WO2009127443A2 - Transcription factors involved in salt stress in plants - Google Patents

Transcription factors involved in salt stress in plants Download PDF

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Publication number
WO2009127443A2
WO2009127443A2 PCT/EP2009/002979 EP2009002979W WO2009127443A2 WO 2009127443 A2 WO2009127443 A2 WO 2009127443A2 EP 2009002979 W EP2009002979 W EP 2009002979W WO 2009127443 A2 WO2009127443 A2 WO 2009127443A2
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Prior art keywords
seq
polynucleotide
plant
loc
sequence
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PCT/EP2009/002979
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French (fr)
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WO2009127443A3 (en
Inventor
Bernd Müller-Röber
Slobodan Ruzicic
Camila Caldana
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Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V
Universität Potsdam
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Publication of WO2009127443A2 publication Critical patent/WO2009127443A2/en
Publication of WO2009127443A3 publication Critical patent/WO2009127443A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • Salinity is a major environmental stress, affecting plant growth and development and it represents an increasing threat to plant agriculture. The impact of these factor increases continuously and leads on the global level to a decline of average yields by more than 50%. High salt depositions alter the basic structure of the soil reducing its porosity and consequently its water potential and aeration, making it difficult for plants to acquire water and nutrients, and it also increases the sensitivity to diverse biotic stresses.
  • better agricultural practices i.e., water and soil management
  • the introduction of salt-tolerant varieties in the affected areas have been approached.
  • the improvement of irrigation management practices in salt-affected areas is usually uneconomical and difficult to implement on large scale.
  • desalinization programmes cause environmental injuries.
  • Soil salinity is thus one of the more important variables that determines where a plant may thrive.
  • sizable land areas are uncultivable due to naturally high soil salinity.
  • salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture. The latter is compounded by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population.
  • Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt level in the whole soil profile.
  • QTLs quantitative trait loci
  • TF transcription factor
  • Transcription factors are key controlling elements of biological pathways, therefore altering their expression levels can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or biomolecules in plants or improvement in other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits, including traits that improve a plant's survival and yield during salt stress other abiotic stresses.
  • the present invention relates to an isolated polynucleotide capable of giving a plant tolerance to abiotic stress, particularly salt stress and/or osmotic stress, which consists of a polynucleotide 5 sequence selected from the group comprising SEQ ID NO. 1 to SEQ ID NO. 253.
  • the present invention also relates to an expression vector comprising the said polynucleotide and/or a promoter capable of giving a plant tolerance to abiotic stress, preferred osmotic stress and/or salt stress, and to a host cell transformed or transfected by the said expression vector.
  • the present invention further relates to a use of said polynucleotide or promoter sequence in improvement of plant tolerance to abiotic 0 stress, preferred osmotic stress and/or salt stress.
  • SEQ ID NO 1 LOC_Os01g08710.2: ATGTTCCCATCACCAGGGAGGGCGGTGATGGCGCTAGGCCACCACGGCGCCGCCCGCCAACCGGCGA
  • SEQ ID NO 18 LOC_Os01g52540.1: ATGGC TGAAGTAATGAGCATATTGGATGTTGACAAAGTGACAGTTGAATTATTCTGTGCAATGCTTGTTTTCTATAAATGGAATGTGGATGCGGTGGCA GAAGACTTTGACATATGTAGGGGCAAACCGCAAATTCAGAACCTGTTTCTGAAGCACAAACTTCAr ⁇ TCAATTTGATATTGTAAAGAGGAAACT
  • SEQ ID NO 66 LOC_Os03g19630 1 CGACCTGTTCCTCGGCCCCGRGCTGGACCTGCTGCTGGACTACCTGGCCGACACCGACCCGAACCGTCAGGGCACGCCGCCGGCCAGGAA
  • LOC_Os03g49880 1 ATGCTTCCTTTCTTCGATTCCCC SEQ ID NO 74: LOC_Os03g50310 1 ATGG
  • SEQ ID NO 102 LOC_Os04g47860 5 ATGGCATC ATGGCATCGAATTCATCGGCTGCAGCTGCGG
  • SEQ ID NO 110 LOC_Os04g51400.4: ATGGATGA
  • SEQ ID NO 111 LOC_Os04g51400.5: ATGGATGATC
  • SEQ ID NO 115 LOC_Os05g03900.1 : ATGGAAAA AATCAACAAAGI I I I I GCTGATGAGAATGAAGCTTTTGACTTCTACAATGGTTATGCTTATATGGTTGGTTTCTCTACATGCAAAGCTAGCAATT
  • LOC_Os06g41060.1 ATGGAGCTTCTCAAG
  • SEQ ID NO 154 LOC_Os07g047005 ATGAACA GTCAGATGTTGAGAAAGAGAAACTTGCTAAAGGAATAAAGCAGCAATACCAAGAGGCATTGATTTTGAACTCGATCAATAAAGGAAGTTCCACC
  • SEQ ID NO 158 LOC_Os07g 2 ATGCAGCCGGTACGGCCATCTGACGGAGTTCGA
  • SEQ ID NO 160 LOC_Os07g37210 1 A
  • LOC_Os08g45110 1 ATGATTCTGATAC
  • LOC_Os09g28210 1 ATGGACTTCGACTTGTTCA AAGCGCCACGTGCTCAAGGTGTGGATGCACAACAACAAGCACACCCTGGGCAAGAAGCTGCCATGA SEQ ID NO 198: LOC_Os09g29930.2:
  • SEQ ID NO 206 LOC_Os10g02910.1: A CAATCACTAGAGCTTGTGTTAAAGTATATGAAGATGAGAAAGAAAAGCTTAAGAAATTTTTTAAGGACAATTGTGTAAGAGTTTGCCTCACAACT
  • LOC_Os11g02530.1 ATGAAGA ATGGTGGAGCCTGTGAAATCGGAGCTTGGCAGC ID NO 225: LOC_Os11gO841O 1 ATGCCGAAGCCGA CTAAGAATCCTTTTGCCGTGCAAATAATGATGGAGTCATATGTCTATGTTGGATTTTTCATGAATATCCCATGTGAATTTGTCCGTGAGTGTCTT GTGCACATCTACAGAGTTGTCCCAGAAATTACTCCGCACAAACTCCGTTCTGACCCGAAGTAA SEQ ID NO 227: LOC_Os11g11220 1 ATGG
  • LOC_Os12g38950 1 ATGGCTTCTGATGTTCCTCAAGATGACGTGCAATGCCATTTTTGTGGC ATACCAACATCTGCTACAAGAGCGACGCATTCAGAAATGGCTTCTGATGTTCCTCAAGGTTACGTGCAATGTCATTTTTGTGACACCTACTTAA
  • ID NO 248 LOC_Os12g40070 1 ATGGGTGATCAAAAA
  • TTCTACTGGGCTAA SEQ ID NO 250: LOC_Os12g424Q02 ATGATGAGCTTCAACAAGAGCCAAGAAGGATTTGGGCAGGTTGCTGCTGTGGC
  • LOC_Os12g42400 3 ATGATGAGCTTCAA CCATTTGCAGAATATAATGGCTGTTTTGAGCTGGGCCTTGGTCAATCTGTGGTTCCCTCTAATTATCCTTATGCTGACCAGCACTATGGCCTAC
  • an isolated polynucleotide selected from the group comprising: (a) polynucleotide consisting of a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,
  • a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90, especially preferred 98% sequence identity to a sequence selected from the group comprising SEQ DD NO. 1 , SEQ DD NO. 2, SEQ DD NO. 14 ro SEQ DD NO.
  • Polynucleotides of the present invention that are variants of the polynucleotides provided herein will generally demonstrate significant identity with the polynucleotides provided herein.
  • polynucleotide homologs having at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, and 5 more preferably at least about 90%, 95% or even greater, such as 98% or 99% sequence identity with polynucleotide sequences described herein.
  • sequence identity refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences.
  • a partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid.
  • the inhibition 5 of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (in other words, the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are 0 such that non-specific binding is permitted; low stringency condiii ⁇ ns require that the binding of two sequences to one another be a specific (in other words, selective) interaction.
  • the absence of nonspecific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (for example, less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • the polynucleotides of the present invention may be present in the form of DNA, such as cDNA or genomic DNA, or as RNA, for example mRNA.
  • the polynucleotides of the present invention may be single or double stranded and may represent the coding, or sense strand of a gene, or the non-coding, antisense, strand. i0
  • isolated refers to a polynucleotide or polypeptide molecule separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified
  • 15 molecule may be greater than 60% free, preferably 75% free, more preferably 90% free, and most preferably 95% free from the other molecules (exclusive of solvent) present in the natural mixture.
  • isolated is also used herein in reference to polynucleotide molecules that are separated from nucleic acids which normally flank the polynucleotide in nature. Thus, polynucleotides fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered isolated herein. Such molecules are considered isolated even when present, for example in the chromosome of a host cell, or in a nucleic acid solution.
  • isolated and purified as used herein are not intended to encompass molecules present in their native state.
  • abiotic stress is the negative impact of non-living factors on the living organisms.
  • the non-living variable influences the environment beyond its normal range of variation to adversely affect the population performance or individual physiology of the organism.
  • Abiotic stress factors, or stressors are naturally occurring, often intangible, factors such as intense sunlight or wind that may cause harm to the plants and animals in the area affected. Abiotic stress comes in many forms.
  • the stressors include: high winds, extreme temperatures, heat, cold, strong light, water deficit, drought, flood, and other natural disasters, such as tornados and wildfires, poor edaphic conditions like rock content and pH, high radiation, compaction, contamination, non-optimal nutrient or salt levels, non-optimal light levels and other, highly specific conditions like rapid rehydration during seed germination.
  • tolerant or “tolerance” refers to the ability of a plant to overcome, completely or to some degree, the detrimental effect of an environmental stress or other limiting factor.
  • the transgenic plants are preferred tolerant to conditions including, but not limited to osmotic stress, particularly salt stress.
  • “Expression” means the production of a protein or nucleotide sequence in the cell itself or in a cell-free system. It includes transcription into an RNA product, post-transcriptional modification and/or translation to a protein product or polypeptide from a DNA encoding that product, as well as possible post-translational modifications.
  • an isolated polynucleotide selected from the group comprising:
  • nucleotide sequence encoding a polypeptide, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 14 to SEQ DD NO.
  • SEQ ID NO. 111 SEQ ID NO. 1 17, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ ID NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 139 to SEQ ID NO. 141, SEQ ID NO. 144, SEQ ID NO. 146 to SEQ ID NO. 148, SEQ ED NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NO167, SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID NO. 180, SEQ ID NO. 183 to SEQ ID NO.
  • nucleotide sequence encoding a polypeptide, wherein said polypeptide is selected from the group consisting of SEQ ID NO. 254, SEQ ID NO. 255, SEQ ID NO. 267 to SEQ ID NO. 270, SEQ ID NO. 273 to SEQ ID NO. 276, SEQ ID NO. 278, SEQ ID NO. 280,SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 297 to SEQ ID NO. 299, SEQ ID NO. 301, SEQ ID NO.
  • (c) a variant of any of the nucleotide sequences of (a) or (b) that has at least 70%, preferred 80%, more preferred 90%, especially 98% sequence identity to a sequence of (a) or (b)
  • the invention relates to said isolated polynucleotide, wherein the polynucleotide is selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ED NO. 20 to SEQ ED NO. 23, SEQ ED NO. 25, SEQ ED NO. 27, SEQ ED NO. 40, SEQ ED NO. 41, SEQ ED NO. 44 to SEQ ED NO. 46, SEQ ID NO. 48, SEQ ED NO. 52, SEQ ED NO. 56 to SEQ ED NO. 58, SEQ ID NO. 61 to SEQ ED NO. 67, SEQ ED NO. 69, SEQ ID NO.
  • these sequences can be used to stabilize the photosynthetic activity during salt stress and/or osmotic stress, which results in a stabilized photosynthetic yield.
  • These sequences were highly responsive and therefore very suitable for transfection.
  • the invention relates to an isolated polynucleotide selected from the group comprising SEQ ID NO. 144, SEQ ID NO. 146 to SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NOl 67, SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO.
  • sequences can be used to alter a plants tolerance to salt and osmotic stress particularly well. Plants comprising these sequences or modifications thereof showed a better growth during salt and/or osmotic stress compared to control plants.
  • the polynucleotides) ⁇ f the present invention find particular use in generation ot transgenic plants to provide for increased or decreased expression of the polypeptides encoded by the polynucleotides provided herein.
  • plants, particularly crop plants, having improved properties are obtained.
  • Crop plants of interest in the present invention include, but are not limited to soy, cotton, canola, maize, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turf grass.
