr Phe Pro Gln Gly Phe Gln His Arg Ile 
                165                 170                 175 

Tyr Ala Ser Glu Ser Cys Asp Gly Leu Phe Cys Ile His Ser Leu Lys 
            180                 185                 190 

Thr Gln Ala Ile Tyr Val Val Asn Pro Ala Thr Arg Trp Phe Arg Gln 
        195                 200                 205 

Leu Pro Pro Ala Arg Phe Gln Ile Leu Met Gln Lys Leu Tyr Pro Thr 
    210                 215                 220 

Gln Asp Thr Trp Ile Asp Ile Lys Pro Val Val Cys Tyr Thr Ala Phe 
225                 230                 235                 240 

Val Lys Ala Asn Asp Tyr Lys Leu Val Trp Leu Tyr Asn Ser Asp Ala 
                245                 250                 255 

Ser Asn Pro Asn Leu Gly Val Thr Lys Cys Glu Val Phe Asp Phe Arg 
            260                 265                 270 

Ala Asn Ala Trp Arg Tyr Leu Thr Cys Thr Pro Ser Tyr Arg Ile Phe 
        275                 280                 285 

Pro Asp Gln Val Pro Ala Ala Thr Asn Gly Ser Ile Tyr Trp Phe Thr 
    290                 295                 300 

Glu Pro Tyr Asn Gly Glu Ile Lys Val Val Ala Leu Asp Ile His Thr 
305                 310                 315                 320 

Glu Thr Phe Arg Val Leu Pro Lys Ile Asn Pro Ala Ile Ala Ser Ser 
                325                 330                 335 

Asp Pro Asp His Ile Asp Met Cys Thr Leu Asp Asn Gly Leu Cys Met 
            340                 345                 350 

Ser Lys Arg Glu Ser Asp Thr Leu Val Gln Glu Ile Trp Arg Leu Lys 
        355                 360                 365 

Ser Ser Glu Asp Ser Trp Glu Lys Phe Asp Met Asn Ser Asp Gly Val 
    370                 375                 380 

Trp Leu Asp Gly Ser Gly Glu Ser Pro Glu Val Asn Asn Gly Glu Ala 
385                 390                 395                 400 

Ala Ser Trp Val Arg Asn Pro Asp Glu Asp Trp Phe Asn Asn Pro Pro 
                405                 410                 415 

Pro Pro Gln His Thr Asn Gln Asn Asp Phe Arg Phe Asn Gly Gly Phe 
            420                 425                 430 

Pro Leu Asn Pro Ser Glu Asn Leu Leu Leu Leu Leu Gln Gln Ser Ile 
        435                 440                 445 

Asp Ser Ser Ser Ser Ser Ser Pro Leu Leu His Pro Phe Thr Leu Asp 
    450                 455                 460 

Ala Ala Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Gln Ser 
465                 470                 475                 480 

Phe Leu Ala Thr Lys Ala Cys Ile Val Ser Leu Leu Asn Val Pro Thr 
                485                 490                 495 

Ile Asn Asn Asn Thr Phe Asp Asp Phe Gly Phe Asp Ser Gly Phe Leu 
            500                 505                 510 

Gly Gln Gln Phe His Gly Asn His Gln Ser Pro Asn Ser Met Asn Phe 
        515                 520                 525 

Thr Gly Leu Asn His Ser Val Pro Asp Phe Leu Pro Ala Pro Glu Asn 
    530                 535                 540 

Ser Ser Gly Ser Cys Gly Leu Ser Pro Leu Phe Ser Asn Arg Ala Lys 
545                 550                 555                 560 

Val Leu Lys Pro Leu Gln Val Met Ala Ser Ser Gly Ser Gln Pro Thr 
                565                 570                 575 

Leu Phe Gln Lys Arg Ala Ala Met Arg Gln Ser Ser Ser Ser Lys Met 
            580                 585                 590 

Cys Asn Ser Glu Ser Ser Ser Glu Met Arg Lys Ser Ser Tyr Glu Arg 
        595                 600                 605 

Glu Ile Asp Asp Thr Ser Thr Gly Ile Ile Asp Ile Ser Gly Leu Asn 
    610                 615                 620 

Tyr Glu Ser Asp Asp His Asn Thr Asn Asn Asn Lys Gly Lys Lys Lys 
625                 630                 635                 640 

Gly Met Pro Ala Lys Asn Leu Met Ala Glu Arg Arg Arg Arg Lys Lys 
                645                 650                 655 

Leu Asn Asp Arg Leu Tyr Met Leu Arg Ser Val Val Pro Lys Ile Ser 
            660                 665                 670 

Lys Met Asp Arg Ala Ser Ile Leu Gly Asp Ala Ile Asp Tyr Leu Lys 
        675                 680                 685 

Glu Leu Leu Gln Arg Ile Asn Asp Leu His Thr Glu Leu Glu Ser Thr 
    690                 695                 700 

Pro Pro Ser Ser Ser Ser Leu His Pro Leu Thr Pro Thr Pro Gln Thr 
705                 710                 715                 720 

Leu Ser Tyr Arg Val Lys Glu Glu Leu Cys Pro Ser Ser Ser Leu Pro 
                725                 730                 735 

Ser Pro Lys Gly Gln Gln Pro Arg Val Glu Val Arg Leu Arg Glu Gly 
            740                 745                 750 

Lys Ala Val Asn Ile His Met Phe Cys Gly Arg Arg Pro Gly Leu Leu 
        755                 760                 765 

Leu Ser Thr Met Arg Ala Leu Asp Asn Leu Gly Leu Asp Val Gln Gln 
    770                 775                 780 

Ala Val Ile Ser Cys Phe Asn Gly Phe Ala Leu Asp Val Phe Arg Ala 
785                 790                 795                 800 

Glu Gln Cys Gln Glu Asp His Asp Val Leu Pro Glu Gln Ile Lys Ala 
                805                 810                 815 

