WO2006006236A1 - Regulation of environmental stress-tolerance in plants using modified dreb2a gene - Google Patents

Abstract

The present invention relates to a modified transcriptional factor gene DREB2A and its use for regulation of environmental stress-tolerance in plants.

Description

DESCRIPTION

Regulation of environmental stress-tolerance in plants using modified DREB2A gene

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a modified transcription factor gene DREB2A and its use for regulation of environmental stress-tolerance in plants.

2. Prior Art

In the natural world, plants are living under various environmental stresses such as dehydration, high temperature, low temperature or salt. Unlike animals, plants cannot protect themselves from stresses by moving. Thus, plants have acquired various stress tolerance mechanisms during the courses of their evolution. For example, low temperature tolerant plants (Arabidopsis thaliana, spinach, lettuce, garden pea, barley, beet, etc.) have less unsaturated fatty acid content in their biomembrane lipid than low temperature sensitive plants (maize, rice, pumpkin, cucumber, banana, tomato, etc.). Therefore, even when the former plants are exposed to low temperatures, phase transition is hard to occur in their biomembrane lipid and, thus, low temperature injury does not occur easily.

To date, dehydration, low temperature or salt tolerant lines have been selected and crossed in attempts to artificially create environmental stress tolerant plants. However, a long time is needed for such selection, and the crossing method is only applicable between limited species. Thus, it has been difficult to create a plant with high environmental stress tolerance.

As biotechnology progressed recently, trials have been made to create dehydration, low temperature or salt tolerant plants by using transgenic technology which introduces into plants a specific, heterologous gene. Those genes which have been used for the creation of environmental stress tolerant plants include synthesis enzyme genes for osmoprotecting substances (mannitol, proline, glycine betaine, etc.) and modification enzyme genes for cell membrane lipid. Specifically, as the mannitol synthesis enzyme gene, Escherichia coli-derived mannitol 1-phosphate dehydrogenase gene [Science 259:508-510 (1993)] was used. As the proline synthesis enzyme gene, bean-derived Δ ^""^"-proline-δ-carboxylate synthetase gene [Plant Physiol. 108:1387-1394 (1995)] was used. As the glycine betaine synthesis enzyme gene, bacterium-derived choline dehydrogenase gene [Plant J. 12:1334-1342 (1997)] was used. As the cell membrane lipid modification enzyme gene, Arabidopsis thaliana-derived 0) -3 fatty acid desaturase gene [Plant Physiol. 105:601-605 (1994)] and blue-green alga- derived Δ9 desaturase gene [Nature Biotech. 14:1003-1006 (1996) were used. However, the resultant plants into which these genes were introduced were instable in stress tolerance or low in tolerance level; none of them have been put into practical use to date.

Further, it is reported that a plurality of genes are involved in the acquisition of dehydration, low temperature or salt tolerance in plants [Plant Physiol., 115:327-334 (1997)]. Therefore, a gene encoding a transcription factor capable of activating simultaneously the expression of a plurality of genes involved in the acquisition of stress tolerance has been introduced into plants, yielding plants with high stress tolerance. However, when a gene which induces the expression of a plurality of genes is introduced into a host plant, the genes are activated at the same time. As a result, the energy of the host plant is directed to production of the products of these genes and intracellular metabolism of such gene products, which often brings about delay in the growth of the host plant or dwarfing of the plant .

The present inventors had i solated the genes DREBlA, DREBlB , DREBlC , DREB2A, and DREB2B encoding the transcription f actors which bind to a stress responsive element and specif ical ly activate the transcription of genes located downstream of the element f rom Arabi dopsi s thaliana ( Lie Q . et al . , The Plant Cel l , Vol . 10 , 1391-1406 , August 1998 , JP10 - 228457 ) . They reported that introduction and overexpression of the genes in a plant enabled impartment of stress tolerance without causing retardation of a plant (Lie Q . et al . , The Plant Cell , Vol . 10 , 1391-1406 , August 1998 , JP10 - 292348 ) .

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide a transgenic plant having improved tolerance to environmental stresses (such as dehydration, low temperature and salt) and being free from dwarfing.

Toward the solution of the above problem, the present inventors transformed a plant using various modified DREB2A gene . As a result, the inventors have succeeded in creating a plant which has remarkably improved environmental stress- tolerance without dwarfing.

The present invention provides the following (1) to (26) .

(1) A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence as shown in SEQ ID NO : 4 with the deletion of amino acids from positions 136-165 thereof , said DNA being operably linked downstream of a stress responsive promoter.

(2) The transgenic plant of (1) above, wherein said protein can bind to said stress responsive promoter.

(3) The transgenic plant of (1) above, wherein a protein consisting of the amino acid sequence as shown in SEQ ID NO: 4 with the amino acids from positions 136-165 deleted therefrom provides at least a three fold increase in transactivation activity of a reporter gene when compared with that of a full length DREB2A protein.

(4) An isolated nucleic acid molecule that encodes a DREB2A protein as shown in SEQ ID NO:4 with the deletion of amino acids from positions 136 to 165 thereof.

(5) A transgenic plant which comprises a DNA comprising the isolated nucleic acid molecule of (4) above, the DNA operably linked downstream of a stress responsive promoter.

(6) An isolated protein having the sequence as shown in SEQ ID NO:4 with the deletion of the amino acids from positions 136 to 165 thereof.

(7) A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence of positions 254-335 of SEQ ID NO: 4, said DNA being operably linked downstream of a stress responsive promoter.

(8) The transgenic plant of (7) above further comprising a DNA that encodes DNA binding domain and a nuclear localization signal.

(9) The transgenic plant of (7) above, wherein said protein can bind to said stress responsive promoter.

(10) A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence of positions 254-335 of SEQ ID NO: 4.

(11) The transgenic plant of (10) above, wherein said protein provides an increase in transactivation activity of a reporter gene of from about 5 to about 9 times greater than in the absence of a protein consisting of the amino acid sequence as shown in SEQ ID NO: 4.

(12) A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence selected from the group consisting of positions 254-317, 136-335, 318-335, 166, 335, and 282-335 of SEQ ID NO: 4, said DNA being operably linked downstream of a stress responsive promoter.

(13) The transgenic plant of (12) above further comprising a DNA that encodes DNA binding domain and a nuclear localization signal.

(14) An isolated nucleic acid molecule encoding a translational activation domain of the DREB2A protein comprising the amino acid sequence of positions 254 to 335 of SEQ ID NO: 4.

(15) A transgenic plant comprising a DNA comprising the isolated nucleic acid molecule of (14) above, the DNA operably linked downstream of a stress responsive promoter.

(16) An isolated protein with DREB2A protein activity and having the amino acid sequence of positions 254 to 335 of SEQ ID NO:4.

(17) A transgenic plant comprising a DNA that encodes a protein consisting of a fragment of the amino acid sequence as shown in SEQ ID NO: 4 with the deletion of amino acids from positions 136-165 thereof, wherein said fragment comprises the amino acid sequence of positions 254-335 and DNA binding domain and a nuclear localization signal, said DNA being operably linked downstream of a stress responsive promoter.

(18) A transgenic plant comprising a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 with the deletion of the nucleotides from positions 572-661 thereof, said DNA being operably linked downstream of a stress responsive promoter.

(19) The transgenic plant of (18) above, wherein a protein encoded by said DNA can bind to said stress responsive promoter.

(20) The transgenic plant of (18) above, wherein said DNA encodes a protein that can bind to said stress responsive promoter.

(21) An isolated nucleic acid molecule as shown in SEQ ID NO: 3 with the deletion of the region from nucleotide positions 572-661 thereof.

(22) A transgenic plant which comprises a DNA comprising the isolated nucleic acid molecule of (21) above, said DNA being operably linked downstream of a stress responsive promoter.

(23) A transgenic plant comprising a DNA comprising the nucleotide sequence as shown in positions 926-1171 of SEQ ID NO: 3, said DNA being operably linked downstream of a stress responsive promoter.

(24) The transgenic plant of (23) above, wherein a protein encoded by said DNA can bind to said stress responsive promoter.

(25) An isolated nucleic acid molecule comprising the nucleotide sequence shown in positions 926 to 1171 of SEQ ID NO: 3.

(26) A transgenic plant which comprises a DNA comprising the isolated nucleic acid molecule of (25) above, the DNA being operably linked downstream of a stress responsive promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the result of domain analysis of the C-terminal region of the DREB2A protein by using protoplasts prepared from Arabidopsis T87 cells.