  • nucleotide construct comprising said polynucleotide, wherein said polynucleotide is operably linked-to a promoter that drives expression in a plant cell.
  • the polynucleotides of the present invention find particular use in generation of transgenic plants to provide for increased or decreased expression of the polypeptides encoded by the polynucleotides provided herein.
  • plants, particularly crop plants, having improved properties are obtained.
  • Crop plants of interest in the present invention include, but are not limited to soy, cotton, canola, maize, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turf grass.
  • the invention relates to a nucleotide construct comprising a one of the mentioned polynucleotide, wherein said polynucleotide is operably linked to a promoter that drives expression in a plant cell.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one 10 additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
  • Such constructs are useful for production of transgenic plants having at least one improved property as the result of expression of a polypeptide of this invention.
  • Improved properties of interest include 15 stress tolerance, preferred abiotic stress tolerance preferred osmotic stress tolerance and/or salt stress tolerance, yield, disease resistance and growth rate.
  • a construct will generally include a plant promoter to direct transcription of the protein-encoding region or the antisense sequence of choice or gene-specific antisense region of sequence or gene- 20 specific region of sequence appropriate for the design of gene-specific artificial micro RNA.
  • a plant promoter to direct transcription of the protein-encoding region or the antisense sequence of choice or gene-specific antisense region of sequence or gene- 20 specific region of sequence appropriate for the design of gene-specific artificial micro RNA.
  • Numerous promoters which are active in plant cells, have been described in the literature. Further preferred is the nucleotide construct, wherein said promoter is a constitutive promoter., especially preferred a tissue-preferred promoter.
  • nucleotide construct wherein said promoter is an inducible promoter, preferred a stress-inducible promoter.
  • the invention relates to a polypeptide encoded by said polynucleotide and/or said nucleotide construct.
  • polypeptides consisting of a sequences selected from the group comprising SEQ ID NO. 254 to SEQ ID NO. 506.
  • SEQ ID NO. 254 LOC_Os01g08710.2: MFPSPGRAVMALGHHGAARQPPTTMAAAASSSTTSAAAAPATATTTVAFSFQHPTPTPSHHHHHHGVL
  • EGRHVHSPSRDDDDAARASAEMTFIW* SEQ ID NO. 255: LOC_Os01g08710.1: MALGHHGAARQPPTTMAAAASSSTTSAAAAPATATTTVAFSF ⁇ f ⁇ ⁇
  • EQ ID NO. 256 LOC_Os01g14010.1 MATSEAAAISNPFAPLTNHQQEHPPPPPPPAKKKRNLPGTPDPEAEVIALSPRTLMATNRFVCEICGKGFQR
  • LOC_Os01g29840 1 MGEQQQQVERQPDLPPGFRFHPTDEEIITFYLAPKWDSRGFCVAAIGEVDLNKCEPWDLPGKAKMNGEKEWYFYCQKDR SEQ ID NO. 260: LOC_Os01g32920 1 ID NO. 261: LOC_Os01g39020 1 MLKPQTPRARRAA
  • SEQ ID NO. 265 LOC_Os01g45730
  • SEQ ID NO. 274 LOC_O LOC_Os01g56070 2 MENRVGESSATAVDGGGGAKDSG GGLFPLLSFQVHGFPQAAAYGPAAGFPYGYGHSFHGWHGHGFPHQAPQGQHVDVFLKVLLVLVGVLVIASLIVF* SEQ ID NO. 277: LOC_Os01g6 RLYYRCSYREDRQCLASKLVQQENDDDPPLYRVTYTYEHTCNTTPVPTPDWAEQPPPGAAGDAYLLRFGSSAGGGGGGAHQQQTERERQQN
  • GSSYGNTMN SEQ ID NO. 295 LOC_Os01g73460 1 MGRRALPPSSSSSSSSSTTTTSPELRRKRTAAPPPPPSPRRYRSISDVMRRSLPVDAAP
  • SEQ ID NO. 303 LOC_Os02g35560.
  • GALP SEQ ID NO. 316 LOC_Os03g08470 2 MCGGAILAEFIPAPSRAAAATKRVTASHLWPAGSKNAARGKSKSKRQQRSFADVDDFEAAFEQF
  • LOC_Os03g51690 3 MCRGGLQVGAPPEVAARLTAVAQDLELRQRTALGVLGAATEPELDQFMEAYHEMLVKYREELTRPLQEA HYKWPYPSESQKVALAESTGLDLKQINNWFINQRKRHWKPSDEMQFVMMDGYHPTNAAAFYMDGHFINDGGLYRLG* SEQ ID NO. 332 LOCJDs RLRAENRELAARLHAVARHGLAARCQNARLRAEAAALARRLLALQRLARGRHMMITASPPQFSRR'SEQ ID NO. 333 LOC_Os03g59460 1 MASS
  • LOC_Os04g47860 MASNSSAAAAAAFF SMPARADEQ* SEQ ID NO 358 LOC_Os04g47860 7 MASNSSAAAAAAFFGISRDGDQHDQIKPLISHQQHQHQQQQLAASLTGVATAAPTAASS QGAPPAAPPAKKKRNLPGNPSNQPKYPFTISAMHAYISVLRDLVSIDWSLIICFLTVKASYRSHRRA* SEQ ID NO 359 LOC_Os04g48350 1 MEW
  • LOC_Os05g50350 1 MASAAGSKQQQAMMSLPSSRGGGGGGWTQRQNKQFECALAVYDKETPDRWHNIARYM GGAKSADEVRRHFDHLVEDVSRIESGRVPFPRYSSSSSSRGADDGNRSRYLKYQ * SEQ ID NO. 384 LOC_Os05g50700 1 MTITHASSLSRFHPL
  • LOC_Os09g28210 1 MDFDLFNSYPESQLDL TTPVDWQMGADEAGSNQLWDGLQDLMKLDEADTWFPPFSGAASSF* SEQ ID NO. 450 LOC_Os09g29130 1 MDFDDHDDGDEEMPPMPVSS PSGSGSGKKRFRTKFTQEQKDKMLAFAERVGWRIQKHDEAAVQQFCDEVGVKRHVLKVWMHNNKHTLGKKLP * SEQ ID NO.451 LOC_Os09g2 99302 SEQ ID NO.452 LOC_Os09g29930 3 MDSGSRS 1 MGIQGNKATTREHDFLSLYTTAAKDPSLQLH
  • LOC_Os12g42400 1 MMSFNKSQEGFGQ
  • a polypeptide consisting of a sequence selected from the group comprising SEQ ID NO. 254, SEQ ID NO. 255, SEQ ID NO. 267 to SEQ ID NO. 270, SEQ ID NO. 273 to SEQ ID NO. 276, SEQ ID NO. 278, SEQ ID NO. 280,SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 297 to SEQ ID NO. 299, SEQ ID NO. 301 , SEQ ID NO. 305, SEQ ID NO. 309 to SEQ ID NO.
  • polypeptide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a sequence selected from the group comprising SFQ ID NO. 254, SEQ ID NO. 255, SEQ ID NO. 267 io SEQ ID NO. 270, SEQ
  • polypeptide means an unbranched chain of amino acid residues that are covalently linked by an amide linkage between the carboxyl group of one amino acid and the amino group of another.
  • polypeptide can encompass whole proteins (i.e. a functional protein encoded by a particular gene), as well as fragments of proteins.
  • proteins Of particular interest are polypeptides of the present invention which represent whole proteins or a sufficient portion of the entire protein to impart the relevant biological activity of the protein.
  • protein also includes molecules consisting of one or more polypeptide chains. Thus, a polypeptide of the present invention may also constitute an entire gene product, but only a portion of a functional oligomeric protein having multiple polypeptide chains.
  • polypeptides involved in one or more important biological properties in plants are polypeptides involved in one or more important biological properties in plants.
  • Such polypeptides may be produced in transgenic plants to provide plants having improved phenotypic properties and/or improved response to stressful environmental conditions.
  • decreased expression of such polypeptides may be desired, such decreased expression being obtained by use of the polynucleotide sequences provided herein, for example in antisense or cosuppression methods.
  • Polypeptides of the present invention that are variants of the polypeptides provided herein will generally demonstrate significant identity with the polypeptides provided herein.
  • polypeptides having amino acid sequences provided herein reference polypeptides
  • functional homologs of such reference polypeptides wherein such functional homologs comprises at least 50 consecutive amino acids having at least 90% identity to a 50 amino acid polypeptide fragment of said reference polypeptide.
  • protein(s) when used herein refer to amino acids in a polymeric form of any length. Said terms also include known amino acid modifications such as disulphide bond formation, cysteinylation, oxidation, glutathionylation, methylation, acetylation, farnesylation, biotinylation, stearoylation, formylation, lipoic acid addition, phosphorylation, sulphation, ubiquitination, myristoylation, palmitoylation, geranylgeranylation, cyclization (e.g. pyroglutamic acid formation), oxidation, deamidation, dehydration, glycosylation (e.g. pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses, sialic acid etc.), acylation and radiolabels
  • Polypeptides useful for improved stress tolerance under a variety of stress conditions include polypeptides involved in gene regulation, such as ion antiporters, ion transporters, H + pyrophosphatases, H + ATPases, aquaporines, CNGCs, glutamate receptors, Ca 2+ - ATPases, transcription factors, serine/threonine-protein kinases, MAP kinases, MAP kinase kinases, and MAP kinase kinase kinases; polypeptides that act as receptors for signal transduction and regulation, such as receptor protein kinases; intracellular signaling proteins, such as protein phosphatases, GTP binding proteins, and phospholipid signaling proteins; polypeptides involved in arginine biosynthesis; polypeptides involved in ATP metabolism, including for example ATPase, adenylate transporters, and polypeptides involved in ATP synthesis and transport; polypeptides involved in glycine be
  • polypeptide wherein said polypeptide is a transcription factor.
  • Transcription factors play a key role in plant growth and development by controlling the expression of one or more genes in spatial, temporal and physiological specific patterns. Enhanced or reduced activity of such polypeptides in transgenic plants will provide significant changes in gene transcription patterns and provide a variety of beneficial effects in plant growth, development and response to environmental conditions.
  • Transcription factors of interest include, but are not limited to ABBVPl, C3H, HRT, SBP, Alfin-like, CAMTA, HSF, Sigma70-like, AP2-EREBP, CCAAT, LFY, SRS, ARF, CPP, LM, TAZ, ARR-B, CSD, MADS, TCP, BBR/BPC, DBP, MYB, Trihelix, BESl, E2F-DP, MYB-related, TUB, bHLH, EIL, NAC, ULT, bZIP, FHA, Orphans, VOZ, C2C2-CO-like, G2-like, PBF-2-like, WRKY, C2C2-Dof, GeBP, PLATZ, zf-HD, C2C2-GATA, GRAS, Pseudo ARR-B, ZIM, C2C2-YABBY, GRF, RWP-RK, C2H2, HB, Sl Fa-like, AR
  • any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homologue polypeptides of the invention. Due to the degeneracy of the genetic code, many diffident polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (i.e., peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code, are also within the scope of the invention.
  • amino acids have analogous physicochemical properties so that these amino acids advantageously can be replaced by each other.
  • these include the group of nonpolar (hydrophobic) amino acids (a) glycine, alanine, valine, leucine and/or isoleucine; or the hydroxy amino acids (b) serine, threonine and/or tyrosine, the amides of amino dicarboxylic acids (c) asparagine and glutamine, the amino dicarboxylic acids (d) aspartic acid and glutamic acid; the basic amino acids (e) lysine, arginine and/or ornithine as well as the group of aromatic amino acids (f) phenylalanine, tyrosine and/or tryptophan.
  • nonpolar amino acids a) glycine, alanine, valine, leucine and/or isoleucine
  • hydroxy amino acids b) serine, threonine and/or tyrosine
  • amino acids by structural similar amino acids.
  • this is the case in the group with a ⁇ - functional group (g) cysteine, methionine, serine, ⁇ - aminobutyric acid and selenocysteine as well as the turn-inducing group (h) proline, 1 -amino-2-carboxy cyclohexane, pipecolic acid and an ortho- aminobenzoic acid.
  • a-h the same group
  • peptide sequences will have a sufficient homology to be an analogous to an amino acid sequence of the peptides of the invention.
  • the amino acids can be replaced by modified amino acids or specific enantiomers.
  • the invention relates to a vector comprising said polynucleotide and/or said nucleotide construct.
  • the vector is a viral expression vector, a phage display vector, a bacterial expression vector, a yeast expression vector, a vector for expression in insects cells, a vector for in-vitro expression, a mammalian expression vector, a fungus expression vector, an algae expression vector or a plant expression vector.
