WO2008075352A1 - Stress response genes useful for generating stress tolerant plants - Google Patents

Abstract

The present invention provides genetically modified plants having increased tolerance to at least one abiotic stress, particularly to heat, salt and osmotic stress. The plants of the invention comprise at least one DEAD-box RNA helicase encoding gene that has been disrupted such that the expression and/or activity of the DEAD-box RNA helicase protein is impaired.

Description

STRESS RESPONSE GENES USEFUL FOR GENERATING STRESS

TOLERANT PLANTS

FIELD OF THE INVENTION The present invention relates to mutated DEAD-box RNA helicases conferring increased tolerance to abiotic stresses in plants, and to transgenic plants expressing at least one mutated gene having an increased tolerance to at least one type of stress related to salinity, osmotic and heat stresses.

BACKGROUND OF THE INVENTION

As a sedentary organism, a plant's ability to adapt to abiotic stresses such as heat, cold, drought and high salinity is crucial for its survival. Global changes in the environment are leading to a significant increase in areas with environmental conditions that adversely affect plant growth and limit agricultural crop production worldwide. Minimizing these losses is a major area of concern for all countries.

Plant responses to abiotic stresses involve a complex variety of tolerance mechanisms that are activated and controlled by the expression of thousands of genes (Chen W et al., 2002 Plant Cell 14: 559-574; Kreps JA et al, 2002 Plant Physiol 130: 2129-2141; Seki M et al., 2002 Plant J 31: 279-292). These genes encode proteins involved in numerous biological processes as well as a large number of proteins of unknown function. Furthermore, the expression of many genes with regulatory functions such as transcription factors, RNA-binding proteins, calcium-binding proteins, kinases, phosphatases etc, is altered by stress. These genes are probably involved not only in regulating downstream stress responses but also in stress perception and signaling (reviewed, for example, Yamaguchi-Shinozaki K and Shinozaki K, 2006 Plant Cell Physiol 33: 217-224).

In recent years, much progress has been made in identifying and characterizing components of stress signaling networks in the model plant Arabidopsis. Promoter analyses of cold- and dehydration-responsive genes such as RD29A have revealed two cis-acting elements mediating stress-induced expression, the DRE/CRT and ABRE elements (Shinozaki K et al., 2003 Curr Opin Plant Biol 6: 410-417). The DRE/CRT element, which has the core sequence, CCGAC, is essential for regulating gene expression in response to cold and hyperosmotic stresses and this control is independent of the plant hormone abscisic acid (ABA) (Yamaguchi-Shinozaki K and Shinozaki K₅ 1994 Plant Cell 6: 251-264). Members of the DRE Binding protein/C-repeat Binding Factor (DREB/CBF) family of transcription factors specifically bind the DRE/CRT element and activate transcription of downstream stress-inducible genes in response to cold, osmotic and salt stresses (Stockinger EJ et al., 1997 Proc Natl Acad Sci USA 94: 1035-1040; Liu Q et al., 1998 Plant Cell 10: 1391-1406). Ectopic or inducible expression of DREB/CBF genes leads to enhanced expression of downstream stress- inducible genes and increased tolerance to freezing, drought and salt stresses. The cold- induced expression of at least one of the DREB/CBF genes as well as that of other cold- responsive transcription factors, is controlled by the constitutively expressed transcription factor, INDUCER OF CBF EXPRESSION 1 (ICEl), the most upstream transcription factor in the cold stress signaling subnetwork identified to date (Chinnusamy V et al., 2003 Genes and Dev 17: 1043-1054; Lee BH et al., 2005 Plant Cell 17: 3155-3175).

The ABRE cis-acting element, PyACGTGGC, controls ABA-regulated gene expression and can bind bZIP transcription factors known as ABRE-binding (AREB) proteins or ABRE-binding factors (ABFs) (Choi HI et al., 2000 J Biol Chem 275: 1723- 1730; Uno Y et al., 2000 Proc Natl Acad Sci USA 97: 11632-11637). Expression of genes encoding several of these proteins are up-regulated by ABA, drought, and high salinity and the proteins themselves can act as transcriptional activators in protoplast transient expression assays (Uno et al., 2000 ibid; Fujita Y et al., 2005 Plant Cell 17: 3470-3488). Over expression of ABF3 and AREB/ABF4 confers ABA-hypersensitive germination and seedling phenotypes, enhanced expression of ABA-regulated genes and tolerance to drought. Several other cis-elements also act in ABA-dependent expression. For instance, RD22 expression is dependent on ABA for its drought- inducible induction (Abe H et al., 1997 Plant Cell 9: 1859-1868) but contains no ABRE element in its promoter. Instead, drought-inducible RD22 expression is mediated by MYC and MYB cis-elements which can bind the AtMYC2 (RD22BP1) and AtMYB2 transcription factors, respectively (Abe H et al., 2003 Plant Cell 15: 63-78). These two transcription factors are synthesized after ABA accumulates and cooperatively activate RD22 expression

Post-transcriptional and post-translational control of stress gene expression is increasingly being recognized as playing a major role in regulating plant stress responses. For example, in unstressed Arabidopsis lines over expressing the Na⁺/H⁺ antiporter SOSl, levels of SOSl transcript are similar to wild-type levels. Only under salt stress, does SOSl transcript accumulate to high levels in the plant over expressing the antiporter, suggesting that SOSl mRNA is unstable in unstressed conditions (Shi H et al, 2003 Nat Biotechnol 21 : 81-85). Phosphorylation/dephosphorylation also appears to be important in stress responses. In ABA signaling, for instance, phosphorylation of AREB/ ABFs by ABA-activated SNFl -related protein kinases may activate these transcription factors. Control of stress-responsive gene expression at the level of protein degradation had also been demonstrated. HOSl, a negative regulator of cold responses, is a RING finger protein that has E3 ligase activity and can mediate ubiquitination of ICEl (Dong CH et al., 2006 Proc Natl Acad Sci USA 103: 8281-8286). Moreover, degradation of ICEl is induced by cold stress and requires HOSl activity.

Evidence is beginning to accumulate that small RNAs such as microRNAs and short interfering RNAs (siRNAs) may regulate gene expression in response to environmental stresses. The expression of a substantial number of small RNAs is altered in response to abiotic stress (Sunkar R and Zhu JK, 2004 Plant Cell 16: 2001-2019) and at least one siRNA is involved in regulating a salt stress response (Borsani O et al., 2005 Cell 123: 1279-1291). RNA helicases are RNA binding proteins that catalyze the unwinding of energetically stable duplex RNA secondary structures. Most RNA as well as DNA helicases are members of the DEAD-box RNA helicases superfamily, and about 50 helicases have been identified in Arabidopsis (Boudet N et al., 2001 Genome Res 11 : 2101-2114). In non-plant systems, these proteins are involved in many aspects of RNA metabolism particularly within supramolecular complexes. These processes include ribosome biogenesis, transcription, pre-mRNA splicing, mRNA export, RNA degradation, translation initiation and organellar gene expression. They are thought to function either as RNA chaperones that promote the formation of optimal RNA structure by local unwinding activity or by mediating RNA-protein association/dissociation. However, very little is known about the function of the superfamily of RNA helicases in plants, The two exceptions are the DExH-box RNA helicase, Carpel Factory/Dicer-Like 1 (DCLl) and the DEAD-box RNA helicase,

LOS4. DCLl is involved in processing micro-RNAs and has been shown to be involved in at least two stress-related mechanisms. DCLl can form a complex with the dsRNA- binding protein, HYPONASTIC LEAVES 1 (HYLl), which functions to assist DCLl in efficient and precise cleavage of pri-miRNAs (Kurihara Y et al, 2006 RNA 12: 206- 212). HYLl, itself, is also involved in ABA signaling and the Arabidopsis hyll mutant is hypersensitive to ABA and exhibits enhanced AB A-induction of downstream stress- responsive genes (Lu C and Fedoroff N, 2000 Plant Cell 12: 2351-2365). DCLl is further involved in processing of the natural antisense siRNAs derived from Al- Pyrroline-5-Carboxylate Dehydrogenase (P5CDH) and SRO5 transcripts (Borsani et al., 2005 ibid). Under salt stress, this system acts to degrade the P5CDH transcript to allow accumulation of the compatible osmolyte, proline, while SRO5 acts to counteract the increased ROS production caused by decreased P5CDH activity. The Ios4-1 mutant exhibits severely reduced cold-induction of DREBICBF expression and its target genes and is more sensitive to cold stress (Gong Z et al., 2002 Proc Natl Acad Sci USA 99: 11507-11512). In contrast, the los4-2 mutation causes enhanced cold-induced DREBl Cl CBF2 expression and its target genes and leads to plants that are more tolerant to freezing stress but more sensitive to heat stress (Gong Z et al., 2005 Plant Cell 17: 256-267). The LOS4 DEAD-box RNA helicase protein is enriched in the nuclear rim and mRNA export is blocked at low and warm temperatures in the Ios4-1 mutant but only at warm temperatures in the los4-2 mutant. The inventors of the present invention disclosed after the priority date of the present application (Kant P et al., 2007 Plant Physiol 145: 814-830) two genes encoding Arabidopsis DEAD-box RNA helicases, identified by employing a functional genomics screen, as being down-regulated by multiple abiotic stresses. Mutations in either gene caused increased tolerance to salt, osmotic and heat stresses suggesting that the helicases suppress responses to abiotic stress.

U.S. Patent Application publication No. 20020081730 discloses a new gene, HVDl from Barley that is induced by salt stress and that was revealed to encode an RNA helicase. That invention further discloses that a salt resistance would be conferred upon a plant incorporating said gene, through stabilizing conformation of RNA. The upstream stress-responsive transcription factors such as ICEl, the

DREB/CRT family, the AREB/ABFs, ATMYC2 and AtMYB2 as well as proteins mediating post-transcriptional regulation of gene expression, ultimately control the expression of many downstream stress-responsive genes involved in the response to multiple abiotic stresses (e.g. Maruyama K et al., 2004 Plant J 38: 982-983; Sakuma Y et al., 2006 Plant Cell 18: 1292-1309). However, there is a need to identify new upstream components of abiotic stress signaling networks that control plant responses to multiple abiotic stresses.

SUMMARY OF THE INVENTION

The present invention provides genes involved in the upstream regulation of multiple abiotic stress responses, and their use in producing stress-resistant or stress tolerant transgenic plants .

The present invention discloses the identification of abiotc-stress related genes from the model plant Arabidopsis. A functional genomic-based screen was performed to identify genes that may function as upstream regulators of multiple abiotic stress responses employing a microarray analysis of early Arabidopsis heat stress-responsive genes combined with analysis of data from published microarray analyses examining Arabidopsis early responses to a variety of abiotic stresses.

The present invention is based in part on the unexpected discovery of two plant mutants exhibiting increased tolerance to salt stress that contained a T-DNA insertion in genes encoding different DEAD-box RNA helicases. The present invention now shows that the wild type non-mutated genes are down regulated by salt, osmotic and heat stress, and the genes were therefore designated as STRESS RESPONSE SUPPRESSOR 1 (STRSl) and STRS2. The present invention further shows that plants expressing the mutated genes exhibit enhanced tolerance not only to salt stress but also to additional abiotic stresses, including salt, osmotic and heat stresses. Without wishing to be bound by any particular theory or mechanism of action this enhanced tolerance may be attributed to the release of the negative regulation by STRSl and STRS2 on upstream abiotic stress transcriptional regulators. The absence of functional STRSl or STRS2 proteins in plants harboring the mutated respective genes enables the expression and function of various stress-induced regulatory factors, which, in turn, induce the expression of stress-related proteins essential for the function of the plant cells under the stressful conditions and enhance the plant tolerance to the abiotic stress. Thus, according to one aspect the present invention provides a genetically modified plant wherein at least one of the plant endogenous genes encoding a DEAD- box RNA helicase protein has been disrupted. The disruption inhibits expression or activity of at least one helicase protein compared to a corresponding control unmodified plant.

According to certain embodiments, the genetically modified plant has an increased tolerance to at least one abiotic stress compared to an unmodified plant. According to one embodiment, the abiotic stress is selected from the group consisting of salt stress, heat stress and osmotic stress. According to certain embodiments, the genetically modified plant grows in a concentration of a salt that inhibits growth of a corresponding unmodified plant.

According to one embodiment, the salt concentration is from about 100 mM to about 200 mM. According to another embodiment, the salt concentration is from about 100 mM to about 150 mM. According to other embodiments, the genetically modified plant grows in a medium having an osmotic potential that inhibits growth of a corresponding unmodified plant. According to one embodiment, the genetically modified plant grows in a medium having an osmotic potential of about -0.75 MPa.

According to yet further embodiments, the genetically modified plant has an increased tolerance to heat stress compared to an unmodified plant. It is to be understood that the super-optimal temperature that will result in heat stress may vary significantly among different plant species and, according to the teachings of the present invention, is not limited to a specific range.