Val Leu Leu Asp Thr Ala Gly Tyr Ala Gly Leu Val 
            820                 825 

 
           
             4  
             351  
             PRT  
             Arabidopsis thaliania  
           
            4 

Met Glu Leu Ser Thr Gln Met Asn Val Phe Glu Glu Leu Leu Val Pro 
1               5                   10                  15 

Thr Lys Gln Glu Thr Thr Asp Asn Asn Ile Asn Asn Leu Ser Phe Asn 
            20                  25                  30 

Gly Gly Phe Asp His His His His Gln Phe Phe Pro Asn Gly Tyr Asn 
        35                  40                  45 

Ile Asp Tyr Leu Cys Phe Asn Asn Glu Glu Glu Asp Glu Asn Thr Leu 
    50                  55                  60 

Leu Tyr Pro Ser Ser Phe Met Asp Leu Ile Ser Gln Pro Pro Pro Leu 
65                  70                  75                  80 

Leu Leu His Gln Pro Pro Pro Leu Gln Pro Leu Ser Pro Pro Leu Ser 
                85                  90                  95 

Ser Ser Ala Thr Ala Gly Ala Thr Phe Asp Tyr Pro Phe Leu Glu Ala 
            100                 105                 110 

Leu Gln Glu Ile Ile Asp Ser Ser Ser Ser Ser Pro Pro Leu Ile Leu 
        115                 120                 125 

Gln Asn Gly Gln Glu Glu Asn Phe Asn Asn Pro Met Ser Tyr Pro Ser 
    130                 135                 140 

Pro Leu Met Glu Ser Asp Gln Ser Lys Ser Phe Ser Val Gly Tyr Cys 
145                 150                 155                 160 

Gly Gly Glu Thr Asn Lys Lys Lys Ser Lys Lys Leu Glu Gly Gln Pro 
                165                 170                 175 

Ser Lys Asn Leu Met Ala Glu Arg Arg Arg Arg Lys Arg Leu Asn Asp 
            180                 185                 190 

Arg Leu Ser Met Leu Arg Ser Ile Val Pro Lys Ile Ser Lys Met Asp 
        195                 200                 205 

Arg Thr Ser Ile Leu Gly Asp Ala Ile Asp Tyr Met Lys Glu Leu Leu 
    210                 215                 220 

Asp Lys Ile Asn Lys Leu Gln Asp Glu Glu Gln Glu Leu Gly Asn Ser 
225                 230                 235                 240 

Asn Asn Ser His His Ser Lys Leu Phe Gly Asp Leu Lys Asp Leu Asn 
                245                 250                 255 

Ala Asn Glu Pro Leu Val Arg Asn Ser Pro Lys Phe Glu Ile Asp Arg 
            260                 265                 270 

Arg Asp Glu Asp Thr Arg Val Asp Ile Cys Cys Ser Pro Lys Pro Gly 
        275                 280                 285 

Leu Leu Leu Ser Thr Val Asn Thr Leu Glu Thr Leu Gly Leu Glu Ile 
    290                 295                 300 

Glu Gln Cys Val Ile Ser Cys Phe Ser Asp Phe Ser Leu Gln Ala Ser 
305                 310                 315                 320 

Cys Ser Glu Gly Ala Glu Gln Arg Asp Phe Ile Thr Ser Glu Asp Ile 
                325                 330                 335 

Lys Gln Ala Leu Phe Arg Asn Ala Gly Tyr Gly Gly Ser Cys Leu 
            340                 345                 350 

 
           
             5  
             315  
             PRT  
             Arabidopsis thaliania  
           
            5 

Met Glu Thr Glu Leu Thr Gln Leu Arg Lys Gln Glu Ser Asn Asn Leu 
1               5                   10                  15 

Asn Gly Val Asn Gly Gly Phe Met Ala Ile Asp Gln Phe Val Pro Asn 
            20                  25                  30 

Asp Trp Asn Phe Asp Tyr Leu Cys Phe Asn Asn Leu Leu Gln Glu Asp 
        35                  40                  45 

Asp Asn Ile Asp His Pro Ser Ser Ser Ser Leu Met Asn Leu Ile Ser 
    50                  55                  60 

Gln Pro Pro Pro Leu Leu His Gln Pro Pro Gln Pro Ser Ser Pro Leu 
65                  70                  75                  80 

Tyr Asp Ser Pro Pro Leu Ser Ser Ala Phe Asp Tyr Pro Phe Leu Glu 
                85                  90                  95 

Asp Ile Ile His Ser Ser Tyr Ser Pro Pro Pro Leu Ile Leu Pro Ala 
            100                 105                 110 

Ser Gln Glu Asn Thr Asn Asn Tyr Ser Pro Leu Met Glu Glu Ser Lys 
        115                 120                 125 

Ser Phe Ile Ser Ile Gly Glu Thr Asn Lys Lys Arg Ser Asn Lys Lys 
    130                 135                 140 

Leu Glu Gly Gln Pro Ser Lys Asn Leu Met Ala Glu Arg Arg Arg Arg 
145                 150                 155                 160 

Lys Arg Leu Asn Asp Arg Leu Ser Leu Leu Arg Ser Ile Val Pro Lys 
                165                 170                 175 

Ile Thr Lys Met Asp Arg Thr Ser Ile Leu Gly Asp Ala Ile Asp Tyr 
            180                 185                 190 

Met Lys Glu Leu Leu Asp Lys Ile Asn Lys Leu Gln Glu Asp Glu Gln 
        195                 200                 205 

Glu Leu Gly Ser Asn Ser His Leu Ser Thr Leu Ile Thr Asn Glu Ser 
    210                 215                 220 

Met Val Arg Asn Ser Leu Lys Phe Glu Val Asp Gln Arg Glu Val Asn 
225                 230                 235                 240 

Thr His Ile Asp Ile Cys Cys Pro Thr Lys Pro Gly Leu Val Val Ser 
                245                 250                 255 

