WO2011108795A1

WO2011108795A1 - Gene implicated in drought stress tolerance and growth acceleration and transformed plants with the same

Info

Publication number: WO2011108795A1
Application number: PCT/KR2010/007468
Authority: WO
Inventors: Woo Taek Kim; Eun Yu Kim; Ji Ho Seo
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2010-03-04
Filing date: 2010-10-28
Publication date: 2011-09-09
Also published as: KR101291365B1; KR20110100417A

Abstract

The present invention relates to a composition for improving a tolerance to a drought stress of a plant, a plant transformed with the composition, a method for preparing a plant transformed with the composition, a method for improving a tolerance to a drought stress, and a composition promoting flowering or growing of a plant. The nucleotide sequences of the present invention encode CaSRPl protein and are involved in the drought stress-tolerance and growing capacity, therefore may be effectively used for cultivating the plants with novel function of rapid growing or bolting.

Description

GENE IMPLICATED IN DROUGHT STRESS TOLERANCE AND GROWTH ACCELERATION AND TRANSFORMED PLANTS WITH THE SAME

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a gene implicated in drought stress tolerance and growth acceleration and transformed plants with the same.

BACKGROUND OF TECHNIQUE

Due to their sessile nature, higher plants are constantly faced with various adverse environmental factors, including drought, high salt, heavy metals, cold, heat shock, and ozone, during their whole life span. These abiotic stresses are a limiting factor for the growth and development of crop plants. Water deficiency causes dramatic reduction of crop production globally, and the decreasing availability of fresh water may pose a future threat to humans and higher plants. Plants have diverse defense strategies to enhance their tolerance to transient and long-term water shortages by triggering signaling network pathways and inducing stress- responsive genes. The cellular and genetic defense mechanisms in response to water stress have been widely documented (Shinozaki and Yamaguchi-Shinozaki, 2007). However, for stress tolerance or sensitivity, our knowledge concerning the biological functions of stress-related genes in higher plants is still rudimentary. Therefore, it is important to study the functions of stress responsive genes to increase the productivity and distribution of crop plants.

Hot pepper {Capsicum annuum L), belonging to the solanaceous species, is a widely cultivated crop for its commercially important hot-tasting fruits. The present inventors had been focusing on the response of hot pepper plants to water-deficit stress. Previously, the present inventors isolated and characterized a broad spectrum of genes induced by dehydration in hot peppers using subtractive hybridization and differential-display polymerase chain reaction (PCR) methods (Park et al. 2003; Hong and Kim 2005). Among these cDNAs, Ca-LEALl {Capsicum annuum late embryogenesis-abundant-like protein I) (Park et al. 2003), Ca-DREBLPl {Capsicum annuum dehydrationresponsive elementbinding-factor-like protein I) (Hong and Kim 2005), CaPUBl {Capsicum annuum putative U-box protein I) (Cho et al. 2006a), CaXTHs {Capsicum annuum xyloglucan endotransglucosylase/hydrolase homologs) (Cho et al. 2006b), and CaRmalHl {Capsicum annuum RING membrane-anchor ubiquitin ligase homolog I) (Lee et al. 2009) were rapidly induced by drought stress and may be functionally involved in drought stress responses as positive or negative regulators in hot pepper plants.

Ca-DSR5 {Capsicum annuum drought stress-responsive 5) (GenBank accession No. CK327621) is one of the previously identified partial genes, the mRNA of which was induced in response to dehydration in hot pepper roots (Hong and Kim 2005). The derived partial Ca-DSR5 protein displayed a significant homology to the putative stress-related proteins from soybean and Arabidopsis and, thus, was renamed CaSRPl (Capsicum annuum stress-related protein 1). In this invention, the present inventors isolated a full-length CaSRPl cDNA clone (GenBank accession No. GU373985). The predicted full-length CaSRPl protein also showed amino acid sequence identity with the small rubber particle proteins (SRPPs) found in rubber trees. To study the cellular functions of CaSRPl, transgenic Arabidopsis plants (35S:CaSRPl) that constitutively expressed the CaSRPl gene were constructed.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains. SUMMARY OF THE INVENTION

The present inventors have made intensive studies to identify genes for improving a tolerance to a drought stress of a plant. As results, we have discovered that the CaSRPl gene in hot pepper {Capsicum annuum cv. Pukang) plays a crucial role in a tolerance to a drought stress and growth acceleration, and that plants with enhanced drought stress tolerance may be obtained by transformation with the CaSRPl gene.

Accordingly, it is an object of this invention to provide a composition and a method for improving a tolerance to a drought stress of a plant.

It is another object of this invention to provide a plant cell or a plant exhibiting improved tolerance to a drought stress, transformed with the composition according to this invention.

It is still another object of this invention to provide a method for preparing transformed plant exhibiting improved drought stress tolerance.

It is further object of this invention to provide a composition and a method for promoting flowering or growing of a plant.

It is still further object of this invention to provide a plant cell and a plant exhibiting promoted flowering or growing, transformed with the composition of this invention.