According to other embodiments, the gene encoding the DEAD-box helicase comprises a nucleic acid sequence as set forth in any one of SEQ ID NO:1 (STRSl; AtI g31970), SEQ ID NO:2 (STRS2; At5g08620) and a functional variant, fragment or homolog thereof. According to further embodiments, the DEAD-box helicase protein has an amino acid sequence as set forth in any one of SEQ. ID NO:3 (STRSl), SEQ ID NO. '4 (STRS2) and a functional variant, fragment or homolog thereof. Any method for gene disruption as is known in the art can be used according to the teaching of the present invention, including, but not limited to, gene insertions, gene deletions, anti-sense based disruption, point mutations and siRNA. In exemplary embodiments, the helicase encoding gene is disrupted by at least one transferred DNA (T-DNA) insertion. According to certain embodiments, the genetically modified plant comprises a DEAD-box RNA helicase encoding gene disrupted by a DNA insertion in a position selected from the group consisting of the gene promoter, first exon, 6^th exon, and the 9^th exon.

The present invention also encompasses seeds of the genetically modified plants, wherein plants grown from the seeds has at least one disrupted DEAD-box RNA helicase encoding gene. According to certain embodiments, the plants grown from said seeds have an increased tolerance to at least one abiotic stress compared to plants grown from seeds of unmodified plants. The present invention further encompasses fruit, leaves or any part of the genetically modified plant, as well as tissue cultures derived thereof and plants regenerated therefrom.

The genetically modified plants of the present invention can be either homozygous or heterozygous to the disrupted gene. When the plant cell comprises more than one disrupted stress response suppressor (STRS) gene, the plant can be independently homozygous or heterozygous for each gene.

The plant of the invention can be a dicot or a monocot. According to certain embodiments, the plant of the invention is an inbred open line. According to other embodiments, the plant is a hybrid.

The present invention is further directed to isolated polynucleotides encoding STRS genes, and their use for detecting plants susceptible to abiotic stresses, and furthermore, as a means for producing genetically modified plants having increased tolerance to at least one such stress, particularly salt, heat and osmotic stress. According to another aspect the present invention provides an isolated polynucleotide encoding a DEAD-box RNA helicase protein having a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a variant, a fragment and a homolog thereof.

According to one embodiment, the isolated polynucleotide comprises a polynucleotide having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a sequence complementary thereto and variants, fragments and homologues thereof. The polynucleotides of the invention can comprise or be contained within a DNA construct or a vector (e.g., a viral vector). The DNA construct or vector can comprise a promoter (e.g., a constitutive, tissue-specific, or inducible promoter) operably linked to the polynucleotide. A polynucleotide of the invention can be linked to the promoter in an anti-sense orientation or a sense orientation, be configured for RNA silencing or interference, and the like.

According to a further aspect, the present invention provides a method of producing a genetically modified plant having an increased tolerance to at least one abiotic stress comprising (a) disrupting in at least one plant cell at least one endogenous DEAD-box RNA helicase encoding gene, wherein the disruption inhibits the expression or activity of at least one DEAD-box RNA helicase protein; and (b) regenerating the plant cell into a plant having an increased tolerance to at least one abiotic stress.

According to certain embodiments, the endogenous DEAD-box RNA helicase protein has an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and an active fragment, variant or homolog thereof.

According to other embodiments, the gene encoding the endogenous DEAD-box RNA helicase comprises a nucleic acid sequence as set forth in any one of SEQ ID NO:1, SEQ ID NO:2, and a functional variant, fragment or homolog thereof.

According to certain embodiment, the disruption step comprises introducing at least one mutation into the sequence of the at least one helicase-encoding gene.

According to one embodiment, the at least one mutation is a point mutation. According to another embodiment, the mutation is a deletion of at least two contiguous nucleic acids.

According to other embodiments, the disruption step comprises transforming at least one plant cell with at least one exogenous polynucleotide targeted to disrupt the at least one gene encoding the DEAD-box RNA helicase.

According to other embodiments, the polynucleotide targeted to the at least one helicase gene has a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1-2, a variant, fragment, or homolog thereof, or complementary sequence thereto. According to one embodiment, the polynucleotide is configured for RNA silencing or interference.

According to another embodiment, the polynucleotide is configured in an anti-sense configuration. According to further embodiments, the polynucleotide is a transposable element. According to further embodiments, the polynucleotide is T-DNA configured for insertion into the helicase gene.

Transformation of plants with a polynucleotide or a DNA construct may be performed by various means, as is known to one skilled in the art. Common methods are exemplified by, but are not restricted to, Agrobacterium-mediated transformation, microprojectile bombardment, pollen-mediated transfer, plant RNA virus-mediated transformation, liposome-mediated transformation, direct gene transfer (e.g. by microinjection) and electroporation of compact embryogenic calli.

Genetically modified plants comprising the polynucleotide or DNA construct of the present invention may be selected employing standard methods of molecular genetics, as are known to a person of ordinary skill in the art. Alternatively, the genetically modified plants are selected based on their tolerance to at least one stress.

According to another aspect the present invention relates to the genetically modified plants generated by the methods of the present invention as well as to their seeds, fruits, roots and other organs or isolated parts thereof.

It is to be understood explicitly that the scope of the present invention encompasses homologs, analogs, variants and derivatives, including shorter and longer sequences of the polynucleotide and proteins of the present invention. It is also to be understood that the methods of disrupting the genes of the present invention are brought as examples only and are not be construed as limiting.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a schematic description of the Arαbidopsis STRS 1 and STRS2 and the disruption by T-DNA insertion mutants. FIG. IA: Alignment of conserved motifs specific to DEAD-box helicases that are present in STRSl and STRS2 with the consensus sequences of the DEAD-box family. Numbers in parenthesis represent the amino acid position of the first residue in each motif. For the consensus sequences, capital letters denote amino acids that are conserved at least 80% while lower case letters denote amino acids that are conserved 50 -79%. FIG. IB: Scheme of the STRSl and STRS2 genes. Solid boxes represent exons and lines symbolize introns. The position and orientation of the T-DNA insertion is depicted (not to scale). LB, left border sequence; RB, right border sequence. FIG. 1C and FIG. ID, Real-time PCR analysis of STRSl and STRS2 expression, respectively, in wild-type and two independent T-DNA insertion mutants for each gene. Relative transcript levels were determined by real-time PCR according to the 2^~ΔΔC _T method using UBIQUITIN 10 (JJBQlO) as an internal control. Gene expression was normalized to the wild-type expression level, which was assigned a value of one. Data represent the average of three independent experiments ± SD. Upstream, RT-PCR carried out with primers complementary to sequences upstream of the T-DNA insertion; Downstream, RT-PCR carried out with primers complementary to sequences downstream of the T-DNA insertion; N.D, Not detectable.

FIG. 2 demonstrates the altered salt and osmotic stress tolerance of the strsl and strs2 mutants. FIG. 2A: Increased tolerance to salt stress. Seeds were germinated and grown on MS plates with and without 125 mM NaCl. Photographs were taken on the 10th day after stratification. WT, wild-type. FIG. 2B: Percentage of germination of wild-type and two independent alleles of strsl and strs2 on MS plates with and without 125 mM NaCl. Data are mean ± SD (n=4). Fisher's protected least significant difference (LSD) test showed no significant difference in germination percent of WT and mutants without NaCl. With NaCl, strsl, strsl, strsla, and strs2a exhibited significantly higher germination percent than WT (P<0.05). FIG. 2C: Fresh weight of wild type and two independent alleles of strsl and strs2, 10 days after stratification on MS plates with and without 125 mM NaCl. Data are mean ± SD (n=4). FW, fresh weight. Bars with different letters indicate significant difference at P<0.05 (Fisher's protected LSD test). FIG. 2D: Increased tolerance to osmotic stress. Seeds were germinated and grown on MS plates with and without 300 mM mannitol. Photographs were taken at the 10th day after stratification. FIG.2E: Percentage of germination of wild-type and two independent alleles of strsl and strs2 on MS plates with and without 300 mM mannitol. Data are mean ± SD (n=4). Fisher's protected LSD test showed no significant difference in germination percent between WT and mutants without mannitol. With mannitol, strsl and strs2 exhibited significantly higher germination than WT at 4 and 5 days after stratification, strsla and strs2a exhibited significantly higher germination percent than WT at 4, 5 and 6 days after stratification (P<0.05). FIG. 2F: Fresh weight of wild type and two independent alleles of strsl and strs2, 10 days after stratification on MS plates with and without 300 mM mannitol. Data are mean ± SD (n=4). Bars with different letters indicate significant difference at P<0.05 (Fisher's protected LSD test).

FIG. 3 S hows altered basal and acquired thermotolerance of strsl and strs2 mutants. FIG. 3A: Basal thermotolerance. Stratified seeds sown on MS plates were exposed to 45⁰C for 3 h and then allowed to germinate and grow at 22⁰C. A representative plate is shown six days after transfer to 22⁰C. WT, wild-type; hotl-3, a mutant of HSPlOl that is defective in basal and acquired thermotolerance. FIG. 3B: Quantification of basal thermotolerance by percentage of germination of seeds treated at 45⁰C for 3 h. The results from two independent alleles of strsl and strs2 are shown. Data are mean ± SD (n=3). Fisher's protected LSD test showed that all strs mutant lines exhibited a significantly higher germination percent compared to WT and hotl-3 (P<0.05). FIG. 3C: Quantification of basal thermotolerance by hypocotyl elongation assay using two independent alleles of strsl and strs2. Seeds were treated at 45⁰C for the indicated time periods and allowed to germinate in the dark on vertical plates. Hypocotyl length was measured 6 days after transfer to 22⁰C. Data are mean ± SD (n=4). Each replicate consisted of approximately 20 seedlings. Bars with different letters indicate significant difference at P<0.05 (Fisher's protected LSD test). FIG. 3D: Acquired thermotolerance. Seedlings were grown on vertical plates in the dark for 3 days. Con: Control seedlings maintained at 22⁰C; PT: Pre-treatment of 38⁰C for 90 min; HS: Heat stress of 45 ⁰C for 2 h; PT + 2: Pre-treatment followed by 2 h at 22⁰C and then 45⁰C for 2 h; PT + 3: Pre-treatment followed by 2 h at 22⁰C and then 45⁰C for 3 h. After heat treatment, seedlings were grown for a further 3 days before measurement of the post-stress increase in hypocotyl length. Data are mean ± SD (n=4). Each replicate consisted of approximately 15 to 20 seedlings. Bars with different letters indicate significant difference at P<0.05 (Fisher's protected LSD test).

FIG. 4 demonstrates expression of stress-responsive genes in wild-type and strs mutant plants subjected to salt, drought and cold treatments. Two week-old soil-grown plants were exposed to various stress treatments. Relative transcript levels were determined by real-time PCR according to the 2^~ΔΔ T method using UBQlO as an internal control. Gene expression was normalized to the wild-type unstressed expression level, which was assigned a value of one. Data represent the average of three independent experiments ± SD. FIG 4A₃ 4D, 4G, 4J₃ 4L, 4N and 4P: Salt treatment, 200 mM NaCl.

FIG. 4B, 4E₃ 4H₃ 4K₃ 4M, 40 and 4Q: Drought treatment, plants were removed from the soil and allowed to dry under 60% humidity. FIG 4C, 4F, 41 and 4R: Cold treatment, 4⁰C.

FIG. 5 demonstrates expression of heat-stress-responsive genes in wild-type and strsl and strs2 mutants. FIG. 5 A, 5B, 5C and 5D: Two week-old plants were exposed to 4O⁰C for the indicated time periods. Relative transcript levels were determined by real-time PCR according to the 2^~ΔΔCχ method using UBQlO as an internal control. Gene expression was normalized to the wild-type unstressed expression level, which was assigned a value of one. Data represent the average of three independent experiments ± SD. FIG. 6 shows ABA-responsive gene expression and ABA sensitivity in wild-type and strsl and strs2 mutants. Seedlings were grown on vertical MS plates for 4 days after germination and then transferred to fresh treatment plates. Relative transcript levels were determined by real-time PCR according to the 2^~ΔΔCχ method using UBQlO as an internal control. Gene expression was normalized to the wild-type control expression level, which was assigned a value of one. Data represent the average of four independent experiments ± SD (n=4). FIG. 6A: Expression of RD26 in wild-type and strs mutant seedlings transferred to MS plates with 100 μM ABA. FIG. 6B: Expression of RD26 in wild-type and strs mutant seedlings transferred to MS plates without ABA. FIG. 6C: Expression of STRSl and STRS2 in wild-type seedlings transferred to MS plates with 100 μM ABA. FIG. 6D: Expression of STRSl and STRS2 in wild-type seedlings transferred to MS plates with 300 mM NaCl. FIG. 6E: Expression of STRSl and STRS2 in aba2-l, ABA-deficient, mutant seedlings transferred to MS plates with 300 mM NaCl. FIG. 6F: Percent germination of wild-type and strsl and strs2 mutant seedlings after 6 days incubation on MS media containing different concentrations of ABA. Data are mean ± SD (n=3). Fisher's protected LSD test showed that all strs mutant lines exhibited a significantly higher germination percent than WT upon exposure to ABA (P≤O.01).

