WO2005085279A1 - Stress-inducible osasr1 gene and protein enhancing tolerance to abiotic stress - Google Patents

Abstract

The present invention relates to abiotic stress-inducible OsAsrl gene and protein enhancing plant tolerance to cold, salt or drought stress, where OsAsri is overexpressed in the plant. The OsAsrl gene and protein of the present invention is very useful for enhancing plant tolerance to cold, salt or drought stress.

Description

STRESS-INDUCIBLE OsAsrl GENE AND PROTEIN ENHANCING TOLERANCE TO ABIOTIC

STRESS

FIELD OF THE INVENTION The present invention relates to abiotic stress-inducible OsAsrl gene and protein enhancing plant tolerance to cold, salt or drought stress .

BACKGROUD OF THE INVENTION As sessile organisms, plantsmust copewithenvironmental stresses, e.g. , low temperatures, drought and salt stress. After such exposures, a series of changes occurs in their patterns of gene expression (Guy, 1999) , a response that affects growth rates, productivity, and species distribution. Rice, a cereal crop of tropical or subtropical origin, frequently suffers from chilling injury, and plants exhibit various symptoms such as chlorosis, necrosis, or growth retardation (De Datta, 1981) . In temperate regions, rice frequently confronts chilling at the seedling stage as well as cool-summer damage during flowering. To understand the abiotic stress-response mechanism, researchers have isolated a number of genes that encode abiotic stress-inducible proteins in several plant species (Guy, 1999; Thomashow, 1999) . CalcineurinB-like 1 { CBLl) gene is highly inducible by drought, salt and cold stresses. In addition, CBLl-overexpressing plants showed enhanced tolerance to salt and drought (Cheong, Y.H., Kim, K.N. , Pandey, G.K., Gupta, R., Grant, J.J., andLuan, S.2003. CBLl, a calciumsensor that differentially regulates salt, drought, and cold responses in Arabidopsis . Plant Cell 15:1833-1845) . The genes induced under low temperatures are thought to function in protecting cells by producing various gene products. For example, AraiDidopsis APkinases (ATMPK4 andATMPK6) andC-repeat/DRE binding factor (CBF/DREB) transcription factors have roles in cold-stress signal transduction (Mizoguchi et al . , 1993; Stockinger et al . , 1997), while lipid desaturase is involved in membrane modifications during chilling (Gibson et al . , 1994) . Cold-induced pathogenesis-relatedproteins, e.g. , chitinase-likeand thaumatin-like proteins, function as antifreeze proteins (Hiilovaara-Teijo et al . ,

1999) . Moreover, chaperones, late embryogenesis abundant (LEA) proteins, calmodulin-related proteins, and 14-3-3 proteins might contribute to enhanced freezing tolerance (Thomashow, 1999) : The enzymes required for biosynthesis of various osmoprotectants , such as sugars, prolines, and betaines are also important to a plant' s osmotic adjustment (Kishor et al., 1995; Igarashi et al . , 1997; Scott et al . , 1999). The Asr genes, which are responsive to ABA, osmotic stress, and ripening, have been identified in various species, including tomato, potato, apricot, loblolly pine, lily, maize, pummelo, grape and rice (Iusem et al . , 1993; Canel et al . , 1995; Silhavy et al . , 1995; Wang et al . , 1998; Chang et al . , 1996; Mbeguie-A-Mbeguie et al . , 1997;

Vaidyanathan et al . , 1999; Hong et al . , 2002; Jeanneau et al . , 2002; Cakir et al . , 2003) . High levels of the tomato Asrl mRNA can be detected in ripe fruit and in leaves subjected to water stress (Hagit et al . , 1995) . The level of lily ASR proteins is also increased through desiccation during pollen maturation (Wang et al . , 1998). The physiological roles of ASR proteins are unclear yet. The localization of tomato ASRl proteins in nucleus proposed the nature of tomato ASR proteins as nonhistone chromosomal proteins (Rossi and Iusem, 1994) . Also, the structural and functional similarity of some ASR proteins with Late Embryo Abundant (LEA) or dehydrin proteins suggests a possible role of ASR in the seed development (Maskin et al . , 2001; Silhavy et al . , 1995) . Many known ASR proteins contain two conserved regions of aputative Zn-binding site at the N-terminal region andaputative nuclear localization sequence (NLS) at the C-terminal region of ~70 amino acids (Cakir et al . , 2003; Silhavy et al . , 1995) . These facts as well as the binding of grape ASR (VvMSA) to the promoter sequence of a monosaccharide transporter gene suggested that VvMSA is a component of the transcription-regulating complex involved in sugar and ABA signaling (Cakir et al . , 2003) . Vaidyanathan et al . (1999) have identified an ASR cDNA, OsAsrl , from 'Pokkali' rice, where OsAsrl was both ABA- and osmotic (NaCl) stress-inducible. However, the functions of OsAsrl having hydrophilic alpha helix structure is almost unknown.

Throughout this application, various patents and publications are referenced and citations are provided inparentheses . The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains .

DETAILED DESCRIPTION OF THIS INVENTION The present inventors have made intensive study to find genes capable of enhancing tolerance to abiotic stress. As a result, the inventors have found that OsAsrl ( Oryza sativa responsive toABA, osmotic stress, and ripening gene 1) gene was induced by stress in Oryza sativa and a plant transformed with the gene showed an enhanced tolerance to cold, salt or drought stress. Accordingly, it is anobject of this inventiontoprovide anabiotic stress-inducible OsAsrl protein enhancing plant tolerance to cold, salt or drought stress. It is another object of this invention to provide a nucleic acid which comprises a nucleotide sequence encoding the abiotic-stress inducible OsAsrl protein. It is other object of this invention to provide a vector comprising a nucleic acid comprising a nucleotide sequence encoding the abiotic-stress inducible OsAsrl protein. It is other object of this invention to provide a transformant transformed with the vector of the present invention. It is other object of this invention to provide a method for preparing a transgenic plant showing an enhanced tolerance to cold, salt, or drought stress. It is other object of this invention to provide a transgenic plant showing an enhanced tolerance to cold, salt, or drought stress.

Other objects and advantages of the present invention will become apparent from examples to follow, appended claims and drawings.

Inone aspect of this invention, there is providedanOsAsrl protein, comprising amino acid sequence of SEQ ID NO 2, which is inducible by abiotic stress , enhancingplant tolerance to cold, salt ordrought stress , when the protein is overexpressed in the plant, or a homologue protein sharing at least 80% amino acid sequence identitywith the OsAsrl protein. The present inventors have made intensive study to find genes capable of enhancing tolerance to abiotic stress. As a result, the inventors have found that OsAsrl (Oryza sativa responsive toABA, osmotic stress, and ripening gene 1) gene was induced by stress in Oryza sativa and a plant transformed with the gene showed an enhanced tolerance to cold, salt or drought stress. The term "abiotic stress" as used herein refers to stress caused by abiological factors such as cold, salt and drought. The term "cold stress" as used herein refers to stress caused by low temperatures, preferably below 15C more preferably 12°C and most preferably IOC The abiotic-stress inducible OsAsrl protein of the present invention is considered to include acid sequences which have substantial identity to the aforementioned amino acid sequence and are capable of enhancing tolerance to cold, salt or drought stress. The phrase "substantial identity" refers to that an amino acid sequence has at least 80%, preferably at least 90%, most preferably at least 95% amino acid sequence identity, when the aforementioned amino acid sequence of the present invention is compared and aligned for maximum correspondence with an amino acid sequence, as measured using conventional sequence comparison program (for example: Clustal X and PROSIS) . Meanwhile, the stress-inducible OsAsrl protein of the present invention may be defined as follows: (a) protein isolated from Oryza sativa L. ; (b) protein having a Zn-binding motif in the region of N-end; and (c) protein having a nuclear localization domain in the region of C-end.

In one aspect of this invention, there is provided a nucleic acid which comprises a nucleotide sequence encoding the abiotic-stress inducible OsAsrl protein.