It is still further object of this invention to provide a method for preparing transformed plant exhibiting promoted flowering or growing.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents sequence analysis of hot pepper CaSRPl. Fig. la indicates the schematic structure of CaSRPl cDNA clone (Genbank accession No. GU373985). The gray bar represents the coding region and the black lines indicate 5'- and 3'- untranslated regions. Restriction sites for Ecc l, Ps&, Ncol, and Spel are indicated. Fig. lb shows multiple alignments of 10 SRP1 homologs. The predicted amino acid sequence of CaSRPl is compared with those of proteins from poplar (Genbank accession No. XP_002319520), grape (Genbank accession No. XP_002283697), Arabidopsis (Genbank accession Nos. At3g05500, At2g47780, and Atlg67360), alfalfa (Genbank accession No. ABD28680), soybean (PvSRP; Genbank accession No. AAB00555), rubber tree (HbSRPP; Genbank accession No. AAC82355), and Parthenium argentatum Gray (GHS; Genbank accession No. AAQ11374). Amino acid residues identical in at least six of the ten sequences are shaded. Amino acid sequences conserved in all ten proteins are indicated in black. Fig. lc shows the phylogenetic relationship of the ten SRP homologs from hot pepper, poplar, grape, Arabidopsis, alfalfa, rubber tree and Parthenium argentatum Gray.

Fig. 2 represents the induction of CaSRPl gene expression in hot pepper leaves in response to dehydration. Light-grown 2-week-old hot pepper plants were subjected to dehydration stress by exposing whole plants to a stream of air in a clean bench for increasing times (0, 30, and 90 min). Total RNA was isolated from leaves and analyzed by RT-PCR using gene-specific primers designed for CaSRPl, CaLEALl (positive control) and CaACT (negative control). As results, it is confirmed that CaSRPl is induced time-dependent manner. The CaLEALl gene also showed the tendency of time-dependent induction, which indicates that the drought stress had been treated to hot pepper efficiently using the CaLEALl gene as a positive marker.

Fig. 3 represents the phenotypic characterizations of T₃ 35S:CaSRPl transgenic Arabidopsis plants. The presence and expression levels of CaSRPl were examined by RT-PCR in wild-type and various transgenic lines. As results, all of the independent T₃ 35S:CaSRPl (#1, #2, #3, #4, and #7) overexpressed CaSRPl gene compared with the wild type (Fig. 3a). These overexpressors contained considerably longer (1.2 to 1.3-fold) roots relative to the wild type roots (Fig. 3b and 3c). In addition, both blade length and width of the second leaf of 2-week-old transgenic plants were increased by 1.3 to 2.1-fold and 1.2 to 1.8-fold, respectively, as compared to the wild type plants (Figs. 3d and 3e). The petiole lengths were also increased to the similar extent as the blade length and width. Such phenotypic features are the most prominent in transgenic line #7 to show increase in leaf area up to 4 folds, which indicates the characteristic of rapid growing.

Fig. 4 shows that CaSRPl overexpressors bolted earlier than did wild-type plants. 28 days after germination, 35S:CaSRPl transgenic Arabidopsis plants bolted 4-5 days earlier as compared to wild type plants (Fig. 4a and 4b). Such phenotypic features are the most prominent in transgenic line #7 to show increase in height up to 10 cm, which indicates the characteristic of early bolting, as well as rapid growing.

Fig. 5 represents the comparison of the expression levels of cell division- and cell elongation-associated genes in wild-type (WT) and T₃ 35S:CaSRPl transgenic Arabidopsis plants. To examine whether the elevation of leaf and root growth in 35S:CaSRPl transgenic plants was due to changes in cell expansion and/or proliferation pathways, the expression profiles of genes in WT and T₃ 35S:CaSRPl transgenic Arabidopsis seedling were investigated by RT-PCR. AtCYCD3 and AtCDC2b as cell cycle activity marker genes were increased in transgenic plants, while the expansin gene ΑίΕΧΡ5ά\ύ not show any significant difference between WT and transgenic plants. These results suggest that the rapid growing of the 35S:CaSRPl transgenic Arabidopsis plants relates to the changes in cell cycle pathways.

Fig. 6 shows that overexpression of CaSRPl is involved in improvement of a drought stress tolerance. Because CaSRPl is rapidly induced by dehydration in hot pepper, we assumed that CaSRPl also participates in drought stress responses. To verify this, the cut rosette water loss (CRWL) rate (Bouchabke et al. 2008) of 35S:CaSRPl plants was examined. After 1 hr of incubation, CRWL rates of wild type and G-^Sft ^-overexpressing leaves were already distinguishable, and after 4 hr of incubation, the difference became clearer; the average fresh weight of wild type leaves was reduced to about 27% of the starting weights, while the fresh weight of the transgenic leaves were reduced to only 42-51% (Fig. 6a). These results suggest that C^/^i-overexpressors contain more water than wild types under drought stress. Thereafter, wild type and 35S:CaSRPl Arabidopsis plants were grown for 3 weeks under normal growth conditions then allowed to dry without watering for 7 days and the survival rate was analyzed. As results, the survival rate of CaSRPl- overexpressors was 3-9 folds higher than that of wild type (Fig. 6b). These results suggest that CaS/W-overexpressors are more tolerant to water deficit.