FIG. 7 shows stress-responsive and circadian clock-controlled expression of STRSl and STRS2. Two week-old wild-type soil-grown plants were exposed to various stress treatments. Relative transcript levels were determined by real-time PCR according to the 2^~AΔCτ method using UBQlO as an internal control. Expression was normalized to unstressed expression level of the respective gene, which was assigned a value of one. Data represent the average of three independent experiments ± SD. FIG. 7A: Salt treatment, 200 niM NaCl. FIG. 7B: Drought treatment, plants were removed from the soil and allowed to dry under 60% humidity. FIG. 7C: Heat treatment, 4O⁰C. FIG. 7D: Cold treatment, 4⁰C. FIG. 7E: Circadian clock-control. Seven-day old wild-type seedlings were entrained in a 12 h light/12 h dark photoperiod for 4 days and then released into continuous light. Data are representative of similar results from two independent experiments. Light and dark shaded bars represent subjective day and subjective night, respectively.

DETAILED DESCRIPTION OF THE INVENTION

In nature as well as in open agricultural fields, plants are exposed to various environmental stresses including dehydration, high temperature, low temperature or salt. As a result, plants have developed a complex variety of tolerance mechanisms, elucidation of which may enable production of desirable plant species having increased tolerance to stress.

The present invention discloses two genes encoding Arabidopsis DEAD-box RNA helicases as being down-regulated by multiple abiotic stresses. Mutations in either gene caused increased tolerance to salt, osmotic and heat stresses suggesting that the helicases suppress responses to abiotic stress. The genes were therefore designated STRESS RESPONSE SUPPRESSOR (STRS) 1 and STRS2. STRS 1 and STRS2 attenuate the expression of stress-responsive transcriptional activators and function in ABA- dependent and ABA-independent abiotic stress signaling networks.

The present invention provides genetically modified plants in which the expression or activity of the STRESS RESPONSE SUPPRESSOR proteins is inhibited such that the genetically modified plants have increased tolerance to abiotic stresses compared to unmodified plants in which the suppressors are active. The present invention further provides nucleic acids encoding the stress response suppressors, constructs and vectors comprising same, and to a method of producing a genetically modified plant having an increased tolerance to abiotic stress by disrupting at least one endogenous gene encoding a DEAD-box RNA helicase. Also provided by the present invention are plants produced by the methods of the present invention, and plant seeds and progeny obtained from the genetically modified plants. The present invention makes a significant contribution to the art by disclosing the function of DEAD-box RNA helicases as negative regulators of multiple abiotic stress responses. Furthermore, the present invention discloses that these negative regulators act as upstream regulators, affecting the expression of transcription elements necessary for activating the plant response to the induced stress. Thus, the present invention provides means capable of attenuating plant response to abiotic stress at a very early stage of the response.

Before describing the invention in detail, it is to be understood that this invention is not limited to particular devices or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

Definitions

The term "plant" as used herein refers generically to any of whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat), fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like), tissue culture callus, and plant cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. It also refers to a plurality of plant cells that are largely differentiated into one of the above-defined structures or tissues that is present at any stage of a plant's development. The term also includes, but is not limited to, any species of woody, herbaceous, perennial or annual plant. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues.

As used herein, the term "dicot" refers to a dicotyledonous plant. Dicotyledonous plants belong to large subclass of Angiosperms that have two seed-leaves (cotyledon).

As used herein, the term "monocot" refers to a monocotyledonous plant, which in the developing plant has only one cotyledon.

As used herein, the term "abiotic stress-inducible" or "abiotic stress-responsive" refers to a protein or gene which is influenced by an altered environmental condition including salt, water and temperature. For example, a salt stress-inducible or salt stress- responsive gene or protein may be over-expressed or its expression may be inhibited as a result of a rise in salt concentration. Alternatively, the enzymatic activity of a stress- inducible or stress-responsive protein may be altered as a response to a rise or fall in the environment temperature.

As used herein, the term "salt concentration" refers particularly to "NaCl concentration". However, it is to be understood that the teachings of present invention encompasses any equivalent salt that may be present in a plant growth medium, including, for example, KCl, and CaCl₂.

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA or a polypeptide. A polypeptide can be encoded by a full-length coding sequence or by any part thereof. The term "parts thereof when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleic acid sequence comprising at least a part of a gene" may comprise fragments of the gene or the entire gene.

The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "RNA helicase" as used herein refers to RNA-binding proteins that catalyze the unwinding of energetically stable duplex RNA secondary structures. "DEAD-box RNA helicase" refers to those enzymes containing the contiguous amino acid sequence D-E-A-D (Asp-Glu-Ala-Asp). As used herein, the term STRESS RESPONSE SUPPRESSOR (STRS) refers to a DEAD-box RNA helicase, particularly to an RNA helicase encoded by a polynucleotide having a nucleic acids sequence as set forth in any one of SEQ ID NO:1 (STRSl; gi 30692626) and SEQ ID NO:2 (STRS2; gi 42567744). As used herein, the terms "upstream regulation" or "downstream regulation" refers in general to the hierarchy of gene expression in response to external signaling, particularly to gene expression in the stress signaling pathway. As used herein, the term

"upstream regulation" refers to the regulation of stress-responsive genes by genes upstream in the stress signaling pathways.

The term "nucleic acid" as used herein refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.

An "isolated" nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid

(i.e., sequences encoding other polypeptides). Preferably, an "isolated" nucleic acid is free of some of the sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in its naturally occurring replicon. For example, a cloned nucleic acid is considered isolated. A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection. Moreover, an

"isolated" nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

The term "sub-sequence" or "fragment" is any portion of an entire sequence.

A polynucleotide sequence is said to "encode" a sense or antisense RNA molecule, or RNA silencing or interference molecule or a polypeptide, if the polynucleotide sequence can be transcribed (in spliced or unspliced form) and/or translated into the RNA or polypeptide, or a sub-sequence thereof.

The terms "expression of a gene" or "expression of a nucleic acid" as used herein means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent modification of the polypeptide, e.g., posttranslational modification), or both transcription and translation, as indicated by the context.

The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

The term "vector" as used herein encompasses both expression and transformation vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources. In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter and an enhancer that control or influence the transcription of the gene, a nucleic acid or nucleic acid fragment according to the present invention and a terminator that directs the termination of transcription; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked. By "operatively linked" is meant that said regulatory elements are capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment. The terms "promoter element," "promoter," or "promoter sequence" as used herein, refer to a DNA sequence that is located at the 5' end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.

The term "genetically modified" when used in reference to a plant or seed (i.e., a

"genetically modified plant" or a "genetically modified seed") refers to a plant or seed that contains at least one heterologous polynucleotide in one or more of its cells. The term "genetically modified plant material" refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous polynucleotide in at least one of its cells.

The terms "transformants" or "transformed cells" include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.

Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more exogenous polynucleotides into a cell in the absence of integration of the exogenous polynucleotide into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the exogenous polynucleotides. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g. β-glucuronidase) encoded by the exogenous polynucleotide. The term "transient transformant" refers to a cell which has transiently incorporated one or more exogenous polynucleotides. In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration of one or more exogenous polynucleotides into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the exogenous polynucleotides. Alternatively, stable transformation of a cell may also be detected by enzyme activity of an integrated gene in growing tissue or by the polymerase chain reaction of genomic DNA of the cell to amplify exogenous polynucleotide sequences. The term "stable transformant" refers to a cell which has stably integrated one or more exogenous polynucleotides into the genomic or organellar DNA. It is to be understood that a plant or a plant cell transformed with the nucleic acids, constructs and/or vectors of the present invention can be transiently as well as stably transformed.

The terms "in vitro growth" or "grown in vitro" as used herein refer to regeneration and/or growth of plant material in tissue culture. Specifically, according to the present invention, a transformed plant cell or tissue is placed in a sterile, (usually gel-based) nutrient medium, supplemented with the adequate additives to induce differentiation and plantlet growth. The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "endogenous" as used herein relates to any gene or nucleic acid sequence that is already present in a cell.

The term "transposable element" (TE) or "transposable genetic element" refers to a DNA sequence that can move from one location to another in a cell. Movement of a transposable element can occur from episome to episome, from episome to chromosome, from chromosome to chromosome, or from chromosome to episome. Transposable elements are characterized by the presence of inverted repeat sequences at their termini. Mobilization is mediated enzymatically by a "transposase." Structurally, a transposable element is categorized as a "transposon," ("TN") or an "insertion sequence element," (IS element) based on the presence or absence, respectively, of genetic sequences in addition to those necessary for mobilization of the element. A mini- transposon or mini-IS element typically lacks sequences encoding a transposase.

According to one aspect, the present invention provides a genetically modified plant wherein at least one of the plant endogenous genes encoding DEAD-box RNA helicase protein has been disrupted. The disruption inhibits expression or activity of at least one helicase protein compared to a corresponding control unmodified plant.

According to other embodiments, the gene encoding the DEAD-box helicase comprises a nucleic acid sequence as set forth in any one of SEQ ID NO:1 and SEQ ID NO:2. A functional genomics-based screen was taken as a method of choice to identify genes that may function as upstream regulators of multiple abiotic stress responses. A microarray analysis of early Arabidopsis heat stress-responsive genes was performed and the resulting data was combined in a "stress gene" database with data from published microarray analyses examining Arabidopsis responses to a variety of abiotic stresses. The database was queried for a set of regulatory genes whose expression was affected early by multiple abiotic stresses and Arabidopsis T-DNA insertion mutants defective in each gene were screened for altered sensitivity to abiotic stresses. A preliminary screen of mutants homozygous for the T-DNA insertion identified two mutants exhibiting increased tolerance to salt stress that contained a T-DNA insertion in genes encoding different DEAD-box RNA helicases (Figure IA and B). Analysis of microarray data in the above-described database indicated that these genes are down- regulated by salt, osmotic and heat stress. The two proteins were designated as STRESS RESPONSE SUPPRESSOR 1 (STRSl) and STRS2.

The STRSl gene is predicted to encode a protein of 537 amino acid residues with an estimated molecular mass of 59.5 kDA while the STRS2 gene is predicted to be a protein of 563 amino acids with an estimated molecular mass of 62.5 kDA. Databases searches revealed that both proteins possess all nine conserved motifs that are characteristic of the DEAD-box protein family as well as an upstream conserved phenylalanine (Figure IA; de Ia Cruz J et al, 1999 Trends Biochem Sci 24: 192-198; Rocak S and Linder P, 2004 Nat Rev MoI Cell Biol 5: 232-241). It has been estimated that the Arabidopsis genome encodes over 50 DEAD-box RNA helicases (Boudet et al., 2001 ibid), 32 of which were classified as AtRHl to AtRH32. Genomic and cDNA sequence analysis indicated that STRSl (SEQ ID NO:3) is identical to AtRH5 while STRS2 (SEQ ID NO:4) is identical to AtRH25. The N- and C-terminal extensions of DEAD-box RNA helicases are of variable length and it is hypothesized they confer substrate specificity. Counting upstream from the conserved phenylalanine and downstream from motif VI for N-termini and C-termini, respectively, STRSl and STRS2 have N-terminal regions of 117 and 81 amino acids and C-terminal regions of 76 and 130 amino acids, respectively. Alignment of STRSl and STRS2 protein sequences (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) showed that the two proteins exhibit 32% sequence identity and 49% sequence similarity. However, all the sequence identity/similarity resides in the core helicase region of the protein containing the conserved motifs whereas there is no significant sequence similarity between the N- terminal and C-terminal regions. This identity/similarity assignment of the proteins reveals hitherto unknown aspects of the function of these DEAD-box RNA helicases and present novel tools and concepts for enhancing abiotic stress tolerance in plants. In non-plant systems, DEAD-box RNA helicase proteins are involved in many aspects of RNA metabolism particularly within supramolecular complexes. These processes include ribosome biogenesis, transcription, pre-mRNA splicing, mRNA export, RNA degradation, translation initiation and organellar gene expression. They are thought to function either as RNA chaperones that promote the formation of optimal RNA structure by local unwinding activity or by mediating RNA-protein association/dissociation. However, very little is known about the function of the superfamily of RNA helicases in plants. The two exceptions are the DExH-box RNA helicase, Carpel Factory/Dicer-Like 1 (DCLl) and the DEAD-box RNA helicase, L0S4. DCLl is involved in processing micro-RNAs (Park W et al., 2002 Curr Biol 12: 1484-1495; Reinhart BJ et al., 2002 Genes Dev 16: 1616-1626) and has been shown to be involved in at least two stress-related mechanisms. DCLl can form a complex with the dsRNA-binding protein, HYPONASTIC LEAVES 1 (HYLl), which functions to assist DCLl in efficient and precise cleavage of pri-miRNAs (Kurihara Y et al., 2006 ibid). HYLl, itself, is also involved in ABA signaling and the Arabidopsis hyll mutant is hypersensitive to ABA and exhibits enhanced ABA-induction of downstream stress- responsive genes. DCLl is further involved in processing of the natural antisense siRNAs derived from Δl-Pyrroline-5-Carboxylate Dehydrogenase (P5CDH) and SRO5 transcripts (Borsani O et al., 2005 ibid). Under salt stress, this system acts to degrade the P 5CDH transcript to allow accumulation of the compatible osmolyte, proline, while SRO5 acts to counteract the increased ROS production caused by decreased P5CDH activity.