  • vector is meant a DNA sequence, which can be introduced in an organism by transformation and can be stably maintained in said organism.
  • Vector maintenance is possible in e.g. cultures of Escherichia coli, Agrobacterium tumefaciens, Saccharomyces cerevisiae or Schizosaccharomyces pombe.
  • Other vectors such as phagemids and cosmid vectors can be maintained and multiplied in bacteria and/or viruses.
  • Vector sequences generally comprise a set of unique sites recognised by restriction enzymes, the multiple cloning site (MCS), wherein one or more non-vector sequence(s) can be inserted.
  • MCS multiple cloning site
  • “Expression vectors” form a subset of vectors which, by virtue of comprising the appropriate regulatory sequences enabling the creation of an expressible format for the inserted non- vector sequence(s), thus allowing expression of the protein encoded by said non-vector sequence(s).
  • Expression vectors are known in the art enabling protein- (gene-) expression in organisms including bacteria (e.g. Escherichia coli), fungi (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris), insect cells (e.g. baculoviral expression vectors), animal cells (e.g. COS or CHO cells) and plant cells (e.g. potato virus X-based expression vectors, see e.g. Vance et al. 1998— WO9844097).
  • the current invention clearly includes any vector or expression vector comprising a non- vector DNA sequence encoding a polypeptide of the invention, homologue and/or derivative.
  • Vectors may also include a screenable marker.
  • Screenable markers may be used to monitor transformation.
  • Exemplary screenable markers include antibiotic resistant genes or genes expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP), a beta- glucuronidase or uidA gene (GUS), which encodes an enzyme for which various chromogenic substrates are known or an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues.
  • GFP green fluorescent protein
  • GUS beta- glucuronidase
  • uidA gene GUS
  • Other possible selectable and/or screenable marker genes will be apparent to those of skill in the art.
  • the invention relates to a host cell comprising said polynucleotide, said nucleotide construct, said polypeptide and/or said vector.
  • the host cell wherein the host cell is selected from the group comprising a bacterial cell, a yeast cell, a fungus cell, a mammalian cell, an insect cell, an algae cell and/or a plant cell.
  • the invention relates to a transgenic plant cell having stably incorporated into its genome at least one nucleotide construct comprising said polynucleotide, operably linked to a promoter that drives expression in said cell.
  • transgenic organism is one whose genome has been altered by the incorporation of foreign genetic material or additional copies of native genetic material, e.g. by transformation or recombination.
  • a "transgenic plant” refers to a plant that contains genetic material not found in a wild type plant of the same species, variety or cultivar.
  • the genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty.
  • the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
  • a transgenic plant may contain an expression vector or cassette.
  • the expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide.
  • the expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant.
  • a plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • Transgenic plant in terms of the invention does also relate to cisgenic plants.
  • Plant or "Plants” comprise all plant species which belong to the superfamily Viridiplantae.
  • the present invention is applicable to any plant, in particular monocotyledonous plants and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp., Camellia sinensis, Carina indica, Capsicum
  • Polynucleotides or DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional
  • Agrobacterium tumefaciens host vector The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Microinjection techniques are known in the art and well described in the scientific and patent literature.
  • the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984).
  • Electroporation techniques are described in Fromm et al Proc. Natl. Acad. Sci. USA 82:5824 (1985).
  • Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).
  • Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
  • the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plant having an altered tolerance to abiotic stress, preferred osmotic stress and/or salt stress compared to a wild-type plant, wherein the transgenic plant comprises at least one modified polynucleotide, wherein the modified polynucleotide is selected from the group comprising an overexpressed polynucleotide, a suppressed polynucleotide and/or a knocked out polynucleotide.
  • transgenic plant having an altered tolerance to abiotic stress preferred osmotic stress and/or salt stress compared to a control plant, wherein the transgenic plant comprises at least one of said polynucleotides, and the control plant does not overexpress a polypeptide encoded by the polynucleotide.
  • transgenic plant comprising at least one said polypeptides.
  • Salt stress tolerance can be assayed according to any of a number of well-know techniques. For example, plants can be grown under conditions in which more than optimum salt concentration is provided to the plant. Salt stress tolerance can be determined by any of a number of standard measures including turgor pressure, growth, yield and the like, yield of photosynthesis, stomatal conductivity, transpiration rate, osmotic potential etc.
  • transgenic plant wherein the transgene comprises a polynucleotide sequence that hybridizes under stringent conditions to the said complement polynucleotide.
  • hybridize refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (in other words, the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T n , of the formed hybrid, and the G:C ratio within the nucleic acids.
  • T n refers to the “melting temperature” of a nucleic acid.
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • the equation for calculating the T m of nucleic acids is well known in the art.
  • stringent conditions refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted.
  • transgenic plant wherein the plant is selected from the group consisting of poales, preferred poaceae, more preferred ehrhartoideae and/or panicideae, especially preferred rice and/or maize.
  • transgenic plant wherein said plant is a crop plant or a monocot or a cereal, such as maize, wheat, barley, millet, rye, sorghum, oats preferred rice; or a plant cell derived from said transgenic plant.
  • a crop plant or a monocot or a cereal such as maize, wheat, barley, millet, rye, sorghum, oats preferred rice; or a plant cell derived from said transgenic plant.
  • transgenic plant wherein the abiotic stress is osmotic stress, preferred salt stress.
  • Rice is one of the most important alimentary corps.
  • the tolerance to abiotic stress, preferred osmotic stress and/or salt stress is particularly important for rice.
  • transgenic plant wherein the transgenic plant is a cultured host cell.
  • the invention also relates to a seed produced from the transgenic plant, preferred a transformed seed.
  • the invention relates to a seed produced fi ⁇ rn the transgenic plant.
  • the invention also relates to a method for producing one of said plants, said method comprising the steps of transforming a target plant with an expression vector comprising a polynucleotide, encoding a transcription factor polypeptide.
  • the transformed plant has a morphology that is substantially similar to a control plant.
  • a method for altering a plant stress response comprising stably introducing into the genome of a plant at least one nucleotide construct comprising a polynucleotide operably linked to a promoter that drives expression in a plant cell, wherein said polynucleotide is selected from the group comprising:
  • polynucleotide consisting of a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ED NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO.
  • SEQ ID NO. 84 SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ ID NO. 111, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ DD NO. 133, SEQ DD NO. 135, SEQ DD NO. 136, SEQ DD NO. 139 to SEQ DD NO. 141, SEQ DD NO. 144, SEQ DD NO. 146 to SEQ DD NO. 148, SEQ DD NO. 152, SEQ DD NO. 157 to SEQ DD NO. 159, SEQ DD NO. 162, SEQ DD NO. 165 to SEQ DD NO167, SEQ ID NO. 170, SEQ DD NO. 171, SEQ DD NO.
  • a method for improving the yield of a plant comprising stably incorporating into the genome of said plant at least one nucleotide construct comprising a polynucleotide operably linked to a promoter that drives expression in a plant cell, wherein said polynucleotide is selected from the group comprising:
  • polynucleotide consisting of a sequence selected from the group comprising SEQ ID • NO. 1, SEQ DD NO. 2, SEQ DD NO. 14 to SEQ DD NO. 17, SEQ DD NO. 20 to SEQ DD NO. 23, SEQ ID NO. 25, SEQ DD NO. 27, SEQ DD NO. 40, SEQ DD NO. 41, SEQ DD NO. 44 to SEQ DD NO. 46, SEQ DD NO. 48, SEQ DD NO. 52, SEQ DD NO. 56 to SEQ
  • Yield refers to increased plant growth, increased crop growth, increased biomass, and/or increased plant product production, and is dependent to some extent on temperature, plant size, organ size, planting density, light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature acclimation and water or nutrient use efficiency.
  • Salt-stressed plants experience a decrease of their photosynthetic efficiency (Munns, 1993). This decrease seems to be associated with the photosystem II (PSII) complex.
  • Salinity stress decreases the PSII activity and promotes the destruction of chlorophyll pigments via accumulation of ions that may inhibit the quantum yield of PSII electron transport (Sudhir and Murthy, 2004).
  • the ratio of F VV F M - i.e., F v >: variable fluorescence from light-adapted material; F M -: the maximum fluorescence signal, when all PSII centres are in the closed state
  • F VV F M - i.e., F v >: variable fluorescence from light-adapted material
  • F M - the maximum fluorescence signal, when all PSII centres are in the closed state
  • Figure 1 shows the photosynthetic rate analysed in wild-type and os02gl 3800-1 plants grown at two different salt stress concentrations, i.e., 5OmM NaCl (left panel) and 10OmM NaCl (right panel). Data are mean ⁇ SE of eight replicates plants eachs.
  • High salt concentration represents a water deficit or osmotic stress because of decreased osmotic potential in the soil or hydroponic solution.
  • solutes are accumulated as a response to falling water potential of the cell's environment.
  • the osmotic potential of the cell is lowered, which in turn attracts water into the cell helping to maintain turgor pressure water deficit (Babu et al., 1999).
  • the effects of osmotic stress caused by high salinity were measured in leaves of plants with reduced expression level of the gene Os02g46030 (ps02g46030- ⁇ ) and wild-type plants. Salt-treated plants (i.e., wild-type and transgenic plants) showed a slight decrease in the osmotic potential three days after the onset of the stress
  • Figure 3 shows the response of transpiration and (B) the stomatal conductance in os02gl 3800-1 transgenic (black bars) and Nipponbare wild-type (grey bars) seedlings upon application of salt stress (5OmM NaCl) for up to three days. Data are mean ⁇ SE of four replicates.
  • Leaves of plants with increased expression level of the gene Os06g41060 (35S::Os06g41060) and Nipponbare wild-type plants were compared employing gas exchange as physiological parameter. Transpiration rate and stomatal conductance were analysed in plants 0 and 48h after start of the salt stress (5OmM NaCl; Figure 4). At 48h, leaves of Nipponbare showed a reduction by 35% of the original transpiration rate, whereas the 35S::Os06g41060 displayed a decline of only 21%. Stomatal conductance was less significantly different between the plants, with a reduction of 30 and 38%, respectively, of the initial rates detected in the mutant and the wild-type, respectively.
  • Figure 4 shows the response of transpiration and (B) the stomatal conductance: 35S::Os06g41060 (black bars) and Nipponbare (grey bars) plants treated by salt stress (5OmM NaCl) for two days. Data are means ⁇ SE of six replicates.

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Abstract

The invention relates to isolated polynucleotides, preferred transcription factors, polypeptides encoded by the polynucleotides, vectors and host cells comprising the polynucleotide and transgenic plants comprising the polynucleotide or the polypeptide. The invention also relates to a method for altering a plants stress response, preferred salt stress and/or osmotic stress.

Description

Transcription factors involved in salt stress in plants
Background
Salinity is a major environmental stress, affecting plant growth and development and it represents an increasing threat to plant agriculture. The impact of these factor increases continuously and leads on the global level to a decline of average yields by more than 50%. High salt depositions alter the basic structure of the soil reducing its porosity and consequently its water potential and aeration, making it difficult for plants to acquire water and nutrients, and it also increases the sensitivity to diverse biotic stresses. To cope with the problem of salinity, better agricultural practices (i.e., water and soil management) and the introduction of salt-tolerant varieties in the affected areas have been approached. However, the improvement of irrigation management practices in salt-affected areas is usually uneconomical and difficult to implement on large scale. In addition, desalinization programmes cause environmental injuries. As alternative, for maintaining stable global food production, genetic improvement of salt tolerance of important crops like rice (Oryza sativa), wheat {Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare) seems to be the most practicable and promising strategy. Detrimental effects of salt on plants are a consequence of both water deficit resulting in osmotic stress (similar to drought stress) and the effects of excess sodium ions on critical biochemical processes. As with freezing and drought, high saline causes water deficit; the presence of high salt makes it difficult for plant roots to extract water from their environment (Buchanan et al. (2000) in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.). Soil salinity is thus one of the more important variables that determines where a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. To compound the problem, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture. The latter is compounded by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt level in the whole soil profile.
Most of the stress tolerance traits are quantitative trait loci (QTLs), which make difficult for genetic selection of traits employing the classical breeding approaches.
Of the various general types of plant responses to salinity stress, avoidance mechanisms mainly result from morphological and physiological changes at the whole-plant level. Recently, comparative molecular studies of the salt-sensitive model plant Arabidopsis thaliana and its close relative Thellungiella halophila, which is able to tolerate seawater-strength sodium chloride concentrations, indicated similar mechanisms of salt tolerance, but they are regulated at different levels in the two species. In contrast, cellular and molecular biochemical modifications lead to tolerance mechanisms that lend themselves to biotechnological manipulation. A full elucidation of these molecular mechanisms is required for improve stress tolerance in agricultural plants.