(A) Schematic diagram of the reporter and effector constructs used in cotransfection experiments

(B) Transactivation of the rd29A promoter-GUS fusion gene by DREBlA, DREB2A, or C-terminal region deletion mutants of DREB2A

Fig. 2 shows a result of transcriptional activation with the C- terminal region of DREB2A fused to the GAL4-binding domain.

(A) Schematic diagram of the reporter and effector constructs

(B) Transactivation of the 6AL4 binding site-GUS fusion gene by the fusion proteins of the GAL4 DNA binding domain and the GAL4 activation region or C-terminal regions of the DREB2A protein as indicated by numbers of amino acid residues Fig. 3 shows effects of overexpressing the constitutive active form of DREB2A in transgenic plants.

(A) Shown are 30-day-old seedlings carrying the 35S-constitutive active form of the DREB2A construct with growth retardation (DREB2A CA-a, b and c) , those carrying the 35S-full length DREB2A construct (DREB2A FL) and those carrying pBI121 (wt) .

(B) Comparison of growth retardation among 5-week-old DREB2A related transgenic plants

(C) Close-up view of a plant carrying pBI 121 (wt) and a plant carrying the 35S:constitutive active form of DREB2A (DREB2A-a) shown in (B)

(D) RNA gel blot analysis of the DREB2A and rd29A genes in the transgenic plants

Fig. 4 shows photographs showing expression of the DREB target genes in plants carrying pBI121 (wt) , the 35S:constitutive active form of DREB2A (35S:DREB2A CA) and 35SrDREBlA constructs.

Fig. 5 shows freezing and drought tolerance of the plants carrying the 35S:constitutive active form of DREB2A and 35S:DREB1A constructs.

(A) Photographs of plants before and after stress treatments

(B) Survival rates of plants exposed to freezing and drought stress

(C) Photographs of plants before and after 10-day dewa^'tering when they were planted in single pot

DETAILED DESCRIPTION OF THE INVENTION

Hereinbelow, the present invention will be described in detail.

The transgenic plant of the invention is a environmental stress tolerant, transgenic plant created by introducing a gene in which a DNA

(called "DREB gene") encoding a transcription factor that binds to a dehydration responsive element (DRE) and activates the transcription of genes located downstream of DRE is ligated downstream of a stress responsive promoter.

The DREB genes used in the invention can be cloned as described below. Of these DREB genes, DRE-binding protein IA gene is called DREBlA gene; DRE-binding protein IB gene is called DREBlB gene; DRE-binding protein 1C gene is called DREBlC gene; DRE-binding protein 2A gene is called DREB2A gene; and DRE-binding protein 2B gene is called DREB2B gene.

1. Cloning of DREB Gene

1-1. Preparation of iriRNA and a cDNA Library from Arabidopsis thaliana As a source of mRNA, a part of the plant of Arabidopsis thaliana such as leaves, stems, roots or flowers, or the plant as a whole may be used. Alternatively, the plant obtained by sowing seeds of Arabidopsis thaliana on a solid medium such as GM medium, MS medium or #3 medium and growing the resultant seedlings aseptically may be used. The rriRNA level of DREBlA gene in Arabidopsis thaliana plants increases when they are exposed to low temperature stress (e.g. 10 to -4°C) . On the other hand, the mRNA level of DREB2A gene increases when plants are exposed to salt stress (e.g. 150-250 mM NaCl) or dehydration stress (e.g. dehydrated state) . Therefore, Arabidopsis thaliana plants which have been exposed to such stress may also be used. mRNA is prepared, for example, by exposing Arabidopsis thaliana plants grown on GM medium to the dehydration stress, low temperature stress or salt stress mentioned above and then freezing them with liquid nitrogen. Subsequently, conventional techniques for mRNA preparation may be used. For example, the frozen plant are ground in a mortar. From the resultant ground material, a crude RNA fraction is extracted by the glyoxal method, the guanidine thiocyanate-cesium chloride method, the lithium chloride- urea method, the proteinase K-deoxyribonuclease method or the like. From this crude RNA fraction, poly(A) ^"*" RNA (mRNA) can be obtained by the affinity column method using oligo dT-cellulose or poly U-Sepharose carried on Sepharose 2B or by the batch method. The resultant mRNA may further be fractionated by sucrose gradient centrifugation or the like.

Single-stranded cDNA is synthesized using the thus obtained mRNA as a template; this synthesis is performed using a commercial kit (e.g. ZAP- cDNA Synthesis Kit: Stratagene) , oligo(dT)_2O and a reverse transcriptase. Then, double-stranded cDNA is synthesized from the resultant single- stranded cDNA. An appropriate adaptor such as EcoRI-Notl-BamHI adaptor is added to the resultant double-stranded cDNA, which is then ligated downstream of a transcriptional activation domain (such as GAL4 activation domain) in a plasmid (such as pAD-GAL4 plasmid: Stratagene) containing such a domain to thereby prepare a cDNA library.

1-2. A Host to Be Used in the Cloning of DREB Gene

DREB gene can be cloned, for example, by one hybrid screening method using yeast. Screening by this method may be performed using a commercial kit (e.g. Matchmaker One Hybrid System: Clontech) .

In the cloning of DREB gene using the above-mentioned kit, first, it is necessary to ligate a DNA fragment comprising DRE sequences to which a protein encoded by DREB gene (i.e. DREB protein) binds to both plasmids pHISi-1 and pLacZi contained in the kit. Then, the resultant plasmids are transformed into the yeast contained in the kit (Saccharoπtayces cerevisiae YM4271) to thereby prepare a host yeast for cloning.

The host yeast for cloning can biosynthesize histidine by the action of HIS3 protein which is expressed leakily by HIS3 minimum promoter. Thus, usually, this yeast can grow in the absence of histidine. However, since the promoter used for the expression of the gene encoding HIS3 protein is a minimum promoter which can only maintain the minimum transcription level, HIS3 protein produced in cells is extremely small in quantity. Therefore, when the host yeast is cultured in the presence of 3-AT (3-aminotriazole) that is a competitive inhibitor against HIS3 protein, the function of HIS3 protein in cells is inhibited by 3-AT in a concentration dependent manner. When the concentration of 3-AT exceeds a specific level, HIS3 protein in cells becomes unable to function and, as a result, the host yeast becomes unable to grow in the absence of histidine. Similarly, lacZ gene is also located downstream of CYCl minimum promoter. Thus, jS -galactosidase is produced only in extremely small quantity in the yeast cells. Therefore, when the host yeast is plated on an Xgal containing plate, colonies appearing thereon do not have such Xgal degrading ability that turns the colonies into blue as a whole. However, when a transcription factor that binds to DRE sequences located upstream of HIS3 and lacZ genes and activate the transcription thereof is expressed in the host yeast, the yeast becomes viable in the presence of a sufficient amount of 3-AT and, at the same time, Xgal is degraded to turn the colonies into blue.

As used herein, the term "dehydration responsive element (DRE) " refers to a cis-acting DNA domain consisting of a 9 bp conserved sequence 5' - TACCGACAT-3' located upstream of those genes which are expressed upon exposure to dehydration stress, low temperature stress, etc.

A DNA fragment comprising DRE can be obtained by amplifying the promoter region of rd29A gene (from -215 to -145 based on the translation initiation site of the gene) by polymerase chain reaction (PCR) , rd29 gene being one of dehydration tolerance genes [Kazuko Yamaguchi-Shinozaki and Kazuo Shinozaki, The Plant Cell 6:251-264 (1994)] . As a template DNA which can be used in this PCR, genomic DNA from Arabidopsis thaliana is given. 1-3. Cloning of DREBlA Gene and DREB2A Gene

DREBlA gene and DREB2A gene can be obtained by transforming the cDNA library obtained in subsection 1-1 above into the host obtained in subsection 1-2 above by the lithium acetate method or the like, plating the resultant transformant on LB medium plate or the like containing Xgal (5-bromo-4-chloro-3-indolyl- j3 -D-galactoside) and 3-AT (3-aminotriazole) , culturing the transformant, selecting blue colonies appearing on the plate and isolating the plasmids therefrom.