In the present invention, the term "nucleic acid" is considered to include DNA (gDNA and cDNA) and RNA. Nucleoside, a basic unit constituting nucleic acid, includes the natural nucleosides as well as the nucleosides having modified base moieties and/or modified sugar moieties (Scheit, Nucleotide Analogs, JohnWiley, NewYork, 1980) ; Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)) Most preferably, the nucleic acid of the present invention comprises 67-480 nucleotide sequence in SEQ ID N0:1. The nucleic acid of the present invention encoding abiotic stress-inducible OsAsrl protein is considered to include nucleotide sequences which have substantial identity to the aforementioned nucleic acid. The phrase

"substantial identity" refers to that an nucleotide sequence has at least 80%, preferably at least 90%, most preferably at least 95% nucleotide sequence identity, when the aforementioned nucleotide sequence of the present invention is compared and aligned for maximum correspondence with an nucleotide sequence, as measured using conventional sequence comparison program (for example: Clustal X and PROSIS) .

In one aspect of this invention, there is provided a vector comprising a nucleic acidwhich comprises a nucleotide sequence encoding the abiotic-stress inducible OsAsrl protein. According to a preferred embodiment, the vector of the present invention comprises (a) nucleotide sequence encoding abiotic stress-inducible OsAsrl protein and (b) promoter operably linked to the nucleotide. The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.

The vector system of this invention may be constructed according to the known methods in the art as described in Sambrook et al . , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) , which is incorporated herein by reference. Typically, the vector may be constructed for cloning or expression. In addition, the vector may be constructed for use in prokaryotic or eukaryotic host cells. For example, where the vector is constructed for expression in prokaryotic cells, it generally carries a strong promoter to initiate transcription (e.g., pLλ promoter, trp promoter, lac promoter, T7 promoter and tac promoter) , a riboso e binding site for translation initiation and a transcription/translation termination sequence. In particular, where E. coli is used as a host cell, a promoter and operator i in operon for tryptophan biosynthesis in E. coli (Yanofsky, C, J.

Bacteriol . , 158:1018-1024(1984)) and a leftward promoter of phage λ

(pLλ promoter, Herskowitz, I. and Hagen, D., Ann . Rev. Genet . , 14:399-445(1980)) may be employed as a control sequence. The vector used in the present invention may be prepared from plasmid (e.g.: pSClOl, ColEl, pBR322, pϋC8/9, pHC79, pGEX series, pET series and pUC19) , phage (e.g.: λgt4 ^• λB, λ-Charon, λ Δ zl and Ml3 etc) or virus (e.g.: SV40) used conventionally in the art. Where the vector of the present invention is anexpression vector constructed foreukaryotic host cell, a promoter derived the genome of mammalian cells (e.g., metallothionein promoter) or mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tit promoter of HSV) ma be used.

The vector generally contains a polyadenylation site as a transcription termination sequence. The vector of this invention may further comprises a nucleotide sequence to conveniently purify the abiotic stress-inducible OsAsrl protein expressed, which includes but not limited to, glutathione S-transferase (Pharmacia, USA) , maltose binding protein (NEB, USA) , FLAG (IBI, USA) and 6X His (hexahistidine; Quiagen, USA) . Due to the additional sequence, the protein expressed in host can be purified with affinity chromatography in a rapid and feasible manner. It is preferable that the expression vector of this invention carries one or more markers which make it possible to select the transformed host, for example, genes conferring the resistance to antibiotics such as ^' ampicillin, gentamycine, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticine, neomycin, geneticin and tetracycline. Since the gene of the present invention has been isolated from plant and is capable of enhancing plant tolerance to abiotic stress, it is most useful for plants. Accordingly, the vector of the present invention comprises (i) nucleotide sequence encoding abiotic stress-inducible OsAsrl protein and (ii) a promoter that functions in plant cells to cause the production of an RNA molecule operably linked to the nucleotide sequence of (i) ; and (iii) a 3' -nontranslated region that functions in plant cells to cause the polyadenylation of the 3 '-end of said RNA molecule. According to a preferred embodiment of the present invention, where the expression vector is constructed for a plant cell, numerous plant-functional promoters known in the art may be used, including the maize ubiquitin promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthetase (nos) promoter, the Figwort mosaic virus 35S promoter, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of the ribulose-1, 5-bis-phosphate carboxylase (ssRUBISCO) , the rice cytosolic triosephosphate isomerase (TPI) promoter, the adenine phosphoribosyltransferase (APRT) promoter of

Arabidopsis, and octopine synthase promoters. According to a preferred embodiment of the present invention, the 3 ' -non-translated region causing polyadenylation in this invention may include that from the nopaline synthase, gene of Agrobacterium tumefaciens (nos 3' end) (Bevan et al . , Nucleic Acids Research,

11(2) :369-385 (1983) ) , that from the octopine synthase gene of

Agrobacterium tumefaciens, the 3 '-end of the protease inhibitor I or II genes from potato or tomato, the CaMV 35S terminator. The vector may alternatively further carry a gene coding for reporter molecule (e.g. luciferase and P-glucuronidase) . The vector may contain antibiotic (e.g. neomycin, carbenicillin, kanamycin, spectinomycin and hygromycin) resistance genes (e.g. neomycin phosphotransferase (nptll) , hygromycin phosphotransferase

(hpt) as selective markers.

In further aspect of this invention, there is provided a transformant transformed with the above-describedvector of the present invention. The hosts useful incloning andexpressing thevector of the present invention are well known to those skilled in the art. For example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thurigensis ,

Salmonella typhimuriu , Serratia marcescens and various Pseudomonas may be employed. As eukaryotic cell, yeast (Saccharomyce cerevisiae) , insect cell, human cell (e.g., CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. Since OsAsrl gene of the present invention is useful for plant, the transformants include callus derived from plant cells or tissues. The transformation of a host cell can be carried out by a large number of methods known to one skilled in the art. For example, in case of using prokaryotic cells as host, CaCl₂ method (Cohen, S.N. et al . , Proc. lVatl. Acac . Sci. USA, 9:21102114(1973)) , Hanahan method (Cohen, S.N. et al. , Proc. Matl . Acac. Sci. USA, 9:2110-2114 (1973) ; and Hanahan, D., J. Mol. Biol., 166 : 5S7-580 (1983) ) and electroporation (Dower, W.J. et al., Nucleic. Acids Res.. 16:6127-6145 (1988))' can be used for transformation. Also, in case of using eukaryotic cells as host, microinjection (Capecchi, M.R., Cell, 22:479(1980)) , calciumphosphate precipitation (Graham, F.L. et al . , virology, 52:456(1973)), electroporation (Neumann, E. et al . , EMBO J., 1:841(1982)), liposome-mediated transfection (Wong, T.K. et al . , Gene, 10:87(1980)), DΞAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190(198®)), and particle bombardment (Yang et al . , Proc. Natl. Acad. Sci. , 87:9568-9572 (1990)) can be use for transformation. The vector introduced to host cell is expressed in the host cell, thereby, a mass of abiotic stress-inducible OsAsrl is obtained. In one aspect of this invention, there is provided a method for preparing transgenic plant showing an enhanced tolerance to cold, salt or drought stress, which comprises the steps of: (a) transforming aplant cell or tissue with the vector of the present invention; (b) selecting a transformed plant cell or tissue; and (c) regenerating the transformed plant cell or tissue to obtain a transgenic plant.