To survive and develop normally, plants must constantly perceive changes in their environment and respond properly through a variety of molecular mechanisms. In high salinity, ionic and osmotic impacts on the cell may be recognized by their own cognate receptors, which may operate to elicit downstream signalling events.
Regulation of gene expression is expected to play a major role in the control of plant responses to salt stress. The link between cellular mediators and gene expression is provided by transcriptional regulatory networks, which e.g. involves the interaction of transcription factor (TF) proteins with the genes they regulate. Thus, the role of TFs in signal transduction is, besides others, to respond to abiotic stress signals and accordingly change the transcription of genes, enabling cells to synthesize proteins they need to adapt and to overcome the stress condition. Although TFs are important in regulating plant responses to environmental stress, specific functions of most of the genes encoding TFs are still unclear. Therefore, the investigation of the salt stress response system at the level of transcription is of high interest to understand the mechanisms of plants salt tolerance.
Despite of the diversity and the fundamental role of TFs in biological processes, only a small fraction (around 10%) has been characterized functionally. There is still very little known about the modes of TF action especially with the respect to genes they regulate and the mechanisms they use to achieve this regulation. The combinatorial nature of transcriptional regulation also adds to the complexity of this research field.
Transcription factors are key controlling elements of biological pathways, therefore altering their expression levels can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or biomolecules in plants or improvement in other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits, including traits that improve a plant's survival and yield during salt stress other abiotic stresses.
So far abiotic stress, particularly salt stress, is a major problem for agriculture. Breeding of more resistant plants is to slow and time consuming. Currently, knowledge about TFs involved in the salt- and osmotic-stress dependent signalling is still limited. Therefore it was an object of the invention to identify polynucleotides, preferred coding transcription factors, whose modification results in an altered stress tolerance in plants.
The present invention relates to an isolated polynucleotide capable of giving a plant tolerance to abiotic stress, particularly salt stress and/or osmotic stress, which consists of a polynucleotide 5 sequence selected from the group comprising SEQ ID NO. 1 to SEQ ID NO. 253. The present invention also relates to an expression vector comprising the said polynucleotide and/or a promoter capable of giving a plant tolerance to abiotic stress, preferred osmotic stress and/or salt stress, and to a host cell transformed or transfected by the said expression vector. The present invention further relates to a use of said polynucleotide or promoter sequence in improvement of plant tolerance to abiotic 0 stress, preferred osmotic stress and/or salt stress.
Before the embodiments of the present disclosure are described in detail, it is to be understood that unless otherwise indicated the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such may vary. It is also to be understood 5 that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps may be executed in different sequence where this is logically possible.
SEQ ID NO 1: LOC_Os01g08710.2: ATGTTCCCATCACCAGGGAGGGCGGTGATGGCGCTAGGCCACCACGGCGCCGCCCGCCAACCGGCGA
20 CCACCATGGCGGCGGCGGCCTCCTCGTCGACGACCTCCGCCGCCGCCGCCCCGGCCACGGCCAUCACCACCGTCGCCTTCTCCTTCCAGC
SEQ ID NO 2: LOC_Os01g08710.1: ATGGCGCTAG
SEQ ID NO 3: LOC_Os01g14
SEQ ID NO 4: LOC Os01g16810.1 : ATGGCTCACGAG
Figure imgf000004_0001
SEQ ID NO 6 LOC_Os01g29840 1 ATGGGAGA
1 ATGCTTAAACCCCAAACACCTCGCGCTCGCCGC
Figure imgf000005_0001
CGTGGATTCACAACTAAGAAATAAAGCAAGTGTTGTTTTTGGACAGCCTGAGGCTCCTGGTGAGGTTGTGAGCTTGAAGAGAGACCGTGCAG
CCCTCAGAGCTGAGGTCATCATGCTTAAGCAGCAGTACAACGCCTGTAAATCTCAGCTCATTGCCATGGACCAGATGGTCCGCAAL-ATCGAG
Figure imgf000005_0002
Figure imgf000005_0003
SEQ ID NO 12 LOC_Os01g4573
Figure imgf000006_0001
GGTGAAGATGGAGAGCTTCGATTGGGAGTGCGCCGAGCTGCTCAGCTAAAAAATGCATCTCCTΠTCCTGCACTTCATAACCAGATCTCAAAT ACTAGTAGTCTTAGTGAAGTTGCACATGCTGTGGCCGTGAAAAGTATTΠTCACATCTACTACAATCCCAGTTGTACTCACAGGTTAAGTCAGT CTGAGTTCATTATACCATATTGGAAGTTTATGAGAAGCTTCAGTCAACCCTTTTCTGTTGGAATGCGATTCAAATTGAGATATGAGAGTGAGGAT
CCCCATTCAAAACGGCTGAMTCATGCTTCCCCCAAGTTAATCCAGATATTGTGCTTCCAAATGGAAGTGTTTCTTCAGATTTTGCGGAGTCTG CCAGATTCCACAAGGTCTTGCAAGGTCAAGAATTGTTGGGTTTAAAAACTCGTGATRGTACTGTTAATACAGCTTCTCAL.UCGACTGAGGCAA
GAAATnTCAGTArACTGaTCV1CGAAGTTGCTCTATCAACATGAGTAACAATATCTTAGGGGTTCCGAGACTTGGGGTGAAAACTCCAAGTGG
AAACCCTGGGTTTTCCTACCATTGCTCAGGCTTTGGGGAATCTCAAAGATTCCAAGAGGTCTTGCAAGGTCAAGAAGTGTTTCGTCCTTACCG
SEQ ID NO 15 LOC_Os01g48Q60 2 ATGGTGG
Figure imgf000006_0002
GCACATGCTGTGGCCGTGAAAAGTATTTTTCACATCTACTACAATCCCAGGTTAAGTCAGTCTGAGTTCATTATACCATATTGGAAGTTTATGAG AAGCTTCAGTCAACCCTTTTCTGTTGGAATGCGATTCAAATTGAGATATGAGAGTGAGGATGCTTCTGAAAGAAGGCGTACTGGAATAATAATT
CCAAGTTAATCCAGATATTGTGCTTCCAAATGGAAGTGTTTCTTCAGATTTTGCGGAGTCTGCCAGATTCCACAAGGTCTTGCAAGGTCAAGAA TTGTTGGGTTTAAAAACTCGTGATGGTACTGTTAATACAGCTTCTCAGGCGACTGAGGCAAGAAATTTTCAGTACACTGATGAACGAAGTTGCT
CTATCAACATGAGTAACAATATCTTAGGGGTTCCGAGACTTGGGGTGAAAACTCCAAGTGGAAACCCTGGGTTTTCCTACCATTGCTCAGGCT
Figure imgf000006_0003
ATGCATCTCCTTTTCCTGCACTTCATAACCAGATCTCAAATACTAGTAGTCTTAGTGAAGTTGCACATGCTGTGGCCGTGAAAAGTATTTTTCAC ATCTACTACAATCCCAGGTTAAGTCAGTCTGAGTTCATTATACCATATTGGAAGTTTATGAGAAGCTTCAGTCAACCCTTTTCTGTTGGAATGCG ATGGGACGGCACGCGTGCTCTGCCGCCGG
SEQ ID NO 18: LOC_Os01g52540.1: ATGGC
Figure imgf000007_0001
TGAAGTAATGAGCATATTGGATGTTGACAAAGTGACAGTTGAATTATTCTGTGCAATGCTTGTTTTCTATAAATGGAATGTGGATGCGGTGGCA GAAGACTTTGACATATGTAGGGGCAAACCGCAAATTCAGAACCTGTTTCTGAAGCACAAACTTCArπTCAATTTGATATTGTAAAGAGGAAACT
CTGCAATGTGACTTGACAACAGAGAAGTGTAGGCTAGTTGATGAGCATGATTTATGTAATTTTTCTCAGAAAAAGAGAAGGAAGCGAGGTTCAT
Figure imgf000007_0002
CTGCTGATGTAATTACAGTTGTTGTGCAGAAAGAAATTTTTAAATGGAATTGTTGTCTTAAGGATAGGGATGCTCAAAGGATAGTGAATGCGTTA CTGGAACATGCTAGAAAAATCAAGGAGATGCACAATTTTAACTCGGAGATGCGGAAGGAAGAGTTTTCTGCAAAGCTGAAGGTTCATTTGAAG
ATGATGGGCGGCGAGTUCAAGGT
Figure imgf000007_0003
GGCGGTCGCTGGGGGAACTACACTTTCTCGGCGGCCATTGGGGGGCTTTTCCCATTGCTGAGCTTCCAGGTTCATGGGTTCCCGCAGGCGG
GGGCCAGCATGTTGATGTGTTCTTGAAGGTATTGCTTGTTCTGGTTGGTGTTCTTGTGATTGCCAGCTTGATTGTGTTTTAG SEQ ID NO 21: L
ID NO 22: LOC_Os01 g56070.2: ATGGAGAACCGCGT
Figure imgf000007_0004
SEQ ID NO 25: LOC_Os01g61080.1: ATGACAACC
ATGCATGACCCACGCGGCTTCCCCATCC
Figure imgf000008_0001
CGACGAGGCGTTOGACACGTTCAGCGCGTCCAGCGATCTGAGCCCCGGCATCCTGGCGGCGGCGTCGGCGCAGTTCGACCCGAACCAGGA GGCAGCCTGTCGTCGTGGTGCTCGATCGTGCCGAGCTTGTCGACATGGGAGGAGCCCAAGTACGACTCGTTGGATTCGTTTCCCGACGACG
ATGGACGCCTCCGCCGGCTCGTCGGC
Figure imgf000008_0002
Figure imgf000008_0003
ATGGACGCCTCCGCCGGCTCGTCGGCCC
Figure imgf000009_0001
CATCCCCATGGAAGACATCGGGACGTCTCAGGTGTGGAACATGCGGTACCGGTTTTGGCCCAACAACAAAAGCAGGATGTATCTTCTCGAAA
Figure imgf000009_0002
SEQ ID NO 39: LOC_Os01g72370.1: AT
SEQ ID NO 41: LOC_Os01g72490.2:
Figure imgf000010_0001
SEQ ID NO 44: LOC_Os02g13800
Figure imgf000011_0001
1 ATGCGGAGGTCCAAAAAGCGTCA
Figure imgf000011_0002