The Ios4-1 mutant exhibits severely reduced cold-induction of DREBICBF expression and its target genes and is more sensitive to cold stress (Gong Z et al., 2002 ibid). In contrast, the los4-2 mutation causes enhanced cold-induced DREBl C/CBF2 expression and its target genes and leads to plants that are more tolerant to freezing stress but more sensitive to heat stress (Gong Z et al., 2005 ibid). The LOS4 DEAD-box

RNA helicase protein is enriched in the nuclear rim and mRNA export is blocked at low and warm temperatures in the Ios4-1 mutant but only at warm temperatures in the Io s4-2 mutant.

The present invention now discloses the direct involvement of the STRSl and STRS2 DEAD-box RNA helicases in upstream inhibition of stress responsive genes. As described in greater details in the Example section hereinbelow, strsl and strs2 mutants showed higher expression of the well characterized stress-responsive gene RD29A. Furthermore, the present invention now shows that the mutants enhance the expression of transcription factors of the DREB family that mediates stress-responsive expression

OΪRD29A via DRE elements in the RD29A promoter. Without wishing to be bound by any specific mechanism or mode of action, it is proposed that STRSl and STRS2 are involved in degrading the stress-induced mRNAs, since the abundance of the STRS transcripts decreases as the abundance of the stress marker transcripts increases. If the STRS proteins themselves are short-lived and their abundance parallels that of their transcripts then a decline in STRS protein would allow accumulation of the stress-induced transcripts. Nevertheless, STRSl and STRS2 may directly affect transcription, pre-mRNA processing, mRNA stability or other aspects of stress transcription factor RNA metabolism. Alternatively, they may function by regulating transcripts of enhancers or repressors of stress transcription factors. The virtually identical phenotypes of the strsl and strs2 mutants plus the close pattern of expression of the two genes in response to the various stresses suggest that STRSl and STRS2 may function together in a complex. It should also be noted that although the sirs mutant phenotypes did not result from a perturbation of general gene expression, the STRS proteins may have additional functions to those in abiotic stress responses. This premise is supported by the observation that the strs mutants have a slightly early flowering phenotype at least under long day (16 h light: 8 h dark) conditions and that the highest expression of both genes was detected in flowers as exemplified hereinbelow.

According to certain embodiments, the genetically modified plants of the present invention have an increased tolerance to at least one abiotic stress selected from the group consisting of osmotic, salt and heat stress.

STRS 1 and STRS2 are two of several negative regulators of stress responses that have been identified in recent years. These include HOSl, HOS5, FIERYl and FIER Y2. The hosl mutation leads to enhanced cold-induction of DREBICBF expression and downstream genes but has no effect on ABA, salt or osmotic stress-mediated gene expression. On the other hand, the hos5-l mutation enhances osmotic, salt and ABA stress-induced gene expression but not cold-induced gene expression, similar to the strsl and strs2 mutations.

Attenuation of the stress signaling networks by negative regulators is thus clearly important for proper regulation of the response to abiotic stresses. Constitutive activation of the stress response by ectopic expression of DREB1AICBF3 leads to severe growth retardation in unstressed plants. Only when DREB 'IAi ¹CBF 3 expression is driven by the stress-inducible RD29A promoter is growth retardation greatly reduced. This suggests that attenuators are necessary to prevent over-activation of the stress response. The present invention shows that peak circadian expression of STRSl and STRS2 coincides with peak expression of DREB1A/CBF3 (Figure 7E) which show a similar expression pattern to other downstream stress genes (Harmer SL et al., 2000 Science 290: 2110-2113). Without wishing to be bound by any specific mechanism or model, it is proposed that co-expression of genes encoding stress-transcriptional regulators such as the DREB/CBFs with genes encoding proteins such as STRS proteins, that attenuate expression or activity of those transcription factors might also ensure that transient stress conditions as occur in the field, do not lead to full activation of the stress response machinery. The present invention now shows that the stress- mediated change in stress gene transcript abundance is itself transient (Figure 4). The fact that the stress-mediated transient decline in STRSl and STRS2 expression closely parallels that of the transient activation of stress transcriptional activators and their downstream targets (e.g. as is shown in Figure 7 A in comparison with Figure 4A and 4G) suggests that STRSl and STRS2 in wild type plants are involved in the regulation of transient responses to abiotic stresses.

According to certain embodiments, the genetically modified plant has an increased tolerance to at least one abiotic stress compared to an unmodified plant. According to one embodiment, the abiotic stress is selected from the group consisting of salt stress, heat stress and osmotic stress.

It has been previously suggested that the upstream regulation of salt and osmotic stresses is distinct from the regulation of cold stress. For example, the DREBl proteins and ICEl, the upstream regulators of DREB IAl CBF3 expression, are mainly involved in regulating cold-induced gene expression while DREB2A and DREB2B control salt- and drought-induced gene expression. Overexpression of constitutively active DREB2A leads to increased tolerance of Arabidopsis to drought stresses but only slight tolerance to freezing. However, inducible expression or overexpression of DREBl AICBF3 leads to increased tolerance to cold, salt and drought stresses. The strs mutants of the present invention exhibit tolerance to salt and osmotic stresses (Figure 2). The mutants do not appear to be tolerant to freezing stress when tolerance was examined on detached leaves. STRSl and STRS2 expression is unaffected by cold stress whereas it is down-regulated by salt and drought stresses (Fig. 7). However, enhanced cold-induced gene expression was observed in the strs mutants (Figure 4C₅ 4F and 41) suggesting that mutant plants might indeed show tolerance to cold stress under certain conditions. The strs mutants of the present invention also show increased basal and acquired tolerance to heat (Figure 3) and enhanced expression of the heat shock factor HSPlOl, HSF4 and HSF7 genes (Figure 5). Thus, the present invention provides further evidence for the interactions between heat, salt and osmotic signaling sub-networks. Analysis of the expression of all 21 Arabidopsis HSF genes has demonstrated that many HSFs are induced by multiple abiotic stresses, again illustrating the links between the signaling sub-networks (Miller G and Mittler R, 2006 Ann Bot 98: 279-288). However, few signaling components that may function as nodes linking the heat signaling subnetwork and other abiotic stress signaling subnetworks have been identified. The present invention now shows that (i) STRSl and STRS2 attenuate expression of upstream transcription factors involved in regulation of drought, salt and heat stress-responsive gene expression and (ii) STRSl and STRS2 may function as nodes linking the salt, drought and heat stress signaling sub-networks. Another recently discovered potential node linking heat stress signaling with other abiotic stress signaling subnetworks is the transcriptional activator, MULTI-PROTEIN BRIDGING FACTORIc (MBFIc). Constitutive expression of MBFIc enhances the tolerance of transgenic Arabidopsis plants to heat, salinity and osmotic stresses and causes enhanced accumulation of stress- related transcripts (Suzuki N et al, 2005 Plant Physiol 139: 1313-1322). MBFIc does not appear to be involved in cold responses.

As described in the Example section hereinbelow, STRSl and STRS2 regulate both ABA-dependent and ABA-independent stress signaling sub-networks and their expression is down-regulated by ABA (Figure 6). It has been previously shown that

ABA biosynthesis and signaling mutants are defective in acquired thermotolerance and that addition of exogenous ABA protects Arabidopsis plants from heat-induced oxidative damage. Since ABA is also involved in regulating stress responses to osmotic and salinity stress, these data provide additional evidence of the connection between heat stress responses and responses to other abiotic stresses. ABA signaling may link

STRS control of heat, salt and osmotic stress responses. However, ABA does not appear to be involved in the induction of HSP expression. Thus heat stress-mediated down- regulation of the STRS genes via ABA would, alone, not be sufficient to induce expression of HSPs. This is supported by the finding that absence of functional STRSs is, in itself, insufficient to enhance expression of heat stress genes in unstressed strs mutant plants; positive stress-mediated signals are also required. In summary, the present invention discloses that STRSl and STRS2 are negative regulators of ABA-dependent and ABA-independent upstream abiotic stress transcriptional activators. The present invention further discloses that mutants harboring at least one T-DNA insertion in these genes are more tolerance to heat, salt and osmotic stresses. Consequently, disruption of these genes in plant hosts could mimic the protective effect of the mutants on abiotic stresses, particularly heat, salt and osmotic stress.

Although the invention is demonstrated with reference to the specific genes isolated from the species Arabidopsis and the polypeptide products thereof, it is apparent to a person of skill in the art that STRSl and STRS2 encoding genes isolated from other plant species are also encompassed within the scope of the present invention. It is also apparent to a person skilled in the art that various species and ecotypes of Arabidopsis can be used to obtain the STRSl and STRS2 genes and gene mutants. According to certain embodiments, the genes are isolated from Arabidopsis thaliana.

Polynucleotides of the Invention The present invention features the identification of DEAD-box RNA helicases, their nucleic acid sequences and encoded proteins, and their association with abiotic stress responses. Attenuating the expression of these genes in genetically modified plants enhances the plant's tolerance to various stresses, including heat, water and salt stress. According to certain aspects, the present invention provides an isolated polynucleotide encoding a STRESS RESPONSE SUPPRESSOR selected from the group consisting of Arabidopsis STRSl, STRS2, a variant, a fragment and a homolog thereof.

According to one embodiment, the present invention provides an isolated polynucleotide encoding a stress response suppressor having an amino acid sequence as set forth in any one of SEQ ID NO:3, SEQ ID NO:4, a fragment, a variant, and a homolog thereof.

According to one embodiment, the present invention provides an isolated polynucleotide having a nucleic acid sequence as set forth in any one of SEQ ID NO:1 (STRSl) and SEQ ID NO:2 (STRS2), a fragment, a variant and a homolog thereof. According to other embodiments, the present invention provides STRSl or STRS2 sequences or fragments thereof configured for RNA production, including, but not limited to, mRNA, antisense RNA, sense RNA and RNA silencing and interference configuration.

According to yet other embodiments, the present invention provides an isolated polynucleotide which is a variant of any one of SEQ ID NO:1 and SEQ ID NO:2 and fragments thereof. As used herein, the term "variant" with regard to polynucleotides refers to polynucleotides having nucleic acid sequences that are substantially similar or substantially identical to any one of SEQ ID NO:1, SEQ ID NO:2 and fragments thereof. As used herein, "substantially similar" or "substantially identical" refers to a particular nucleic acid sequence comprising at least one mutation. According to certain embodiments, the mutation includes, but is not limited to, to insertion, deletion and substitution relative to any one of SEQ ID NO:1, SEQ ID NO: 2 and fragments thereof. The present invention further encompasses polynucleotides having a nucleic acid sequence complementary to any one the above-described nucleic acid sequences. In exemplary embodiments, the polynucleotide variants of the present invention comprise at least one T-DNA insertions.

According to another aspect the present invention provides a construct comprising a polynucleotide encoding a STRESS RESPONSE SUPPRESSOR protein having an amino acid sequence as set forth in any one of SEQ ID NO:3, SEQ ID NO:4, a fragment, a variant, and a homolog thereof.

According to one embodiment, the construct of the invention comprises a polynucleotide having a nucleic acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO. "2, a variant, a fragment and a homolog thereof or a construct comprising same. According to a further aspect the present invention provides to a vector comprising a polynucleotide having a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1-2, a variant, a fragment and a homolog thereof.

Preferably the vector is a plant transformation vector. In addition, the vector preferably further includes a promoter and a terminator, wherein the promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked. The vector may be of any suitable type and may be viral or non- viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacteriwn tumefaciens, derivatives of the Ti plasmid from Agrobacteriwn rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable, integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors according to the teachings of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include tissue specificity of the vector, constitutive or inducible expression and the nature of the plant cell to be transformed (e.g. monocot or dicot). Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter and derivatives thereof, the maize UBIQ UITIN promoter, and the rice ACTIN promoter.

A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or from a different gene.

The genetic construct according to the teachings of the present invention can further comprise a reporter gene or a selection marker that is effective in the target plant cells to permit the detection of genetically modified cells, tissues or plants containing the genetic construct. Such selection markers and reporter genes, which are well known in the art, typically confer resistance to one or more toxins or encode for a detectable enzymatic activity, respectively. The nptll gene, whose expression results in resistance to kanamycin or hygromycin antibiotics, which are generally toxic to plant cells at a moderate concentration, can be used as a selection marker. Alternatively, the presence of the desired construct in transgenic cells may be determined by means of other techniques that are well known in the art, including PCR, Southern and Western blots.

Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites. Methods of producing plants with enhanced tolerance to abiotic stresses

According to a further aspect, the present invention provides a method of producing a genetically modified plant having an increased tolerance to at least one abiotic stress comprising disrupting at least one endogenous DEAD-box RNA helicase encoding gene, wherein the disruption inhibits the expression or activity of at least one DEAD-box RNA helicase protein.