In one aspect of this invention, there is provided a transformant which shows an enhanced tolerance to cold, salt or drought stress. The explants used in the present invention are plant cells or tissues. Employing callus is preferred in case of using plant tissues. The transformation of plant cells may be carried out according to the conventional methods known one of skill in the art, including electroporation (Neumann, E. et al . , EMBO J., 51:841 (1982)), particle bombardment (Yang et al . , Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (U.S. Pat. Nos. 5, 004, 863,

5, 349,124 and 5,416, 011) . Among them, Agrobacterium-mediated transformation is the most preferable. The selectionof transformedcellsmaybe carriedout withexposing the transformed cultures to a selective agent such as a metabolic inhibitor, anantibioticandherbicide. Cells whichhavebeentransformed and have a stably integrated marker gene conferring resistance to the selective agent will grow and divide in culture. The exemplary marker includes, but not limited to, a hygromycin phosphotransferase gene, a glyphosphate resistance gene and aneomycinphosphotransferase (np^'tll) system. The development or regeneration of plants from either plant protoplasts or various explants is well known in the art . The development or regeneration of plants containing the foreign gene of interest introduced by Agrobacterium may be achieved by methods well known in the art (U.S. Pat. Nos. 5,004,863, 5,349,124 and 5, 416,011) . The method of the present method may be applicable to various plants, preferably to corps such as rice, barley, wheat and maize, more preferably rice and most preferably japonica cultivars . In the present invention, transformation is preferably performed with Agrobacterium system, more preferably with Agrobacterium tumefaciens-binary vector system. An embodiment of a method using Agrobacterium tumefaciens-binary vector systemcomprises the steps of : (a') inoculatinganexplantmaterial from the plant with Agrobacterium tumefaciens harboring a vector, in which the vector is capable of being inserted into a genome of a cell from the plant and contains the following nucleotide sequences: (i) the nucleotide sequence encoding abiotic stress-inducible OsAsrl protein; (ii) a promoter that functions in plant cells to cause the production of an RNA molecule operably linked to the nucleotide sequence of (i) ; and (iii) a 3 ' -non-translated region that functions in plant cells to cause the polyadenylation of the 3 '-end of the RNA molecule;

(b' ) regenerating the inoculated explant material on a regeneration medium to obtain regenerated transformants . Transformation of plant cells derived is carried out with Agrobacterium tumefaciens harboring Ti plasmid (Depicker, A. et al . , Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York (1983)). More preferably, binary vector system such as pBinl9, pRD400, pRD320, pGA1611 and pGA1991 is used for transformation (An, G. et al . ,

Binary vectors" In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986) ; An et al.1988 ; and Lee et al . , 1999) . The binary vector useful in this invention carries: (i) a promoter capable of operating in plant cell; (ii) a structural gene operably linked to the promoter; and (iii) apolyadenylationsignal sequence . Thevectormayalternatively further carryagene codingforreportermolecule (forexample, luciferase andp-glucuronidase) . Examples of the promoter used in the binary vector include but not limited to cauliflower mosaic Virus 35S promoter, 1' promoter, 2' promoter and promoter nopaline synthetase (nos) promoter. Inoculation of the explant with Agrobacterium tumefaciens involves procedures known in the art. Most preferably, the inoculation involves immersing the explant in the culture of Agrobacterium tumefaciens to coculture. Agrobacterium tumefaciens is infected into plant cells. The explant transformed with Agrobacterium tumefaciens is regenerated in a regeneration medium. The transgenic plant is finally produced on a rooting medium. The transformedplant produced according to the present invention may be confirmed using procedures known in the art. For example, using DNA sample from tissues of the transformed plant, PCR is carried out to elucidate exogenous gene incorporated into a genome of the transformed plant. Alternatively, Northern or Southern Blotting may be performed for confirming the transformation as described in Maniatis et, al . , MolecularCloning, ALaboratoryManual, ColdSpringHarbor 110 Laboratory, Cold Spring Harbor, N.Y. (1989) . The OsAsrl gene andprotein of the present invention is very useful for enhancing plant tolerance to cold, salt or drought stress

BRIF DESCRIPTION OF THE DRAWINGS Figure 1 represents comparison of amino acid sequence of the deduced OsAsrl protein with other known Asr proteins performed with the CLUSTALW program. Putative 2n²⁺ DNA binding site and nuclear localization signal (NLS) were underlined. White letters in a black box indicate 16 out of 16 matches. Figure 2 is a dendrogram representing a phylogenetic analysis of various Asr proteins with rice homologues . The rice homologues were retrieved from TBLASTN search against the indica rice WGS contigs. The. dendrogram was constructed using the ClustalX and Mega2 program. The respective accession number is described next to the plant name. The lengths of horizontal lines reflect the evolutionary distance. Figure 3 represents southern blot analysis of OsAsrl . Twenty βg of genomic DNA from three japonica cultivars, Namyang 21 (1) , Odae (2) , Dong in (3) , was cut with EcoRI (E) , HindiII (H) , and BamEI (B) , and hybridized with radiolabeled OsAsrl probe. Positions and sizes in kb of HindiII digested OsAsrl DNA are indicated. Figures 4a-4e represent OsAsrl expression analysis under the treatments of various abiotic stress and ABA: Figures 4a-4c repesent northern blot analysis of OsAsrl . Thirty (A) or 10#g (B and C) of total RNA was separated, blotted, and hybridized with radiolabeled OsAsrl probe. Figure 4a represents OsAsrl expression in the various organs or tissues. Lanes: 1, callus; 2, shoot of seedling; 3, root of seedling; 4, mature leaf; 5, leaf sheath of flag leaves; 6, highest internode (between node I and II) at prehead stage; 7, 1-2 cm panicle; 8, 3-8 cm panicle; 9, mature panicle prior to anthesis, 10, developing seeds of 3 days after pollination (DAP) ; 11, developing seeds of 6 DAP. Figure 4b represents the cold inducible expression of OsAsrl . Mature plants were treated at 12 ^°C for 4 d. L, leaves; CL, cold-treated leaves; F, florets; CF, cold-treated florets. Figure 4c represents expression of OsAsrl by different temperature and ABA. Seedlings grown at 30^°C were

treated with 4 °C , 12 ^°C , or lOμM ABA for 3 hr, 6 hr and 20 hr . Figure 4d represents analysis of OsAsrl expression under various abiotic stresses and ABA. SalT was used for the drought-, cold-, salt-, and ABA-response controls . Transcript of rice actin gene shows an 'internal control for PCR analysis. Figure 4e represents analysis of OsAsrl expression levels using real-time PCR. Error bars represent standard deviation. 8-d seedlings were treated with 4°C (c) , 250 mM NaCl (s) , drought (d) or 100 μM ABA (A) and then were periodically harvested. Figures 5a and 5b represent in si tu localization of OsAsrl mRNA.

Cross sections of the rice flowers of 4 days before heading and leaves exposed to 12 °C for 4 days were hybridized with digoxygenin-labeled antisense (A, C, E, and G) or sense (B, D, F, and H) OsAsrl probes. Higher magnification of the cross section is shown (C, D, G, and H) . an, anther; lm, lemma; pa, palea; l.e.p, lower epidermis of palea; LVB, large vascular bundle; mc, motor cell; xy, xylem; ph, phloem; me, mesophyll . Bar = 0.3 mm. Figures 6a-6c represent analysis of transgenic plants expressing OsAsrl in sense and antisense oritentation. Figure 6a represents construction of OsAsrl sense (pSK167) and antisense (pSK168) expression vector for rice transformation. P_UBI, maize ubiqutin promoter; P_3Ss/ CaMV

35S promoter; Tnos, terminator sequence of the nopaline synthase gene;

T7, terminator sequence of the transcripts 7; hph, the hygromycin phophotransferase gene for selection of transgenic rice callus; RB and LB, the right and left border sequences, respectively, of the Ti plasmid from Agoro-bacterium tumefaciens . Figure 6b represents southern blot analysis of transgene . Southernblot was conducted using 10 βg of genomic

DNA cut with Hindlll from transgenic plants. The ubiquitin promoter fragment in the transformation vector was used as a probe. Positions and sizes in kb of Hindlll-digested DNA are indicated. Figure 6c represents expression of OsAsrl in the sense transgenic rice ( Ubiquitin : . - OsAsrl) . Leaf total RNA from wild type segregant (WS) and sense transgenic plants under normal growth condition was separated, blotted, and hybridized with radiolabeled OsAsrl probe. Number represents each transgenic line. EtBr-stained rRNA bands indicate an equal amount of loading. Figure 7 represents stress tolerance of Ubiqui tin : : OsAsrl plants as judged by the measurement of chlorophyll fluorescence. Changes in the chlorophyll fluorescence of the extended leaves under cold stress was measured. Functional damage to photosynthesis was estimated by measuring the mean and standard error of Fv/Fm values . Figure 8. represents expression of OsAsrl in CBF1 transgenic rice . Total RNA from mature leaves of two strong CBFl-expressors (18-2 and 18-3) and the wild type non-transgenic line (NT) under control (30 °C)

or cold (4 °C) treated conditionwas hybridized.with radiolabeled OsAsrl probe. EtBr-stained rRNA bands indicate an equal amount of loading. Figure 9 represents salt tolerance of transgenic rice plants expressing OsAsrl in sense (9a) or antisense (9b) orientation. 12-day old seedlings treated with 200 mM NaCl for 1 d (for sense plants) or

12 h (for antisense plants) were recovered to normal condition for 2 d. Figure 10 represents wilting ratio ofricecultivarsandtransgenic

OsAsrl rice plants. 12-day old seedlings treated 200 mM NaCl for 1 d (for sense plants;A) or 12 h (for antisense plants;B) were recovered to normal condition for 2 d. Figure 11 represents drought tolerance transgenic rice plants expressing OsAsrl in sense (11a) or antisense (lib) orientation.14-day old seedlings treated no water for 4 d were returned to normal growth conditions for 7 d. Figure 12. wilting ratio of rice cultivars and transgenic OsAsrl rice plants .14-day old seedlings treated no water for 4 d were recovered to normal condition for 7 d.