Briefly, a positive clone containing DREBlA gene or DREB2A gene contains a fusion gene composed of a DNA region coding for GAL4 activation domain (GAL4 AD) and a DNA region coding for a DRE-binding protein, and expresses a fusion protein (hybrid protein) composed of the DRE-binding protein and GAL4 activation domain under the control of alcohol dehydrogenase promoter. Subsequently, the expressed fusion protein binds, through the DRE-binding protein moiety, to DRE located upstream of a reporter gene. Then, GAL4 activation domain activates the transcription of lacZ gene and HIS3 gene. As a result, the positive clone produces remarkable amounts of HIS3 protein and /3 -galactosidase. Thus, because of the action of the HIS3 protein produced, the positive clone can biosynthesize histidine even in the presence of 3-AT. Therefore, the clone becomes viable in the presence of 3-AT and, at the same time, the Xgal in the medium is degraded by the β -galactosidase produced to turn the colonies into blue.

Subsequently, such blue colonies are subjected to single cell isolation, and the isolated cells are cultured. Then, plasmid DNA is purified from the cultured cells to thereby obtain DREBlA gene or DREB2A gene. 1-4. Homologues to DREBlA Protein or DREB2A Protein

Organisms may have a plurality of genes with similar nucleotide sequences which are considered to have evolved from a single gene. Proteins encoded by such genes are mutually called homologues. They can be cloned from the relevant gene library using as a probe a part of the gene of which the nucleotide sequence has already been known. In the present invention, genes encoding homologues to DREBlA or DREB2A protein can be cloned from the Arabidopsis thaliana cDNA library using DREBlA cDNA or DREB2A cDNA obtained in subsection 1-3 above as a probe.

1-5. Determination of Nucleotide Sequences

The cDNA portion is cut out from the plasmid obtained in subsection 1-3 or 1-4 above using a restriction enzyme and ligated to an appropriate plasmid such as pSK (Stratagene) for sub-cloning. Then, the entire nucleotide sequence is determined. Sequencing can be performed by conventional methods such as the chemical modification method by Maxam- Gilbert or the dideoxynucleotide chain termination method using M13 phage. Usually, sequencing is carried out with an automated DNA sequencer (e.g. Perkin-Elmer Model 373A DNA Sequencer) .

SEQ ID NO: 1 shows the nucleotide sequence of DREBlA gene, and SEQ ID NO: 2 the amino acid sequence of the protein encoded by this gene. SEQ ID NO: 3 shows the nucleotide sequence of DREB2A gene, and SEQ ID NO: 4 the amino acid sequence of the protein encoded by this gene. SEQ ID NO: 5 shows the nucleotide sequence of DREBlB gene, and SEQ ID NO: 6 the amino acid .sequence of the protein encoded by this gene. SEQ ID NO: 7 shows the nucleotide sequence of DREBlC gene, and SEQ ID NO: 8 the amino acid sequence of the protein encoded by this gene. SEQ ID NO: 9 shows the nucleotide sequence of DREB2B gene, and SEQ ID NO: 10 the amino acid sequence of the protein encoded by this gene. As long as a protein consisting of one of the above-mentioned amino acid sequences has a function to bind to DRE to thereby activate the transcription of genes located downstream of DRE, the amino acid sequence may have mutation (such as deletion, substitution or addition) in at least one amino acid. A mutated gene coding for the protein having such mutated, amino acid sequence may also be used in the present invention.

For example, at least 1 amino acid, preferably 1 to about 20 amino acids, more preferably 1 to 5 amino acids may be deleted in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10; at least 1 amino acid, preferably 1 to about 20 amino acids, more preferably 1 to 5 amino acids may be added to the amino acid sequence shown in SEQ ID NO: 2, 4, 8 or 10; or at least 1 amino acid, preferably 1 to about 160 amino acids, more preferably 1 to 40 amino acids may be substituted with other amino acid(s) in the amino acid sequence shown in SEQ ID NO: 2, 4, 8 or 10. A gene coding for a protein having such mutated amino acid sequence may be used in the present invention as long as the protein has a function to bind to DRE to thereby activate the transcription of genes located downstream of DRE.

Also, a DNA which can hybridize with the above-mentioned gene under stringent conditions may be used in the present invention as long as the protein encoded by the DNA has a function to bind to DRE to thereby activate the transcription of genes located downstream of DRE. The "stringent conditions" means, for example, those conditions in which formamide concentration is 30-50%, preferably 50%, and temperature is 37- 50 °C, preferably 42¹C. A mutated gene may be prepared by known techniques such as the method of Kunkel, the gapped duplex method or variations thereof using a mutation introducing kit [e.g. Mutant-K (Takara) or Mutant-G (Takara)] or using LA PCR in vitro Mutagenesis Series Kit (Takara) .

Once the nucleotide sequence of DREB gene has been determined definitely, the gene can be obtained by chemical synthesis, by PCR using the cDNA or genomic DNA of the gene as a template, or by hybridization with a DNA fragment having the above nucleotide sequence as a probe.

The recombinant vectors containing DREBlA gene and DREB2A gene, respectively, were introduced into E^ coli K-12 strain and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba City, Ibaraki, Japan) under accession numbers FERM BP-6654 (E^ coli containing DREBlA gene) and FERM BP-6655 ( E^ coli containing DREB2A gene) on August 11, 1998.

2. Determination of the DRE Binding Ability and Transcription

Activating Ability of the Proteins encoded by DREB Genes 2-1. Analysis of the DRE Binding Ability of the Proteins encoded by

DREB Genes

The ability of the protein encoded by DREB gene (hereinafter referred to as the "DREB protein") to bind to DRE can be confirmed by performing a gel shift assay [Urao, T. et al., The Plant Cell 5:1529-1539 (1993)] using a fusion protein composed of the above protein and GST. A fusion protein composed of DREBlA protein and GST can be prepared as follows. First, DREBlA gene is ligated downstream of the GST coding region of a plasmid containing GST gene (e.g. pGEX-4T-l vector: Pharmacia) so that the reading frames of the two genes coincide with each other. The resultant plasmid is transformed into E. coli, which is cultured under conditions that induce synthesis of the fusion protein. The resultant E. coli cells are disrupted by sonication, for example. Cell debris is removed from the disrupted material by centrifugation. Then, the supernatant is purified by affinity chromatography using a carrier such as glutathione-Sepharose to thereby obtain the fusion protein.

Gel shift assay is a method for examining the interaction between a DNA and a protein. Briefly, a DRE-containing DNA fragment labelled with ³²P or the like is mixed with the fusion protein described above and incubated. The resultant mixture is electrophoresed. After drying, the gel is autoradiographed to detect those bands which have migrated behind as a result of the binding of the DNA fragment and the protein. In the present invention, the specific binding of DREBlA or DREB2A protein to the DRE sequence can be confirmed by making it clear that the above-mentioned behind band is not detected when a DNA fragment containing a varied DRE sequence is used.

2-2. Analysis of the Transcription Activating Ability of the Proteins Encoded by DREB Genes

The transcription activating ability of the proteins encoded by DREB genes can be analyzed by a trans-activation experiment using a protoplast system from Arabidopsis thaliana. For example, DREBlA cDNA is ligated to pBI221 plasmid (Clontech) containing CaMV35S promoter to construct an effector plasmid. On the other hand, 3 cassettes of the DRE-containing 71 base DNA region obtained in subsection 1-2 above are connected tandemly to prepare a DNA fragment, which is then ligated upstream of TATA promoter located upstream of j3 -glucuronidase (GUS) gene in pBI221 plasmid to construct a reporter plasmid. Subsequently, these two plasmids are introduced into protoplasts of Arabidopsis thaliana and then GUS activity- is determined. If GUS activity is increased by the simultaneous expression of DREBlA protein, it is understood that DREBlA protein expressed in the protoplasts is activating the transcription of GUS gene through the DRE sequence.

In the present invention, preparation of protoplasts and. introduction of plasmid DNA into the protoplasts may be performed by the method of Abel et al. [Abel, S. et al., Plant J. 5:421-427 (1994)]. In order to minimize experimental errors resulted from the difference in plasmid DNA introduction efficiency by experiment, a plasmid in which luciferase gene is ligated downstream of CaMV35S promoter may be introduced into protoplasts together with the two plasmids described above, and β - glucuronidase activity against luciferase activity may be determined. Then, the determined value may be taken as a value indicating the transcription activating ability. β -glucuronidase activity can be determined by the method of Jefferson et al. [Jefferson, R.A. et al., EMBO J. 83:8447-8451 (1986)]; and luciferase activity can be determined using PicaGene Luciferase Assay Kit (Toyo Ink) .