Disrupting one or more STRS genes can be performed by any method as is known to a person skilled in the art.

Antisense, Sense, RNA Silencing or Interference Configurations

The one or more STRS genes can be inactivated by introducing and expressing in a plant cell an exogenous polynucleotide having a nucleic acid sequence homologous to the STRS genes or a sub-sequence thereof including, but not limited to, expressible antisense or sense configurations and RNA silencing or interference configurations. The at least one polynucleotide sequence can be introduced into the plant by any transformation technique as is known in the art. Use of antisense nucleic acids is well known in the art. An antisense nucleic acid has a region of complementarity to a target nucleic acid, e.g., an STRS gene, mRNA, or cDNA. The antisense nucleic acid can be RNA, DNA, a PNA or any other appropriate molecule. A duplex can form between the antisense sequence and its complementary sense sequence, resulting in inactivation of the gene. The antisense nucleic acid can inhibit gene expression by forming a duplex with an RNA transcribed from the gene, by forming a triplex with duplex DNA, etc. An antisense nucleic acid can be produced for an STRS gene by a number of well-established techniques (e.g., chemical synthesis of an antisense RNA or oligonucleotide (optionally including modified nucleotides and/or linkages that increase resistance to degradation or improve cellular uptake) or in vitro transcription). Antisense nucleic acids and their use are described, for example, in U.S. Patent Nos. 6,242,258; 6,500,615; 6,498,035; 6,395,544; and 5,563,050.

Catalytic RNA molecules or ribozymes can also be used to inhibit expression of STRS genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. For example, one class of ribozymes is derived from a number of small circular RNAs that are capable of self- cleavage and replication in plants. The RNAs can replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples of RNAs include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes has been described. See, for example, Haseloff J and Gerlach WL, 1988 Nature, 334: 585- 591.

Another method to inactivate an STRS gene of the invention by inhibiting expression is by sense suppression. Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of a desired target gene. (See, for example, Napoli C et al., 1990 The Plant Cell 2: 279-289, and U.S. Patent Nos. 5,034,323; 5,231,020; and 5,283,184.

Genetically modified plants which include one or more ST-ftS-disrupted genes can also be produced by using RNA silencing or interference (RNAi), which can also be termed post-transcriptional gene silencing (PTGS) or co-suppression. "RNA silencing"

(also called RNAi or RNA-mediated interference) and "RNA interference," refer to any mechanism through which the presence of a single-stranded or, typically, a double- stranded RNA in a cell results in inhibition of expression of a target gene comprising a sequence identical or substantially identical to that of the RNA, including, but not limited to, RNA interference, repression of translation of a target mRNA transcribed from the target gene without alteration of the mRNA's stability, transcriptional silencing (e.g., histone acetylation and heterochromatin formation leading to inhibition of transcription of the target mRNA) and to endonucleolytic cleavage and then degradation of the target mRNA.

Use of RNAi for inhibiting gene expression in a number of cell types (including, e.g., plant cells) and organisms, e.g., by expression of a hairpin (stem-loop) RNA or of the two strands of an interfering RNA, for example, is well described in the literature, as are methods for determining appropriate interfering RNA(s) to target a desired gene, e.g., an ACC synthase gene, and for generating such interfering RNAs. For example, RNA interference is described e.g., in U.S. Patent Application Publication Nos. 20020173478; 20020162126; and 20020182223. The STRS polynucleotide sequence(s) or sub-sequence(s) expressed to induce

RNAi according to the teachings of the present invention can be expressed, e.g., under control of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (see, for example, Potrykus, 1, 1991 Annu Rev Plant Physiol Plant MoI Biol 42: 205-225; Shimamoto K. et al, 1989 Nature 1989 338, 274-276).

The principal methods of the stable integration of exogenous DNA into plant genomic DNA include two main approaches:

Agrobacterium-mediated gene transfer: The Agrobαcterium-mediated system includes the use of plasmid vectors that contain defined DNA segments which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobαcterium delivery system. A widely used approach is the leaf-disc procedure, which can be performed with any tissue explant that provides a good source for initiation of whole-plant differentiation (Horsch RB et al., 1988 Plant Molecular Biology Manual A5: 1-9, Kluwer Academic Publishers, Dordrecht). A supplementary approach employs the Agrobαcterium delivery system in combination with vacuum infiltration. The Agrobαcterium system is especially useful for the creation of transgenic dicotyledenous plants.

Direct DNA uptake. There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

The choice of technique will depend largely on the type of plant to be transformed. The exogenous nucleic acid can be introduced into any suitable cell(s) of the plant, such a root cell(s), stem cell(s) and/or leaf cell(s) of the plant.

T-DNA Insertion Mutagenesis

As described hereinabove, Agrobacterivm tumefaciens is widely used as an efficient vector for the genetic modification of plants. This is based on the bacterium's ability to introduce part of its Ti plasmid, the transferred DNA (T-DNA), as a single- stranded nucleoprotein complex into cells of plants. The T-DNA is also used as a tool for gene disruption, wherein the T-DNA is inserted into a gene such that the gene is inactivated. The advantage of using T-DNA for gene mutagenesis is that the foreign DNA not only disrupts the expression of the gene into which it is inserted but also acts as a marker for subsequent identification of the mutation. Additional advantage of using T-DNAs as the insertional mutagen is that T-DNA insertions will not transpose subsequent to integration within the genome and are therefore chemically and physically stable through multiple generations.

Typically, the incoming T-DNA integrates at random positions into the plant genome by a process of non-homologous recombination. However, the T-DNA can be targeted to a specific region of the genome by the incorporation of a segment from this genomic area in the T-DNA (see, for example Offringa R et al., 1990 EMBO J., 9,

3077-3084).

The T-DNA insertion is flanked by T-DNA borders. After activation of the Agrobacterium vir genes, the Ti plasmid is nicked at the T-DNA borders and the T- DNA is transferred as a single stranded DNA to the nucleus of the plant cell. As used herein, "T-DNA borders", "T-DNA border region", or "border region" are meant either right T-DNA border (RB) or left T-DNA border (LB). Such a border typically comprises a core sequence flanked by a border inner region as part of the T- DNA flanking the border and/or a border outer region as part of the vector backbone flanking the border. The core sequences in the right border region and left border region form imperfect repeats. Border core sequences are indispensable for recognition and processing by the Agrobacterium nicking complex consisting of at least VirDl and VirD2. Detection of the T-DNA insertion may be performed by any method as is known to a person skilled in the art. Typically, the border regions are further designed to include PCR-specific primers sequences that would enable the detection of the inserted T-DNA within the genome. Detection and/or identification of the disrupted gene typically further employ the use of gene specific primers. Using T-DNA specific primers together with gene specific primers provide immediate identification of a gene- specific T-DNA insertion mutagenesis. Transposons

The at least one STRS DEAD-box RNA helicase genes can also be inactivated by transposon based gene inactivation. In one embodiment, the inactivating step comprises producing at least one mutation in a DEAD-box RNA helicase gene sequence, wherein at least one mutation in the gene sequence comprises at least one transposon insertion, thereby inactivating said gene. The at least one mutation can be homozygous or heterozygous or a combination of both homozygous disruptions and heterozygous disruptions if more than one gene is disrupted.

Transposons were first identified in maize by Barbara McClintock in the late

1940s. The Mutator families of transposable elements, e.g., Robertson's Mutator (Mu) transposable elements, are typically used in plant gene mutagenesis (for example in maize) because they are present in high copy number and insert preferentially within and around genes.

Transposable elements can be categorized into two broad classes based on their mode of transposition. These are designated Class I and Class II; both have applications as mutagens and as delivery vectors. Class I transposable elements transpose by an

RNA intermediate and use reverse transcriptases, i.e., they are retroelements. There are at least three types of Class I transposable elements, including retrotransposons, retroposons and short interspersed-liked elements (SINE-like elements).

Retrotransposons typically contain long terminal repeats (LTRs)₃ and genes encoding viral coat proteins (gag) and reverse transcriptase, RNaseH, integrase and polymerase (pol) genes. Numerous retrotransposons have been described in plant species. Such retrotransposons mobilize and translocate via a RNA intermediate in a reaction catalyzed by reverse transcriptase and RNaseH encoded by the transposon.

Examples fall into the Tyl-copia and Ty3 -gypsy groups as well as into the SINE-like and LINE-like classifications. A more detailed discussion can be found in Kumar A and

Bennetzen JL, 1999 Annual Review of Genetics 33: 479-532. In addition, DNA transposable elements such as Ac₃ Taml and En/Spm are also found in a wide variety of plant species, and can be utilized in the invention.

Transposons (and other insertion sequence elements) are common tools for introducing mutations in plant cells. These mobile genetic elements are delivered to cells, e.g., through a sexual cross, transposition is selected for and the resulting insertion mutants are screened genetically for the presence of the insertion or for a phenotype of interest. Disrupted STRS gene can be introduced into other plants by crossing the genetically modified plants with a control plant in which the gene is non-disrupted, e.g., by a sexual cross. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. The location of an insertion within a genome of the genetically modified plant can be determined by known methods, for example by sequencing of flanking regions. A PCR reaction can be used to amplify the sequence, which can then be diagnostically sequenced to confirm its origin. Optionally, the insertion mutants are screened for a desired phenotype, such as tolerance to abiotic stress, including salt, water and heat stress. Tilling

TILLING can also be used to inactivate one or more DEAD-box RNA helicase genes according to the teaching of the present invention. TILLING is Targeting Induced

Local Lesions IN Genomics. (See, for example McCallum CM et al., 2000 Plant

Physioll23, 439-442; McCallum CM et al., 2000 Nature Biotechnol 18, 455-457; and, Colbert T et al., 2001 Plant Physiol 126: 480-484.

TILLING combines high density point mutations with rapid sensitive detection of the mutations. Typically, ethylmethanesulfonate (EMS) is used to mutagenize plant seed. EMS alkylates guanine, which typically leads to mispairing. For example, seeds are soaked in an about 10-20 inM solution of EMS for about 10 to 20 hours; the seeds are washed and then sown. The plants of this generation are known as Ml. Ml plants are then self-fertilized. Mutations that are present in cells that form the reproductive tissues are inherited by the next generation (M2). Typically, M2 plants are screened for mutation in the desired gene and/or for specific phenotypes.

For example, DNA from M2 plants is pooled and mutations in an STRS gene are detected by detection of heteroduplex formation. Typically, DNA is prepared from each M2 plant and pooled. The desired STRS gene is amplified by PCR. The pooled sample is then denatured and annealed to allow formation of heteroduplexes. If a mutation is present in one of the plants; the PCR products will be of two types: wild-type and mutant. Pools that include the heteroduplexes are identified by separating the PCR reaction, e.g., by Denaturing High Performance Liquid Chromatography (DPHPLC). DPHPLC detects mismatches in heteroduplexes created by melting and annealing of heteroallelic DNA. Chromatography is performed while heating the DNA. Heteroduplexes have lower thermal stability and form melting bubbles resulting in faster movement in the chromatography column. When heteroduplexes are present in addition to the expected homoduplexes, a double peak is seen. As a result, the pools that carry the mutation in an STRS gene are identified. Individual DNA from plants that make up the selected pooled population can then be identified and sequenced. Optionally, the plant possessing a desired mutation in an STRS can be crossed with other plants to remove background mutations.

Other mutagenic methods can also be employed to introduce mutations in an STRS gene. Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known. For instance, seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, diethyl sulfate, ethylene imine and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as X-rays or gamma rays can be used. Homologous Recombination

Homologous recombination can also be used to inactivate one or more DEAD- box RNA helicase genes of the invention. Homologous recombination has been demonstrated in plants. (See, for example, Swoboda P et al, 1994) EMBO J. 13, 484- 489; Kempin SA et al., 1997 Nature 389, 802-803; and Terada R et al., 2002 Nature Biotechnol 20(10): 1030-1034.

Homologous recombination can be used to induce targeted gene modifications by specifically targeting an STRS gene in vivo. Mutations in selected portions of an STRS gene sequence (including 5' upstream, 3' downstream and intragenic regions) such as those provided herein are made in vitro and introduced into the desired plant using standard techniques. The mutated gene will interact with the target STRS wild-type gene in such a way that homologous recombination and targeted replacement of the wild-type gene will occur in transgenic plants, resulting in suppression of the STRS gene activity.

Other detection methods for detecting mutations in DEAD-box RNA helicase STRS genes can be employed, e.g., capillary electrophoresis including constant denaturant capillary electrophoresis and single-stranded conformational polymorphism. In another example, heteroduplexes can be detected by using mismatch repair enzymology (e.g., CEL-I endonuclease from celery). CEL-I recognizes a mismatch and cleaves exactly at the 3' side of the mismatch. The precise base position of the mismatch can be determined by cutting with the mismatch repair enzyme followed by, e.g., denaturing gel electrophoresis.

The plant containing the mutated STRS gene can be crossed with other plants to introduce the mutation into another plant. This can be done using standard breeding techniques.