Thr Val Ser Thr Leu Glu Thr Leu Gly Leu Glu Ile Glu Gln Cys Val 
            260                 265                 270 

Ile Ser Cys Phe Ser Asp Phe Ser Leu Gln Ala Ser Cys Phe Glu Val 
        275                 280                 285 

Gly Glu Gln Arg Tyr Met Val Thr Ser Glu Ala Thr Lys Gln Ala Leu 
    290                 295                 300 

Ile Arg Asn Ala Gly Tyr Gly Gly Arg Cys Leu 
305                 310                 315 

 
           
             6  
             623  
             PRT  
             Arabidopsis thaliania  
           
            6 

Met Thr Asp Tyr Arg Leu Gln Pro Thr Met Asn Leu Trp Thr Thr Asp 
1               5                   10                  15 

Asp Asn Ala Ser Met Met Glu Ala Phe Met Ser Ser Ser Asp Ile Ser 
            20                  25                  30 

Thr Leu Trp Pro Pro Ala Ser Thr Thr Thr Thr Thr Ala Thr Thr Glu 
        35                  40                  45 

Thr Thr Pro Thr Pro Ala Met Glu Ile Pro Ala Gln Ala Gly Phe Asn 
    50                  55                  60 

Gln Glu Thr Leu Gln Gln Arg Leu Gln Ala Leu Ile Glu Gly Thr His 
65                  70                  75                  80 

Glu Gly Trp Thr Tyr Ala Ile Phe Trp Gln Pro Ser Tyr Asp Phe Ser 
                85                  90                  95 

Gly Ala Ser Val Leu Gly Trp Gly Asp Gly Tyr Tyr Lys Gly Glu Glu 
            100                 105                 110 

Asp Lys Ala Asn Pro Arg Arg Arg Ser Ser Ser Pro Pro Phe Ser Thr 
        115                 120                 125 

Pro Ala Asp Gln Glu Tyr Arg Lys Lys Val Leu Arg Glu Leu Asn Ser 
    130                 135                 140 

Leu Ile Ser Gly Gly Val Ala Pro Ser Asp Asp Ala Val Asp Glu Glu 
145                 150                 155                 160 

Val Thr Asp Thr Glu Trp Phe Phe Leu Val Ser Met Thr Gln Ser Phe 
                165                 170                 175 

Ala Cys Gly Ala Gly Leu Ala Gly Lys Ala Phe Ala Thr Gly Asn Ala 
            180                 185                 190 

Val Trp Val Ser Gly Ser Asp Gln Leu Ser Gly Ser Gly Cys Glu Arg 
        195                 200                 205 

Ala Lys Gln Gly Gly Val Phe Gly Met His Thr Ile Ala Cys Ile Pro 
    210                 215                 220 

Ser Ala Asn Gly Val Val Glu Val Gly Ser Thr Glu Pro Ile Arg Gln 
225                 230                 235                 240 

Ser Ser Asp Leu Ile Asn Lys Val Arg Ile Leu Phe Asn Phe Asp Gly 
                245                 250                 255 

Gly Ala Gly Asp Leu Ser Gly Leu Asn Trp Asn Leu Asp Pro Asp Gln 
            260                 265                 270 

Gly Glu Asn Asp Pro Ser Met Trp Ile Asn Asp Pro Ile Gly Thr Pro 
        275                 280                 285 

Gly Ser Asn Glu Pro Gly Asn Gly Ala Pro Ser Ser Ser Ser Gln Leu 
    290                 295                 300 

Phe Ser Lys Ser Ile Gln Phe Glu Asn Gly Ser Ser Ser Thr Ile Thr 
305                 310                 315                 320 

Glu Asn Pro Asn Leu Asp Pro Thr Pro Ser Pro Val His Ser Gln Thr 
                325                 330                 335 

Gln Asn Pro Lys Phe Asn Asn Thr Phe Ser Arg Glu Leu Asn Phe Ser 
            340                 345                 350 

Thr Ser Ser Ser Thr Leu Val Lys Pro Arg Ser Gly Glu Ile Leu Asn 
        355                 360                 365 

Phe Gly Asp Glu Gly Lys Arg Ser Ser Gly Asn Pro Asp Pro Ser Ser 
    370                 375                 380 

Tyr Ser Gly Gln Thr Gln Phe Glu Asn Lys Arg Lys Arg Ser Met Val 
385                 390                 395                 400 

Leu Asn Glu Asp Lys Val Leu Ser Phe Gly Asp Lys Thr Ala Gly Glu 
                405                 410                 415 

Ser Asp His Ser Asp Leu Glu Ala Ser Val Val Lys Glu Val Ala Val 
            420                 425                 430 

Glu Lys Arg Pro Lys Lys Arg Gly Arg Lys Pro Ala Asn Gly Arg Glu 
        435                 440                 445 

Glu Pro Leu Asn His Val Glu Ala Glu Arg Gln Arg Arg Glu Lys Leu 
    450                 455                 460 

Asn Gln Arg Phe Tyr Ala Leu Arg Ala Val Val Pro Asn Val Ser Lys 
465                 470                 475                 480 

Met Asp Lys Ala Ser Leu Leu Gly Asp Ala Ile Ala Tyr Ile Asn Glu 
                485                 490                 495 

Leu Lys Ser Lys Val Val Lys Thr Glu Ser Glu Lys Leu Gln Ile Lys 
            500                 505                 510 

Asn Gln Leu Glu Glu Val Lys Leu Glu Leu Ala Gly Arg Lys Ala Ser 
        515                 520                 525 

Ala Ser Gly Gly Asp Met Ser Ser Ser Cys Ser Ser Ile Lys Pro Val 
    530                 535                 540 

Gly Met Glu Ile Glu Val Lys Ile Ile Gly Trp Asp Ala Met Ile Arg 
545                 550                 555                 560 