3. Creation of Transgenic Plants

A transgenic plant having tolerance to environmental stresses, in particular, low temperature stress (including freezing stress) , dehydration stress and salt stress, can be created by introducing the gene obtained in section 1 above into a host plant using recombinant techniques. As a method for introducing the gene into a host plant, indirect introduction such as the Agrobacterium infection method, or direct introduction such as the particle gun method, polyethylene glycol method, liposome method, microinjection or the like may be used. When the Agrobacterium infection method is used, a transgenic plant can be created by the following procedures.

3-1. Preparation of a Recombinant Vector to be Introduced into a Plant and Transformation of Agrobacterium

A recombinant vector to be introduced into a plant can be prepared by digesting with an appropriate restriction enzyme a DNA comprising DREBlA, DREBlB, DREBlC, DREB2A or DREB2B gene obtained in section 1 above, ligating an appropriate linker to the resultant DNA if necessary, and inserting the DNA into a cloning vector for plant cells. As the cloning vector, a binary vector type plasmid such as pBI2113Not, pBI2113, pBHOl, pBI121, pGA482, pGAH, pBIG; or an intermediate vector type plasmid such as pLGV23Neo, pNCAT, pMON200 may be used.

When a binary vector type plasmid is used, the gene of interest is inserted between the border sequences (LB, RB) of the binary vector. The resultant recombinant vector is amplified in E^ coli. The amplified recombinant vector is introduced into Agrobacterium tumefaciens C58, LBA4404, EHAlOl, C58ClRif^R, EHA105, etc. by freeze-thawing, electroporation or the like. The resultant Agrobacterium tumefaciens is used for the transduction of a plant of interest.

In addition to the method described above, the three-member conjugation method [Nucleic Acids Research, 12:8711 (1984)] may also be used to prepare DREB gene-containing Agrobacterium for use in plant infection. Briefly, an E^ coli containing a plasmid comprising the gene of interest, an E^ coli containing a helper plasmid (e.g. pRK2013) and an Agrobacterium are mixed and cultured on a medium containing rifampicin and kanamycin. Thus, a zygote Agrobacterium for use in plant infection can be obtained. Since DREB gene encodes a protein which activates transcription, various genes are activated by the action of the expressed DREB protein in a DREB gene-introduced plant. This leads to increase in energy consumption and activation of metabolism in the plant. As a result, the growth of the plant itself may be inhibited. As a means to prevent such inhibition, it is considered to ligate a stress responsive promoter upstream of DREB gene so that the DREB gene is expressed only when a stress is loaded. Specific examples of such a promoter include the following ones: rd29A gene promoter [Yamaguchi-Shinozaki, K. et al., The Plant Cell 6:251-264 (1994)] rd29B gene promoter [Yamaguchi-Shinozaki, K. et al., The Plant Cell 6:251-264 (1994)] rdl7 gene promoter [Iwasaki, T. et al., Plant Physiol., 115:1287 (1997)] rd22 gene promoter [Iwasaki, T. et al., MoI. Gen. Genet., 247:391-398 (1995)]

DREBlA gene promoter [Shinwari, Z.K. et al., Biochem. Biophys. Res. Com. 250:161-170 (1988)] cor6.6 gene promoter [Wang, H. et al., Plant MoI. Biol. 28:619-634 (1995)] corl5a gene promoter [Baker, S.S. et al., Plant MoI. Biol. 24:701-713 (1994)] erdl gene promoter [Nakashima K. et al.. Plant J. 12:851-861 (1997)] kinl gene promoter [Wang, H. et al.. Plant MoI. Biol. 28:605-617 (1995)]

Other promoter may also be used as long as it is known to be stress responsive and to function in plant. These promoters can be obtained by PCR amplification using primers designed based on a DNA comprising the promoter and using relevant genomic DNA as a template.

If necessary, it is also possible to ligate a terminator which demands termination of transcription downstream of DREB gene. As the terminator, cauliflower mosaic virus-derived terminator or nopaline synthase gene terminater may be used. Other terminator may also be used as long as it is known to function in plant.

If necessary, an intron sequence which enhances the expression of a gene may be located between the promoter sequence and DREB gene. For example, the intron from maize alcohol dehydrogenase (Adhl) [Genes S- Development 1:1183-1200 (1987)] may be introduced.

In order to select transformed cells of interest efficiently, it is preferable to use an effective selection marker gene in combination with DREB gene. As the selection marker, one or more genes selected from kanamycin resistance gene (NPTII) , hygromycin phosphotransferase gene (htp) which confers resistance to the antibiotic hygromycin on plants, phosphinothricin acetyl transferase gene (bar) which confers resistance to bialaphos and the like.

DREB gene and the selection marker gene may be incorporated together into a single vector. Alternatively, the two genes may be incorporated into separate vectors to prepare two recombinant DNAs.

3-2. Introduction of DREB Gene into a Host Plant

In the present invention, the term "host plant" means any of the following: cultured plant cells, the entire plant of a cultured plant, plant organs (such as leaves, petals, stems, roots, rhizomes, seeds) , or plant tissues (such as epidermis, phloem, parenchyma, xylem, vascular bundle) . Specific examples of plants which may be used as a host include Arabidopsis thaliana, tobacco, rice and maize.

DREB gene can be introduced into the above-described host plant by introducing a DREB gene-containing vector into plant sections by the Agrobacterium infection method, particle gun method or polyethylene glycol method. Alternatively, a DREB gene-containing vector may be introduced to protoplasts by electroporation.

If a gene of interest is introduced by the Agrobacterium infection method, a step of infecting a host plant with an Agrobacterium containing a plasmid comprising the gene of interest is necessary. This step can be performed by the vacuum infiltration method [CR Acad. Sci. Paris, Life Science, 316:1194 (1993)]. Briefly, Arabidopsis thaliana is grown in a soil composed of vermiculite and perlite (50:50). The resultant plant is dipped directly in a culture fluid of an Agrobacterium containing a plasmid comprising DREB gene, placed in a desiccator and then sucked with a vacuum pump to 65-70 mmHg. Then, the plant was allowed to stand at room temperature for 5-10 min. The plant pot is transferred to a tray and covered with a wrap to maintain the humidity. The next day, the wrap is removed. The plant is grown in that state to harvest seeds.

Subsequently, in order to select those individuals which have the gene of interest, seeds from various plant bodies are sown on MS agar medium supplemented with appropriate antibiotics. Arabidopsis thaliana grown on this medium are transferred to pots and grown there. As a result, seeds of a transgenic plant into which DREB gene is introduced can be obtained.

Generally, a transgene is located on the genome of the host plant. However, due to the difference in the locations on the genome, the expression of the transgene varies among transformants, presenting a phenomenon called position effect. Those transformants in which the transgene is expressed more highly can be selected by assaying mRNA levels in transformants by Northern blot analysis using a DNA fragment from the transgene as a probe.

The confirmation that the gene of interest is integrated in the transgenic plant of the invention and in the subsequent generation thereof can be made by extracting DNA from cells and tissues of those plants by conventional methods and detecting the transgene by PCR or Southern analysis known in the art.

3-3. Analysis of Expression Levels and Expression Sites of DREB Gene in Plant Tissues

Expression levels and expression sites of DREB gene in a transgenic plant into which the gene is introduced can be analysed by extracting RNA from cells and tissues of the plant by conventional methods and detecting the mRNA of DREB gene by RT-PCR or Northern blot analysis known in the art. Alternatively, DREB protein may be analysed directly by Western blotting or the like using an antibody raised against the protein.

3-4. Changes in mRNA Levels of Various Genes in a Transgenic Plant in to which DREB Gene is Introduced

It is possible to identify by Northern blot analysis those genes whose expression levels are believed to have been changed as a result of the action of DREB protein in a transgenic plant into which DREB gene is introduced. Northern blotting can assay those genes by comparing their mRNA levels in the transgenic plant into which DREB gene is introduced and in plants into which the gene is not introduced.

For example, plants grown on GM agar medium or the like are given dehydration and/or low temperature stress for a specific period of time (e.g. .1 to 2 weeks) . Dehydration stress may be given by pulling out the plant from the agar medium and drying it on a filter paper for 10 min to 24 hr. Low temperature stress may be given by retaining the plant at 15 to -4 °C for 10 min to 24 hr. Total RNA is prepared from control plants which did not receive any stress and plants which received dehydration and low temperature stresses. The resultant total RNA is subjected to electrophoresis. Then, genes expressing are assayed by Northern blot analysis or RT-PCR.