Following stable gene disruption performed by any of the methods known to a person skilled in the art and described hereinabove, plant propagation then occurs. The most common method of plant propagation is by seed. The disadvantage of regeneration by seed propagation, however, is the lack of uniformity in the crop due to heterozygosity, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. In other words, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the regeneration be effected such that the regenerated plant has identical traits and characteristics to those of the parent genetically modified plant. The preferred method of regenerating a transformed plant is by micropropagation, which provides a rapid, consistent reproduction of the transformed plants. Micropropagation is a process of growing second-generation plants from a single tissue sample excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue and harboring the modified gene. The newly generated plants are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows for mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars with preservation of the characteristics of the original transgenic or transformed plant. The advantages of this method of plant cloning include the speed of plant multiplication and the quality and uniformity of the plants produced. Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. The micropropagation process involves four basic stages: stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the newly grown tissue samples are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that they can continue to grow in the natural environment.

Any suitable plant can be used to produce the genetically modified plants of the present invention. Non-limiting examples include tobacco, maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, corn, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, solanaceous plants, potato, eggplant, tomato, Vicia species, pea, alfalfa, sorghum, cucumber, lettuce, turf grass, ornamental (e.g., larger flowers, larger leaves), coffee, cacao, tea, Salix species, oil palm coconut, perennial grass and a forage crop. According to certain embodiment, the genetically modified plants have increased tolerance to at least one abiotic stress compared to an unmodified plant.

According to certain embodiments, the genetically modified plants of the present invention are able to grow in a medium containing a salt concentration that inhibits growth of a corresponding non-modified plant, for example a concentration of salt in the range of from about 0.1M to about 0.2M, typically at a salt concentration ranging from about 0.1M to about 0.15M

According to other embodiment, the genetically modified plants of the present invention show an enhanced tolerance to osmotic stress compared to unmodified plants. Plants having increased tolerance to osmotic stress can easily adjust to growth under semi-dry and dry conditions, a trait which is highly desirable due to the growing process of desertification in agricultural areas all over the world. According to one embodiment, the genetically modified plants of the present invention can grow in a medium having an osmotic potential of about -0.75 MPa.

According to yet further embodiments, genetically modified seeds of the present invention show an increased tolerance to heat stress compared to unmodified seeds as measured by germination rate and shoot elongation of the emerging seedling. Growth rate of modified seedling exposed to heat stress was also significantly higher compared to wild type, unmodified plants. Seeds in which at least one STRS gene of the invention has been disrupted survived exposure to 45⁰C for 4 h, whereas such treatment killed the unmodified seeds. Furthermore, genetically modified seedlings from seeds exposed to

3h of heat stress showed 2.5-3 fold higher growth rate compared to unmodified seedlings.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES Experimental; procedures

Plant Materials and Growth

AU SaIk T-DNA insertion mutants were obtained from the Arahidopsis

Biological Resource Centre (ABRC), Ohio, USA. The SaIk ID for each mutant is as follows: strsl- Salk_062509; strsla - Salk_147039; strs2 - Salk_028850; strs2a - Salk_005131 (Alonso JM et al., 2003 Science 301: 653-657). Lines homozygous for the

T-DNA insert were isolated by PCR using gene-specific and T-DNA specific primers (http://signal.salk.edu/tdnaprimers.2.html). The sequences of the gene-specific primers were as follows:

Salk_062509 (F): 5'-ATAGTCGTTGCATCGTTTCTTGCT-S' (SEQ ID NO:5) Salk_062509 (R): 5'-CCAAAACGCTTGGTATTAGTATGT-S' (SEQ ID NO:6) Salk_147039 (F): 5'-ATGCAGGTCTTGGATGAACGTG-S' (SEQ ID NO:7)

Salk_147039 (R): 5'-TGTTCAGTGGCGTGAAGAAGGT-3^l (SEQ ID NO:8) Salk_028850 (F): S'-TTTTCCTAGTGATCTGCTTTGGTT-S' (SEQ ID NO:9) Salk_028850 (R): 5-GCTCTGAAGCTATCTCCGAAAGAA-S' (SEQ ID NO: 10) Salk_005131 (F): 5'-TTGGTAGTTGCAGAGCCTTACA-S' (SEQ ID NO: 11) Salk_005131 (R): 5-TACCTGGAAAACCAAGGAAGAA-S (SEQ ID NO: 12)

The F2 generation of homozygous mutants was used for all experiments. The wild-type Arabidopsis {Arabidopsis thaliana) accession was Columbia. For plate experiments, seeds were surface-sterilized by soaking in a solution of 50% bleach for 10 min and then rinsing five times with sterile water. Seeds were sown on nutrient agar plates containing MS salts (Murashige and Skoog, 1962) pH 5.8, 2% (w/v) sucrose, 0.5 g/liter MES and 0.8% (w/v) agar. Seeds were stratified at 4°C for four days in the dark before being placed in a growth room at 22⁰C₅ 50% relative humidity, and a photoperiod of 16 h light (150 μ mol photons m^'2 s^"!)/8 h dark. For soil experiments, seeds were suspended in 0.12 % agarose and stratified at 4°C for four days in the dark before being sown in a 1:1 mixture of perlite (three mesh) and Arabidopsis growth medium, (Weizmann Institute of Science, Rehovot, Israel) in pots. This soil mixture allowed leaching of nutrient solution to prevent build-up of salts and also permitted easy harvesting of whole plants for drought assays. Plants were irrigated with one third Hoagland's nutrient solution (Hoagland and Arnon, 1950). For analysis of organ- specific gene expression, plants were grown in soil in the growth room for approximately 4 weeks after germination. Plants were harvested after bolting when both inflorescences and siliques were visible. Only green siliques were taken for analysis. Roots were harvested from soil by gently washing in sterile water.

Abiotic Stress Assays For salt and osmotic stress assays, 50 to 100 surface-sterilized wild-type and mutant seeds were sown on plates containing MS media with or without NaCl (salt stress) or mannitol (osmotic stress). Four replicate plates were used per treatment and germination (emergence of radicals) was scored daily for 6 to 7 days until no further germination was observed. Fresh weight of seedlings was measured 10 days after stratification. Thermotolerance assays were performed essentially according to Hong SW and Vierling E 2000 Proc Natl Acad Sci USA 97: 4392-4397). Seeds were surfaced-sterilized, sown on plates containing MS media and stratified at 4°C for four days. For basal thermotolerance, 50 to 100 seeds were heat-treated at 45°C and then allowed to germinate in the growth room. Germination was recorded daily until no further germination was observed. Alternatively, heat-treated plates were placed vertically in the dark at 22°C to facilitate hypocotyl elongation along the plane of the agar. Hypocotyl elongation was measured after 6 days. For acquired thermotolerance, seedlings were grown for 3 days on vertical plates in the dark at 22°C and then heat- stressed by subjecting seedlings to 45⁰C of various duration with and without a prior pre-treatment. The pre-treatment consisted of 38°C for 90 min followed by 2 h at 22⁰C. The increase in hypocotyl elongation following heat stress was measured after 3 days further growth in the dark at 22⁰C. Both basal (hypocotyl measurement) and acquired thermotolerance experiments were performed with four replicate plates per treatment with each plate containing approximately 12 to 15 seedlings. ABA sensitivity was measured by germinating seeds on four replicate MS plates per treatment containing 0, 0.5, 1 or 2 μM ABA (Sigma-Aldrich Corp., St. Louis, MO, USA). AU stress assay experiments were repeated at least twice. For analysis of stress-responsive gene expression, wild-type and mutant plants were grown in soil for 2 weeks and then exposed to various stresses. Salt stress was applied by irrigating with one third Hoagland solution supplemented with 200 mM NaCl. Drought stress was induced by removal of whole plants from the soil, gently washing the roots, blotting dry and then placing the plants in the growth room under 60% humidity (Lu PI et al, 2007 Plant MoI Biol 63: 289-305). Cold stress was applied by placing plants at 4⁰C in the light. Heat stress was applied by exposing plants to 4O⁰C. Each experiment consisted of 3 replicate pots with 10 to 12 plants per pot. ABA-responsive gene expression was measured by transferring 4 day-old seedlings grown on vertical MS plates to MS plates with or without 100 μM ABA. Four replicate plates were used per treatment. Three independent stress or ABA expression experiments were performed. Analysis of Circadian Clock-Controlled STRS expression

Fifty wild-type seedlings were germinated and grown on MS plates in the growth room for 7 days. After this period, seedlings were transferred to a tabletop growth chamber (LE509, MRC, Holon, Israel) at 22⁰C and exposed to a photoperiod of 12 h light (100 μ mol photons m^"2 s^"!)/12 h dark. After four days entrainment, the photoperiod was changed to continuous light and seedlings were harvested every three hours over the third and fourth circadian cycles (Green RM and Tobin EM, 1999 Proc Natl Acad Sci USA 96: 4176-4179).

RNA Isolation, cDNA Preparation, Primer Design and Quantitative Real-Time PCR Total RNA was isolated and cDNA was prepared according to Kant S et al.

(2006 Plant Cell Environ 29: 1220-1234). Primers for amplification of PCR products of between 50 and 120 bp from A. thaliana cDNA were desi gned using A. thaliana sequences from Genebank and the Primer Express 2.0 software (Applied Biosystems, Forster City, CA, USA). Primer sequences for each gene are shown in Table 1. Real- time PCR was performed according to Kant et al. (2006, ibid). The relative quantification (RQ) values for each target gene were calculated by the 2^~ΔACχ method using UBIQUITIN 10 (UBQlO) as an internal reference gene for comparing data from different PCR runs or cDNA samples. To ensure the validity of the 2^~ΔΔCχ method, twofold serial dilutions of cDNA from unstres sed A. thaliana were used to create standard curves, and the amplification efficiencies of the target and reference genes were shown to be approximately equal (Livak KJ and Schmittgen TD, 2001 Methods 25: 402-408). For quantification of unstressed transcript levels, real-time PCR products for each gene were gel purified (QIAEX II Gel Extraction Kit, Qiagen, Valencia, CA, USA) and quantified with a NanoDrop spectrophotometer (ND- 1000; NanoDrop Technologies, Wilmington, DE). The 10-fold serial dilutions of each PCR product were used to create standard curves. At least three values corresponding to the absolute transcript copy number were produced for each sample in three independent experiments. As a loading control, the absolute transcript copy number of UBQlO was also calculated and normalized to the highest UBQlO level, which was assigned a value of 1. The target gene transcript copy number was then adjusted for loading differences by dividing by normalized UBQlO level. Table 1: Primers Used For Real-Time PCR Analysis of Gene Expression.

Example 1: Identification of the sirs Mutants

A functional genomics-based screen was performed to identify genes that may function as upstream regulators of multiple abiotic stress responses (P. Kant et al., Plant Cell, and Environment, In Press). In brief, a microarray analysis of early Arabidopsis heat stress-responsive genes was performed and the resulting data was combined in a "stress gene" database with data from published microarray analyses examining Arabidopsis responses to a variety of abiotic stresses. The database was queried for a set of regulatory genes whose expression was affected early by multiple abiotic stresses and Arabidopsis T-DNA insertion mutants defective in each gene were screened for altered sensitivity to abiotic stresses. A preliminary screen of mutants homozygous for the T- DNA insertion identified two mutants exhibiting increased tolerance to salt stress that contained a T-DNA insertion in genes encoding different DEAD-box RNA helicases (Figure IA and IB). Analysis of microarray data in our database indicated that these genes are down-regulated by salt, osmotic and heat stress. The proteins were designated the proteins as STRESS RESPONSE SUPPRESSOR 1 (STRSl) and STRS2.

STRSl is predicted to encode a protein of 537 amino acid residues with an estimated molecular mass of 59.5 kDA while STRS2 is predicted to be a protein of 563 amino acids with an estimated molecular mass of 62.5 kDA. Databases searches revealed that both proteins possess all nine conserved motifs that are characteristic of the DEAD-box protein family as well as an upstream conserved phenylalanine (Figure IA). Genomic and cDNA sequence analysis indicated that STRSl is identical to the known RH5 DEAD-box protein while STRS2 is identical to the known RH25. Alignment of STRSl and STRS2 protein sequences fhttp://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) showed that the two proteins exhibit 32% sequence identity and 49% sequence similarity. However, all the sequence identity/similarity resides in the core helicase region of the protein containing the conserved motifs whereas there is no significant sequence similarity between the N- terminal and C-terminal regions. Counting upstream from the conserved phenylalanine and downstream from motif VI for N-termini and C-termini, respectively, STRS 1 and STRS2 have N-terminal regions of 117 and 81 amino acids and C-terminal regions of 76 and 130 amino acids, respectively.