Val Glu Ser Ser Lys Arg Asn His Pro Ala Ala Arg Leu Met Ser Ala 
                565                 570                 575 

Leu Met Asp Leu Glu Leu Glu Val Asn His Ala Ser Met Ser Val Val 
            580                 585                 590 

Asn Asp Leu Met Ile Gln Gln Ala Thr Val Lys Met Gly Phe Arg Ile 
        595                 600                 605 

Tyr Thr Gln Glu Gln Leu Arg Ala Ser Leu Ile Ser Lys Ile Gly 
    610                 615                 620 

 
           
             7  
             592  
             PRT  
             Arabidopsis thaliania  
           
            7 

Met Asn Gly Thr Thr Ser Ser Ile Asn Phe Leu Thr Ser Asp Asp Asp 
1               5                   10                  15 

Ala Ser Ala Ala Ala Met Glu Ala Phe Ile Gly Thr Asn His His Ser 
            20                  25                  30 

Ser Leu Phe Pro Pro Pro Pro Gln Gln Pro Pro Gln Pro Gln Phe Asn 
        35                  40                  45 

Glu Asp Thr Leu Gln Gln Arg Leu Gln Ala Leu Ile Glu Ser Ala Gly 
    50                  55                  60 

Glu Asn Trp Thr Tyr Ala Ile Phe Trp Gln Ile Ser His Asp Phe Asp 
65                  70                  75                  80 

Ser Ser Thr Gly Asp Asn Thr Val Ile Leu Gly Trp Gly Asp Gly Tyr 
                85                  90                  95 

Tyr Lys Gly Glu Glu Asp Lys Glu Lys Lys Lys Asn Asn Thr Asn Thr 
            100                 105                 110 

Ala Glu Gln Glu His Arg Lys Arg Val Ile Arg Glu Leu Asn Ser Leu 
        115                 120                 125 

Ile Ser Gly Gly Ile Gly Val Ser Asp Glu Ser Asn Asp Glu Glu Val 
    130                 135                 140 

Thr Asp Thr Glu Trp Phe Phe Leu Val Ser Met Thr Gln Ser Phe Val 
145                 150                 155                 160 

Asn Gly Val Gly Leu Pro Gly Glu Ser Phe Leu Asn Ser Arg Val Ile 
                165                 170                 175 

Trp Leu Ser Gly Ser Gly Ala Leu Thr Gly Ser Gly Cys Glu Arg Ala 
            180                 185                 190 

Gly Gln Gly Gln Ile Tyr Gly Leu Lys Thr Met Val Cys Ile Ala Thr 
        195                 200                 205 

Gln Asn Gly Val Val Glu Leu Gly Ser Ser Glu Val Ile Ser Gln Ser 
    210                 215                 220 

Ser Asp Leu Met His Lys Val Asn Asn Leu Phe Asn Phe Asn Asn Gly 
225                 230                 235                 240 

Gly Gly Asn Asn Gly Val Glu Ala Ser Ser Trp Gly Phe Asn Leu Asn 
                245                 250                 255 

Pro Asp Gln Gly Glu Asn Asp Pro Ala Leu Trp Ile Ser Glu Pro Thr 
            260                 265                 270 

Asn Thr Gly Ile Glu Ser Pro Ala Arg Val Asn Asn Gly Asn Asn Ser 
        275                 280                 285 

Asn Ser Asn Ser Lys Ser Asp Ser His Gln Ile Ser Lys Leu Glu Lys 
    290                 295                 300 

Asn Asp Ile Ser Ser Val Glu Asn Gln Asn Arg Gln Ser Ser Cys Leu 
305                 310                 315                 320 

Val Glu Lys Asp Leu Thr Phe Gln Gly Gly Leu Leu Lys Ser Asn Glu 
                325                 330                 335 

Thr Leu Ser Phe Cys Gly Asn Glu Ser Ser Lys Lys Arg Thr Ser Val 
            340                 345                 350 

Ser Lys Gly Ser Asn Asn Asp Glu Gly Met Leu Ser Phe Ser Thr Val 
        355                 360                 365 

Val Arg Ser Ala Ala Asn Asp Ser Asp His Ser Asp Leu Glu Ala Ser 
    370                 375                 380 

Val Val Lys Glu Ala Ile Val Val Glu Pro Pro Glu Lys Lys Pro Arg 
385                 390                 395                 400 

Lys Arg Gly Arg Lys Pro Ala Asn Gly Arg Glu Glu Pro Leu Asn His 
                405                 410                 415 

Val Glu Ala Glu Arg Gln Arg Arg Glu Lys Leu Asn Gln Arg Phe Tyr 
            420                 425                 430 

Ser Leu Arg Ala Val Val Pro Asn Val Ser Lys Met Asp Lys Ala Ser 
        435                 440                 445 

Leu Leu Gly Asp Ala Ile Ser Tyr Ile Asn Glu Leu Lys Ser Lys Leu 
    450                 455                 460 

Gln Gln Ala Glu Ser Asp Lys Glu Glu Ile Gln Lys Lys Leu Asp Gly 
465                 470                 475                 480 

Met Ser Lys Glu Gly Asn Asn Gly Lys Gly Cys Gly Ser Arg Ala Lys 
                485                 490                 495 

Glu Arg Lys Ser Ser Asn Gln Asp Ser Thr Ala Ser Ser Ile Glu Met 
            500                 505                 510 

Glu Ile Asp Val Lys Ile Ile Gly Trp Asp Val Met Ile Arg Val Gln 
        515                 520                 525 

Cys Gly Lys Lys Asp His Pro Gly Ala Arg Phe Met Glu Ala Leu Lys 
    530                 535                 540 

Glu Leu Asp Leu Glu Val Asn His Ala Ser Leu Ser Val Val Asn Asp 
545                 550                 555                 560 

Leu Met Ile Gln Gln Ala Thr Val Lys Met Gly Ser Gln Phe Phe Asn 
                565                 570                 575 