GAGATTGCTAAGTTTATTATTGCTGAGGATCTACCMTTAGAATGGGGGAAAGCAAACATTTTGAGAGAATGATCΑVAAAAGCTTTTTGCCCAC
\5 AGTTTTTCATTTGCTGTGACATCGGATATCTGGACATCCCAACACCAAAGAACATΓTTACCTTAGTCTTGTTCTTCATTATUITGACAACAATCG
GTCATTAAATΔ.ΔGΛ CACTCATAGGGTTCCAAU I CATGACGTCACACACGGGAGATGCCATAGCCACGACAATCCTAGAGGTATTGAGAGAATT CAATTTACAATCTAGGGTTGTTTCTATTACTCTTGACAATGCATCAGCAAACACAACGGCCATATCGATTTTGGAGTCGGATCTGCGAAGTTAT
>0
TTCCCTCCTTGGAGGTGTTTCCTTGTGGACTCTCTCCCACCTTTGGGTGAAAACCCAGTCCAACTTTTGGGCGGGCGTCAACAATGGCTTTGG
ID NO 49 LOC_Os02g33240 1 ATGGCGGCGACCACC
Figure imgf000011_0003
1 ATGATGCAGCAAGACAGCAAGAG
10
Figure imgf000012_0001
CI I I I I GAAGGGAACTTGCTTGAGAAGGAATrTTTGAAGTGTGAGATATACACACTTGGGGGCGATGAAGGGGATATTTGGAGGCCTGCCGCT GGTGGAGTACCTrrCCGGTTCTACAGTTTTGCTCGTTCTGCTATTTCCAATGCGGTGATGAACAAACTGCAGCCACTTTTCTTCAATGGATATC
LOC_OS02G35690 2 ATGGACGACGACGGCGGCGC
SEQ ID NO 55: LOC_Os02g44790 1 ATGTC
Figure imgf000012_0002
ATGGGAATTAGCGGCGAGAGGAAGGG
ID NO 58: LOC_Os02g51670.1: ATGGCCGCAGCAATA
Figure imgf000013_0001
TAA SEQ ID NO 59: LOC_Os02g55380.1 : ATGGCGAGGCCGCAGCAACGATACCGCGGCGTGCGGCAGCGCCACTGGGGCTCCTGGGTCTCC
GAGATCCGCCACCCTCTCCTGAAGACGCGGATATGGCTGGGCACGTTCGAGACGGCGGAGGATGCGGCGCGGGCGTACGACGAGGCGGC GAGGATCATGTGCRGGCCCCGCGCGCGCACCAACTTCCCGCTCGCCGACGCCACCGCGGCGGCGGCGGCGGCCGCCGCGTCCTCGTCGT
GCTACTA SEQ ID NO 60: LOC_Os03g04890.1: ATGGCGCCGGCGTGGGATCGCGCCAAGCACGCGCTCGCCACCAGGCTCTGCATTCGCTT GTGGCATACGGACAGGAGGACAGGCCTTGTTCACAGCTGAATGCTCCCACGAATTCCATTTTCATTGCATCTCCTCAAATGTCAACCATGGCA GTCTTCCGTACTCCAGAGGCAAGCATTTTCAATGATGATGAGAACATTGATCCTCAGTCTGAAACAGTTGATGATCATAATGCAGTTACTAATTC MGCGTGCCATGAGTTTTGTCATCCAAACACTTGGACCCAATGACCGCCTGTCTGTTGTAGCCTTTTCATCTACAGCACAGAGGCTCTTTCCAC
GGTACAGACCATGATTCGGCTGCCATGCATGCTATTGCTGAGACATCTAATGGCACATTTTCATTTATTGATGCTGAAGGCTCAATCCAAGATG
Figure imgf000013_0002
SEQ ID NO 63: LOC_Os03g08470 2. AT
SEQ ID NO 66: LOC_Os03g19630 1
Figure imgf000014_0001
CGACCTGTTCCTCGGCCCCGRGCTGGACCTGCTGCTGGACTACCTGGCCGACACCGACCCGAACCGTCAGGGCACGCCGCCGGCCAGGAA
50
GGCGGCGGCGGCGATGGTGATGGCGACGGAGGAAGCAGTGGCAGGAGGCGCTGGCTCTCGTGGCCTTTTGGCGGGTTATTCTCGCACAGA ID NO 69: LOC_Os03g22170 1 ATGTGCGGCGGCGC
Figure imgf000014_0002
SEQ ID NO 71: LOC_Os03g42290 1 ATGC
LOC_Os03g49880 1 ATGCTTCCTTTCTTCGATTCCCC SEQ ID NO 74: LOC_Os03g50310 1 ATGG
ID NO 78: LOC_Os03g51690 3 ATGTGTCGTGGTGG
Figure imgf000015_0001
1 ATGCATTGCTGCATGTCGCTTCATCCTCACC
Figure imgf000016_0001
SEQ ID NO 85. LOC_Os04g30870 1 AT
Figure imgf000016_0002
AGGGAGAACCATTTCTTTAGACACAAAAAAAGATGTTAATTTGAAGTACAAGGATGATAGACTAATTTTGGGAAAAGCAAAGATATTCTCCAAAT ATCACAGTGAAATAACTTATTATAATTTTAATTTCTATGAAGCAGGAAGAAGCATTTCTTTGGATACAGGTATCTCAAATGAGTTATCTCAAGAAG
TTAGGAGCTCAAAATTTTTCATATACAAGTCTCATGGAACAAATTATATCATCTCAGCCTTCAATATCTGAAGTTCAGCAAGAAATATCAACAGAA TCAAACTTTGTTGCTACACCAAGAGCATTTCAGAATTCAATGATATTAACAACAAGTAGAACAGAAGATATTCCTTTTGATATGGATTATGCTGAT
TTTATGCTTTGTTGGCTGGATTTTCTGTTGTCATTGCTCACAATTACCATAGTACAAGCAAAAAAAGGAATGGAGAAGTGATTAGAGTAACATTC GCCACAAGTTGTAACTGTGCACTGGTAATTTCTGAGAGAAATTTGATTTGGAGAATTACAAGGGTTAATTTAGATCACAATTATAAGATGAGTCC
AGGCATATGATTGTGATTTTGTCAACTTTGAGAGGAGGGCTGACATCTTTGCCTTATACTAAGAAGGATGTTAGTAATGTGAGAACTTGTATAAA
TAAAGAGACAAGTAGCAATGATATGATGCAAGTTCTTCAATTTTTCAGAAAGAAGAAGGAGAAAGATCCAAAGTTCTTTTATGAATTTGATTTGG
ATGAGAATAAGAAGGTGACAAATTTGTTTRGGACTGATGGAAGGTCAATTGATTGGTACGAGAAGTATGGAGATGTTGTTAGTTTTGATACAAC
ATATTTCACAAATAGATACAATCTTCCATTTGCTCCATΓTGTTGGGATTTCAGGACATGGAAATACAATTGTTTTTGGCTGTGCCTTTCTGCATGA
TGAGACTGCAGAAACATTTAAATGGTTGTTCATAACTTΓTTTGAAAGCAATGTCAAAAAAAGCACCAAAGACAATTATAACTGATCAGGACGGAG
CTATGAGCACAGCTCTAAGTGAAGGTACAAATTCAAGGTTTAAGAAAGATGTTGGTCCACAATACAGTATAACAAGCTTTTTGATAGAATATGC
AGAAATTGAAGACTTATGAAGTTTTTCTTGCATTGAATCAGAAAATAAAAGTAGTAAGAAGAAGGAAGTACCTTGTAATTGTTGACTCTGAAAAG TCCAGAGAAATATATTATCGATCGGTGGAGAAAAAAGGATTACAAAGAAAAATCTGATTTTGAAGATAGAATTATACCTTTATCGGAGAGCTCAG
TTAGAGAGGAATTACAGAGTAAAGAAGTTTATCATTGCAGTCATTGTCAAAGAACAGATCACACTTTTCCAACATGCCCTCTAAAGCACATTGAA SEQ ID NO 87: LOC_Os04
Figure imgf000017_0001
TAGCAGTGTTACATATAGCTTGA SEQ ID NO 89: LOC_Os04g34970 1 ATGGCGTTGTCGGAGCCGGACCTCGCGGCGGAGACGGAGGCGAT SEQ ID NO 90: LOC_OS04
Figure imgf000017_0002
LOC_Os04g42950.1. ATGGCGGCGGCGGAAGTGCAG
SEQ ID NO 95: LOC_Os04g45340
1 ATGAATGGTCGAACACAGCTAGCTAGCTGG
ICCACCACGAC
99: LOC_Os04g478608 ATGGCATCGAATTCATCG SEQ I
SEQ ID NO 102: LOC_Os04g47860 5 ATGGCATC
Figure imgf000018_0001
ATGGCATCGAATTCATCGGCTGCAGCTGCGG
Figure imgf000019_0001
GTGAAAGCATCTTATCGATCTCACAGGCGCGCTTGA SEQ ID NO 106: LOC_Os04g48350.1 : ATGGAGTGGGCGTACTACGGCAGTGGGTACT
GGGGAGGACCAAGTTCAAGGAGACGAGGCACCCGGTGTACAAGGGCGTGCGCAGCAGGAΛCCCCGGGAGGTGGGTCTGCGAGGTGCGCG AGCCGCACGGCAAGCAGAGGATCTGGCTCGGCACCTTCGAGACCGCCGAGATGGCGGCGCGCGCGCACGACGTCGCGGCGATGGCGCTG
ATGGATGATCACATGGGAAGACGGAC
SEQ ID NO 110: LOC_Os04g51400.4: ATGGATGA
Figure imgf000019_0002
SEQ ID NO 111 : LOC_Os04g51400.5: ATGGATGATC
Figure imgf000020_0001
CAAGGGTGTTTTTCGCAGTTCATTGATGGTCTTGATTGGATGACTCAGCCTTCCTACTACCAARACTCCA.ΛTGTAATTCAACCAGCCGGTCiTGT CAGAGAACATTrTGAGTTCTTCTGCAGATATACCTCCCTCAATGArTGCTGATACTATGGAGACTTTTCAGGCTTCATGTCTTTCTGATTGCCTT
TGATGGAAGCAATTTGCCAAGCACGTCCAACTCGTTTGTACAGATGAGTTTTTCTGAAGAGAGTGCAAGTCAGAGTGCAAACTTAAGTGGACTT
SEQ ID NO 114: LOC_Os04g5
Figure imgf000020_0002
TGGCTATCATGCTTCTCAACCAATCTGGTCAAACCCTTGGGAGTCCCCTTAGTTTTCACCAGTCCTCATATTCTAGTATTATCCAAAATGTGAAG
GCTTGCCTCACAAAACCGTTTCAGAGAAATCTGACCTTTCTTCAGCCCCTTCTTGGATCTGTGACAATCAGCAGGTTGGTTTAGAATCGAAACT GGTTGGTTGTGATGAACAAGTAAACTGTGGAAATATTGAAGATTCATCAGGTGCACTTACTCAAGGTAATTTTGTTGGACAGCCGCATGGCCAT
SEQ ID NO 115: LOC_Os05g03900.1 : ATGGAAAA
Figure imgf000020_0003
Figure imgf000021_0001
AATCAACAAAGI I I I I GCTGATGAGAATGAAGCTTTTGACTTCTACAATGGTTATGCTTATATGGTTGGTTTCTCTACATGCAAAGCTAGCAATT
AAGCTGAGATGGTGATAACATTGAAGAGGGGCTTTTGGTACATTACAAGGCTTAATCTAGAGCACAATCATCCACTATCTCCACCAGAAGAAAG AAAATTCCTATGGTCTCACAAACATATGATAGACCAGGAGAAACTTTTGATAAGAACTTTGAACAAAATCAATGTGCCCACAAGAATGATTATGT CTGTTTTGTCATATGTTAGGGGTGGCTTGTTTGCAGTTCCTTACACAAAAAAGGCTATGAGCAATTATAGAGATTTTGTTAGAAGAGAAAGTGG GAAAAATGACATGATGCAGTGTTTGGAI I I I I I I GAGAAGAAAATATCAGAGGATCCATTATTTTATTTCAGATTCAGAACTGATGAGAATAATG TTGTGAAGAGTCTGTTTTGGTCCGATAGGAACAGCAGAAAATTTTATGAGATGTTTGGTGACATAGTTAGTTTTGACACGACGTACAAGACAAA
TGGCAATGAAGGCTGCTATTGTCATAGTTTTTCCGGACACTGTTCATAGGAATTGCATGTTCCATATGTTATCAAATGCAAGGGACAAAACAGG
SEQ ID NO 117: L
ATGTCGACGCCGACGGTGCAGATGGTGGCGCCG
Figure imgf000021_0002
ATGTGTGGGGGAGCGATCATCGCCGACTTCGTC
ATGGCAATGCAGCTGTCCTTGCCTGTCTTGCCAACGG
SEQ ID NO 121: LOC_Os05g34730.1
SEQ ID NO 122: LOC_Os05g34830.1: ATGA
SEQ ID NO 123: LOC_Os05g
Figure imgf000021_0003
SEQ ID NO 124: LOC_Os05g34830 3 A
SEQ ID NO 125: LOC_Os05g36100 1 ATGGCCGA
Figure imgf000022_0001
TCTTGAG I I I I I CCAATCGGAGATTCAGAATCTGTAG SEQ ID NO 130 LOC_Os05g50350 1 ATGGCGTCGGCGGCCGGCTCGAAGCAGCAG
GACGGAAACAGATCTAGGTACCTGAAGTATCAGTGA SEQ ID NO 131: LOC_Os05g50700 1 ATGACCATCACACACGCTAGCTCTCTTTCTCG
GAGTTCGTTGGGCAACCGGTGAGGTCGTCGGCGGCATCGTTTTACGACTTGGAGTACTTGGACCTGTACCACGAGCGGCCTCGGGCGCCGT
ID NO 132: LOC_Os05g51040 1 ATGGCAAACAATC
Figure imgf000022_0002
: ATGGGGCGAGGGAAAGTAGAGCTGAAG
137: LOC_Os06g07440.1: ATGGACGACGATCCTACCTC
Figure imgf000023_0001
Figure imgf000023_0002
GGTGTTTATTATCCAACATCTCCATTAATCATGCATCACATTTTAGAGATTGCTGGACATCTAAACACATATGGCAATGTTCAAAACCTTGCAAA TGTTGTTGGTCCCATGAAAACTAAGTTTATGAACTACTGGTCTAAAATTCCAATTCTTTATTCATTTGCATTTATCTTGGACCCTAGGGCCAAGAT
SEQ ID NO 139: LOC_Os06g10880.1: ATGGAGTTG
SEQ ID NO 140: LOC_Os06g108
ATGAGCAGCACGGCGAAGGCGGCGG
Figure imgf000023_0003
SEQ ID NO 144: LOC_Os06g28630.