Fig. 7 indicates the map of a pBI121 vector carrying CaSRPl cDNA.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of this invention, there is provided a composition for improving a tolerance to a drought stress of a plant, comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

The present inventors have made intensive studies to identify genes for improving a tolerance to a drought stress of a plant. As results, we have discovered that CaSRPl gene in hot pepper Capsicum annuum cv. Pukang) is related to a tolerance to a drought stress and a plant with enhanced drought stress tolerance may be obtained by transforming with the CaSRPl gene.

According to a preferred embodiment, the nucleotide sequence of this invention comprises the nucleotide sequence of SEQ ID NO:l. According to the present invention, SEQ ID NO:l is the nucleotide sequence of CaSRPl gene which is a partial fragment of Ca-DSR5 Capsicum annum-drought stress responsive 5) (GenBank accession No. CK327621).

It would be obvious to the skilled artisan that the nucleotide sequences used in this invention are not limited to those listed in the appended Sequence Listings. The nucleotide sequences described herein are illustrative and their biological equivalents may be also used in this invention for enhancement of tolerance to a drought stress and promotion of flowering or growing in plants. In this regard, the sequence variations should be construed to be covered by the present invention.

For nucleotides, the variations may be purely genetic, i.e., ones that do not result in changes in the protein product. This includes nucleic acids that contain functionally equivalent codons, or codons that encode the same amino acid, such as six codons for arginine or serine, or codons that encode biologically equivalent amino acids.

Considering biologically equivalent variations described hereinabove, the nucleic acid molecule of this invention may encompass sequences having substantial identity to them. Sequences having the substantial identity show at least 80%, more preferably at least 90%, most preferably at least 95% similarity to the nucleic acid molecule of this invention, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Moi. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989) Corpet et al., Nuc. Acids Res. 16:10881-90(1988) Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol. 215:403- 10(1990)] is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html. In another aspect of this invention, there is provided a composition for improving a tolerance to a drought stress of a plant, comprising a recombinant plant expression vector which comprises: (a) the nucleotide sequence of this invention; (b) a promoter which is operatively linked to the nucleotide sequence of (a) and generates RNA molecules in plant cells; and (c) a poly A signal sequence inducing polyadenylation at the 3'-end of the RNA molecules.

In still another aspect of this invention, there is provided a plant cell exhibiting improved tolerance to a drought stress, transformed with the composition of this invention.

The term "operatively 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 nucleotide 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 in accordance with conventional techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001), teachings of which are incorporated herein by reference.

The suitable promoter in the present invention includes any one commonly used in the art, for example SP6 promoter, 77 promoter, T3 promoter, PM promoter, maize-ubiquitin promoter, Cauliflower mosaic virus (CaMV)-35S promoter, Nopalin synthase (nos) promoter, Figwort mosaic virus 35S promoter, Sugarcane bacilliform virus promoter, commelina yellow mottle virus promoter, photo-inducible promoter of small subunit of Ribulose-l,5-bis-phosphate carboxylase (ssRUBISCO), cytosolic triosphosphate isomerase (TPI) promoter in rice, adenine phosphoribosyltransferase (APRT) or octopine synthase promoter in Arabidopsis. Preferably, the promoter used in this invention is CaMV 35S. According to a preferred embodiment, the 3'-non-translated region causing polyadenylation includes 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, and OCS (octopine synthase) terminator. Most preferably, the 3'- non-translated region causing polyadenylation in this invention is NOS.

Optionally, the present vector for plants may further carry a reporter molecule {e.g., genes for luciferase and β-glucuronidase). In addition, the vector may contain antibiotic resistant genes as selective markers (e.g., neomycin phosphotransferase gene (nptll) and hygromycin phosphotransferase gene (hpt)).

According to a preferred embodiment, the plant expression vector of this invention is Agrobacterium binary vectors.

The term "binary vector" as used herein, refers to a cloning vector containing two separate vector systems harboring one plasmid responsible for migration consisting of left border (LB) and right border (RB), and another plasmid for target gene-transferring. Any Agrobacterium suitable for expressing the nucleotide of this invention may be used, and most preferably, the transformation is carried out using Agrobacterium tumefaciens G '3101.

Introduction of the recombinant vector of this invention into Agrobacterium can be carried out by a large number of methods known to one skilled in the art. For example, particle bombardment, electroporation, transfection, lithium acetate method and heat shock method may be used. Preferably, the electroporation is used.

In still another aspect of this invention, there is provided a plant exhibiting improved tolerance to a drought stress, transformed with the composition of this invention. To introduce a foreign nucleotide sequence into plant cells or plants may be performed by the methods {Methods of Enzymology, Vol. 153, 1987) known to those skilled in the art. The plant may be transformed using the foreign nucleotide inserted into a carrier {e.g., vectors such as plasmid or virus) or Agrobacterium tumefaciens as a mediator (Chilton et al., Cell, 11: 263-271 (1977)) and by directly inserting the foreign nucleotide into plant cells (Lorz et al., Mol. Genet, 199: 178-182 (1985); the disclosure is herein incorporated by reference). For example, electroporation, microparticle bombardment, polyethylene glycol-mediated uptake may be used in the vector containing no T-DNA region.