His Asp Gln Leu Lys Val Ala Leu Met Thr Lys Val Gly Glu Asn Tyr 
            580                 585                 590 

 
           
             8  
             610  
             PRT  
             Zea mays  
           
            8 

Met Ala Leu Ser Ala Ser Arg Val Gln Gln Ala Glu Glu Leu Leu Gln 
1               5                   10                  15 

Arg Pro Ala Glu Arg Gln Leu Met Arg Ser Gln Leu Ala Ala Ala Ala 
            20                  25                  30 

Arg Ser Ile Asn Trp Ser Tyr Ala Leu Phe Trp Ser Ile Ser Asp Thr 
        35                  40                  45 

Gln Pro Gly Val Leu Thr Trp Thr Asp Gly Phe Tyr Asn Gly Glu Val 
    50                  55                  60 

Lys Thr Arg Lys Ile Ser Asn Ser Val Glu Leu Thr Ser Asp Gln Leu 
65                  70                  75                  80 

Val Met Gln Arg Ser Asp Gln Leu Arg Glu Leu Tyr Glu Ala Leu Leu 
                85                  90                  95 

Ser Gly Glu Gly Asp Arg Arg Ala Ala Pro Ala Arg Pro Ala Gly Ser 
            100                 105                 110 

Leu Ser Pro Glu Asp Leu Gly Asp Thr Glu Trp Tyr Tyr Val Val Ser 
        115                 120                 125 

Met Thr Tyr Ala Phe Arg Pro Gly Gln Gly Leu Pro Gly Arg Ser Phe 
    130                 135                 140 

Ala Ser Asp Glu His Val Trp Leu Cys Asn Ala His Leu Ala Gly Ser 
145                 150                 155                 160 

Lys Ala Phe Pro Arg Ala Leu Leu Ala Lys Ser Ala Ser Ile Gln Ser 
                165                 170                 175 

Ile Leu Cys Ile Pro Val Met Gly Gly Val Leu Glu Leu Gly Thr Thr 
            180                 185                 190 

Asp Thr Val Pro Glu Ala Pro Asp Leu Val Ser Arg Ala Thr Ala Ala 
        195                 200                 205 

Phe Trp Glu Pro Gln Cys Pro Ser Ser Ser Pro Ser Gly Arg Ala Asn 
    210                 215                 220 

Glu Thr Gly Glu Ala Ala Ala Asp Asp Gly Thr Phe Ala Phe Glu Glu 
225                 230                 235                 240 

Leu Asp His Asn Asn Gly Met Asp Asp Ile Glu Ala Met Thr Ala Ala 
                245                 250                 255 

Gly Gly His Gly Gln Glu Glu Glu Leu Arg Leu Arg Glu Ala Glu Ala 
            260                 265                 270 

Leu Ser Asp Asp Ala Ser Leu Glu His Ile Thr Lys Glu Ile Glu Glu 
        275                 280                 285 

Phe Tyr Ser Leu Cys Asp Glu Met Asp Leu Gln Ala Leu Pro Leu Pro 
    290                 295                 300 

Leu Glu Asp Gly Trp Thr Val Asp Ala Ser Asn Phe Glu Val Pro Cys 
305                 310                 315                 320 

Ser Ser Pro Gln Pro Ala Pro Pro Pro Val Asp Arg Ala Thr Ala Asn 
                325                 330                 335 

Val Ala Ala Asp Ala Ser Arg Ala Pro Val Tyr Gly Ser Arg Ala Thr 
            340                 345                 350 

Ser Phe Met Ala Trp Thr Arg Ser Ser Gln Gln Ser Ser Cys Ser Asp 
        355                 360                 365 

Asp Ala Ala Pro Ala Ala Val Val Pro Ala Ile Glu Glu Pro Gln Arg 
    370                 375                 380 

Leu Leu Lys Lys Val Val Ala Gly Gly Gly Ala Trp Glu Ser Cys Gly 
385                 390                 395                 400 

Gly Ala Thr Gly Ala Ala Gln Glu Met Ser Gly Thr Gly Thr Lys Asn 
                405                 410                 415 

His Val Met Ser Glu Arg Lys Arg Arg Glu Lys Leu Asn Glu Met Phe 
            420                 425                 430 

Leu Val Leu Lys Ser Leu Leu Pro Ser Ile His Arg Val Asn Lys Ala 
        435                 440                 445 

Ser Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys Glu Leu Gln Arg Arg 
    450                 455                 460 

Val Gln Glu Leu Glu Ser Ser Arg Glu Pro Ala Ser Arg Pro Ser Glu 
465                 470                 475                 480 

Thr Thr Thr Arg Leu Ile Thr Arg Pro Ser Arg Gly Asn Asn Glu Ser 
                485                 490                 495 

Val Arg Lys Glu Val Cys Ala Gly Ser Lys Arg Lys Ser Pro Glu Leu 
            500                 505                 510 

Gly Arg Asp Asp Val Glu Arg Pro Pro Val Leu Thr Met Asp Ala Gly 
        515                 520                 525 

Thr Ser Asn Val Thr Val Thr Val Ser Asp Lys Asp Val Leu Leu Glu 
    530                 535                 540 

Val Gln Cys Arg Trp Glu Glu Leu Leu Met Thr Arg Val Phe Asp Ala 
545                 550                 555                 560 

Ile Lys Ser Leu His Leu Asp Val Leu Ser Val Gln Ala Ser Ala Pro 
                565                 570                 575 

Asp Gly Phe Met Gly Leu Lys Ile Arg Ala Gln Phe Ala Gly Ser Gly 
            580                 585                 590 

Ala Val Val Pro Trp Met Ile Ser Glu Ala Leu Arg Lys Ala Ile Gly 
        595                 600                 605 

Lys Arg 
    610 

 
           
             9  
             379  
             PRT  
             Arabidopsis thaliania  
           
            9 

Met Asp Glu Ser Ser Ile Ile Pro Ala Glu Lys Val Ala Gly Ala Glu 
1               5                   10                  15 