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims .

EXAMPLES

MATERIALS AND METHODS

Plant samples and bacterial strains 'Dongjin' , a cultivar of japonica rice ( Oryza sativa) , was used for constructing our seed-coat cDNAlibraryandforgenerating transgenic plants. The other cultivars for cold tolerance are listed in Table 1, 2 and 3. Seeds were surface-sterilized and then germinated either in water or on an MS agar medium (Murashige and Skoog, 1962) . Also, seeds were germinated in Yoshida solution (Yoshida et al . , 1976) . They were grown under hydroponically controlled conditions having 29°C day/21°C night temperatures, 16-h-light/8-h-dark cycle, and 83% relative humidity. The seedlings were transferred to soil and kept in a growth chamber at 30 °C under continuous light (intensity of 60 to 70 μmol rrf² sec^"1) . For the cold treatment, 11-d-old seedlings exposed to 4 °C or 12 °C in the dark were harvested. For the ABA, 5-d-old seedlings transferred to an MS liquid medium containing 10 μM ABA were harvested. For the salt orABA treatments, seedlings were transferred to an Yoshida solution containing either 250 mM NaCl or 100 μM ABA and then were periodically harvested. For drought treatment, the seedlings were dried in the air and then periodically harvested. To study the effect of cold stress at the reproductive stage, field-grown rice plants at ca. 4 d before heading were transferred to the greenhouse (30 °C, 14-h light/10-h dark) for 2 d of adapting. Afterward, they were treated for 4 d at 12 °C under a 14-h photoperiod (ca. 300 μmol m^"2 sec^"1) . All tissue samples were immediately frozen with liquid nitrogen and stored at -70 °C . The E. coli strain, XL-1 Blue MRF' {Δ (mcrA) 183Δ (mcrCB-hsdSMR-mrr) 173 endAl

supE44 thi-1 recAl gyrA96 relAl lac [F' proAB, lacI^qZΔM15 TnlO (Tet^r) ] } was used as a host for molecular cloning.

Construction of cDNA library and EST Analysis The coats from 4- to 8-day-old seeds were manually dissected under a microscope. A cDNA librarywas then constructed frompolyARNAprepared from the seed coats . cDNA clones were randomly selected from the library, and their 5' ends were sequenced. The DNA preparation, sequencing, and computer analysis were preformed as described by Hong et al . (1998) .

Briefly, template DNAs were prepared by the alkaline lysis method, and the inserts were sequenced with an ABI PRISM™ BigDye™ Terminator Cycle

Sequencing kit (Amersham) . Computer software, such as DNAsis, Prosis (Hitachi), ClustalX, ClustalW, and GeneDoc (Thompson et al . , 1994;

Nicholas and Nicholas, 1997) , were used for our sequence analysis. The

Genbank, EMBL, andSwiss-Prot databases were then searched for amino-acid sequence homology using the BLASTX algorithm (Altschul et al . , 1997) .

DNA and RNA gel - blot analysis DNA gel-blot analyses were conducted on three japonica cultivars - 'Namyang 21' , 'Dongjin' , and ^λOdae' . Namyang 21 is cold-sensitive (the cold tolerance index, CTI = 7) , Odae is cold-tolerant (CTI = 3) , and Dongjin is intermediate (CTI = 5) (Lim, 1998) . In the CTI value given between 0 to 9, 0 is most tolerant, 9 most sensitive. Genomic DNA was extracted from their seedlings according to the cetyltrimethylammonium bromide method (Roger and Bendich, 1988) . Ten micrograms of DNA, digested with restriction enzymes for 6 h at 37 °C, was separated on a 0.8% agarose gel, then transferred to a Hybond-N membrane (Amersham) using a vacuum transfer system (Hoefer) . For RNA gel-blot analyses, 10 μg of total RNA was resolved on a 1.3% formalehyde agarose gel and blotted onto a nylon membrane . The DNA and RNA blot analyses were performed using the radiolabeled OsAsrl probe. To prepare this probe, OsAsrl cDNA fragments were radioactively labeledwith [ -³²P] dCTP (3000 cimmol^"1) , followingthe randomprimingmethod. Unincorporated nucleotides were removed via G-50 Sephadex column chromatography. After hybridization, the membrane was washed with 2 x SSC, 0.1% SDS at RT for 15 min; 1 x SSC, 0.1% SDS at RT for 15 min; and 0.1 x SSC, 0.1% SDS at RT for 15 min. Hybridization signals were detected with an image analyzer (BAS-1500, Fuji) and exposed on Hyperfilm™ MP film (Amersham) .

RNA in situ hybridization Rice flowers and leaves were fixed overnight at 4 °C in 2% (wt/vol) paraformaldehyde plus 2.5% (vol/vol) glutaraldehyde in a 50-mM PIPES buffer (pH 7.2). The fixed tissues were dehydrated by graded concentrations of ethanol, then embedded in a paraplast medium (Oxford labware, USA) . The embedded tissues were sliced into 7-μm sections with a rotary microtome (Leica, Germany) , and each section was attached to a silanized glass slide (Matsunami, Japan) . Paraffin was removed through a graded series of ethanol concentrations, and the samples were dried for 1 h. Dioxygenin-labeled sense or antisense RNA probes were prepared from the linearized pBluscript carrying the OsAsrl cDNA, using either T3 or T7 RNA polymerase. The sections were hybridized with the probes at 48 °C for 16 h in a hybridization solution, then washed in a solution

containing 2 x SSC, 1 x SSC, and 0.1 x SSC for 15 min at 50°C. The hybridizing probe was detected colorimetrically using an anti-DIG conjugated alkaline phosphatase (Boehringer Mannheim) . Photographs were taken under bright-field microscopy (Nikon Eclipse 600) .

Production and analysis of transgenic rice plants Full-length OsAsrl cDNA was introduced into the binary vector pGA1611 (An et al . , 1988; Kim et al . , 2003) , in the sense and antisense orientations downstream of the maize ubiqui tin promoter (Christensen et al . , 1992). Rice transformation was performed via the

Agrobacterium-mediated cocultivation method (Jeon et al . ,1999; Lee et al. ,1999) . Rice seeds of which emma and palea have been removed were place on 2N6 medium containing 2 mgL^"12,4-D and cultivated for one month to induce callus. Agrobacterium cultured in AB medium containing 30 mgL^"1 hygromycin B and 3 mgL^"1 tetracyclin for 3 days were cocultured with the induced calli in 2N6-ASB medium at 20°C under dark condition for 3 days. Then, they were placed in 2N6-CH40 medium containing 40 mgL^"1 hygromycin B and 250 mgL^"1 cefotaxime for 3 weeks. Well growing calli were selected and regenerated on MS medium containing 0.1 mgL^"1 NAA, 2 mg L^"1 kinetin, 2% sorbitol, 1.6% fitagar, 50 mgL^"1 hygromycin B and 250 mgL^"1 cefotaxime under continuous light for 3 days. The regenerated individuals were transferred to soil and grown in the greenhouse. All transgenic rice plants were generated on a 40 -mg L^"1 hygromycin B-containing medium, and were transferred to the greenhouse after regeneration. PCR analysis to verify those transgenic plants was performed using the forward primer (5'-CAC CCT GTT GTT TGG TG-3') and the reverse primer (5' -GCG GGA CTC TAA TCATAAAAA CC-3 ' ) . PCR conditions included 1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C, through 30 cycles.