Generally, Agrobacterium-mediated transformation is the most preferable

(U.S. Pat. Nos. 5,004,863, 5,349,124 and 5,416,011), and the skilled artisan can incubate or culture the transformed cells or seeds to mature plants in appropriate conditions.

The term "plant(s)" as used herein, is understood by a meaning including a plant cell, a plant tissue and a plant seed as well as a mature plant.

The plants applicable of the present method include, but not limited to, most dicotyledonous plants including lettuce, Chinese cabbage, potato and radish, and most monocotyledonous plants including rice plant, barley and banana tree. Preferably, the present method can be applied to the plants selected from the group consisting of food crops such as rice plant, wheat, barley, corn, bean, potato, Indian bean, oat and Indian millet; vegetable crops such as Arabidopsis sp., Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welsh onion, onion and carrot; crops for special use such as ginseng, tobacco plant, cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut and rape; fruit trees such as apple tree, pear tree, jujube tree, peach tree, kiwi fruit tree, grape tree, citrus fruit tree, persimmon tree, plum tree, apricot tree and banana tree; flowering crops such as rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and fodder crops such as ryegrass, red clover, orchardgrass, alfalfa, tallfescue and perennial ryograss.

In still another aspect of this invention, there is provided a method for preparing transformed plant exhibiting improved drought stress tolerance, comprising the steps of:

(a) introducing the composition of this invention into a plant cell ; and

(b) obtaining the transformed plant exhibiting improved drought stress tolerance from the plant cell of (a).

In still another aspect of this invention, there is provided a method for improving a tolerance to a drought stress of a plant, comprising introducing the composition of this invention into a plant cell.

Introducing plant expressing recombinant vectors into a plant cell can be carried out by various methods known to those skilled in the art. Selection of the transformed plant cell can be performed by exposing it to selective agents {e.g., metabolic inhibitors, antibiotics or herbicides). Transformed plant cells stably harboring marker genes which give a tolerance to selective agents are grown and divided in above culture.

The exemplary markers include, but not limited to, hygromycin phosphotransferase (hpt), glyphosate-resistance gene and neomycin phophotransferase (nptll) system.

The methods for developing or regenerating plants from plant protoplasms or various ex-plants are well known to those skilled 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).

According to a preferred embodiment, the plant of this invention is selected from the group consisting of food crops such as rice plant, wheat, barley, corn, bean, potato, Indian bean, oat and Indian millet; vegetable crops such as Arabidopsis sp., Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welsh onion, onion and carrot; crops for special use such as ginseng, tobacco plant, cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut and rape; fruit trees such as apple tree, pear tree, jujube tree, peach tree, kiwi fruit tree, grape tree, citrus fruit tree, persimmon tree, plum tree, apricot tree and banana tree; flowering crops such as rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and fodder crops such as ryegrass, red clover, orchardgrass, alfalfa, tallfescue and perennial ryograss. More preferably, the plant of this invention is food crops such as rice plant, wheat, barley, corn, bean, potato, Indian bean, oat and Indian millet.

In still another aspect of this invention, there is provided a composition for promoting flowering or growing of a plant comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.

According to the present invention, CaSRPl overexpressing transgenic plants show longer roots, broader leaves and earlier bolting relative to the wild-type under normal growth conditions, which indicates the characteristic of early bolting, as well as rapid growing. This is another useful feature of the CaSRPl gene allowing application to a plant with novel function and biomass through early bolting and rapid growing.

According to a preferred embodiment, the nucleotide sequence of this invention comprises the nucleotide sequence of SEQ ID NO:l.

In still another aspect of this invention, there is provided a composition for promoting flowering or growing of a plant, comprising a recombinant plant expression vector which comprises: (a) the nucleotide sequence of this invention; (b) a promoter which is operatively linked to the nucleotide sequence of (a) and generates RNA molecules in plant cells; and (c) a poly A signal sequence inducing polyadenylation at the 3'-end of the NA molecules.

In still another aspect of this invention, there is provided a plant cell exhibiting promoted flowering or growing, transformed with the composition of this invention.

In still another aspect of this invention, there is provided a plant exhibiting promoted flowering or growing, transformed with the composition of this invention.

In still another aspect of this invention, there is provided a method for preparing transformed plant exhibiting promoted flowering or growing, comprising the steps of:

(a) introducing the composition of this invention into a plant cell ; and

(b) obtaining the transformed plant exhibiting promoted flowering or growing from the plant cell of (a).

In still another aspect of this invention, there is provided a method for promoting flowering or growing of a plant, comprising introducing the composition of this invention into a plant cell.

As the plant expression vector comprising the nucleotide of the present invention and the method for introducing the same into a plant cell are mentioned above, they are omitted herein to avoid excessive overlaps.