Example 2: Abiotic Stress Tolerance of the strsl and strs2 Mutants The stress-responsive phenotypes of two independent T-DNA insertion lines for each of the above-described gene were analyzed. The two strsl mutant lines designated strsl and strsla contain a T-DNA insertion in exon 6 and exon 9, respectively (Figure IB). Real-time PCR analysis of STRSl gene expression in unstressed wild-type and mutant plants using primers complementary to DNA downstream of the insertion, showed that STRSl transcript was undetectable in both mutant lines (Figure 1C). However, when primers complementary to DNA upstream of the T-DNA insertion were employed, STRSl transcripts were detected in strsl and strsla plants albeit at 25% to 30% of wild-type STRSl transcript levels. This suggests that truncated STRSl transcripts are produced in both strsl mutant lines but that they are less stable than wild- type transcripts. Furthermore, while it is unknown whether the truncated transcripts are translated, any truncated protein that may be produced in the strsl mutants is unlikely to be functional due to the absence of essential protein motifs such as motif V and motif VI (Figure IA). The T-DNA insertion in strs2 is located in the STRS2 promoter region close to the transcription start site whereas the insertion in strs2a is located in the first exon (Figure IB). No STRS2 transcript could be detected by real-time PCR in either line (Figure ID). When grown under unstressed conditions both seedling and adult sirs mutants showed no morphological or developmental differences compared to wild-type except for a very weak early flowering phenotype. Percentage of seed germination of all four mutant lines on Murahing and Skoog (MS) plates in the absence of stress was virtually identical to wild-type. However, germination of the strs mutants on MS plates supplemented with NaCl showed substantial tolerance to salt stress (Figure 2A and 2B). At only 2 days after stratification, the strs lines already showed two- to three-fold greater percentage of germination than wild-type seeds. By five days after stratification, strs seeds exhibited 95% to 99% germination whereas wild-type showed approximately 60% germination. In fact, final percentage of germination of wild-type seeds never reached more than 70%. In addition, all the st}"s lines grew faster than wild-type under salt stress. Quantification of fresh weight (FW) at 7 days after germination demonstrated that strsl and strsl a seedlings exhibited 100% greater FW than wild-type while strs 2 and strs2a seedlings showed 37% and 49% greater FW, respectively, than wild-type (Figure 2C). The strs mutants also showed tolerance to osmotic stress albeit to a lesser extent than their tolerance to salt stress. When seedlings were germinated and grown on MS plates supplemented with mannitol, all four mutant lines showed between 11% and 33% greater germination than WT seedlings by 4 to 5 days after stratification (Figures 2D and 2E). Furthermore, strsl and strsla mutants exhibited over 30% greater FW than wild-type while strs2 and strs2a showed approximately 20% greater FW (Figure 2F).

The strs mutants were next tested for altered basal and acquired thermotolerance. For basal thermotolerance, seeds were sown on MS plates, stratified for 4 days at 4⁰C and then exposed to 1 h to 4 h of 45⁰C (Hong SW and Vierling E, 2000 ibid). Basal thermotolerance was quantified by two methods, (i) seeds were allowed to germinate and grow at 22⁰C with a 16 h photoperiod, and percentage of germination was recorded; (ii) seeds were allowed to germinate and grow for 6 days at 22⁰C in the dark and hypocotyl elongation was measured. Figures 3 A and 3 B show germination results of seeds given 3 h of heat stress. This duration of heat stress killed seeds of hot 1-3, a mutant of HSPlOl that is defective in basal and acquired thermotolerance (Hong and Vierling, 2000 ibid). Although a portion of wild-type seeds were able to germinate, all the strs lines displayed greater germination rate at each time point after transfer to 22⁰C. Mutant seedlings also grew better than wild-type after germination. While 3 h heat stress led to a reduction in hypocotyl elongation in both wild-type and strs lines compared to the control treatment (Figure 3C), strs mutants still showed 2.5- to 3-fold greater hypocotyl elongation than wild-type. Moreover, whereas 4 h heat stress killed wild-type seeds altogether, the strs mutants survived and hypocotyl growth continued.

Acquired thermotolerance results from prior exposure to a pre-treatment such as a sub-lethal high temperature (Lindquist S, 1986 Annu Rev Biochem 55: 1152-1591). To assess whether the strs mutants possessed enhanced acquired thermotolerance, a quantitative hypocotyl assay was performed (Hong SW and Vierling E, 2000 ibid). In brief, seedlings were grown on vertical MS plates in the dark for 2 to 3 days before application of heat stress treatments. Hypocotyl elongation was recorded 3 days after the heat stress was applied. Figure 3D shows that a heat stress of 45⁰C for 2 h killed both wild-type and mutant seedlings as evidenced by lack of hypocotyl elongation. However, a pre-treatment of 38⁰C followed by 45⁰C for 2 h or 3 h allowed both mutant and wild type seedlings to survive, with mutant seedling exhibiting significant higher growth rate compared to wild type. The hot 1-3 mutant was most severely affected by 2 h heat stress, exhibiting an 80% reduction in hypocotyl elongation compared to the control (no heat treatment). Hypocotyl elongation of wild-type seedlings was reduced by 42% whereas strsl and strs2 exhibited a drop of only 4% and 13%, respectively. A heat stress of 3 h killed the hot 1-3 mutant while wild-type and strs seedlings showed a further reduction in hypocotyl elongation, with the mutants displaying approximately half the reduction observed in wild-type. A similar effect was observed for the strsl a and strs2a mutants. Taken together, these results show that the strs mutants exhibit enhanced basal and acquired thermotolerance. Plants were also tested for freezing tolerance but no difference could be observed between wild-type and mutants (data not shown).

Example 3; Expression of Stress-Responsive Genes and their Upstream Regulators The expression of the well-characterized stress-responsive marker gene, RD29A

(Yamaguchi-Shinozaki K and Shinozaki k, 1994 ibid) was investigated in order to gain insight into the molecular basis of the stress-tolerant strs mutant phenotypes. RD29A expression was analyzed by real-time PCR using UBQlO expression as an internal control. The two independent T-DNA insertion lines for each of the STRS genes exhibited identical expression phenotypes and therefore only the results for the strsl and strs2 mutants are shown. In wild-type plants subjected to salt, drought or cold stress, RD29A expression was induced by each stress (Figures 4A, 4B and 4C). RD29A expression peaked at 6 h, 12 h or 24 h after the onset of salt, drought or cold stress, respectively, and then progressively declined in agreement with a previous report (Albrecht V et al., 2003 Plant J 36: 457-470). RD29A expression was also induced by stress in the strs mutants with similar kinetics to that observed in wild-type plants. However, fold-induction of RD29A expression in the mutants was consistently higher than in wild-type plants particularly at, and after, peak expression, suggesting that the STRS proteins function as negative regulators of stress-responsive gene expression. In unstressed plants, no differences in RD29A expression were observed between wild-type and mutant plants (Figures 4A, 4B and 4C) indicating that loss of STRS function alone is not sufficient for enhanced RD29A expression. Therefore, de-repressed expression of upstream transcription factors in the strs mutants might account for the enhanced RD29A expression. Consequently, the expression of two members of the DREB transcription factor family that mediate stress-responsive RD29A expression via DRE elements in the RD29A promoter (Stockinger EJ et al., 1997 ibid; Liu Q et al., 1998 ibid) was analyzed. In wild-type plants, DREBl AICBFS expression was induced by salt, drought and cold stress with peak expression at 6 h after onset of salt stress and 3 h after onset of drought or cold stress (Figures 4D, 4E and 4F). Under salt and drought stress, expression dropped sharply by 6 h after stress and thereafter slowly declined, whereas under cold stress a more gradual decrease in DREBl AICBF3 expression was observed. Furthermore, peak expression of DREB1AICBF3 was an order of magnitude higher under cold stress than in salt and drought stress reflecting the primary role of DREB1A/CBF3 in cold-responsive gene expression (Liu Q et al., 1998 ibid; Shinwari ZK et al., 1998 Biochem Biophys Res Commun 250: 161-170). In the strs mutants, expression kinetics of DREBl Al CBF3 was comparable to wild-type but fold-induction of DREB1AICBF3 expression was higher in the mutant lines compared to wild-type. The DREB2 proteins play a major role in drought and salt stress signaling networks (Liu et al., 1998, ibid; Nakashima K et al., 2000 ibid). DREB2A expression was induced in wild-type plants by salt and drought stress and by cold stress although cold-induced expression levels were two orders of magnitude lower than drought-induced expression (Figures AG, 4H and 41). Peak expression of DREB2A occurred at identical time points to RD29A expression. Enhanced stress-induced expression of DREB2A was also observed in the strs mutants with expression displaying similar kinetics to that observed in wild-type plants. No difference was observed in either DREB IAI CBF 3 or DREB 2 A expression between unstressed wild-type and strs mutant plants. This suggests that the STRS proteins are not acting to repress stress-responsive gene expression in unstressed plants. Rather, the STRS proteins attenuate gene expression once it has been induced by stress.

It was further examined whether STRS genes also regulate stress-responsive genes that are not controlled by the DREB signaling subnetwork by analyzing the salt- and drought-induced expression of two non-DRE element genes RD19 and RD22 (Yamaguchi-Shinozaki K et al., 1992 Plant Cell Physiol 33: 217-224; Abe H et al., 1997 ibid). Salt and drought led to induction of RDl 9 and RD22 expression with similar kinetics in both wild-type and mutant plants (Figures 4 J, 4K, 4L and 4M). However, expression of both genes was enhanced in the strsl and strs2 mutants, particularly at, and after, peak expression. Furthermore, salt and drought-induced expression of AtMYC2, one of the transcription factors that regulate RD22 expression also exhibited increased expression in the strs mutants (Figures 4N and 40). These results suggest that STRSl and STRS2 also function in DREB-independent signaling networks.

Expression of the housekeeping gene, ACTIN2 (ACT2) served as a control to examine the specificity the effects of the strs mutants. Figures 4P, 4Q and 4R demonstrate that ACT2 expression was unaffected by either stress treatments or by absence of STRSl or STRS2, thereby suggesting a specific role for the STRS proteins in Arabidopsis abiotic stress responses. The molecular basis for the increased tolerance to heat stress of the strs mutants was examined by analyzing expression of the gene encoding Heat Shock Protein 101 (HSPlOl). This protein has been shown to be essential for both basal and acquired thermotolerance (Hong SW and Vierling E, 2000 ibid). In wild-type plants exposed to 4O⁰C heat stress, induction of HSPlOl expression was detected at 5 min after application of stress, reaching a peak at 2 h and declining by 3 h of stress (Figure 5A). In both strs mutants, kinetics of stress-induced HSPlOl expression followed closely that observed in wild-type plants, but fold-induction was higher in the mutants than in the wild-type plants, particularly at, and after, the peak in HSPlOl expression. No difference in HSPlOl expression was observed between unstressed wild-type and mutant plants, similar to the other stress-response genes analyzed.

Heat shock proteins are primarily regulated at the transcriptional level by heat shock transcription factors (HSFs). In Arabidopsis, there are 21 putative HSFs and of those that have been studied, some are constitutively expressed while the expression of others is induced by heat stress. HSF4 and HSF7 are two genes whose expression is induced by heat stress (Prandl R et al., 1998 MoI Gen Genet 258: 269-278; AtGenExpress database, http://www.arabidopsis.org/info/expression/ATGenExpress.jsp) and therefore it was examined whether expression of HSF4 and HSF7 is enhanced in the strs mutants. Figures 5B and 5C show that in wild-type and strs mutants the expression of both HSF4 and HSF7 was induced by heat stress, reaching peak expression at 30 minutes after onset of stress. Moreover, their expression was enhanced in the strs mutants predominantly at, and after, peak expression. As a control, the expression of TUBULIN5 (TUB5) in response to the heat treatment was examined. ACT2 expression was not used as a control because its expression is affected by heat stress. TUB5 expression was unaffected by heat stress in either wild-type or strs mutant seedlings (Figure 5D) demonstrating that HSPlOl, HSF4 and HSF7 expression was specifically affected in the strs mutants. Thus, although there is, as yet, no evidence that HSF4 and/or HSF7 directly regulate HSPlOl expression, these results suggest that the enhanced HSPlOl expression observed in the strs mutants is due to de-repression of upstream HSFs. Example 4: ABA Regulation of STRS Expression

Drought-induced RD22 and AtMYC2 expression is mediated by the plant hormone abscicic acid (ABA) and the finding that STRSl and STRS2 negatively regulate RD22 and AtMYC2 expression (Figure 4) suggests that the STRS proteins can function in ABA-dependent stress signaling. To further explore this notion ABA-induced expression of the RD26 gene in wild-type and strs mutant seedlings exposed to ABA was examined. RD26 is a dehydration-induced NAC protein that functions as a transcriptional activator in ABA-dependent stress signaling (Fujita M et al., 2004 Plant J 39: 863-876). Figure 6 A shows that in wild-type seedlings, RD26 exhibited a continual rise in expression at least up to 2 h after induction by ABA, consistent with previously reported findings (Fujita M al., 2004 ibid). On the other hand, fold-induction of ABA-induced RD26 expression was enhanced in the strs mutants with maximum induction occurring at 1 h after transfer to ABA-containing plates. In wild-type and mutant seedlings transferred to MS plates without ABA, no induction of RD26 expression occurred (Figure 6B), thereby demonstrating that the rise in RD26 expression in seedlings exposed to ABA was due to the action of ABA and not due to any stress caused by the transfer procedure itself.