Determination of chlorophyll fluorescence About 5 -cm-long segments of the extended leaves frommature plants were floated on MS liquid media at 4 °C under white fluorescent light

(260 μmol m^"2 sec^"1) for 0 , 6, 12, or 24 h. After 30 min of dark-adaptation, chlorophyll fluorescence signals were measured with a Plant Efficiency Analyzer (Hansatech) . All experiments were repeated four times .

Determination of cold stress Rates of survival were calculated forplants exposed to cold stress by assessing either their degree of wilting (Saijo et al . , 2000) or the amount of their regrowth (Lee et al . , 1993) . Ten day-old seedlings (at the three-leaf stage) , that had been raised in a growth chamber (16-h light/8-h dark; light intensity of 60 μmol m^"2 sec^"1; 30 °C) , were exposed to 4 °C for 3, 4, 5, 6, 7, 10, or 12 d under continuous light (60 μmol m^"2 sec^"1) . Afterward, they were returned to standard growth-chamber conditions for 10 d to allow for recovery. The wilting ratio was based on the level of prominent chlorosis and withering of the leaves. To analyze regrowth, seedlings that had previously been exposed to 4°C for 4 d were returned to the growth chamber for 13 d of recovery. Regrowth was defined as the production of new, fourth leaves on the seedlings.

Determination of drought and salt stress Plants were grown for about 2 weeks in the growth chamber (30 °C, with light/dark cycles of 16/8 h) and then used to measure survival rate after stress treatment . For the drought-survival test , theyreceived nowater for 4 d. Afterwards theywere returnedtonormal growth conditions for 7 d and numbers of wilted and healthy plants were counted (Lee, S.C. , Huh, K.W., An. K., An. G., and Kim, S. R.2004b Ectopic expression of a cold-inducible transcription factor. CBFl/DREBlb, in transgenic rice (Oryza sativa L.) Mol. Cells 18: 107-114). For the salt stress treatment, 10-d-old seedlings were incubated in a nutrient solution of 0.1 % (w/v) Hyponex (Hyponex) for 2 days and the transferred to fresh nutrient solution containing 200 mM NaCl for 1 d (for sense plants) or 12 h (for antisense plants) at 30 °C with a photoperiod of 16 h (50-60 μmol m^"2 sec^"1 light) . After salt stress, the roots of the seedlings were rinsed with water, and then grown in a fresh nutrient solution without NaCl under the normal condition. Wilting after stresses was determined as three and four leaves of the plant completely wilted showing prominent chlorosis .

Results Identification of OsAsrl cDNA from the rice seed-coat library The cDNA clone isolated from our developing seed coats is highly homologous with OsAsrl , found previously by Vaidyanathan et al . (1999) . This clone is 838 bp long and comprises a 66-bp, 5' untranslated region (UTR) , a 417-bp open reading frame, a 325-bp 3' UTR, and a poly (A) -tail (data not shown) . It is 4 bp shorter at the 5' end than the previously reported clone, but its nucleotide sequence exactlymatches OsAsrl except for a T-to-C change at the 3' UTR (304 bp downstream of the termination codon) . Because the previously isolated cDNA was from 'Pokkali', an indica rice, and the clone identified here is from the japonica type 'Dongjin' , we propose that this one-nucleotide variation at the 3' UTR is due to the difference in cultivars. An OsAsrl genomic sequence was retrieved from the indica rice WGS genomic database (Yu et al . , 2002) . In-silico analysis of the clone predicted that core sequences of the ABA response element (ABRE) , ACGT, are present at positions -261, -610 and -616 from the first ATG codon. An important cold-responsive cis element, C-repeat/drought responsive element (CRT/DRE) sequence, GCCGAC, is also found at -714. Putative Myb (-143, -149, and -698) , Myc (-71, -512, and -694) and bZIP (-202 and -711) binding sites are also found. Aputative Zn-bindingHis-residue and the putative NLS domain are present in the N-terminal and the

C-terminal region of OsAsrl protein, respectively (Figure 1) . A comparison of the cDNA and the genomic sequences revealed a 119-bp-long intron in the OsAsrl gene. TBLASTN searches (Altschul et al . , 1997), using the OsAsrl protein sequence (accessionno. AAB96681) as the query, were able to retrieve five homologues in the scaffolds (002913, 037286,

026604, 023736, and 081294) . Their phylogenetic relationship presented as a dendrogram indicates that OsAsrl has the highest homology with ZmAsrl of maize (Figure 2) . ASR proteins had been categorized into 4 main groups (Hong et al . , 2002) and OsAsrl belongs to group I related to ZmAsrl. More than 100 rice OsAsrl cDNA sequences are registered in the GenBank database, indicating that the gene belongs to an abundantly expressed gene family. Moreover, the OsAsrl homologues have been found only in plant species.

DNA gel -blot analysis of OsAsrl The results of our DNA gel-blot analyses on three japonica cultivars showed only one copy of the OsAsrl gene (Figure 3) . This observation matches that previously made with 'Pokkali' (Vaidyanathan et al . , 1999) . No significant polymorphism was found with regard to restriction-fragment lengths for these three cultivars of different cold tolerance.

■Induction of OsAsrl by cold stress The inventors of the present invention used RNA gel-blot analyses to examine gene expression at different tissues under various abiotic condition. The OsAsrl transcript was detectable in all the tissues and organs except in calluses (Figure 4a) . OsAsrl transcript was induced by cold, drought, salt, and ABA treatment. It reached their maximum after 3 h of drought and salt stresses (Figure 4d and 4e) . The transcript size was about 0.9 kb, indicating that the OsAsrl cDNA clone is nearly full-length. The transcript was present at high levels in the shoots and roots of seedlings, sheath of flag leaves, and most abundantly in the internodes between node I and II, demonstrating organ-preferential expression. OsAsrl was expressed at the basal level in the leaves of mature plants, but more abundantly in mature flowers. Interestingly, low temperatures elevated overall transcript levels in both organ types (Figure 4b) . Cold treatment increased the transcript level also at the seedling stage, but more significantly at 12 °C instead of 4 °C (Figure 4c) . The cold-responsive OsAsrl accumulation was restricted to shoots, demonstrating the organ-specific stress response of the gene expression. Because OsAsrl is ABA-inducible (Vaidyanathan et al . , 1999) , we compared the inductionkinetics of cold stress versus that ofABA. Whereas transcript levels reached their maximum after 3 h of cold stress, ABA treatment caused levels to increase more slowly, achieving their highest point only after 6 h (Figure 4c) . The expression of OsAsrl in the physiological range of growth temperature (12, 20, and30°C) was compared between the cold-tolerant cultivar Odae and the cold-sensitive cultivar Namyang 21. However, we did not observe any significant difference of the gene expression between the cultivars. RNA in-situ hybridization was performed to further elucidate the cold-induced expression pattern of the OsAsrl gene at the tissue level . In the leaves, induction was confined to the mesophyll tissues, and was not present in the epidermal or vascular tissues (Figure 5e and g) . Because the flower is a primary target organ of cold stress, hybridization was also performed for the flowers. Immature flowers at 4 d before heading were cold-treated at 12 °C for 4 d. Afterward, OsAsrl transcript was detectable in the parenchyma cells of the palea and lemma from those stressed flowers (Figure 5a and c) . In the control flowers that had not been cold-stressed, theOsAsrlexpressionpatternwassimilar to that of the cold-stressed flowers although the expression level was much lower.