The features and advantages of the present invention will be summarized as follows:

(a) The present invention provides a composition for improving a tolerance to a drought stress of a plant, a method for preparing a plant transformed with the composition, a method for improving a tolerance to a drought stress, and a composition promoting flowering or growing of a plant.

(b) The nucleotide sequences of the present invention are involved in the drought stress-tolerance and growing capacity, therefore may be effectively used for cultivating the plants with novel function of rapid growing or bolting. The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

MATERIALS AND METHODS

1. Plant materials

Dried hot pepper Capsicum annuum cv. Pukang) and Arabidopsis thaliana ecotype Columbia (Col-0) seeds were soaked for 10 min in 30% sodium hypochlorite solution (bleach) and rinsed extensively with sterilized water as previously described by Cho et al. (2006). Seedlings were grown on MS (Murashige and Skoog) medium (Duchefa, Haarlem, Netherlands) that contained 1% sucrose, 12 pg/ml B5 vitamin, and 0.8% agar (pH 5.7) in a 25°C-growth chamber under continuous light condition.

2. Application of dehydration stress

Hot pepper plants were grown in pots for 2 weeks under light-grown conditions. Whole hot pepper plants were then exposed to a stream of air in a clean bench for varying times (0, 30, and 90 min) as described by Kilian et al. (2007). During this incubation time, the plants gradually lost their fresh weight.

3. Isolation of a full-length cDNA clone of CaSRPl Full-length CaSRPl cDNA was PCR-amplified from a λ-uni-Zap II cDNA library constructed from water-stressed leaves of hot pepper plants (Cho et al. 2006b). Vector-specific primers corresponding to the T3 promoter (Table 1) were used to generate the sense oligonucleotide. The antisense oligonucleotide was designed from the sequences corresponding to the 3'-end of the partial Ca-DSR5 cDNA (Table 1 5'-CTTCACACTTCACAGAAC-3'). PCRs used high-fidelity Ex-Taq DNA polymerase (Takara, Otsu, Shiga, Japan) and consisted of 40 amplification cycles with an annealing temperature of 52°C for 30 sec and an elongation temperature of 68°C for 1 min. PCR products were introduced into the pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed into DH5a E coll The cDNA inserts from the plasmids of 12 resulting colonies were sequenced.

Table 1. Primers for PCR, Cloning, and Construction of Transgenic Plants

Gene Forward Primers Reverse Primers

For cloning of CaSRPI cDNA

T3 promoter 5'- attaaccctcactaaaggga- 3'

Ca-DSR53' UTR 5'-cttcacacttcacagaac-3'

For RT-PCR primer

CaSRPI 5' -gcagatctatggctgaatcaaacccc- 3' 5'-gcgagctcctagttggaagccacagg-3'

CaLEAU 5 ' -atggatctgattgacaaggc - 3' 5'-tcatatctctacaacctttgatg-3'

CaACT 5'-ttggactctggtgatggtgtg-3' 5'-aacatggttgagccaccactg-3'

AtACT8 5'-tactgattacctcatgaagatccttac-3' 5'-aaacgatgtctctttagtttagaagc-3'

AtCYCD3 5'-tccggtacctcccatcag-3' 5 ' - gagtggctacgattgccc- 3'

AtCDC2b 5'-gagcagcaatggccgg-3' 5' -cattgctaccacaccattgtg- 3'

AtEXP5 5 ' -ggtacggcaatctgtatagc - 3 ' 5'-gttcccacacatatattcgc-3'

4. RNA Isolation Total RNA from hot pepper and transgenic Arabidopsis plants were extracted using RNAiso Plus reagent as per the manufacturer's instructions (Takara). Isolated RNA was treated with DNase I to remove possible residual DNA contamination as per the manufacturer's instructions (Promega).

5. Reverse transcriptase-polymerase chain reaction (RT-PCR)

RT-PCR was carried out in a total volume of 50 μΙ containing 1 μΙ of the first strand cDNA reaction products, 1 μΜ gene-specific primers (Table 1), 10 mM Tris (pH 8.0), 50 mM KCI, 1.5 mM MgCI_2/ 0.01% gelatin, 200 μΜ deoxynucleotides, and 2.5 units of high-fidelity Ex-Taq polymerase (Takara) as previously described (Jun et al. 2008). Amplification consisted of 27 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C in an automatic thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT, USA). PCR products were separated on 1.0% agarose gels and visualized under UV light.

6. Generation of 35S:CaSRPl transgenic Arabidopsis plant.

The full-length pCaSRPl cDNA was introduced into the corresponding sites of the binary vector pBI121 (Po-Yen Chen, et al., Molecular Breeding, Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants, ll(4):287-293(2003)). The resulting 35S:CaSRPl fusion gene was transferred to Agrobacterium tumefaciens strain GV3101 by electroporation. Agrobacterium cells containing the 35S:CaSRPl construct were transformed into Arabidopsis plants (Col-0) by means of the floral-dip method (Zhang et al. 2006). Ti seeds were collected from regenerated T₀ plants. Ti seeds were germinated on 0.5 X Murashige and Skoog (MS) medium with 30 pg/ml kanamycin. Homozygous T₃ lines were obtained by subsequent self-fertilization and used in phenotypic analyses. The presence and expression level of the CaSRPl gene were confirmed by genomic Southern blotting and RT-PCR, respectively. 7. Measurements of dimensional parameters of roots, leaves, and petioles in wild-type and transgenic Arabidopsis seedlings.