3-5. Evaluation of the Tolerance to Environmental Stresses of the Tra nsgenic Plant

The tolerance to environmental stresses of the transgenic plant into which DREB gene is introduced can be evaluated by setting the plant in a pot containing a soil comprising vermiculite, perlite and the like exposing the plant to various stresses such as dehydration, low temperature and freezing, and examining the survival of the plant. For example, tolerance to dehydration stress can be evaluated by leaving the plant without giving water for 2 to 4 weeks and then examining the survival. Tolerance to freezing stress can be evaluated by leaving the plant at -6 to -10"C for 5 to 10 days, growing it at 20 to 25 °C for 5 to 10 days and then examining its survival ratio.

4. Modification of DREB2A

A drought and high-salinity stress inducible transcription factor, DREB2A seems to require some kind of modification for its activation, but the activation mechanism has not been clarified. As results of domain analysis of the DREB2A, we found that transcriptional activation domain of the DREB2A exists between amino acid residue 254 to 335 and a deletion of a region between amino acid residue 136 and 165 transforms the DREB2A to constitutively active. The microarray analysis of transgenic plants overexpressing the constitutive active form of the DREB2A revealed that the DREB2A regulates expression of many water stress inducible genes. However, a part of the DREB2A target genes were not targets of a DREBlA that recognizes same cis-element. Overexpression of the constitutive active form of the DREB2A slightly improved freezing tolerance and significantly improved drought tolerance of the transgenic plants.

PREFERRED EMBODIMENTS OF THE INVENTION

Hereinbelow, the present invention will be described more specifically with reference to the following Examples. However, the technical scope of the present invention is not limited to these Examples.

EXAMPLE 1

Functional analysis of Arabidopsis DREB2A using a constitutive active form mutant

1. MATERIALS AND METHODS 1) Plant Materials

Plants (Arabidopsis thaliana ecotype Columbia) were grown on germination medium agar plates for 3 weeks. For Northern analysis, 3-week- old plants were subjected to stress treatments and then frozen in liquid nitrogen for RNA extraction. For a stress tolerance test, 3-week-old plants were transferred onto soil and grown for one week. Stress treatments were carried out as described above. Arabidopsis T87 suspension cultured cells were maintained as described previously (Axelos et al. , 1992, Plant Physiol. Biochem. 30, 123-128) . 2) Transient Expression Experiments

Effector and reporter plasmids used in the transient transactivation experiment regarding C-terminal deletion mutants of the DREB2A were constructed as described previously (Liu et al, 1998 Plant Cell 10, 1391- 1406) . Effector plasmids that encode the GAL4 DNA-binding domain fused to C-terminal region of DREB2A and a reporter plasmid that contains the GAL4 binding sequence were constructed as described above. Insert fragments used for construction of effector plasmids were amplified by PCR using the primer pairs shown in Table 1 (SEQ ID NOs:11-62) .

Isolation of Arabidopsis T87 cell protoplasts and polyethylene glycol-mediated DNA transfection were performed as described previously

(Abel and Theologis, 1994 Plant J. 5, 421-427) . Five-day-old Arabidopsis T87 suspension cultured cells were collected by filtration and washed with water. Five grams of T87 cells were incubated with enzyme solution (0.4M mannitol, 5 mM MES-KOH (pH 5.7), 8 rriM CaCl₂, 1% [w/v] Cellulase ONOZUKA RlO

(Yakult) , 0.5% [w/v] Macerozyme RlO (Yakult) ) at room temperature for 2 hours with gentle agitation. Cells were passed through 125 μm nylon mesh, recovered by centrifugation at 450 g for 5 minutes at room temperature and washed twice in 30 ml of 0.4 mM mannitol, 70 mM CaCl2, and 5 mM MES-KOH

(pH 5.7). Finally protoplasts were resuspended in MaMg solution (0.4 M mannitol, 15 mM MgCl2, 5 mM MES-KOH (pH 5.7)) and concentration was adjusted to 3x106 cell/ml. The isolated protoplasts were kept on ice until use. Plasmid DNAs used for protoplast transformation were prepared by using a QINGEN plasmid isolation kit (QIAGEN) and dissolved in 10 mM Tris- HCl (pH 8.0), and 1 mM EDTA at lμg/μL. One hundred μL of protoplast suspension was mixed well with 10 μL of effector plasmid, 10 μL of reporter plasmid and 5 μL of 35S:Luciferase internal control plasmid. Then PEG-CMS solution (0.4 M maπnitol, 0.1 M Ca(NO3)2, and 40% [w/v] polyethylene glycol (PEG) 3350 (Sigma) ) were added immediately to this protoplast-plasmid mixture and mixed well. Protoplasts were kept on ice for 20 minuets, and then diluted with 10 mL of 0.4 M mannitol, 125 itM CaCl2, 5 itM KCl, 5 mM glucose, and 1.5 πM MES-KOH (pH 5.7). The diluted protoplasts were harvested by centrifugation at 450 g for 5 min at room temperature. Transformed protoplasts were resuspended in 2.5 mL of culture medium (0.4 M mannitol, 1 x Murashige and Skoog [1962] basal medium) and cultured in the dark at 22⁰C for up to 24 h.

GUS activity was assayed by fluorometric quantification of 4- methylumbelliferone produced from the glucuronide precursor as described above. Luciferase activity was assayed by measuring light emission of the reaction with a PikkaGene luciferase assay kit (Toyo-ink) using a lumino meter (Wallac 1420 ARVOsx) .

Table 1.

Primer pairs used for effector plasmid construction .

Constructs Forward primer 1 Reverse primer 1 Forward primer 2 Reverse primer 2

DREB2A 1-317 SEQ ID NO.11 SEQ ID NO.12

DREB2A 1-281 SEQ ID NO.13 SEQ ID NO.14

DEEB2A 1-253 SEQ ID NO.15 SEQ ID NO.16

DREB2A 1-165 SEQ ID NO.17 SEQ ID NO.18

DREB2A 1-135 SEQ ID NO.19 SEQ ID NO.20

DREB2AΔ136-165* SEQ ID NO.21 SEQ ID NO.22 SEQ ID NO.51 SEQ ID NO.52

DREB2AΔ166-253* SEQ ID NO.23 SEQ ID NO.24 SEQ ID NO.53 SEQ ID NO.54

DREB2AΔ254-281* SEQ ID NO.25 SEQ ID NO.26 SEQ ID NO.55 SEQ ID NO.56

DREB2AΔ282-317* SEQ ID NO.27 SEQ ID NO.28 SEQ ID NO.57 SEQ ID NO.58

DREB2AΔ136-253* SEQ ID NO.29 SEQ ID NO.30 SEQ ID NO.59 SEQ ID NO.60

DREB2AΔ135-165, Δ318-335* SEQ ID NO.31 SEQ ID NO.32 SEQ XD NO.61 SEQ ID NO.62

GAL4-BD-DREB2A 254-281 SEQ ID NO.33 SEQ ID NO.34

GAL4-BD-DREB2A 254-317 SEQ ID NO.35 SEQ ID NO.36

GAL4-HD-DREB2A 254-335 SEQ ID NO.37 SEQ ID NO.38

GAL4-ED-DREB2A 136-253 SEQ ID . NO.39 SEQ ID NO.40

GAL4-BD-DREB2A 136-335 SEQ ID NO.41 SEQ ID NO.42

GAL4-HD-DREB2A 318-335 SEQ ID NO.43 SEQ ID NO.44

GAL4-ED-DREB2A 166-253 SEQ ID NO.45 SEQ ID NO.46

GAL4-HD-DREB2A 282-317 SEQ ID NO.47 SEQ ID NO.48

GAL4-BD-DREB2A 282-335 SEQ ID NO.49 SEQ ID NO.50

♦These fragments were generated by two-step PCR. First PCR used primer pair of the forward primer 1 and the reverse primer 1 or that of the forward primer 2 and the reverse primer 2 was carried out individually, amplified fragments in the first PCRs were used for second PCR as template with the forward primer 1 and the reverse primer 2.

3) Plant Transformation

Plasmids used for the transformation of Arabidopsis were constructed with a mutant DREB2A fragment that lacks the region from amino acid residues 136 to 165. The fragment was digested by Notl from the Δ136-165 effector plasmid used in the transactivation experiment and inserted into the Notl site of pBluescript II SK- (Stratagene) . Then the fragment was cut out from the plasmid with EcoRV and Sad, and subcloned into the Smal- Sacl site of pBE2113Not vector (Liu et al., 1998) in sense orientation. The constructed plasmid was introduced into Agrobacterium tumefaciens C58 by electro-transformation. Plant transformation was carried out as described above.