SEQ ID NO 146: LOC_Os06g34860.1: ATGAGTAT
ID NO 147: LOC_Os06g41060.1: ATGGAGCTTCTCAAG
CATCGGCCTTGAGA
Figure imgf000024_0001
GGAAAAACTTGGTAGTGGCCGATGTGTTAGGAAGGGTCCCTTTGAATGTTTCTTTTAG SEQ ID NO 149: LOC_Os06g44010.1: ATGGCTAAGA
GCGCArrTCGGCTCTCGCCTTCTTCGCCTTCTTCTCCTCCTCCTCCTGTTCTTGATTTTCAGTATATTCAATTCATGGATTCGTGGATTGAGCAG
Figure imgf000024_0002
Figure imgf000025_0001
50
AGCAGCAATACCAAGAGGCATTGATTTTGAACTCGATCAATAAAGGAAGTTCCACCGGTGACTTCAGTGCTGTAGATATGGCATATGCACTGA CGAATACTGCTGGCAATTTTGAGGTTACTCCCGAAAGTCTTAGATCGGTCCTGCCATTAATAAACTGGGACAAAATAGCTGCTATGTACCTGCC
NO 153: LOC_Os07g047004: ATGAACACAAAAACTG
Figure imgf000025_0002
SEQ ID NO 154: LOC_Os07g047005 ATGAACA
Figure imgf000026_0001
GTCAGATGTTGAGAAAGAGAAACTTGCTAAAGGAATAAAGCAGCAATACCAAGAGGCATTGATTTTGAACTCGATCAATAAAGGAAGTTCCACC
GGTGACTTCAGTGCTGTAGATATGGCATATGCACTGACGAATACTGCTGGCAATTTTGAGGTTACTCCCGAAAGTCTTAGATCGGTCCTGCCA
SEQ ID NO 155: LOC_Os07g04700
Figure imgf000026_0002
GTACAAAATGGTACTGAAGCAACTCCCTATGTCTCTTCAGAAGCAACCGTGGTCAGΔTGTTGAG.ΛAAGAGAAACTTGCTAAAUGAATAAAGCA
SEQ ID NO 157: LOC_Os07g26720 1 ATGAGGGT
SEQ ID NO 158: LOC_Os07g
Figure imgf000026_0003
2 ATGCAGCCGGTACGGCCATCTGACGGAGTTCGA
SEQ ID NO 160: LOC_Os07g37210 1 A
Figure imgf000027_0001
ATAAATGGATATGGCAGACCATTGTCATTATTCCTCGGAGTGAATCATCACAAACAAACAATTGTTTTTGGTGCAGCTATGCTTTATGATGAATC ArrTGAGTCATATAGGTGGCTGTTTGAGAGTTTTAAGATTGCTATGCATGGAAAACAACCAGCGGTAGCTTTAGTAGATCAATCTATTCCACTTG
Figure imgf000027_0002
2 ATGCGAGTGTGGCTTliOCAGGGTT
Figure imgf000027_0003
Figure imgf000027_0004
ATGCAGATTGATGAAGACGACAAGCTAACCAACTTCTTTTGGGCTGACCCAAAATCTAGAGAAGACTTCAACTACTTTGGTGATGTCCTCTGTC TAGACACAACTTACAAAATAAATGGATATGGCAGACCATTGTCATTATTCCTCGGAGTGAATCATCACAAACAAACAATTGTTTTTGGTGCAGCT ATGCTTTATGATGAATCATTTGAGTCATATAGGTGGCTGTTTGAGAGTTTTAAGATTGCTATGCATGGAAAACAACCAGCGGTAGCTTTAGTAG
GCACCTTAATCATGTTTTCCAAGGTTCAAAAACATTΓGCAMGGATTTTAGCAGATGTGTATTTGGCTATGAGGAAGAGGAAGAATTCTTGTTTG AGAGACATATATTTTGTGCTGACATAATAAGTGCACTTCAAGCTGAAAGCTTCAGTAGTGTTCTGAAAAAGTTCTTGGGCCCTCAACTGGATTTA
GTGAGAAGAAAATTTCGCTACTCCGCGTTCACAAAAT
Figure imgf000027_0005
I I I I ICTTGTG
ATGGGCAGGTCGTTCAGGGACTCGCTGAAGGTGCT
SEQ ID NO 169: LOC_Os07g4
Figure imgf000028_0001
SEQ ID NO 171: LOC_Os08g09800 1 ATGGAG
Figure imgf000028_0002
I I I I I GCTGGCATCTGTCCTACATATAATATT
GTGCCACAGGCTGCAATTGAAGAGAAGTTCCTCAGCATΠTTCAGAATGAAAAGATGAAGCTAAAAGAGGAAATAGCACTTAAACCTGGAGGA
GATCATAAGATTCCAATTTTCTGGAGATGAGGCCCTTGAAGCTGAGCACTATGTCAGTATTCTGTCCAACTGGAAGTCCTTCTCGAATATAAGA
^GCTTTTAGGAA
GAGTACATGACATGCTCAGCAATTCGTCTGGAAAAATACAAGGAAATTTTGTTGCGCCTGCACCTAAGCCAACCATCTTTTGGTTCACAGAAAT
ACCATTTCCTGAAAGACTAGAAGCAACAAAAAACTTTTGTGATCTTGCAAGGCCAATTTACCATGCAATTGATGTACTTTCCAGACAGAACGTG
NO 172: LOC_Os08g 13840 1 ATGGCCGTGGACCTCA
Figure imgf000028_0003
ID NO 174: LOC_Os08g 15050 1 ATGATGGCAAGC
Figure imgf000029_0001
TCCCTATGCCAACGCGAGAACGAAGACTGCCATCATTTGCTTGTGTCCTGTGATTATACGGCGGCGGTTTGGCGCAAGCTGAGACGTTGGTG
CAACATTAACATTGCAATCCCTGCGGAAGATGGCAGGCCGCTTGCAGATTGGTGGATCACGACAAGATGGCGTTTTCAGAACACTAGCAAGAA
AGTTAAGAAAGAGGTCCATCCTTCTTCCCAGTACATTCATGAGCACAATGCACTTAATAAAGCAAGACAAGGTTTTCCTGTTGAACTGGGAGGC
ATGATCTGAAGTTTCAGGGCTTAGTTAAAGAGCCCACTCAGGTGTGTTTTGCAGCAGAAGCCATGGTACAACCTTTCTGA SEQ ID NO 176: LO C_Os08g34360 1 ATGGCCAAGCGACGCAGCAACGGCGAGACCGCCGCCGCGAGCAGCGACGACTCTAGCTCCGGCGTCTGCGGCGGCGG CGGCGGCGGTGAGGTTGAGCCGAGGCGGCGGCAGAAGCGGCCGCGGAGGAGCGCCCCGCGRGΔTTGCCCCTCCCAGCGCAGCTCCGCU TTCCGCGGCRTCΔCΔCGGCΛCCCGTGGACGGGGCGGTTCUAGGCGCATCTCTGGGACAAGAACACCTGGAACGAGTCGCAGAGCAAGAAG
ID NO 178: LOC_Os08g37290 1 ATGGTGACGGACG
1 ATGGTGCCGCCGGCGGCGCACGCGCCGA
Figure imgf000029_0002
2 ATGGGGAGGGGTCGGGTGGAGCTGAAGAG
SEQ ID NO 183: LOC_Os08g43210 1 ATGTGTACG
1 ATGGAGCGAGCGGCTGGTACTGGTAGCGGCGGCG ID NO 185. LOC_Os08g45110 1 ATGATTCTGATAC
SEQ ID NO 186
ID NO 187: LOC_Os09g16510 1 ATGGAGAGCATGG
Figure imgf000030_0001
Figure imgf000030_0002
SEQ ID NO 190: LOC_Os09g25
ATGTTGACTACAAAGCTTGTCCAACATGATGGACCT
Figure imgf000031_0001
ATGACGCTGAAGCCnGCA-AGATCCGTGGCAAGAAAGCt-AAGGTCAACTTTCCTGATGAACCAGCTGTTGCTCAGAAGCTCTCCCTGAAGCAA 195 LOC_Os09g28210 1 ATGGACTTCGACTTGTTCA
Figure imgf000031_0002
Figure imgf000032_0001
AAGCGCCACGTGCTCAAGGTGTGGATGCACAACAACAAGCACACCCTGGGCAAGAAGCTGCCATGA SEQ ID NO 198: LOC_Os09g29930.2:
I I I I I ATCCTCCCAAGCCAGCGCTGCTTGA
I CCAGGGA ATGGCAGGCTTCTCACACCTCCCACAGCAGATGGAG
Figure imgf000032_0002
NO 205: LOC_Os09g38300.1: ATGCCGCGAGCTCTAC
SEQ ID NO 206: LOC_Os10g02910.1: A
Figure imgf000033_0001
CAATCACTAGAGCTTGTGTTAAAGTATATGAAGATGAGAAAGAAAAGCTTAAGAAATTTTTTAAGGACAATTGTGTAAGAGTTTGCCTCACAACT
55 I I I 1 1 I IGGTTAAGGGACATAGAGGGGAGGACATTGGAAAATCATTAGAGAACTGCTTGGCTGAGTGGGGCATTGACAAGGTmTACAATAAC
Figure imgf000033_0002
AATTAGAATTAGAAGGAGAGAATGGTCCAGGGGTACCAACAAGAGCAGATTGGGAGAAGGCTAGGAAGATGGCTGATTTTCTTGAACACTTTT SEQ ID NO 207: LOC_Os10g22430.2: ATGTCAGCTA ID NO 208: LOC_Os10g22430.1: ATGTCAGCTAGGGC
Figure imgf000033_0003
NO 212: LOC_Os10g26270 1 ATGGCGTGTGCAGAGA
Figure imgf000034_0001
ID NO 213: LOC_Os10g26500 1 ATGGCCAGCAATGG SEQ ID NO 214: LOC_Os10g30420 1 ATGGTCTC
Figure imgf000034_0002
TTGTAGCTGAGTTTGCAGACCCCAACAATAATTTTGCATCACCTGATCCTGACAACCCTAACACACCACAATTTGATGAGAAAAATATACGACG
70
Figure imgf000034_0003
AGACAGCGTGACCCGGCAATGGCAGCTCCCGATCTTTCGTACGCTGGATGGCGGCATTTTCGATGACGATGAACAGTTGGATCTTCATCCTG
Figure imgf000034_0004
LOC_Os10g32750.1: ATGGAGAGAGAGACCGATCCG
I CGGCGTCAGCTCGAGGACG SEQ ID NO 220: LOC_Os10g424
Figure imgf000035_0001
ATCA
Figure imgf000036_0001
GCTGGCTTCCrrTTACCCATCAGCAGGTGTTTGAGCTTCTTGCAGATGAACAACAGCGCTGCCAGCTTGAGATTTTGTCAAACGGCGGCTCCC
SEQ ID NO 222: LOC_Os11g02530.1: ATGAAGA ATGGTGGAGCCTGTGAAATCGGAGCTTGGCAGC ID NO 225: LOC_Os11gO841O 1 ATGCCGAAGCCGA
Figure imgf000036_0002
Figure imgf000037_0001
CTAAGAATCCTTTTGCCGTGCAAATAATGATGGAGTCATATGTCTATGTTGGATTTTTCATGAATATCCCATGTGAATTTGTCCGTGAGTGTCTT GTGCACATCTACAGAGTTGTCCCAGAAATTACTCCGCACAAACTCCGTTCTGACCCGAAGTAA SEQ ID NO 227: LOC_Os11g11220 1 ATGG
CATGGGTTTTAGCATTCGAAGAAGCAGTTACCATTATGTGAAAGATTCCACCATAATAAAGAATCGGACATTTTGTTGTTCTCGTGCAGGTACT CGTGGTCATGATAAAAGAGAAGACCAAATTTCAAATTACGGTCAGTGTTTTAGTAGGCCAGAAACACGATGTATGTGCCGAGCATGCATGAAG
GATGGTCGTCCATTTGGTTTGCTTGTTGGTGTGGATAATCATAAGAAAAGCGTTG i I I I I GGTGCTGCACTΠTGTATGATGAAACTGCTGATA GTTTTGTΓTGGCTTTTCAAGACATTTTTAAAAGCCATGTCTGGAAAGAAGCCACAGACTATATTGACTGATGAAGACGCAGCAATGGCCAAAGC
SEQ ID NO 228: LOC_Os11g47870 1 ATGGCC
Figure imgf000037_0002
GGCGAGAGGGGACGCGACGCAGAGGCTGGCGTGCTGCTTCGCCGAGGGGCTGGAGGCGCGGCTCGCCGGCACGGGGAGCCAGGTGTAC
1 ATGGAGACCATGTCATACCCTTGC
Figure imgf000037_0003
Figure imgf000038_0001
GTCCAAAAGAAAAAACCATCTTAG SEQ ID NO 234: LOC_Os12g13130 3 ATGCCGAGGCGGGAGGGTGGTCGCTCTCGCTCCGCCGCCTAC CTCGTCCTCTTCGCTTCCTGCCTTTTAGCTGTTGCGGCGGCATCACACCAGGAATTTCATGAAGCTGCTGGATCCAGAACTCTTCTTATGTCAC
Figure imgf000038_0002
TCTGTGCCTTAACAATAGTACTTTTGCCACTGTTCTATCTCATAGTATTCTTGCACCAGAGGGAAATGAAGAAAGGCGGTCAAAATTTAAGACG AATTTCGAAAGTTGTCCAAAAGAAAAAACCATCTTAG SEQ ID NO 235: LOC_Os12g131304 ATGCCGAGGCGGGAGGGTGGTCGCTCTCGC TCCGCCGCCTACCTCGTCCTCTTCGCTTCCTGCCTTTTAGCTGTTGCGGCGGCATCACACCAGGAATTTCATGAAGCTGCTGGATCCAGAACT
LOC_Os12g18120 1 ATGGCGGACGCCGACGCGA
Figure imgf000038_0003
ITGACCTGAACTTTCTC
Figure imgf000038_0004
CATCAGCAGGTTCCTTCCGAGGAATACCCAGAGAGACCTGGCCAACCTGMTGTCAGCATTTTGTGAAGAGTGGTTTTTGCAAATTTAGGATG
ACATACTATGGTCGCTATGGGGTCTGCAAATTTGGGCCAGCTTGCGCATACAATCACCCCTTCAATTTTAGCCCTGTACCTGCAGCGGGACCT 238: LOC_Os12g18120 4 ATGGCGGACGCCGACGCGA
Figure imgf000038_0005
CAAGGAATGTATCCACCTCCTGAATGGAATGGGTATCATCAGGTTCCGTTGAATCCATATTATCCTCCTGGAGTCCCTTTCCAACATTTTCCAG
CTGMTGTCAGCATTTTGTGAAGAGTGGTTTTTGCAAATTTAGGATGAAGTGCAAATATCACCACCCAAGGTCACCGGTGCCTCCAGCAGGAG CTCTTAGCCCTCTTGGCTTGCCTATAAMCCTGTATGTTCCTTCTTCCATTTATTTCCTCMTTTTAG SEQ ID NO 239: LOC_Os12g18120 3 AT GGCGGACGCCGACGCGAGGGCCCCGCCCAMTCGGACCCCGGCGCCACGCCGATCGGTTCGATCTCCCCTTCCTCCGCCGCCCCCGCCG NO 240: LOC_Os12g22630.1: ATGGCGACCAACCTCC
ATGGCCAGCCTCGATCTTCTCCTCAAGCTAGGGTT
CCAGGA
Figure imgf000039_0001
CGTCGGGGAATGTGTTGTGA SEQ ID NO 246: LOC_Os12g38950 1: ATGGCTTCTGATGTTCCTCAAGATGACGTGCAATGCCATTTTTGTGGC ATACCAACATCTGCTACAAGAGCGACGCATTCAGAAATGGCTTCTGATGTTCCTCAAGGTTACGTGCAATGTCATTTTTGTGACACCTACTTAA