Root and leaf growth of wild type and transgenic Arabidopsis plants were monitored as described by Seo et al. (2008) with minor modifications. To examine root growth, vertically oriented agar plates (0.8% select agar; Life Technology, Rockville, MD, USA) were incubated at 22°C under continuous light for 1-7 days. During incubation, the advancing root tips were monitored with the image-analyzing program SCIONIMAGE (Scion Corp., Frederic, MD, USA). Leaves and petioles of 2- week-old wild-type and transgenic Arabidopsis plants were removed from the plants. The surface areas, lengths, and widths were also determined using SCIONIMAGE software.

8. Survival rate determination of wild-type and transgenic Arabidopsis plants after drought stress.

Three-week-old wild type and 35S:CaSRPl transgenic plants, which had been grown under normal growth conditions, were subjected to drought stress by withholding water for 7 days. The plants were then re-watered and their phenotypes were examined after 3 days. Survival was defined as the ability to resume growth when returned to normal conditions following water stress (Cho et al. 2008).

RESULTS

1. Isolation and characterization of full-length CaSRPl cDNA

In our previous study, a partial hot pepper Ca-DSR5 (Capsicum annuum- drought stress responsive 5) gene, which was rapidly induced by drought stress in root tissue, was identified (Hong and Kim 2005). The derived partial Ca-DSR5 protein showed homology to the putative stress-related proteins from soybean (PvSRP) and Arabidopsis. Thus, we referred to this gene as CaSRPl for Capsicum annuum stress-related protein 1 and proceeded to isolate its full-length cDNA. Total recombinant lambda DNA was obtained from the λ-uni-Zap II cDNA library constructed from water-stressed leaves of hot pepper plants (Cho et al. 2006b). The entire coding region of CaSRPl was PCR-amplified using the lambda DNA as a template with primers corresponding to the 5'-end of the library vector sequence and the 3 -untranslated region of the partial Ca-DSR5 cDNA clone (Table 1).

Figure la shows the restriction enzyme map of pCaSRPl. The pCa-SRPl clone (GenBank accession No. GU373985) is 978 bp long, consisting of a 68-bp 5'- untranslated region, a 684-bp coding region encoding 228 amino acids and a 226-bp 3'-untranslated region. The predicted molecular mass and calculated isoelectric point of CaSRPl are 24.9 kDa and 9.27, respectively. A database search showed that CaSRPl is 60.5 and 58.5% identical to grape and poplar proteins, respectively, whose cellular functions are not yet known (Fig. lb and lc). In addition, CaSRPl is 51.8-28.3% homologous to the putative stress-related proteins in Arabidopsis (At3g05500, At2g47780 and Atlg67360), soybean (PvSRP), and alfalfa. Functions of these proteins are also unknown. Interestingly, CaSRPl shares a significant degree of sequence identity (-46%) with the small rubber particle proteins (SRPP) of rubber tree Hevea brasiliensis) and the guayule homolog of SRPP (GHS) from Parthenium argentatum Gray, both of which produce natural rubber (cis-1, 4- polyisoprene) (Oh et al. 1999; Sookmark et al. 2002; Kim et al. 2004). Hevea brasiliensis currently supplies most of the commercially used high-molecular weight natural rubber, while Parthenium argentatum Gray is potential rubber tree for an alternative source of natural rubber (Mooibroek and Cornish 2000). 2. Drought induction of the CaSRPl gene in hot pepper leaves

The CaSRPl gene was induced rapidly in roots of hot pepper plants in response to dehydration (Hong and Kim 2005). To examine whether CaSRPl is also up-regulated in leaf tissue in response to dehydration, we monitored the steady- state level of CaSRPl transcripts in water-stressed leaves. For drought stress treatment, light-grown 2-week-old hot pepper plants were exposed to a stream of air in a clean bench for increasing times (0, 30, and 90 min) (Kilian et al. 2007). Total RNA was then isolated from the treated leaves and subjected to RT-PCR using gene-specific primers. Figure 2 indicates that the low basal-level of CaSRPl transcripts already began to increase after 30 min of dehydration. mRNA levels were continuously elevated for at least 90 min. The CaLEALl gene, which encodes abiotic stress-induced late embryogenesis-abundant-like protein 1 (Park et al. 2003), was chosen for the RT-PCR experiment as a positive control for water deficit. The amount of CaLEALl mRNA was also elevated concomitantly in response to water loss. In contrast, the expression of the hot pepper actin gene {CaACT) remained unchanged during incubation. These results, along with those of Hong and Kim (2005), indicate that CaSRPl is induced by dehydration in both roots and leaves (Fig. 2) of hot pepper plants.