4) Microarray Analysis

Total RNA was isolated using the TRIZOL Reagent (Invitrogen) from 3- week-old plants having pBI121 or overexpressing a constitutive active form of DREB2A. rnRNAs were prepared using the PolyATract mRNA isolation system III (Promega) . Preparation of fluorescent probes, microarray hybridization, and scanning have been described previously (Seki et al., 2002, Plant J. 31, 279-292) .

5) Northern Blot Analysis

Total RNA was extracted using the TRIZOL Reagent. Northern blot analysis was performed as described above.

2. RESULTS

1) Transcriptional activation activity of deletion mutants of DREB2A

Because the C-terminal region of DREB2A is rich in acidic amino acids, the translational activation domain of the DREB2A protein has been predicted to exist in this region (Liu et al., 1998). To identify the translational activation domain, we carried out domain analysis of DREB2A in detail. Effector constructs containing a variety of C-terminal region deletion mutants of DREB2A were cotransfected with a β-glucuronidase (GUS) reporter construct driven by DRE sequences into protoplasts prepared from Arabidopsis T87 suspension cultured cells (Fig. 1) . Overexpression of full length DREB2A resulted in five to nine times higher transactivation of the reporter gene as compared with the case of the empty effector control. Deletion from the C-terminal end to amino acid residue (a.a.) 254 (DREB2A: 1-253). decreased the DREB2A-mutant-dependent transcativation to a level as same as in the case of the control. Qn the other hand, an internal deletion mutant lacking the region between a.a. 136 and a.a. 253 showed same level of reporter gene activation as the full length of DREB2A. These results suggest that the translational activation domain exists between a.a. 254 and the C-terminal end.

Interestingly, deletion of the region between a.a. 136 and 165 significantly increased its activity. Expression of the reporter gene by DREB2A Δ136-165, which was over 30 times higher than the basal level, and more than 3 times higher than the case of the with full length of DREB2A. This induction was equivalent to the DREBlA effector construct. The region between a.a. 136 and 165 seems to have a negative roll in regulation of DREB2A activity. Deletions of the other regions did not result in significantly modulated trasnactivation activity.

2) The region from 254 to the C-terminal end contains a necessary and sufficient domain for translational activation, and the region between a.a. 136 and a.a. 165 has a negative role in DREB2A activity.

For further domain analysis of DREB2A, we prepared effector constructs that contained variety of fragments from DREB2A fused to the GAL4 DNA binding domain (GB) (Ma et al, 1988, Nature 334, 631-633) . The effector plasmid was cotransfected into protoplasts of the Arabidopsis T87 cell with a reporter plasmid that contained nine copies of a GAL4-binding site fused to the minimal promoter of CaMV35S and the GUS reporter gene (Fig. 2) . As in the case of the results in Fig. 1, effector constructs of GB-DREB2A 136-253 and GB-DREB2A 166-253 that lacked the region from a.a. 254 the to C-terminal end of DREB2A did not induce expression of the reporter gene. GB-DREB2A 254-335, the effector construct containing the region from a.a. 254 to the C-terminal end, induced highest expression of the reporter gene among all effector constructs. This region consists of three sub-regions. Each sub-region could not or could only . weakly stimulate the expression of the reporter gene when it fused to the GAL4 binding domain independently (Fig. 2B, GB-DREB2A 254-281, 282-317 and 318- 335) , and a deletion of at least one sub-domain significantly decreased activity of the GUS reporter compared with GB-DREB2A 254-335 (Fig. 2B, GB- DREB2A 254-317 and 282-335) . Even if the fragment of GB-DREB2A 254-335 was extended toward the N-terminal such as GB-DREB2A 166-335 or 136-335, no further increase of reporter activity was observed. Although the effector construct, GB-DREB2A 166-335, induced reporter gene expression at almost the same level as GB-DREB2A 254-335, the transactivation by GB-DKEB2A 136- 335 that contained the region a.a. 136-165 was about six times lower than that by GB-DREB2A 254-335. These results indicate that the region between a.a. 254 and a.a. 335 is a necessary and sufficient activation domain of DREB2A, and that the region between a.a. 136-165 negatively controls translational activation ability of the DREB2A protein.

3) Overexpression of the constitutive active form of DREB2A in Arabidopsis To analyze the function of DREB2A, the DREB2A deletion mutant, DREB2A Δ136-165 (Fig. 1) that showed highest activity in the transient transactivation experiments was overexpressed in Arabidopsis plants. The gene encoding the constitutive active form of DREB2A was overexpressed under the control of the CaMV35S promoter (Mitsuhara et al., 1996). The tobacco mosaic virus (TMV) Ω sequence (Gallie et al., 1987) was inserted upstream of the mutant DREB2A fragment to increase the translation level. Fifty five transgenic Arabidopsis plants were generated by using the vacuum infiltration method. Expression levels of the transgene in transgenic T2 plants were analyzed by northern blot analysis, and we selected three lines of DREB2A, CA-a, -b and -c, that showed strong, moderate and weak transgene expression, respectively, for further analysis. Growth and expression of the DREB2A target gene of T2 transformants were confirmed. The growth patterns of the DREB2A CA plants were compared with control plants having the pBI121 vector (wt) and DREB2A FL plants overexpressing the full length DREB2A cDNA. All the DREB2A CA plants showed retarded growth (Fig. 3A and B) . The severest growth retardation was observed in the DREB2A CA-a plants in which the transgene was strongly expressed, and the levels of growth retardation of DREB2A CA-c in which the transgene was weakly expressed were mild. By contrast, no growth retardation was observed in DREB2A FL overexpresssing the full length of DREB2A. The DREB2A CA plants had a round shape and slightly dark green leaves with short leaf stems. These phenotypes appeared in DREB2A CA-a to a greater extent than in the case of DREB2A-C (Fig. 3C) . The environment stress-responsive gene, rd29A, has the DRE motives in the promoter region and it is confirmed that the DKEB2A protein can bind to this DRE sequence as described above. Accumulation levels of the rd29A iriRNA were increased in correlation with the expression levels of the constitutive active form of DREB2A.

4) Microarray analysis of the transgenic Arabidopsis plants overexpressing the constitutive active form of DREB2A

In order to understand which genes were under the control of DREB2A, we compared accumulation of rtiRNAs of ^"7000 genes between wild type plants and DREB2A CA plants by using an Arabidopsis full-length cDNA microarray. Cy3-labeled and Cy5-labeled cDNA probes were prepared by using rriRNA that was isolated from DREB2A CAs and control plants without stress treatment, respectively. These probes were hybridized with the cDNA microarray, and the expression profiles of the ^"7000 genes were analyzed. The experiments were repeated three times and the genes regarding which signal intensities exceeded 2000 in at lest one experiment were further analyzed. We choose genes that showed an expression ratio 5 or more times greater in the DREB2A CA-a plants than in wild type plants as candidates for the DREB2A target genes (Table 2) .

Table 2 .

Significantly upregulated transcπpits in DREB2A CA-a plants'¹

Ralιo^d

Gene Name^b AGI code^c DREβ ABREβ Description¹¹

DREB2A CA-a DREB1A^Θ rd29A At5g52310 160 147^f -265 to -260 -55 to -50 Late embryogenesis abundant protein -215 to -210 -158 to -153 -121 to -116

ΛtGo/S3 At1gO93SO 143 10 6 ^f -800 to -805 -265 to -260 Galaotinol synthase -376 to -381 -772 to -777 rd29B At5g52300 125 2 8 -162 to 157 -168 to -163 Late embryogenesis abundant protein

-63 to -58 -704 to -709

-77 to -82

-35 to -30

RAFL06-13-J20 At1g52690 12 0 0 8 -37 to -32 -220 to -225 Late embryogenesis-abundant protein -625 to -620 -617 to -622 -228 to -223 -190 to -185 rd17 At1g20440 11 2 8 3 ' -985 to -980 -909 to -904 Dehydrin -151 to 146 -330 to -335 -956 to 951 cor15A At2g42540 107 131 ' -350 to -345 -121 to -116 Late embryogenesis abundant protein -173 to -168 -113 to -118 -402 to -407 -289 to -294 -60 to -55

RAFL02-04-G03 At4g33720 103 0 8 -16 to -11 Pathogenesis-related protein 1 -195 to -200

AtMT-K At3g09390 100 20 -172 to -167 Metallothionein-like protein

RAFL05-13-A17 At1g32860 94 1 4 -61 to -56 -572 to -577 Unknown protein -«05 to -600