Lys Lys Glu Leu Gln Gly Leu Leu Lys Thr Ala Val Gln Ser Val Asp 
            20                  25                  30 

Trp Thr Tyr Ser Val Phe Trp Gln Phe Cys Pro Gln Gln Arg Val Leu 
        35                  40                  45 

Val Trp Gly Asn Gly Tyr Tyr Asn Gly Ala Ile Lys Thr Arg Lys Thr 
    50                  55                  60 

Thr Gln Pro Ala Glu Val Thr Ala Glu Glu Ala Ala Leu Glu Arg Ser 
65                  70                  75                  80 

Gln Gln Leu Arg Glu Leu Tyr Glu Thr Leu Leu Ala Gly Glu Ser Thr 
                85                  90                  95 

Ser Glu Ala Arg Ala Cys Thr Ala Leu Ser Pro Glu Asp Leu Thr Glu 
            100                 105                 110 

Thr Glu Trp Phe Tyr Leu Met Cys Val Ser Phe Ser Phe Pro Pro Pro 
        115                 120                 125 

Ser Gly Met Pro Gly Lys Ala Tyr Ala Arg Arg Lys His Val Trp Leu 
    130                 135                 140 

Ser Gly Ala Asn Glu Val Asp Ser Lys Thr Phe Ser Arg Ala Ile Leu 
145                 150                 155                 160 

Ala Lys Thr Val Val Cys Ile Pro Met Leu Asp Gly Val Val Glu Leu 
                165                 170                 175 

Gly Thr Thr Lys Lys Asn Gly Lys Glu His Gln Gln Val Lys Thr Ala 
            180                 185                 190 

Pro Ser Ser Gln Trp Val Leu Lys Gln Met Ile Phe Arg Val Pro Phe 
        195                 200                 205 

Leu His Asp Asn Thr Lys Asp Lys Arg Leu Pro Arg Glu Asp Leu Ser 
    210                 215                 220 

His Val Val Ala Glu Arg Arg Arg Arg Glu Lys Leu Asn Glu Lys Phe 
225                 230                 235                 240 

Ile Thr Leu Arg Ser Met Val Pro Phe Val Thr Lys Met Asp Lys Val 
                245                 250                 255 

Ser Ile Leu Gly Asp Thr Ile Ala Tyr Val Asn His Leu Arg Lys Arg 
            260                 265                 270 

Val His Glu Leu Glu Asn Thr His His Glu Gln Gln His Lys Arg Thr 
        275                 280                 285 

Arg Thr Cys Lys Arg Lys Thr Ser Glu Glu Val Glu Val Ser Ile Ile 
    290                 295                 300 

Glu Asn Asp Val Leu Leu Glu Met Arg Cys Glu Tyr Arg Asp Gly Leu 
305                 310                 315                 320 

Leu Leu Asp Ile Leu Gln Val Leu His Glu Leu Gly Ile Glu Thr Thr 
                325                 330                 335 

Ala Val His Thr Ser Val Asn Asp His Asp Phe Glu Ala Glu Ile Arg 
            340                 345                 350 

Ala Lys Val Arg Gly Lys Lys Ala Ser Ile Ala Glu Val Lys Arg Ala 
        355                 360                 365 

Ile His Gln Val Ile Ile His Asp Thr Asn Leu 
    370                 375 

 
           
             10  
             432  
             PRT  
             Arabidopsis thaliania  
           
            10 

Met Glu Gln Val Phe Ala Asp Trp Asn Phe Glu Asp Asn Phe His Met 
1               5                   10                  15 

Ser Thr Asn Lys Arg Ser Ile Arg Pro Glu Asp Glu Leu Val Glu Leu 
            20                  25                  30 

Leu Trp Arg Asp Gly Gln Val Val Leu Gln Ser Gln Ala Arg Arg Glu 
        35                  40                  45 

Pro Ser Val Gln Val Gln Thr His Lys Gln Glu Thr Asn Gln Glu Thr 
    50                  55                  60 

Val Gln Lys Pro Asn Tyr Ala Ala Leu Asp Asp Gln Glu Thr Val Ser 
65                  70                  75                  80 

Trp Ile Gln Tyr Pro Pro Asp Asp Val Ile Asp Pro Phe Glu Ser Glu 
                85                  90                  95 

Phe Ser Ser His Phe Phe Ser Ser Ile Asp His Leu Gly Gly Pro Glu 
            100                 105                 110 

Lys Pro Arg Met Ile Glu Glu Thr Val Lys His Glu Ala Gln Ala Met 
        115                 120                 125 

Ala Pro Pro Lys Phe Arg Ser Ser Val Ile Thr Val Gly Pro Ser His 
    130                 135                 140 

Cys Gly Ser Asn Gln Ser Thr Asn Ile His Gln Ala Thr Thr Leu Pro 
145                 150                 155                 160 

Val Ser Met Ser Asp Arg Ser Lys Asn Val Glu Glu Arg Leu Asp Thr 
                165                 170                 175 

Ser Ser Gly Gly Ser Ser Gly Cys Ser Tyr Gly Arg Asn Asn Lys Glu 
            180                 185                 190 

Thr Val Ser Gly Thr Ser Val Thr Ile Asp Arg Lys Arg Lys His Val 
        195                 200                 205 

Met Asp Ala Asp Gln Glu Ser Val Ser Gln Ser Asp Ile Gly Leu Thr 
    210                 215                 220 

Ser Thr Asp Asp Gln Thr Met Gly Asn Lys Ser Ser Gln Arg Ser Gly 
225                 230                 235                 240 

Ser Thr Arg Arg Ser Arg Ala Ala Glu Val His Asn Leu Ser Glu Arg 
                245                 250                 255 

Arg Arg Arg Asp Arg Ile Asn Glu Arg Met Lys Ala Leu Gln Glu Leu 
            260                 265                 270 