3. CaSWl-overexpressing transgenic Arabidopsis plants grew more rapidly than control plants

The structure (Fig. 1) and expression profile (Fig. 2) of the CaSRPl gene raises the possibility that CaSRPl may be involved in cellular responses to drought stress in hot pepper plants. Thus, we wanted to investigate the cellular functions of CaSRPl with the aid of transgenic approach. It was previously reported that construction of transgenic hot pepper plants was extremely difficult (Seo et al. 2008). Specifically, transformation and regeneration yields were too low to obtain sufficient independent transgenic lines. Alternatively, hot pepper genes appeared to be fully functional in heterologous Arabidopsis cells (Cho et al. 2006a; Cho et al. 2006b; Seo et al. 2008; Lee et al. 2009). We indeed failed to obtain transgenic hot pepper plants that constitutively expressed CaSRPl gene. Thus, in this invention, we generated transgenic Arabidopsis plants that overexpressed CaSRPl under the control of the 35S CaMV promoter by means of Agrobacter/um-med\ated transformation. Several independent primary transformants were identified due to kanamycin resistance. Transgenic plants were subsequently regenerated and used for phenotype analyses. The presence and expression levels of transgenes were elucidated by RT-PCR. Figure 3a indicates that several independent T₃ transgenic lines (#1, #2, #3, #4 and #7) possessed varied amounts of the CaSRPl transcripts under normal growth conditions. Transgenic line #3 contained the highest level of CaSRPl mRNA and line #4 contained the lowest level (Fig. 3a).

In pursuing the phenotypic analysis, we first found that light-grown 4-day- old CaSft/ -overexpressing seedlings contained considerably longer (1.2- to 1.3-fold) roots relative to the wild type roots under normal growth conditions (Fig. 3b). The lengths of transgenic and wild type roots were similar in very early seedlings (1 day after germination), but began to differ 2 days after germination. Thereafter, the differences in root length became greater during the period 5 to 7 days after germination (Fig. 3c). In addition, although the morphology of transgenic leaves appeared to be somewhat similar in 5- to 7-day-old-seedlings (Fig. 3a), the 2-week- old transgenic leaves were markedly larger than those of wild type Arabidopsis plants (Fig. 3d). Both blade length and width of the second leaf were increased 1.3- to 2.1-fold and 1.2- to 1.8-fold, respectively, in transgenic plants as compared to the wild type plants (Fig. 3e). The petiole lengths were also increased to the similar extent that blade length and width were (Fig. 3e). An increase in blade length was paralleled with that in blade width, resulting in the uniformly larger shape in 35S:CaSRPl lines. Consequently, the leaf-blade area of the independent transgenic CaSR i-overexpressing lines was 1.6- to 3.6-fold greater than that of wild type leaves.

The faster-growing phenotype of 35S:CaSRPl plants consequently resulted in earlier bolting as compared to wild type plants. The CaSR^i-overexpressing transgenic lines bolted 20 to 21 days after germination, whereas control plants bolted approximately 25 days after germination (Fig. 4a). At 28-day-after germination, the inflorescence length of CaS^i-overexpressors was from 7.7 to 12.9 cm depending on the transgenic lines, while that of control plants averaged 1.0 cm (Fig. 4b). Taken together, these results suggest that the ectopic expression of the hot pepper-originated CaSRPl gene caused faster growth in heterologous Arabidopsis plants that is coupled with enhanced development of both vegetative and reproductive organs.

4. Expression profiles of cell cycle- and elongation-controlled genes in CaSft/y-overexpressing transgenic lines

Based on the aforementioned results of phenotypic analyses, we hypothesized that CaSRPl was associated with a subset of cell and tissue development. To examine whether the elevation of leaf and root growth in 35S:CaSRPl transgenic plants was due to changes in cell expansion and/or proliferation pathways, we next investigated expression profiles of genes that are intimately tied with cell division and elongation, respectively. The D-type cyclin /4ftT c7?J(Riou-Khamlichi et al. 1999) and cell cycle-dependent kinase-related gene AtCDC2b (Yoshizumi et al. 1999) were selected as marker genes for cell cycling activity. The expansin gene AtEXP5( et al. 2002, GenBank accession No. NM_113824) was included as a marker for cell elongation. Total RNA was extracted from light-grown 7-day-old wild type and 35S:CaSRPl whole seedlings (lines #2 and #7) and analyzed by RT-PCR with gene- specific primers (Table 1). The results showed that mRNA levels of AtCYCD3 and AtCDC2b were markedly enhanced in the C S^ -overexpressor lines #2 and #7 (Fig. 5). In contrast, the amount of AtEXPS transcript was nearly identical in transgenic and control seedlings (Fig. 5). Thus, these results allowed the suggestion that the faster-growing phenotypes of 35S:CaSRPl transgenic plants correlated with enhanced expression of cell cycle progression genes, rather than elongation-related genes.