The STRS proteins could attenuate ABA-dependent stress-responsive gene expression either by modulating ABA signaling to its target genes or by acting directly in the ABA signaling pathways. In the latter case, it would be expected that STRS expression would respond to ABA. Figure 6C shows that exposure of wild-type seedlings to ABA led to rapid suppression of STRS expression, indicating that STRS 1 and STRS2 can function as components of the ABA-dependent signaling subnetwork. However, the fact that the STRS proteins regulate stress-mediated DREB expression as well (Figure 4) suggests that STRSl and STRS2 also function in the ABA-independent stress signaling subnetwork. To test this hypothesis, stress-mediated expression of STRSl and STRS2 in wild-type and the ABA-deficient aba2-l mutant (Leon- Kloosterziel et al., 1996) was examined. Figures 6D and 6E show that salt stress led to a reduction in STRS expression in both wild-type and aba2-l plants, thus demonstrating that STRSl and STRS 2 can respond to stress signals in the absence of ABA. This finding shows that STRSl and STRS2 regulate both the ABA-independent and ABA-dependent stress signaling subnetworks. Because the absence of STRSl and STRS2 led to enhanced expression of ABA- responsive genes (Figures. 4L, 4M, 4N, 40 and 6A), the strsl and strs2 mutants might exhibit an ABA hypersensitive phenotype. The effect of the strs mutations on ABA- inhibition of seedling germination was therefore examined. Wild-type and mutant seed (both independent strsl and strs2 T-DNA insertion lines) were germinated on MS plates containing 0, 0.5, 1 or 2 μM ABA. On the control plates, no difference in germination could be observed between wild-type and mutant plants (Figure 6F). Surprisingly, however, the strs mutant lines exhibited an ABA-insensitive phenotype, as was shown by the more severe inhibition of the germination rate of the wild-type seeds compared to the strs mutants at each ABA concentration. This finding is similar to that found in the hos5 and jryl mutants which also exhibit up-regulated stress gene expression but display an ABA-insensitive germination phenotype.

Example 5: Abiotic Stresses and STRS Expression

Based on the screening method that revealed the STRS gene of the present invention along with results obtained by examining STRS expression in wild-type and aba2~l plants (Figures 6D and 6E) it was suggested that STRSl and STRS2 expression is down-regulated by salt, osmotic and heat stress. To further confirm this observation and to examine the detailed temporal expression of STRSl and STRS2, STRS gene expression in wild-type plants in response to salt, drought, heat and cold stresses was analyzed. Salt and drought stresses led to over 50% reduction in STRSl and STRS2 expression 1 h after the onset of the stress (Figures 7A and 7B). Expression continued to decline to about 20% and 10% of control levels under salt and drought stress, respectively, with STRS expression progressively rising thereafter. However, the later rise in STRS expression levels was considerably less under drought stress than under salt stress. Furthermore, under salt stress, STRSl expression exhibited greater stress- mediated repression than STRS2. Down-regulation of STRS expression was even more rapid after onset of heat stress (Figure 7C). Expression levels reached their nadir 2 h after exposure to heat stress and began rising again after 3 h from exposure.

The lowest STRS expression coincided with peak expression of RD29A and DREB2A, at 6 h and 12 h after exposure to salt or drought stress, respectively (Figure

7A vs. Figures 4A and 4G; Figure 7B vs. Figures 4B and 4H). Furthermore, the fast post-peak recovery of STRS expression under salt and the slow recovery of expression under drought were also reflected in the kinetics of the post-peak decline of RD29A and DREB2A expression. Similarly, minimal STRS expression coincided with peak expression of HSPlOl 2 h after exposure to heat stress (Figure 7C vs. Figure 5A). These results show a remarkable temporal correlation between low expression of the STRS genes and the peak expression of downstream stress-responsive genes within a particular stress treatment.

In contrast to salt, drought and heat stress, cold treatment had no effect on the expression of the STRS genes (Figure 7D). This result is in agreement with the finding that the strs mutants did not exhibit enhanced freezing tolerance (data not shown) and confirmed the notion that STRSl and STRS2 are not involved in attenuation of cold stress-regulated gene expression. However, the strs mutants did display enhanced expression of the DREB genes and a downstream target gene in response to cold stress (Figures 4C, 4F and 41). This finding suggests that in the absence of STRSl and STRS2, stress-induced expression of genes normally attenuated by the STRS proteins, will show enhanced expression in response to any signal that triggers stress-responsive gene expression. This will occur even if that signal does not normally down-regulate STRS expression.

One limitation of using relative real-time PCR quantification is that, unlike Northern analysis, a visualization of overall expression levels between various genes is not possible. Therefore, in order to determine expression levels of STRSl and STRS2 compared to other stress-responsive genes, STRS transcript copy number in unstressed plants was quantified and compared to the transcript copy numbers of DREBl AICBF3, RD29A and HSPlOl (Table 2). Quantification of transcript copy number was performed by relating the real-time PCR signal for each gene to a standard curve. The target gene transcript copy number was then adjusted for loading differences by dividing by normalized UBQlO level. The table represents the average results from three independent experiments ± SD. Table 2: Comparison of STRS transcript levels with transcripts of other stress- responsive genes in unstressed wild-type plants

STRSl and STRS2 exhibited comparable amounts of unstressed transcript levels to each other but these were an order of magnitude higher than RD29A and HSPlOl and two orders of magnitude higher than DREB1AICBF3 transcript levels. STRSl and STRS2 expression was detected in all organs from unstressed plants that were examined (Table 3). However, transcript copy numbers differed according to the organ analyzed. Highest STRS expression was observed in flowers and lowest levels in siliques and roots. Quantification of transcript copy number was performed as described for Table 2 hereinabove.

Table 3 : OrRan-specific STRS transcript copy number in unstressed wild-type plants

Example 6; Expression of STRSl and STRS 2 and the Orcadian Clock

Transcript profiling has shown that the expression of many stress-responsive genes is under the control of the circadian clock (Harmer SL et al., 2000 ibid). Regulation by the circadian clock may be an important means of coordinating plant stress responses to ensure optimum expression at periods when stresses are most likely to occur, thereby allowing anticipation of stress even in its absence. It was examined whether STRSl and STRS2 expression is under the control of the circadian clock by exposing wild-type seedlings to a photoperiod of 12-h-light:12-h-dark followed by a period of continuous light. Samples for RNA extractions were taken every 3 hours. Figure 7E shows that both STRSl and STRS2 exhibited circadian rhythms in transcript accumulation with peak expression at mid-to late subjective afternoon. Because the STRS proteins attenuate stress-responsive gene expression, it was expected that peak STRS expression would be close to peak expression of downstream stress-responsive genes. Indeed, peak STRSl and STRS2 expression coincided with peak expression of DREBl AICBFS and other downstream stress genes (Harmer wt al., 2000 ibid).

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A genetically modified plant, wherein at least one of the plant endogenous genes encoding a DEAD-box RNA helicase protein has been disrupted.

2. The genetically modified plant of claim 1, wherein the expression of the DEAD-box RNA helicase protein is inhibited compared to a corresponding unmodified plant.

3. The genetically modified plant of claim 1, wherein the activity of the DEAD- box RNA helicase protein is inhibited compared to a corresponding unmodified plant.

4. The genetically modified plant of claim 1, wherein the plant has an increased tolerance to at least one abiotic stress compared to a corresponding unmodified plant.

5. The genetically modified plant of claim 4, wherein the abiotic stress is selected from the group consisting of salt stress, heat stress and osmotic stress.

6. The genetically modified plant of claim 5, wherein said plant grows in a concentration of a salt that inhibits growth of a corresponding unmodified plant.

7. The genetically modified plant of claim 6, wherein the salt concentration is from about 100 mM to about 200 mM.

8. The genetically modified plant of claim 7, wherein the salt concentration is from about 100 mM to about 150 mM.

9. The genetically modified plant of claim 5, wherein said plant grows in a medium having an osmotic potential that inhibits growth of a corresponding unmodified plant.

10. The genetically modified plant of claim 9, wherein the medium osmotic potential is about -0.75 MPa.

11. The genetically modified plant of claim 5, wherein said plant has an enhanced tolerance to heat stress compared to an unmodified plant.

12. The genetically modified plant of claim 1, wherein the gene encoding the DEAD-box RNA helicase comprises a nucleic acid sequence as set forth in any one of SEQ ID NO:1, SEQ ID NO:2 and a functional variant, fragment or homolog thereof.

13. The genetically modified plant of claim 1, wherein the DEAD-box RNA helicase protein has an amino acid sequence as set forth in any one of SEQ. ID NO:3, SEQ ID NO:4 and a functional variant, fragment or homolog thereof.

14. The genetically modified plant of claim 1, wherein the DEAD-box RNA helicase encoding gene is disrupted by a method selected from the group consisting of gene insertions, gene deletions, anti-sense based disruption, point mutations and siRNA.

15. The genetically modified plant of claim 14, wherein the DEAD-box RNA helicase encoding gene is disrupted by a DNA insertion in a position selected from the group consisting of the gene promoter, first exon, 6^th exon, and 9^th exon.

16. A plant seeds produced by the plant of claim 1.

17. The plant seed of claim 16, wherein said seed is used for breeding a plant having an enhanced tolerance to at least one abiotic stress.

18. The plant seed of claim 17, wherein the abiotic stress is selected from salt stress, heat stress and osmotic stress.

19. A tissue culture comprising at least one genetically modified plant cell or protoplast, wherein at least one of the plant cell or protoplast's endogenous genes encoding a DEAD-box RNA helicase protein has been disrupted.

20. The tissue culture of claim 19, wherein the at least one plant cell or protoplast is obtained from a plant part selected from the group consisting of leaves, stems, bolts, pollen, embryos, roots, root tips, anthers, flowers, fruit and seeds.

21. The tissue culture of claim 19, wherein said tissue culture regenerates genetically modified plants having an enhanced tolerance to at least one abiotic stress as compared to a corresponding unmodified plant.

22. A plant regenerated from the tissue culture of claim 19.

23. An isolated polynucleotide encoding a DEAD-box RNA helicase protein having a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a variant, a fragment and a homolog thereof.

24. The isolated polynucleotide of claim 23, having a nucleic acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO:2, a fragment, a variant and a homolog thereof.

25. An isolated polynucleotide complementary to any one of the polynucleotide of claim 24.

26. A DNA construct comprising at least one polynucleotide of any one of claims 23 to 25.

27. The DNA construct of claim 26, further comprising an expression regulatory element.

28. The DNA construct of claim 27 wherein the regulatory element is selected from the group consisting of a promoter, an enhancer, and a termination sequence.

29. A vector comprising the DNA construct of any one of claims 26-28.

30. A host cell comprising the vector of claim 29.

31. A method of producing a genetically modified plant having an increased tolerance to at least one abiotic stress comprising (a) disrupting in at least one plant cell at least one endogenous DEAD-box RNA helicase encoding gene, wherein the disruption inhibits the expression or activity of at least one DEAD- box RNA helicase protein; and (b) regenerating the plant cell into a plant having an increased tolerance to at least one abiotic stress.

32. The method of claim 31, wherein the DEAD-box RNA helicase protein has an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4, and an active fragment, variant or homolog thereof.

33. The method of claim 31, wherein the gene encoding DEAD-box RNA helicase comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and a functional variant, fragment or homolog thereof.

34. The method of claim 31, wherein the disruption of the DEAD-box RNA helicase encoding gene is performed by introducing at least one mutation into the sequence of said gene.

35. The method of claim 34, wherein the mutation is a point mutation.

36. The method of claim 34, wherein the mutation is a deletion of at least two contiguous nucleic acids.

37. The method of claim 31, wherein the disruption of the DEAD-box RNA helicase encoding gene is performed by transforming into the at least one plant cells at least one polynucleotide targeted to disrupt said gene.

38. The method of claim 37, wherein the at least one polynucleotide has a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a variant, a fragment and a homolog thereof.

39. The method of claim 39, wherein the at least one polynucleotide has a nucleic acid sequence complementary to any one of SEQ ID NO:1, SEQ ID NO:2, a variant, a fragment and a homolog thereof.

40. The method of claim 37, wherein the polynucleotide is configured for RNA silencing or RNA interference.

41. The method of claim 37, wherein the polynucleotide is configured in an anti- sense configuration.

42. The method of claim 37, wherein the polynucleotide is a T-DNA configured for insertion into the DEAD-box RNA helicase encoding gene.

43. The method of claim 31, wherein the abiotic stress is selected from the group consisting of salt stress, heat stress and osmotic stress.

44. The method of claim 43, wherein the regenerated plant grows in a salt concentration of from about 100 mM to about 200 mM.

45. The method of claim 44, wherein the regenerated plant grows in a salt concentration of from about 100 mM to about 150 mM.

46. The method of claim 43, wherein the regenerated plant grows in a medium having osmotic potential of about -0.75 MPa.

47. The method of claim 43, wherein the regenerated plant grows in a super- optimal temperature.

48. A plant produced by the method of claim 31.