Ile Pro His Cys Ser Arg Thr Asp Lys Ala Ser Ile Leu Asp Glu Ala 
        275                 280                 285 

Ile Asp Tyr Leu Lys Ser Leu Gln Met Gln Leu Gln Val Met Trp Met 
    290                 295                 300 

Gly Ser Gly Met Ala Ala Ala Ala Ala Ala Ala Ala Ser Pro Met Met 
305                 310                 315                 320 

Phe Pro Gly Val Gln Ser Ser Pro Tyr Ile Asn Gln Met Ala Met Gln 
                325                 330                 335 

Ser Gln Met Gln Leu Ser Gln Phe Pro Val Met Asn Arg Ser Ala Pro 
            340                 345                 350 

Gln Asn His Pro Gly Leu Val Cys Leu Asn Pro Val Gln Leu Gln Leu 
        355                 360                 365 

Gln Ala Gln Asn Gln Ile Leu Ser Glu Gln Leu Ala Arg Tyr Met Gly 
    370                 375                 380 

Gly Ile Pro Gln Met Pro Pro Ala Gly Asn Gln Thr Val Gln Gln Gln 
385                 390                 395                 400 

Pro Ala Asp Met Leu Gly Phe Gly Ser Pro Ala Gly Pro Gln Ser Gln 
                405                 410                 415 

Leu Ser Ala Pro Ala Thr Thr Asp Ser Leu His Met Gly Lys Ile Gly 
            420                 425                 430 

 
           
             11  
             430  
             PRT  
             Arabidopsis thaliania  
           
            11 

Met Glu His Gln Gly Trp Ser Phe Glu Glu Asn Tyr Ser Leu Ser Thr 
1               5                   10                  15 

Asn Arg Arg Ser Ile Arg Pro Gln Asp Glu Leu Val Glu Leu Leu Trp 
            20                  25                  30 

Arg Asp Gly Gln Val Val Leu Gln Ser Gln Thr His Arg Glu Gln Thr 
        35                  40                  45 

Gln Thr Gln Lys Gln Asp His His Glu Glu Ala Leu Arg Ser Ser Thr 
    50                  55                  60 

Phe Leu Glu Asp Gln Glu Thr Val Ser Trp Ile Gln Tyr Pro Pro Asp 
65                  70                  75                  80 

Glu Asp Pro Phe Glu Pro Asp Asp Phe Ser Ser His Phe Phe Ser Thr 
                85                  90                  95 

Met Asp Pro Leu Gln Arg Pro Thr Ser Glu Thr Val Lys Pro Lys Ser 
            100                 105                 110 

Ser Pro Glu Pro Pro Gln Val Met Val Lys Pro Lys Ala Cys Pro Asp 
        115                 120                 125 

Pro Pro Pro Gln Val Met Pro Pro Pro Lys Phe Arg Leu Thr Asn Ser 
    130                 135                 140 

Ser Ser Gly Ile Arg Glu Thr Glu Met Glu Gln Tyr Ser Val Thr Thr 
145                 150                 155                 160 

Val Gly Pro Ser His Cys Gly Ser Asn Pro Ser Gln Asn Asp Leu Asp 
                165                 170                 175 

Val Ser Met Ser His Asp Arg Ser Lys Asn Ile Glu Glu Lys Leu Asn 
            180                 185                 190 

Pro Asn Ala Ser Ser Ser Ser Gly Gly Ser Ser Gly Cys Ser Phe Gly 
        195                 200                 205 

Lys Asp Ile Lys Glu Met Ala Ser Gly Arg Cys Ile Thr Thr Asp Arg 
    210                 215                 220 

Lys Arg Lys Arg Ile Asn His Thr Asp Glu Ser Val Ser Leu Ser Asp 
225                 230                 235                 240 

Ala Ile Gly Asn Lys Ser Asn Gln Arg Ser Gly Ser Asn Arg Arg Ser 
                245                 250                 255 

Arg Ala Ala Glu Val His Asn Leu Ser Glu Arg Arg Arg Arg Asp Arg 
            260                 265                 270 

Ile Asn Glu Arg Met Lys Ala Leu Gln Glu Leu Ile Pro His Cys Ser 
        275                 280                 285 

Lys Thr Asp Lys Ala Ser Ile Leu Asp Glu Ala Ile Asp Tyr Leu Lys 
    290                 295                 300 

Ser Leu Gln Leu Gln Leu Gln Val Met Trp Met Gly Ser Gly Met Ala 
305                 310                 315                 320 

Ala Ala Ala Ala Ser Ala Pro Met Met Phe Pro Gly Val Gln Pro Gln 
                325                 330                 335 

Gln Phe Ile Arg Gln Ile Gln Ser Pro Val Gln Leu Pro Arg Phe Pro 
            340                 345                 350 

Val Met Asp Gln Ser Ala Ile Gln Asn Asn Pro Gly Leu Val Cys Gln 
        355                 360                 365 

Asn Pro Val Gln Asn Gln Ile Ile Ser Asp Arg Phe Ala Arg Tyr Ile 
    370                 375                 380 

Gly Gly Phe Pro His Met Gln Ala Ala Thr Gln Met Gln Pro Met Glu 
385                 390                 395                 400 

Met Leu Arg Phe Ser Ser Pro Ala Gly Gln Gln Ser Gln Gln Pro Ser 
                405                 410                 415 

Ser Val Pro Thr Lys Thr Thr Asp Gly Ser Arg Leu Asp His 
            420                 425                 430 

 
           
             12  
             165  
             PRT  
             Danio rerio  
           
            12 

Met Ser Asp Asn Asp Asp Ile Glu Val Asp Ser Asp Ala Asp Ser Pro 
1               5                   10                  15 

Arg Phe His Gly Val Ala Asp Lys Arg Ala His His Asn Ala Leu Glu 
            20                  25                  30 