5. Overexpression of CaSRPl conferred increased drought tolerance.

Because CaSRPl is rapidly induced by dehydration in hot pepper, we considered the possibility that, in addition to a subset of cell and tissue development, CaSRPl also participates in drought stress responses. To test this possibility, we first examined the cut rosette water loss (CRWL) rate (Bouchabke et al. 2008) of 35S:CaSRPl plants. The rosette leaves were detached from 3-week-old wild type and transgenic plants, placed on open-lid Petri dishes, and incubated for different time periods (0-4 h) at room temperature under dim light. Decreases in fresh weight were monitored. As shown in Figure 6a, detached rosette leaves from 35S:CaSRPl plants (lines #1, #2, #3, and #7) lost water more slowly than those from wild type plants. After 1 h of incubation, CRWL rates of wild type and CaSRPl- overexpressing leaves were already distinguishable, with that of the transgenic leaves being significantly lower than that of control leaves. After 4 h of incubation, the difference became clearer; the average fresh weight of wild type leaves was reduced to about 27% of the starting weights, while the fresh weight of the transgenic leaves were reduced to only 42-51% depending on the independent lines (Fig. 6a). As a next experiment, the response of transgenic lines to drought stress was examined at the whole-plant level. Wild type and 35S:CaSRPl Arabidopsis plants were grown for 3 weeks in pots under normal growth conditions. The soil was then allowed to dry without watering for 7 days. After this drought condition, wild type plants appeared to be severely wilted, their leaves were rolling, and they ceased to grow (Fig. 6b). After re-watering for 3 days, most of the wild type plants were unable to re-grow and died with a survival rate of 9.8% (11 out of 112 plants). In contrast, significant numbers of 35S:CaSRPl leaves remained turgid and green after withholding water for 7 days. At 3 days of re-watering, many CaSRPl- overexpressors continued to grow and their survival rates were between 27.3% and 87.1% depending on the transgenic lines [#1; 27.3% (27 out of 99 plants), #2; 73.0% (65 out of 89 plants), #3; 60.2% (50 out of 83 plants), and #7; 87.1% (108 out of 124 plants)] (Fig. 6b). Thus, these results are in agreement with the slower CRWL rate of 35S:CaSRPl leaves as compared to the wild type leaves (Fig. 6a) and indicate that CaSR 'i-overexpressing transgenic plants were more tolerant to water deficits than were the control plants. Overall, our data in Figures 3, 4 and 6 are consistent with the notion that CaSRPl is involved not only in a subset of cell and tissue development but also in the drought stress response of transgenic Arabidopsis plants. On the other hand, the phenotypes of the independent 35S:CaSRPl lines were not necessarily paralleled with the levels of CaSRPl transcripts (Fig. 3a), suggesting that CaSRPl protein levels may be subject to control in transgenic Arabidopsis plants. Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents. References

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Claims

What is claimed is:

1. A composition for improving a tolerance to a drought stress of a plant, comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.

2. The composition according to claim 1, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:l.

3. A composition for improving a tolerance to a drought stress of a plant, comprising a recombinant plant expression vector which comprises: (a) the nucleotide sequence according to claim 1 or 2; (b) a promoter which is operatively linked to the nucleotide sequence of (a) and generates RNA molecules in plant cells; and (c) a poly A signal sequence inducing polyadenylation at the 3'-end of the RNA molecules.

4. A plant cell exhibiting improved tolerance to a drought stress, transformed with the composition according to any one of claims 1 to 3.

5. A plant exhibiting improved tolerance to a drought stress, transformed with the composition according to any one of claims 1 to 3.

6. A method for preparing transformed plant exhibiting improved drought stress tolerance, comprising the steps of:

(a) introducing the composition according to any one of claims 1 to 3 into a plant cell ; and

7. The composition according to claim 6, wherein the plant is selected from the group consisting of food crops such as rice plant, wheat, barley, corn, bean, potato, Indian bean, oat and Indian millet; vegetable crops such as Arabidopsis sp., Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welsh onion, onion and carrot; crops for special use such as ginseng, tobacco plant, cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut and rape; fruit trees such as apple tree, pear tree, jujube tree, p each tree, kiwi fruit tree, grape tree, citrus fruit tree, persimmon tree, plum tree, apricot tree and banana tree; flowering crops such as rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and fodder crops such as ryegrass, red clover, orchardgrass, alfalfa, tallfescue and perennial ryograss.

8. A method for improving a tolerance to a drought stress of a plant, comprising introducing the composition according to any one of claims 1 to 3 into a plant cell.

9. A composition for promoting flowering or growing of a plant comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.

10. The composition according to claim 1, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: l.

11. A composition for promoting flowering or growing of a plant, comprising a recombinant plant expression vector which comprises: (a) the nucleotide sequence according to claim 9 or 10; (b) a promoter which is operatively linked to the nucleotide sequence of (a) and generates RNA molecules in plant cells; and (c) a poly A signal sequence inducing 3'-terminal polyadenylation of the RNA molecules.

12. A plant cell exhibiting promoted flowering or growing, transformed with the composition according to claim 9 or 10.

13. A plant exhibiting promoted flowering or growing, transformed with the composition according to claim 9 or 10.

14. A method for preparing transformed plant exhibiting promoted flowering or growing, comprising the steps of:

(a) introducing the composition according to any one of claims 9 to 11 into a plant cell ; and

15. A method for promoting flowering or growing of a plant, comprising introducing the composition according to any one of claims 9 to 11 into a plant cell.