Figure imgf000039_0002
SEQ ID NO 2
ID NO 248: LOC_Os12g40070 1 ATGGGTGATCAAAAA
Figure imgf000040_0001
GCCCAAAGTCATCAGAATATACCCTTTCCACCAAGAGTCCTACACGTAGAGTTCCTAAGCGGGACTATTTTGCAACAAATCTTACTAATCTAAC
GA SEQ ID NO 249: LOC_Os12g40120 1 ATGGGTGAGAAGGGGTGTGAGAGTTGCAGAGAGTGGCAGGAGCACTGCTACAGGGAGCACATG GATGTGAGCAGGATTCGCTTCTTCAGGCTCATGACTGGAGATTTTGCTCATGGCATTAGCATACCGGAGAAAGTTGCGGACAGATTCAGTGGT
AGGATGGGAGGATTTTGCCAAGGCTCATGAATTGCAGGAGAATGACCTCCTGTTCTTCACTTGCAATGGCCGTTGCAATGGCAGCTTCTCCTT
Figure imgf000040_0002
TTCTACTGGGCTAA SEQ ID NO 250: LOC_Os12g424Q02 ATGATGAGCTTCAACAAGAGCCAAGAAGGATTTGGGCAGGTTGCTGCTGTGGC
CAGGTAAAGGAGAAAAATGTTCTGAGCACTCAACTACTATTGCTCTGCAGTCACCATTTGCAGAATATAATGGCTGTTTTGAGCTGGGCCTTGG CTTATTCCACCAAATATGCCAGCTGATGCACrAATTTΔTGTGΔ.-.τGc.Λ«ΛΛACAGTGTTCAGCCATCATTCGACGTCt.CCATGCTCGTGCCAAGG
ID NO 251: LOC_Os12g42400 3 ATGATGAGCTTCAA CCATTTGCAGAATATAATGGCTGTTTTGAGCTGGGCCTTGGTCAATCTGTGGTTCCCTCTAATTATCCTTATGCTGACCAGCACTATGGCCTAC
1 ATGGCAGCCAATGTTGGGGAATCCACAAG
Figure imgf000040_0004
CAGATGCAACCGCCTATGGGCAGCCTGCTGGTTTTCCCTACGGATATGGACACGGCCACGGTCACGGTCATGGGCACGCGTTCCACGGCGG GGTTTTCTTGTGATTGCAAGCCTCATCACATTCTAG
Suφrisingly the invention solves the above technical problem by providing an isolated polynucleotide selected from the group comprising: (a) polynucleotide consisting of a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71,
SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ ID NO. 111, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ ID NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 139 to SEQ DD NO. 141, SEQ ID NO. 144, SEQ ID NO. 146 to SEQ ID
NO. 148, SEQ ID NO. 152, SEQ ID NO. 157 to SEQ DD NO. 159, SEQ DD NO. 162, SEQ DD NO. 165 to SEQ DD NOl 67, SEQ DD NO. 170, SEQ DD NO. 171, SEQ DD NO. 180, SEQ DD NO. 183 to SEQ DD NO. 186, SEQ DD NO. 190 to SEQ DD NO. 194, SEQ DD NO. 197 to SEQ DD NO. 200, SEQ DD NO. 205, SEQ DD NO. 208, SEQ DD NO. 207, SEQ DD NO. 214, SEQ DD NO. 216, SEQ DD NO. 217, SEQ DD NO. 219,
SEQ DD NO. 225, SEQ DD NO. 228, SEQ DD NO. 231 to SEQ DD NO. 239, SEQ DD NO. 242 to SEQ DD NO.SEQ DD NO. 249,
(b) a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90, especially preferred 98% sequence identity to a sequence selected from the group comprising SEQ DD NO. 1 , SEQ DD NO. 2, SEQ DD NO. 14 ro SEQ DD NO.
17, SEQ DD NO. 20 to SEQ DD NO. 23, SEQ DD NO. 25, SEQ DD NO. 27, SEQ DD NO. 40, SEQ DD NO. 41, SEQ DD NO. 44 to SEQ ID NO. 46, SEQ DD NO. 48, SEQ ID NO. 52, SEQ DD NO. 56 to SEQ DD NO. 58, SEQ DD NO. 61 to SEQ DD NO. 67, SEQ DD NO. 69, SEQ DD NO. 71, SEQ DD NO. 75, SEQ DD NO. 78, SEQ DD NO. 79, SEQ DD NO. 81, SEQ DD NO. 84, SEQ DD NO. 85, SEQ DD NO. 87, SEQ DD NO. 88,
SEQ DD NO. 95, SEQ DD NO. 98 to SEQ DD NO. 111 , SEQ DD NO. 117, SEQ DD NO. 120, SEQ DD NO. 122 to SEQ DD NO. 124, SEQ DD NO. 131 to SEQ DD NO. 133, SEQ DD NO. 135, SEQ DD NO. 136, SEQ DD NO. 139 to SEQ DD NO. 141, SEQ ID ..
NO. 144, SEQ DD NO. 146 to SEQ DD NO. 148, SEQ DD NO. 152, SEQ DD NO. 157 to SEQ DD NO. 159, SEQ DD NO. 162, SEQ DD NO. 165 to SEQ DD NO 167, SEQ DD
NO. 170, SEQ DD NO. 171, SEQ DD NO. 180, SEQ DD NO. 183 to SEQ DD NO. 186, SEQ DD NO. 190 to SEQ DD NO. 194, SEQ DD NO. 197 to SEQ DD NO. 200, SEQ DD NO. 205, SEQ DD NO. 208, SEQ DD NO. 207, SEQ DD NO. 214, SEQ DD NO. 216, SEQ DD NO. 217, SEQ DD NO. 219, SEQ DD NO. 225, SEQ DD NO. 228, SEQ DD NO. 231 to SEQ DD NO. 239, SEQ DD NO. 242 to SEQ ID NO.SEQ DD NO. 249,
(c) a polynucleotide of (a) and/or (b), wherein said sequence is modified, to alter an abiotic stress tolerance of a plant, preferred osmotic tolerance and/or salt tolerance. Polynucleotides of the present invention that are variants of the polynucleotides provided herein will generally demonstrate significant identity with the polynucleotides provided herein. Of particular interest are polynucleotide homologs having at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, and 5 more preferably at least about 90%, 95% or even greater, such as 98% or 99% sequence identity with polynucleotide sequences described herein.
The term "homology" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (in other words, identity). "Sequence identity" 0 refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences. A partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid. The inhibition 5 of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (in other words, the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are 0 such that non-specific binding is permitted; low stringency condiiiυns require that the binding of two sequences to one another be a specific (in other words, selective) interaction. The absence of nonspecific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (for example, less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
.5
Depending on the intended use, the polynucleotides of the present invention may be present in the form of DNA, such as cDNA or genomic DNA, or as RNA, for example mRNA. The polynucleotides of the present invention may be single or double stranded and may represent the coding, or sense strand of a gene, or the non-coding, antisense, strand. i0
The term "isolated" is used herein in reference to purified polynucleotide or polypeptide molecules. As used herein, "purified" refers to a polynucleotide or polypeptide molecule separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified
15 molecule may be greater than 60% free, preferably 75% free, more preferably 90% free, and most preferably 95% free from the other molecules (exclusive of solvent) present in the natural mixture. The term "isolated" is also used herein in reference to polynucleotide molecules that are separated from nucleic acids which normally flank the polynucleotide in nature. Thus, polynucleotides fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered isolated herein. Such molecules are considered isolated even when present, for example in the chromosome of a host cell, or in a nucleic acid solution. The terms "isolated" and "purified" as used herein are not intended to encompass molecules present in their native state.
In terms of the invention abiotic stress is the negative impact of non-living factors on the living organisms. The non-living variable influences the environment beyond its normal range of variation to adversely affect the population performance or individual physiology of the organism. Abiotic stress factors, or stressors, are naturally occurring, often intangible, factors such as intense sunlight or wind that may cause harm to the plants and animals in the area affected. Abiotic stress comes in many forms. The stressors include: high winds, extreme temperatures, heat, cold, strong light, water deficit, drought, flood, and other natural disasters, such as tornados and wildfires, poor edaphic conditions like rock content and pH, high radiation, compaction, contamination, non-optimal nutrient or salt levels, non-optimal light levels and other, highly specific conditions like rapid rehydration during seed germination.
As used herein, the terms "tolerant" or "tolerance" refers to the ability of a plant to overcome, completely or to some degree, the detrimental effect of an environmental stress or other limiting factor. In embodiments of the present disclosure, the transgenic plants are preferred tolerant to conditions including, but not limited to osmotic stress, particularly salt stress.
"Expression" means the production of a protein or nucleotide sequence in the cell itself or in a cell-free system. It includes transcription into an RNA product, post-transcriptional modification and/or translation to a protein product or polypeptide from a DNA encoding that product, as well as possible post-translational modifications.
Further preferred is an isolated polynucleotide selected from the group comprising:
(a) a nucleotide sequence encoding a polypeptide, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 14 to SEQ DD NO.