Expression of OsAsrl cDNA in transgenic plants Binary vectors containing OsAsrl cDNA were constructed in the sense (pSK167) or antisense (pSK168) orientation under the maize ubiquitin promoter (Figure 6a) , to determine the function of OsAsrl . Twenty transgenic plants were generated via the Agrobacterium co-cultivation method, and integration of the transgene into the rice genome was examinedby DNA gel-blot analyses. The copy number was usually one to two, although three or more copies of the introduced genes were detected (Figure 6b) . Transgenic plants showed no significant morphological changes in their TI and T2 generations. To examine whether the changed expression of OsAsrl has a role in the cold tolerance of the transgenic rice, RNA gel-blot analyses were conducted (Figure 6c) . Overall, the OsAsrl transcript level was higher in the transgenic plants compared with the wild-type segregants . Size of the hybridized transcript in the transgenic plants was slightly bigger than those of wild type. Two strong over-expressers, S2 and S15, were selected for further analyses .

Chlorophyll fluorescence of transgenic plants under cold stress Chlorophyll fluorescence was measured as an indicator of chilling tolerance after cold treatment (4°C) . The ratio of Fv to Fm, which represents the activity of Photosystem II, is used to assess functional damage in plants (Genty et al . , 1989) . For our wild-type segregants, Fv/Fm progressively decreased following chilling. This decline illustrates the extent of photoinhibition causedby cold stress (Krause, 1994) . Values for Fv/Fm had been 0.84+0.01 before stress was induced. Following the 6-h cold treatment, Fv/Fm decreased slightly, and no significant difference in values was found between the transgenic and the wild-type segregants. After 24 h of treatment, however, the Fv/Fm values for the wild-type were reduced significantly, to 0.25+-0.021 (Figure 7) . In contrast, the Fv/Fm values for the over-expressers S2 and S15 were 0.53 ± 0.049 and 0.60 ± 0.067, respectively. These ratios were about two-fold higher those calculated for the wild-type (Figure 7) , which indicates that the transgenic plants had a higher degree of cold tolerance.

Survival test The rate of survival for transgenic plants at the seedling stage was measured in wilting and regrowth tests. The critical length of the cold treatment was first determined by assessing the amount of wilting (i.e. , the ratio of the number of wilted seedlings to the total number of cold-treated seedlings) after stress. The cold-tolerant cultivars, 'Odae' and 'Stejaree 45', had wilting ratios of 10/18 (55.6%) and 8/20 (40%) , respectively, after 5 dof exposure to low temperatures (Table 1) . This wilting frequency increased to about

75% after 6 d of stress. In contrast, the parental line of our OsAsrl transformation, 'Dongjin' , exhibitedwilting symptoms in 12 of 17 plants (70.6%) after 5 d of stress. The wilting frequency increased to about

94.% after 6 d of stress. A cold-sensitive indica x japonica cultivar, ^xMilyang23' , showedl00%wilting after only 4 d. Therefore, itis concluded that the critical length of time for tolerating low-temperature stress by 'Dongjin' was about 5 to 6 d. Based on that assumption, the inventors chose to cold-treat our T2 transgenic plants for 6 d. Of the 34 seedlings obtained from the S2 plants, 23 (68%) showed wilting after chilling while 10 (67%) of 15 seedlings fromthe S15plants exhibitedthose symptoms . In contrast, 63 (96%) of 66 seedlings derived from the wild-type segregating plants had wilted. The antisense transgenic plants were examined as well, with the occurrence of wilting in A3, A12, and A14 being 9/9 (100%), 9/11 (82%), and 10/11 (91%), respectively. The regrowth test, conducted in the T3 generation, involved analyzing homozygous lines for the transgene after hygromycin selection. Of the 35 seedlings from S15 plants, 14 (40%) showed active development of a fourth leaf. In contrast, the S18 plants, which are from a weaker OsAsrl-expressing line, presented only 7 of 35 plants (20%) with signs of regrowth. Nevertheless, the wild type-segregating control plants had even lower rates of regrowth, i.e., 9%, or 9 out of 105 plants. Similar results were seen from the A3 plants, in which only 7 of 70 (10%) showed any response. Taken together, these results indicate that the transgenic plants over-expressing the OsAsrl transcript gained increased tolerance to cold stress. However, the level of tolerance did not change noticeably when OsAsrl expression was suppressed in the antisense plants,

Table 1

Cold stress tolerance of rice cultivars and rice transgenic OsAsr 1 rice

Expression of OsAsrl cDNA in transgenic rice plants expressing Arabidopsis CBFl Since a CRT/DRE core sequence is located in the putative promoter region of OsAsrl, the gene may be controlled by CBFl. To this end, we have generated transgenic rice plants that ectopically expressed Arabidopsis CBFl cDNA. RNA gel-blot experiments showed that expression of the OsAsrl gene was elevated in transgenic plants (18-.2 and 18-3) that strongly expressed the CBFl transcription factor (Figure 8) . Expression of OsAsrl in the transgenic plants was further increased by cold stress .

Salt tolerance of transgenic OsAsrl plants The rate of survival for transgenic plants at the seedling stage was measured in wilting tests . For accurate experiments, I showed the salt tolerance with two wild type rice cultivars, a japonica cultivar, Dongjin and an indica- cultivar, ^λTachung Native 1' (TNI) . Ten-d-old seedlings were transferred to a nutrient solution, 0.1% (w/v) Hyponex solution, containing 200mM NaCl. Sense transgenic plants were treated for 1 d and antisense transgenic plants were treated for 12 h at 30 °C, and then returned into a fresh nutrient solution without NaCl under normal condition for 2 d. Wilting after stresses was determined as three and four leaves of the plant completely wilted showing prominent chlorosis. The salt-tolerant cultivar, TNI, hadwilting ratios of 36.1+8.4 % after 2 d of recovery (Figure 9, Figure 10, Table 2) . In contrast, the parental line of our OsAsrl transformation, 'Dongjin' , exhibitedwilting symptoms in 66 of 86 plants (77+22.5 %) after 2 d of recovery.39 (67+4.1%) of 58 seedlings derived from the wild-type segregating plants had wilted after 2 d of recovery. Two strong over-expressers, S2 and S15, were selected for stress tolerance tests. The plants of each T3 line were homozygous plants. Homozygous T3 transgenic plants are salt stressed for 1 d for sense and 12 h for antisense plants. These salt-treated times were referred to the literature (Kim S.H., PhD thesis, Sogang University, 2004) . Of the 94 seedlings obtained from the S2-1 plants, 26 (27+11%) showed wilting. Also, of the 88 seedlings obtained from the S2-2 plants, 27 (31+8%) showed wilting. However, 33 (45+22%) of

73 seedlings from the S15-lplants exhibited those symptoms and34 (39+5%) of 88 seedlings from the S15-2 plants showed wilting. In contrast, the antisense transgenic plants were examined after 12 h of stress, with theoccurrenceof wilting in A3-1 and A3-2 being 38/53 (72+3%) and 30/67

(45+23%) , respectively. After 5 d of recovery, the wilting ratio was increased in 64/73 (73±4%) plants of TNI, 86/86 (100+0%) plants of Dongj in,

58/58 (100+0%) plants of NT-S2, 87/94 (93±3%) plants of S2-1, 75/88

(85±2%) plants of S2-2, 71/73 (97+4%) plants of S15-1, and 87/88 (99+2%) plants of S15-2. All experiments were repeated three times. Based on these data, it is concluded that overexpression of OsAsrl could confer salt tolerance on rice plants and that the OsAsrl antisense lines show increased salt sensitivity than wild type.