RAFL05-16-B15 At1g69B70 9 0 25 -332 to -327 -92 to -87 Unknown protein -190 to -185

RAFL06-16-L13 At5g54960 7 0 1 6 -288 to -293 -934 to -929 Pyruvate decarboxylase -357 to -362

RAFL05-17-B13 Al1g01470 6 3 7 1 ' -404 to -399 -631 to -636 Late embryogenesis-abundant protein -56 to -51 -132 to -137 -369 to -374 -88 to -93

RAFL04-10-U13 At2g23120 60 50 ' -117 to -112 -65 to -60 Unknown protein -85 to -SO

RAFL05-21-K17 At5g54170 56 0 8 Membrane related protein-like kιn1 At5g15960 55 11 7 ' -109 to -104 -346 to -351 Late embryogenesis abundant protein -67 to -62 -132 to -137

RAFL04-18-B07 At5g62350 4 6 f -550 to -545 Unknown protein -30 to -25 -182 to -177

RAFL03-05-E08 At3g53990 53 23 -968 to -973 Unknown protein -181 to -186

AtGRP7 At2g21660 52 3 3 ' -77 to -72 -292 to -297 Glycine-rich RNA binding protein kιπ2 At5g15970 5 2 33 ' -121 to -116 -54 to -59 Late embryogenesis abundant protein -62 to -57 -71 to -76 -79 to -74 -145 to -149 -365 to -370 ^amRNAs from DREB2A CA-a and pBI121 plants were used for preparation of Cy3-labeled and Cy5-labeled cDNA probes These cDNA probes were mixed and hybridized with the cDNA microarray In this study, we used lambda DNA as internal control because its fluoresence level is almost the same in both the plants ^bGene names are fulWeπgth cDNA clones (Seki et al 2002) ^CAGI code for cDNAs used in this study ri Fluorescence Intensity (Fl) of each cDNA of 35S DREB2A CA -a plants _ Fl of lambda DNA of DREB2A CA-a plants Fl of each cDNA of pBH 21 plants Fl of lambda DNA of pBI121 plants

Nineteen genes exhibited expression that increased more than 5 times in DREB2A CA-a plants than in wild type plants. Among DREB2A CA-a, b and c, expression levels of these genes correlated with the accumulation of mRNA of the constitutive active form of DREB2A (data not shown) . Many of these genes are those encoding water-stress-related proteins such as LEA protein. To confirm whether the promoter regions of the nineteen genes contained the DRE and ABA responsive element (ABRE) or not, we surveyed the promoter region from ATG to 1 fcb upstream. Seventeen of the nineteen genes had the DRE sequence(s), fifteen genes had the ABRE sequence(s) and fourteen genes had both cis-elements in their promoter region. These facts suggest that most of DREB2A target genes are important in water stress tolerance and therefore these genes are .regulated by both the DREB pathway and the ABA pathway. On the other hand, interestingly, only ten genes in the list were identified as the DREBlA target genes.

For further analysis of the genes that up-regulated by overexpression of the constitutive active form of the DREB2A, we carried out the Northern blot analysis. The total RNAs isolated from plants of the wild type, DREBLA-b and DREB2A CA a-c with or without stress treatment of 4⁰C for 5 hours or dehydration for 5 hours were used for the Northern blot analysis (Fig. 4) . Accumulations of mRNA of eleven genes were increased in both the DREBlA-b and the DREB2A CAs plants compared with the wild type plants (Fig. 4B) . DREBlA-b was the transgenic plants overexpressing DREBlA under the control of the CaMV35S promoter. The DREBlA-b plants showed moderate phenotypic change among the DREBlA overexpressors. Though AtGolS3 has the DRE sequence in the promoter region, it is known that this gene is cold inducible but not drought inducible. Thus the promoter region of this gene is predicted to contain a novel cis-element that negatively regulates the gene expression under drought conditions (Taji et al., 2002, Plant J. 29, 417-426) . The expression of AtGolS3 in the DREB2A CA plants was stronger in control and cold treatment, and weaker in dry treatment. This fact strongly supports the hypothesis described above, and the AtGolS3 may not be the target gene of DREB2A physiologically, though the DREB2A protein can bind to the AtGolS3 promoter region. In spite of the fact that At2g02100 and Atlg29395 are target genes of DREBlA and these have DREs in their promoter regions, expression of these genes was not induced in the DREB2A CA plants compared with the wild type plants. However, expression of Atlg29395 was induced by drought in the wild type plants. Four of the ABRE existed in the promoter region of the AtIg 29395, and thus ABA seems to be involved with drought inducible expression of Atlg29395. Results of the northern blot analysis regarding the genes for which expression was induced in DREB2A CA plants but not in DREBlA-b plants were showed in Fig. 4D. Most of these genes are shown inducible. In particular, rd29B, Atlg52690, At3g09390, Atlg69870 and Atlg22985 showed distinct drought specific gene expression. It is suggested that regulation of the expression of these genes via DRE is regulated by DREB2A, but not by DREBlA. Because the promoter region of At5g54170 did not contain the DRE sequence, drought specific gene expression of this gene may be an indirect affect of DREB2A. Atlg22985 encodes a transcriptional factor containing the ERF/AP2 domain. The genes like the Atlg22985 seem to control the DREB2A target genes that do not contain DRE in their promoter region, such as At5g54170. At4g33720 did not show stress inducible gene expression. Moreover, this gene did not contain DRE in its promoter region. Thus, an increase of expression of At4g33720 was probably an indirect and artificial effect of the strong expression of the constitutive active form of DREB2A. 5) Freezing and drought stress tolerance of the transgenic Arabidopsis plants overexpressing the constitutive active form of DREB2A

The tolerance for freezing and drought stresses of the DREB2A CA plants were compared with those of the DREBlA-b and wild type plants (Fig. 5) . The plants were grown on germination medium agar plates for 3 weeks, then transferred onto pots that filled with soil and grown for one week at 22°C. For drought stress treatment, water was withheld from the plants for 2 weeks. They were then watered and grown under control conditions for 3 days. This treatment blighted all wild type plants, whereas about 60% of the DREBlA-b plants survived this treatment. As in the case of the DREBlA- b, 62.8%-83.3% of the DREB2A CA plants survived this treatment. For the freezing stress treatment, plants were exposed to a temperature of -6⁰C for 30 h, and returned to 22°C for 5 days. This treatment blighted all wild type plants, whereas about 40% of the DREBlA-b plants survived this treatment. By contrast to the drought stress tolerance, just 5.0%-11.7 % of the DREB2A CA plants remained after freezing treatment. In another experiment, wild type plants and transgenic plants (DREBlA-a, DREB2A CA-a, DREB2A CA-b, and DREB2A CA-c) were planted in single pot and their survival after 10-day dewatering was compared. Wild type plants blighted after 10-day dewatering, whereas transgenic plants grew well. These results indicate that the target genes of DREB2A play an important role in acquirement of tolerance to drought stress, but these are riot sufficient for resistance to freezing stress.

3. DISCUSSION

Though overexpression of DREBlA under the control of the CaMV35S promoter caused phenotype changes in the transgenic plants, transgenic plants overexpressing full-length cDNA of DREB2A showed almost same phenotype as wild type plants. Therefore, it seems that the translated DREB2A protein is an inactive form and the DREB2A protein requires some kind of modification for its activation.

In this study, it is revealed that the translational activation domain of the DREB2A protein exists at the C-terminal, in the a.a. 254-335 region. The fusion protein of this region and GAL4 DNA binding domain showed significant transactivation of the reporter gene (Fig. 2, GD-DREB2A 254- 335) . These results suggest that this region has transcriptional activation ability without any modification and there is another region that negatively controls activity of the DREB2A protein. Actually, the deletion of the a.a.136-165 region significantly increased DREB2A activity. This fact indicates that this region has a negative roll in regulation of DREB2A protein activity. The a.a. 254-335 and a.a. 136-165 regions are encoded by the positions 572-661 and 926-1171 of SEQ ID NO: 3, respectively.