Arg Lys Arg Arg Asp His Ile Lys Asp Ser Phe His Ser Leu Arg Asp 
        35                  40                  45 

Ser Val Pro Ala Leu Gln Gly Glu Lys Gln Ser Ile Lys Gln Ala Ser 
    50                  55                  60 

Arg Ala Gln Ile Leu Asp Lys Ala Thr Glu Tyr Ile Gln Tyr Met Arg 
65                  70                  75                  80 

Arg Lys Asn His Thr His Gln Gln Asp Ile Asp Asp Leu Lys Arg Gln 
                85                  90                  95 

Asn Ala Leu Leu Glu Gln Gln Val Arg Ala Leu Glu Lys Val Lys Gly 
            100                 105                 110 

Thr Thr Gln Leu Gln Ala Asn Tyr Ser Ser Ser Asp Ser Ser Leu Tyr 
        115                 120                 125 

Thr Asn Pro Lys Gly Gln Ala Val Ser Ala Phe Asp Gly Gly Ser Asp 
    130                 135                 140 

Ser Ser Ser Gly Ser Glu Pro Glu Glu Gln Arg Thr Arg Lys Lys His 
145                 150                 155                 160 

Arg Pro Glu Asp Ser 
                165 

 
           
             13  
             440  
             PRT  
             Homo sapiens  
           
            13 

Met Pro Leu Asn Val Thr Ile Thr Asn Lys Asn Tyr Asp Leu Asp Tyr 
1               5                   10                  15 

Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr 
            20                  25                  30 

Gln Gln Gln Gln Gln Ser Asp Leu Gln Pro Pro Ala Pro Ser Glu Asp 
        35                  40                  45 

Ile Trp Lys Lys Phe Glu Leu Leu Leu Pro Asn Pro Pro Leu Ser Pro 
    50                  55                  60 

Ser Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro 
65                  70                  75                  80 

Phe Ser Leu Arg Gly Asp Asn Asp Asp Gly Gly Gly Asn Phe Ser Thr 
                85                  90                  95 

Ala Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val 
            100                 105                 110 

Asn Gln Asn Phe Ile Cys Asp Pro Gly Asp Glu Thr Phe Ile Lys Asn 
        115                 120                 125 

Ile Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys 
    130                 135                 140 

Leu Val Ser Glu Lys Val Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser 
145                 150                 155                 160 

Gly Ser Pro Asn Pro Ala Arg Gly His Ser Val Ser Ser Thr Ser Ser 
                165                 170                 175 

Leu Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro 
            180                 185                 190 

Ser Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Arg Ser Pro Lys Ser 
        195                 200                 205 

Cys Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu 
    210                 215                 220 

Leu Ser Ser Thr Glu Ser Ala Pro Gln Gly Ser Pro Glu Pro Leu Val 
225                 230                 235                 240 

Phe His Glu Glu Thr Ser Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu 
                245                 250                 255 

Gln Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln 
            260                 265                 270 

Ala Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His 
        275                 280                 285 

Ser Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser 
    290                 295                 300 

Thr His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr 
305                 310                 315                 320 

Pro Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln 
                325                 330                 335 

Ile Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu 
            340                 345                 350 

Glu Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg 
        355                 360                 365 

Asn Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu 
    370                 375                 380 

Leu Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala 
385                 390                 395                 400 

Thr Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser 
                405                 410                 415 

Glu Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu 
            420                 425                 430 

Glu Gln Leu Arg Asn Ser Cys Ala 
        435                 440 

 
           
             14  
             32  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            14 

tcatggatcc accatttgtt aatgcatgat gg                                   32 

 
           
             15  
             26  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            15 

gctcaagctt tctgttctag ttcagg                                          26 

 
           
             16  
             24  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            16 

ttcgattttt atttccattt ttgg                                            24 

 
           
             17  
             22  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            17 

ccaaacgtcc ttgagtcttg at                                              22 

 
           
             18  
             26  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            18 

taaaactcag attattattt ccattt                                          26 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            19 

gaggagccac gtagagggcc                                                 20 

 
           
             20  
             24  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            20 

cgtggatcac agcaatacag agcc                                            24 

 
           
             21  
             25  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            21 

cctcctgcac ttccacttcg tcttc                                           25 

 
           
             22  
             37  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            22 

agggatccgg accaccgtca ataacatcgt taagtag                              37 

 
           
             23  
             40  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            23 

cgaattctaa ccgccattaa ctatgtctcc tctctatctc                           40 

 
           
             24  
             37  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            24 

agggatccgg accaccgtca ataacatcgt taagtag                              37 

 
           
             25  
             34  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            25 

cgaattcgcc aaagttgaca cctttacccc aaag                                 34 

 
           
             26  
             28  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            26 

gcgatgggtc ttgacggaaa caatggtg                                        28 

 
           
             27  
             31  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            27 

tcagatcata ccagcatacc ctgctgtatc g                                    31 

 
           
             28  
             21  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            28 

gtcaagaggt tctcagcagt a                                               21 

 
           
             29  
             21  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            29 

tcaccttctt gatccgcagt t                                               21 

 
           
             30  
             36  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            30 

gctctagagc gatgggtctt gacggaaaca atggtg                               36 

 
           
             31  
             39  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            31 

ggggtacctc agatcatacc agcataccct gctgtatcg                            39 

 
           
             32  
             36  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            32 

aggaattcgc gatgggtctt gacggaaaca atggtg                               36 

 
           
             33  
             39  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            33 

ctggatcctc agatcatacc agcataccct gctgtatcg                            39 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            34 

accccaccat ttgttaatgc                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            35 

acaattacaa ctgcatgctt                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            36 

aatgttacat ttgatcattc                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            37 

ctctggacac atggcagatc                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            38 

cattttacaa ttgcttcgct                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            39 

atataattaa ctacttttat                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            40 

gactcgtttc gcgatccgat                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            41 

ctctggacac atggcagatc                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic DNA  
             
           
            42 

ctctggaacc agtgcagatc                                                 20