Table 2

Salt stress tolerance of the rice cultivars and transgenic OsAsrl rice Line Wilted Total Wilting ^a (%) TNI 32 88 36+8 Dongj in 66 86 77+23 NT-: 32 39 58 67+4 S2 • -1 26 94 27+11 S2 ■ -2 27 88 31+8 S15 -1 33 73 45+22 S15 -2 34 88 39+5

A3 • -1 38 53 72+3 A3 • -2 30 67 45+23 NT-S2 17 50 ₃₄+₄

^aNumbers of wiltingplants/ numbers of totalplants (percentage ofnumbers of wilting plants) . Numbers of wilting plants indicate three and four leaves of plants wilted 2d recovery after salt stress. Drought tolerance of transgenic OsAsrl plants Drought tolerance of OsAsrl transgenic plants were assessed. For accurate experiments, the inventors showed that the drought tolerance with two wild type rice cultivars, Sangnambatbyeo and Dongjin (Table 3 , Figures 1 and 12) .14-d-old seedlings were grown in the growth chamber (30 °C, with light/dark cycles of 16/8 h) and then received no water for 4 d. We chose to drought-treat our T3 transgenic plants for 4 d. Wilting after stresses was determined as three and four leaves of the plant completely wilted showing prominent chlorosis . These drought-treated times are referred to the literature (Lee, S.C., Huh, K.W., An. K., An. G. , and Kim, S. R. 2004b Ectopic expression of a cold-inducible transcription factor. CBFl/DREBlb, in transgenic rice ( Oryza sativa L.) Mol. Cells 18: 107-114) . Of the 60 S2-1 seedlings, 11 (18+12%) showedwilting. Also, among the 61 S2-2 seedlings, 15 (25±10%) showed wilting. However, 14 (27+13%) of 51 seedlings from the S15-1 plants exhibited those symptoms and 17 (29±4%) of 58 seedlings from the S15-2 plants showed wilting after 4 d of stress. The inventors also examined the antisense transgenic plants for the drought tolerance. The antisense plants A3-1 and A3-2 showed sensitivity of 41/45 (91+12%) and 38/51 (75+3%) , respectively. All experiments were repeated three times. These data suggest that overexpression of OsAsrl could confer drought tolerance on rice plants and that the OsAsrl antisense lines show more drought sensitivity than wild type,

Table 3

Drought stress tolerance of the rice cultivars and transgenic OsAsrl rice plants. Line Wilted Total Wilting (%) SNB 25 57 44+16 Dong j in 64 100 64+ 6 NT- ! 32 35 59 59+ 3 S2 ^■ - 1 11 60 18+12 S2 - 2 15 61 25+10 S 15 - 1 14 50 28+13 S 15 - - 2 17 58 29+4 A3 ^■ - 1 38 53 90+12 A3 - 2 30 67 79+3

^a Numbers of WT and T3 plant, three and four leaves of which completely wilted 7 d recovery after drought stress .

As described above, the present invention provides abiotic stree-inducible OsAsrl protein enhancing tolerance to cold, salt, or drought stress. In addition, the present invention provides nucleic acid which comprises a nucleotide sequence encoding the abiotic-stress inducible OsAsrl protein. The present invention provides a vector and a transformant. Furthermore, the present invention provides transgenic plants showing an enhanced tolerance to cold, salt, or drought stress and a method for preparing thereof. The OsAsrl gene and protein of the present invention is very useful for enhancing plant tolerance to cold, salt or drought stress.

Having described specific examples of the present invention, it is to be understood that such examples are only preferred embodiments and should not be construed as limiting the scope of the invention. Therefore, the substantive scope of the invention may be determined by appended claims and their equivalents

REFERENCES

Altschul, S.F., Madden, T.L., Schafler, A.A. , Zhang, J. , Zhang, Z., Miller, W. , and Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs . Nucleic Acids Res . 25: 3389-3402. An, G., Ebert, P.R., Mitra, A., and Ha, S.B. 1988. Binary vectors . InPlant MolecularBiologyManual . EditedbyGelvin, S.B. , Schilperoort, R.A. The Netherlands, pp. A3 1-19. Kluwer Academic Publishers, Dordrecht , The Netherlands . Boothe, J.G., Sonnichsen, F.D., deBeus, M.D., and Johnson-Flanagan, A.M. 1997. Purification, characterization, and structural analysis of a plant low-temperature-induced protein. Plant Physiol. 113: 367-376. Cakir, B., Agasse, A., Gaillard, C, Saumonneau, A., Delrot, S., and Atanassova, R.2003. A grape ASR protein involved in sugar and abscisic acid signaling. Plant Cell 15: 2165-2180. Canel, C, Bailey-Serres, J.N., and Roose, M.L. 1995. Pummelo fruit transcript homologous to ripening-induced genes . Plant Physiol .108 :

1323-1325.

Chandler, P.M., and Robertson, M. 1994. Gene expression regulated by abscisic acid and its relation to stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 113-141.

Chang, S., Puryear, J.D., Dias, M.A.D.L., Funkhouser, E.A., Newton,

R.J., and Cairney, J. 1996. Gene expression under water deficit in loblolly pine ( Pinus taeda L.) : isolation and characterization of cDNA clones. Physiol. Plant 97: 139-148. Christensen, A.H., Sharrock, R.A., and Quail, P.H. 1992. Maize polyubiquitm gene: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675-689.

De Datta, S.K. 1981. The climatic environment and its effects on rice production. In Principles and Practices of Rice Production, Editied by De Datta, S.K., pp. 9-40. John Wiley & Sons, Chichester UK.

Dure, L. 3rd. 1993. A repeating 11-mer amino acid motif and plant desiccation. Plant J. 3: 363-369.

Furumoto, T., Hata, S., and Izui, K. 2000. Isolation and characterization of cDNAs for differentially accumulated transcripts between mesophyll cells and bundle sheath strands of maize leaves.

Plant Cell Physiol. 41: 1200-1209.

Gale, M.D., andDevos, K.M.1997. Comparative genetics in the grasses .

Plant Mol. Biol. 35: 3-15. Genty, B., Briantais, J.M., and Baker, N.R. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys . Acta 990:

87-92. Gibson, S., Arondel, V., Iba, K. , and Somerville, C. 1994. Cloning of a temperature-regulated gene encoding a chloroplast omega-3-desaturase from Arabidopsis thaliana . Plant Physiol. 106:

1615-1621. Gilmour, S.J., Artus, N.N. , and Thomashow, M.F. 1992. cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana . Plant Mol. Biol. 18: 13-21.

Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D., and

Thomashow, M.F. 2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124:1854-1865. Guy, C. 1999. Molecular responses of plants to cold shock and cold acclimation. J. Mol. Microbiol . Biotechnol. 1: 231-242. Hagit, A.Z., Pablo, A.S., and Dudy, B.Z. 1995. Tomato Asrl mRNA and protein are transiently expressed following salt stress, osmotic stress and treatment with abscisic acid. Plant Sci. 110: 205-213. Hiilovaara-Teijo, M. , Hannukkala, A., Griffith, M., Yu, X.M., and Pihakaski-Maunsbach, K.1999. Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity. Plant Physiol. 121: 665-74.

Hong, S.T., Chung, J.E., An, G., and Kim, S.R. 1998. Analysis of 176 expressed sequence tags generated from cDNA clones of hot pepper by single-pass sequencing. J. Plant Biol. 41: 116-124. Hong, S.H., Kim, I.J., Yang, D.C. and Chung, W.I. 2002. Characterization of an abscisic acid responsive gene homologue from Cucumis melo . J. Exp. Bot . 53: 2271-2272.

Houde, M., Dhindsa, R.S., and Sarhan, F. 1992. A molecular marker to select for freezing tolerance in Gramineae. Mol. Gen. Genet. 234: 43 -48 .

Igarashi, Y. , Yoshiba, Y. , Sanada, Y. , Yamaguchi-Shinozaki, K. , Wada,

K. , and Shinozaki, K. 1997. Characterization of the gene for deltal-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. Plant

Mol. Biol. 33: 857-865.

Iusem, N.D., Bartholomew, D.M., Hitz, W.D., and Scolnik, P.A. 1993.

Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiol. 102: 1353-1354. Jeanneau, M., Gerentes, D., Foueillassar, X., Aivy, M., Vidal, J. , Toppan, A., and Perez, P. 2002. Improvement of drought tolerance in maize: towards the functional valication of the Zm-Asrl gene and increase of wateruse efficiencyby over-expressingC4-PEPC. Biochimie 84: 1127-1135. Jeon, J.S., Chung, Y.Y., Lee, S., Yi, G.H. , Oh, B.G., and An, G. 1999. Isolation and characterization of an anther-specific gene, RA8 , from rice ( Oryza sativa L.). Plant Mol. Biol. 39: 35-44. Joachim, V.B., Francesco, S., and Christiane, G. 1994. Transcripts accumulating during cold storage of potato ( Solanum tuberosum L.) tubers are sequence related to stress-responsive genes . Plant Physiol . 104: 445-452.