DNA binding domain and a nuclear localization signal (NLS) in N- terminal region of DREB2A gene are also essential for exertion of transactivation activity of DREB2A. In fact, effecter constructs containing various C-terminal region deletion mutants of DREB2A gene shown in Fig. 1 contain the DNA binding domain and nuclear localization signal in N-terminal region. Also, effecter constructs shown in Fig. 2 contain exogenous GAL4 DNA binding domain, which includes NLS therein, instead of endogenous DNA binding domain and NLS of DREB2A gene. Liu et al. describes "We searched DNA and protein databases for sequences homologous to those of the DREBlA and DREB2A proteins and found that each DREB protein has a conserved DNA binding domain of 58 amino acids present in a large family of plant genes for DNA binding proteins, including EREBPs of tobacco and AP2 of Arabidopsis. The deduced amino acid sequences of DREBlA and DREB2A showed no significant sequence identity except in the conserved DNA binding domain. However, each DREB protein contains a basic region in its N-terminal region that might function as a nuclear localization signal and an acidic C-terminal region that might act as an activation domain for transcription. These data suggest that each DREB cDNA encodes a DNA binding protein that might function as a transcriptional activator in plants.

In order to determine the positions of NLS and DNA binding domain of DR EB2A, we have conducted the following analysis and database search. An analy sis by a PSORT program (http://psort.ims.u-tokyo.ac.jp/) revealed that the D REB2A protein has bipartite nuclear localization signals (NLS) at N-terminal region as RKRK from amino acid position 19 and KKRK from amino acid position 52. A search of CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) revealed that the DREB2A protein has three DNA binding domains within the reg ions of a.a. 78-138, a.a. 77-137, and a.a. 79-135, respectively.

The DREBlA protein and the DREB2A proteins have appeared to recognize the same cis-element, DRE. However, it is revealed in this study that the set of the DREBlA target genes and the set of the DREB2A target genes were not completely consistent. Moreover, although the Corl5A and B were recognized by both DREBs, expression levels of these genes were significantly different between DREBlA-b plants and DREB2A CA-a (Fig. 4B) . This inconsistency regarding the sets of the target genes between DREB proteins was perhaps a reason for the fact that freezing tolerance of the DREB2A CA plants was weaker than the DREBlA -b plants, although these two kinds of transgenic plants showed same levels of drought stress tolerance. In the previous report, we revealed that both the DREBlA and the DREB2A proteins recognized the same core sequence of RCCGAC (Sakuma et al., 2002) . However, recently we have elucidated that the DREBlA protein has the most affinity to an RCCGACxT sequence by detailed analysis of the promoter region of the DREBlA target genes (Maruyama et al., 2003). To find the reason why the sets of the target genes of DREBlA and DREB2A did not coincide, we analyzed the promoter region within 500 bp upstream from ATG of the genes whose expression level in microarray analysis is increased more than 5 times in the DREB2A CA-a plants but increased less than 3 times in the DREBlA-b plants compared, with that in the wild type plants. Eight of the DRE sequences were found, but only two (25%) DRE sequences have the RCCGACxT sequences. The DREB2A protein probably can bind to a DRE sequence other than RCCGACxT and control the expression of genes the expression of which is hardly controlled by DREBlA. We have also attempted to identify the sequence that is a prerequisite to binding of the DREB2A protein. However, remarkable bias of the nucleotide abundance ratio was not observed. To achieve this goal, more DREB2A-specific target genes may be necessary.

Expression of DREB2A is induced by drought and high-salinity stress and the DREB2A protein specifically binds to the DRE element, and therefore, DREB2A is expected to be involved with ABA independent water stress inducible gene expression. However, distinct evidence to confirm this hypothesis has not been obtained because overexpression of DREB2A did not induce any phenotypic change in the transgenic plants. Also, the DREB2 gene family consists of at least two members (DREB2A and DREB2B) and thus the function of the DREB2 genes may be redundant. In addition, most of water stress inducible genes have both the DRE and the ABRE in their promoter region and ABA signals may be also transmitted to the DRE via DREB1D/CBP4. This study clearly showed that DREB2A certainly plays a roll in the dehydration stress inducible signal transduction pathway. This fact means that we have obtained a novel tool for the molecular improvement of plant tolerance to environmental stresses.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

EFFECT OF THE INVENTION

According to the present invention, there is provided a transgenic plant containing a gene in which a DNA coding for a protein that binds to a stress responsive element and regulates the transcription of genes located downstream of the element is ligated downstream of a stress responsive promoter, the transgenic plant having improved tolerance to environmental stresses (such as dehydration, low temperature and salt) and being free from dwarfing.

Claims

Claims

1. A transgenic plant comprising a DNA that encodes a protein comprising the amino acid sequence of positions 254-335 of SEQ ID NO: 4.

2. The transgenic plant of claim 1, wherein said protein provides an increas e in transactivation activity of a reporter gene of from about 5 to about 9 t imes greater than in the absence of a protein consisting of the amino acid se quence as shown in SEQ ID NO: 4.

3. A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence as shown in SEQ ID NO: 4 with the deletion of amino a cids from positions 136-165 thereof, said DNA being operably linked downstre am of a stress responsive promoter.

4. The transgenic plant of claim 3, wherein said protein can bind to said st ress responsive promoter.

5. The transgenic plant of claim 3 or 4, wherein the protein consisting of t he amino acid sequence as shown in SEQ ID NO: 4 with the deletion of amino ac ids from positions 136-165 thereof provides at least a three fold increase in transactivation activity of a reporter gene when compared with that of a ful 1 length DREB2A protein.

6. An isolated nucleic acid molecule that encodes a DREB2A protein as shown in SEQ ID NO:4 with the deletion of amino acids from positions 136 to 165 the reof.

7. A transgenic plant Which comprises a DNA comprising the isolated nucleic acid molecule of claim 6, the DNA operably linked downstream of a stress resp onsive promoter.

8. An isolated protein having the sequence as shown in SEQ ID NO:4 with the deletion of the amino acids from positions 136 to 165 thereof.

9. A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence of positions 254-335 of SEQ ID NO: 4, said DNA being operably linked downstream of a stress responsive promoter.

10. The transgenic plant of claim 9 further comprising a DNA that encodes DN A binding domain and a nuclear localization signal.

11. The transgenic plant of claim 9 or 10, wherein said protein can bind to said stress responsive promoter.

12. A transgenic plant comprising a DNA that encodes a protein consisting of the amino acid sequence of positions selected from the group consisting of p ositions 254-317, 136-335, 318-335, 166-135 and 282-335 of SEQ ID NO: 4, said DNA being operably linked downstream of a stress responsive promoter.

13. The transgenic plant of claim 12 further comprising a DNA that encodes D NA binding domain and a nuclear localization signal.

14. An isolated nucleic acid molecule encoding a translational activation d omain of the DREB2A protein comprising the amino acid sequence of positions 2 54 to 335 of SEQ ID NO: 4.

15. A transgenic plant comprising a DNA comprising the isolated nucleic acid molecule of claim 14, the DNA operably linked downstream of a stress respons ive promoter.

16. An isolated protein with DREB2A protein activity and having the amino ac id sequence of positions 254 to 335 of SEQ ID NO:4.

17. A transgenic plant comprising a DNA that encodes a protein consisting of a fragment of the amino acid sequence as shown in SEQ ID NO: 4 with the delet ion of amino acids from positions 136-165 thereof, wherein said fragment com prises the amino acid sequence of positions 254-335 and DNA binding domain an d a nuclear localization signal, said DNA being operably linked downstream o f a stress responsive promoter.

18. A transgenic plant comprising a DNA comprising the nucleotide sequence s hown in SEQ ID NO: 3 with the deletion of the nucleotides from positions 572- 661 thereof, said DNA being operably linked downstream of a stress responsiv e promoter.

19. The transgenic plant of claim 18, wherein a protein encoded by said DNA can bind to said stress responsive promoter.

20. The transgenic plant of claim 18, wherein said DNA encodes a protein tha t can bind to said stress responsive promoter.

21. An isolated nucleic acid molecule as shown in SEQ ID NO: 3 with the dele tion of the region from nucleotide positions 572-661 thereof.

22. A transgenic plant which comprises a DNA comprising the isolated nucleic acid molecule of claim 21, said DNA being operably linked downstream of a st ress responsive promoter.

23. A transgenic plant comprising a DNA comprising the nucleotide sequence a s shown in positions 926-1171 of SEQ ID NO: 3, said DNA being operably linked downstream of a stress responsive promoter.

24. The transgenic plant of claim 23, wherein a protein encoded by said DNA can bind to said stress responsive promoter.

25. An isolated nucleic acid molecule comprising the nucleotide sequence sh own in positions 926 to 1171 of SEQ ID NO: 3

26. A transgenic plant which comprises a DNA comprising the isolated nucleic acid molecule of claim 25, the DNA being operably linked downstream of a str ess responsive promoter.