Kasuga, M. , Liu, Q. , Miura, S. , Yamaguchi-Shinozaki, K. , andShinozaki, K. 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnol. 17: 287-291.

Kim, S.-R., Lee, S., Kang, H.-G., Jeon, J.-S., Kim, K.-M. and An, G. 2003. A complete sequence of the pGA1611 binary vector. J. Plant Biol. 46: 211-214. Kirsten, R. , Jaglo-Ottosen, S.J. , Gilmour, D.G. , Zarka, O.S. , Michael,

F., and Thomashow, M.F.1998. Arabidopsis CBFl overexpression induces

COR genes and enhances freezing tolerance. Science 280: 104-106.

Kishor, P.B.K., Hong, Z., Miao, G.H., Hu, C.A.A., and Verma, D.P.S. 1995. Overexpression of delta-pyrolline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol. 108: 1387-1394.

Krause, G.H. 1994. Photoinhibition of photosynthesis : an evaluation of damaging andprotective mechanism. Physiol. Plantarum 74 : 566-574. Kurkela, S., and Borg-Franck, M. 1992. Structure and expression of kin2, one of two cold- and ABA-induced genes of Arabidopsis thaliana .

Plant Mol. Biol. 19: 689-692.

Lee, S., Jeon, J.S., Jung, K.H., and An, G. 1999. Binary vectors for efficient trans ormation of rice. J. Plant Biol. 42: 310-316. Lee, T.M. , Lur, H.S . , andChu, C.1993. Role of abscisic acid in chilling tolerance of rice ( Oryza sativa L.) seedlings. I. Endogenous abscisic acid levels. Plant Cell Environ. 16: 481-490.

Lin, C, and Thomashow, M.F. 1992. DNA sequence analysis of a complementary DNA for cold-regulated Arabidopsis gene corl5 and characterizationof the COR15polypeptide. Plant Physiol .99 : 519-525.

Lim, J.N.1998. Report of a research on new crop cultivar development .

Rural development administration, Korea, pp. 191-193.

Maskin, L., Gubesblat, G. E., Moreno, J.E., Carrari , F.0., Frankel,

N. , Sambade, A., Rossi, M., and Iusem, N.D. 2001. Differential expressionof themembers of theAsrgene family intomato (Lycopersicon esculentum) . Paint Sci. 161: 739-746.

Mbeguie-A-Mbeguie, D., Gomez, R.M., and Fils-Lucaon, B. 1997.

Molecular cloning and nucleotide sequence of an abscisic acid-stress-ripening induced (ASR) -like protein from apricot fruit (ace . No. U93164) . Gene expressionduring fruit ripening. (PGR97-166) .

Plant Physiol. 115: 1288.

Mizoguchi, T., Hayashida, N., Yamaguchi-Shinozaki, K. , Kamada, H., and Shinozaki, K. 1993. ATMPKs : a gene family of plant MAP kinases in Arabidopsis thaliana . FEBS Lett. 336: 440-444.

Monroy, A.F. , Castonguay, Y. , Laberge, S. , Sarhan, F. , Verzina, L.P. , andDhindsa, R.S.1993. A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature. Plant Physiol. 102: 873-879.

Murashige, T. , and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15:

473-495.

Nicholas, K.B., and Nicholas, H.B. 1997. GeneDoc : analysis and visualization of genetic variation.

Http : //www.cris . com/~Ketchup/genedoc .html .

Nishiyama, I. 1984. Climatic influence of pollen formation and fertilization. In S Edited by Tsunoda N. T. pp. 153-171. Biology of Rice, Japan Sci. Soc. Press, Tokyo. Nylander, M., Svensson, J. , Palva, E.T., and Welin, B.V. 2001. Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana . Plant Mol. Biol. 45: 263-279. Roger, S.O., and Bendich, A.J. 1988. Extraction of DNA from plant tissues. In Plant Molecular Biology Manual, Edited by Gelvin, S.B. , and Schilperoort, R.A. pp. A6 1-10 Kluwer Academic Publishers, Dordrecht, The Netherlands.

Rossi, M., and Iusem, N.D. 1994. Tomato (Lycopersicon esculentum) genomic clone homologous to a gene encoding an abscisic acid-induced protein. Plant Physiol. 104: 1073-1074.

Saijo, Y., Hata, S., Kyozuka, J., Shimamoto, K., and Izui, K. 2000.

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J. 23: 319-327.

Schneider, A. , Salamini, F. , andGebhardt, C.1997. Expressionpatterns and promoter activity of the cold-regulated gene ci21A o.f Potato.

Plant Physiol. 113: 335-345.

Schreiber, U. , Bilger, W., and Neubauer, C. 1994. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In Ecological Studies, Vol. 110: Ecop. Scott, D.M. , Michael, L.N. , andAndrew, D.H.1999. Betaines andrelated osmoprotectants . Targets for metabolic engineering of stress resistance. Plant Physiol .120: 945-949. multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl . Acids Res. 22: 4673-4680. Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K. , Carninci, P., Hayashizaki, Y., and Shinozaki , K.2001. Monitoring the expression pattern of 1300 arabidopsis genes under drought and cold stresses by using a full-length cDNA icroarray. Plant cell 13 : 61-72.

Silhavy, D . , Hutvagnet, G. , Barta, E. , andBanfalvi, Z .1995. Isolation and characterization of a water stress-inducible cDNA clone from Solanum chacoence . Plant Mol. Biol. 27: 587-595. Steponkus, P.L. 1984. Role of the plasma membrane in freezing injury and cold acclimation. Annu. Rev. Plant Physiol. 35: 543-584. Stockinger, E . J. , Gilmour, S . J. , andThomashow, M.F.1997. Arabidopsis thaliana CBFl encodes an AP2 domain-containing transcriptional 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.

Taj i , T . , Ohsumi , C . , Iuchi , S . , Seki , M . , Kasuga, M . , Kobayashi , M., Yamaguchi-Shinozaki, K. , and Shinozaki, K.2002. Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana . Plant J. 29: 417-426.

Thomashow, M.F.1999. Plant coldacclimation: freezing tolerance genes and regulatory mechanisms . Annu. Rev. Plant Physiol. Plant Mol . Biol. 50: 571-599.

Thompson, J.D., Higgins, D.G., and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivityofprogressive hysiologyof Photosynthesis , Edited by Schulze, E.D. andCaldwell, M.M.pp.49-70. Springer Veriag, Berlin. Yu, J. et al . , 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296: 79-92.

Vaidyanathan, R. , Kuruvilla, S. , andThomas, G.1999. Characterization and expression pattern of an abscisic acid and osmotic stress responsive gene from rice. Plant Sci. 140: 25-36. Wang, C.S., Liau, Y.E., Huang, J.C., Wu, T.D., Su, CC. and Lin, CH. 1998. Characterization of a desiccation relatedprotein in lilypollen during development and stress. Plant Cell Physiol. 39: 1307-1314.

Claims

What is claimed is:

1. An OsAsrl protein comprising amino acid sequence of in SEQ ID NO 2, which is inducible by abiotic stress , enhancing plant tolerance to cold, salt or drought stress when the protein is overexpressed in the plant, or a homologue protein sharing at least 80% amino acid sequence identity with the OsAsrl protein.

2. A nucleic acid which comprises a nucleotide sequence encoding the abiotic-stress inducible OsAsrl protein according to claim 1.

3. The nucleic acid according to claim 2 , wherein the nucleotide sequence corresponds to 67-480 nucleotide sequence in SEQ ID NO 1.

4. A vector comprising the nucleic acid according to claim 2 or 3.

5. A transformant transformed with the vector according to claim 4.

6. Amethod forpreparing a transgenicplant showing an enhanced tolerance to cold, salt or drought stress, which comprises the steps of: (a) transforming a plant cell or tissue with the vector according to claim 4;

(b) selecting a transformed plant cell or tissue; and

(c) regenerating the transformed plant cell or tissue to obtain a transgenic plant.

7. The method according to claim 6, wherein the plant is rice, barley, wheat or maize. A transgenic plant prepared by the method according to claim 6 or which shows an enhanced tolerance to cold, salt or drought stress.