17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ
ID NO. 111, SEQ ID NO. 1 17, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ ID NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 139 to SEQ ID NO. 141, SEQ ID NO. 144, SEQ ID NO. 146 to SEQ ID NO. 148, SEQ ED NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NO167, SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ ID NO. 217, SEQ ID NO. 219, SEQ ID NO. 225, SEQ ID NO. 228, SEQ ID NO. 231 to SEQ ID NO.
239, SEQ ID NO. 242 to SEQ ID NO.SEQ ID NO. 249,
(b) a nucleotide sequence encoding a polypeptide, wherein said polypeptide is selected from the group consisting of SEQ ID NO. 254, SEQ ID NO. 255, SEQ ID NO. 267 to SEQ ID NO. 270, SEQ ID NO. 273 to SEQ ID NO. 276, SEQ ID NO. 278, SEQ ID NO. 280,SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 297 to SEQ ID NO. 299, SEQ ID NO. 301, SEQ ID NO.
305, SEQ ID NO. 309 to SEQ ID NO. 311, SEQ ID NO. 314 to SEQ ID NO. 320, SEQ ID NO. 322, SEQ ID NO. 324, SEQ ID NO. 328, SEQ ID NO. 331, SEQ ID NO. 332, SEQ ID NO. 334, SEQ ID NO. 337, SEQ ID NO. 338, SEQ ID NO. 340, SEQ ID NO. 341, SEQ ID NO. 348, SEQ ID NO. 351 to SEQ ID NO. 364, SEQ ID NO. 370, SEQ ID NO. 373, SEQ ID NO. 375 to SEQ ID NO. 377, SEQ ID NO. 384 to SEQ ID NO. 386, SEQ ID NO. 388, SEQ
ID NO. 389, SEQ ID NO. 392 to SEQ ID NO. 394, SEQ ID NO. 397, SEQ ID NO. 399 to SEQ ID NO. 401, SEQ ID NO. 405, SEQ ID NO. 410 to SEQ ID NO. 412, SEQ ID NO. 415, SEQ ID NO. 418 to SEQ ID NO. 420, SEQ ID NO. 423, SEQ ID NO. 424, SEQ ID NO. 433, SEQ ID NO. 436 to SEQ ED NO. 439, SEQ ID NO. 443, SEQ ID NO. 444 to SEQ ID NO. 447, SEQ ID NO. 450 to SEQ ID NO. 453, SEQ ID NO. 458, SEQ ID NO. 461 , SEQ ID NO.
460, SEQ ID NO. 467, SEQ ID NO. 469, SEQ ID NO. 470, SEQ ID NO. 472, SEQ ED NO. 478, SEQ ID NO. 481, SEQ ID NO. 484 to SEQ ID NO. 492, SEQ ID NO. 495 to SEQ ID NO. 502,
(c) a variant of any of the nucleotide sequences of (a) or (b) that has at least 70%, preferred 80%, more preferred 90%, especially 98% sequence identity to a sequence of (a) or (b)
(d) a nucleotide sequence that hybridizes to any of the nucleotide sequence of (a) or (b) under stringent conditions
(e) a polynucleotide of (a) and/or (b), wherein said sequence is modified, to alter an abiotic stress tolerance of a plant, preferred salt tolerance and/or osmotic tolerance.
In another preferred embodiment the invention relates to said isolated polynucleotide, wherein the polynucleotide is selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ED NO. 20 to SEQ ED NO. 23, SEQ ED NO. 25, SEQ ED NO. 27, SEQ ED NO. 40, SEQ ED NO. 41, SEQ ED NO. 44 to SEQ ED NO. 46, SEQ ID NO. 48, SEQ ED NO. 52, SEQ ED NO. 56 to SEQ ED NO. 58, SEQ ID NO. 61 to SEQ ED NO. 67, SEQ ED NO. 69, SEQ ID NO. 71, SEQ ED NO. 75, SEQ ED NO. 78, SEQ ED NO. 79, SEQ ED NO. 81, SEQ ED NO. 84, SEQ ED NO. 85, SEQ ED NO. 87, SEQ ID NO. 88, SEQ ED NO. 95, SEQ ID NO. 98 to SEQ ED NO. 111 , SEQ ED NO. 117, SEQ ED NO. 120, SEQ ED NO. 122 to SEQ ID NO. 124, SEQ ED NO. 131 to SEQ ED NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ED NO. 139 to SEQ ID NO. 141. It was very surprising that these sequences can be used to alter a plant's tolerance to salt stress and/or osmotic stress. These sequences showed particularly good results in rice.
Surprisingly these sequences can be used to stabilize the photosynthetic activity during salt stress and/or osmotic stress, which results in a stabilized photosynthetic yield. These sequences were highly responsive and therefore very suitable for transfection.
In an also preferred embodiment the invention relates to an isolated polynucleotide selected from the group comprising SEQ ID NO. 144, SEQ ID NO. 146 to SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NOl 67, SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ ID NO. 217, SEQ ID NO. 219, SEQ ID NO. 225, SEQ ID NO. 228, SEQ ID NO. 231 to SEQ ID NO. 239, SEQ ID NO. 242 to SEQ ID NO.SEQ ID
NO. 249. These sequences can be used to alter a plants tolerance to salt and osmotic stress particularly well. Plants comprising these sequences or modifications thereof showed a better growth during salt and/or osmotic stress compared to control plants.
The polynucleotides) υf the present invention find particular use in generation ot transgenic plants to provide for increased or decreased expression of the polypeptides encoded by the polynucleotides provided herein. As a result of such biotechnological applications, plants, particularly crop plants, having improved properties are obtained. Crop plants of interest in the present invention include, but are not limited to soy, cotton, canola, maize, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turf grass.
Also preferred is a nucleotide construct comprising said polynucleotide, wherein said polynucleotide is operably linked-to a promoter that drives expression in a plant cell.
The polynucleotides of the present invention find particular use in generation of transgenic plants to provide for increased or decreased expression of the polypeptides encoded by the polynucleotides provided herein. As a result of such biotechnological applications, plants, particularly crop plants, having improved properties are obtained. Crop plants of interest in the present invention include, but are not limited to soy, cotton, canola, maize, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turf grass. In another preferred embodiment the invention relates to a nucleotide construct comprising a one of the mentioned polynucleotide, wherein said polynucleotide is operably linked to a promoter that drives expression in a plant cell.
5 By "operably linked" is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The cassette may additionally contain at least one 10 additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
Such constructs are useful for production of transgenic plants having at least one improved property as the result of expression of a polypeptide of this invention. Improved properties of interest include 15 stress tolerance, preferred abiotic stress tolerance preferred osmotic stress tolerance and/or salt stress tolerance, yield, disease resistance and growth rate.
A construct will generally include a plant promoter to direct transcription of the protein-encoding region or the antisense sequence of choice or gene-specific antisense region of sequence or gene- 20 specific region of sequence appropriate for the design of gene-specific artificial micro RNA. Numerous promoters, which are active in plant cells, have been described in the literature. Further preferred is the nucleotide construct, wherein said promoter is a constitutive promoter., especially preferred a tissue-preferred promoter.
25 Also preferred is the nucleotide construct, wherein said promoter is an inducible promoter, preferred a stress-inducible promoter.
In another preferred embodiment the invention relates to a polypeptide encoded by said polynucleotide and/or said nucleotide construct. 30
Preferred are polypeptides consisting of a sequences selected from the group comprising SEQ ID NO. 254 to SEQ ID NO. 506.
SEQ ID NO. 254: LOC_Os01g08710.2: MFPSPGRAVMALGHHGAARQPPTTMAAAASSSTTSAAAAPATATTTVAFSFQHPTPTPSHHHHHHGVL
EGRHVHSPSRDDDDAARASAEMTFIW* SEQ ID NO. 255: LOC_Os01g08710.1: MALGHHGAARQPPTTMAAAASSSTTSAAAAPATATTTVAFSF Λ f\ Λ
EQ ID NO. 256: LOC_Os01g14010.1 MATSEAAAISNPFAPLTNHQQEHPPPPPPPAKKKRNLPGTPDPEAEVIALSPRTLMATNRFVCEICGKGFQR
45 NVPYDVLSAVRHHAGLKDAGGVGREETRDFLGVGVQALCSSSIHGWI' SEQ ID NO. 257: LOC_Os01g16810 1 MAHEMMGGFFGHPPPPPATA
RGEPPGAVDQRGGAQATTDSHTIPFFDFLGVGAT* SEQ ID NO. 258 LOC_θs01g18440 1 MARKKIVLDRIANDATRRATFKKRRRGLLKKASEL
GGNDQNAWLMNVARNGGDLGALVYSAFASSSSSNTGGAGTSAAGAAAPGPDMMDLANPDMPGFGCPWDDDSAGPSFPPM* SEQ ID NO. 259 LOC_Os01g29840 1 MGEQQQQVERQPDLPPGFRFHPTDEEIITFYLAPKWDSRGFCVAAIGEVDLNKCEPWDLPGKAKMNGEKEWYFYCQKDR SEQ ID NO. 260: LOC_Os01g32920 1 ID NO. 261: LOC_Os01g39020 1 MLKPQTPRARRAA
SEQ ID NO. 265: LOC_Os01g45730
LOC_Os01g47560 1 MGVAVHWRRAGDSLHMGGEP
Figure imgf000047_0001
VLSSFLPRAAAAHHGMTTMGGAAATTTTSHGLNSAISGGGGVSSETTSAVTVAASAQPSSPAALQMQHFMAQDLGLLQDMLLPSFIHGTNQP' SE Q ID NO. 267: LOC_Os01g48060 1 MVGIDLNTVEEEEDEEEGGATGTVTAPAEARAGGAVCLELWHACAGPVAPLPRKGSAWYLPQGHLEHLGA
RRAAEDCFPPLDYSLQRPFQELVAKDLHGTEWRFRHIYRGQPRRHLLTTGWSCFINKKKLVSGDAVLFLRGEDGELRLUVRRAAQLKNASPFPALH
SEQ ID NO. 268: LOC_Os
ID NO. 269: LOC_Os01g48060 3 MVGIDLNTVEEE
SEQ ID NO. 271: LOC_Os01g52540 1 MA
SEQ ID NO. 274: LOC_O LOC_Os01g56070 2 MENRVGESSATAVDGGGGAKDSG
Figure imgf000047_0002
GGLFPLLSFQVHGFPQAAAYGPAAGFPYGYGHSFHGWHGHGFPHQAPQGQHVDVFLKVLLVLVGVLVIASLIVF* SEQ ID NO. 277: LOC_Os01g6 RLYYRCSYREDRQCLASKLVQQENDDDPPLYRVTYTYEHTCNTTPVPTPDWAEQPPPGAAGDAYLLRFGSSAGGGGGGAHQQQTERERQQQN
GGDDNY* SEQ ID NO. 278: LOC Os01g61080 1 MTTSSSGSVETSANSRLGTFSFASASFTDLLGGNAGAGGGGVSRYKAMTPPSLPLSPPPVSP SSFFNSPIGMNQADFLGSPVLLTSSIFPSPTTGAFASQHFDWRPEVAAAQSADQGGKDEQRNSYSDFSFQTAPASEEAVRTTTFQPPVPPAPLGDE
SEQ ID NO. 281: LOC_Os01 g64590 1 MLPYAPRPP
LOC_Os01 g683704 MDASAGSSAPAAEIRGEAARDDV
SEQ ID NO. 287 LOC_Os01g70110 1 MAAEAASGGG
SEQ ID NO. 289 LOC_Os01g70310 2 MDEAEAAAAAA
SEQ ID NO. 291: LOC_Os01g 72370 2 ME
Figure imgf000048_0001
EGLHFISSSTSSGFGNRTFYSIHLQRSEGTINEECPAFCERLEKWRNKAKL* SEQ ID NO. 292 LOC_Os01g72370 1 MEQLFVDDPAFASSMSSL 75 TFYSIHLQRSEGTINEECPAFCERLEKWRNKAKL* SEQ ID NO. 293 LOC_Os01 g72490 1 MADVGMVWTPAASFHHTHHHHHHHEAAAAAAAA
GGGSGGGPHILGGSSYGNTMN* SEQ ID NO.294 LOC_Os01g72490 2 MADVGMVWTPAASFHHTHHHHHHHEAAAAAAAAAAAAADPIFPLLS
30
GSSYGNTMN* SEQ ID NO. 295 LOC_Os01g73460 1 MGRRALPPSSSSSSSSSTTTTSPELRRKRTAAPPPPPSPRRYRSISDVMRRSLPVDAAP
?5
ESHVLLVACRDIACGEKLYYDYNGYEHEYPTHHFV* SEQ ID NO. 296 LOC_Os02g08070 1 MAVPAAAVAADWDFGYAAPMPPPYVGFDPAGM NO. 297: LOC_Os02g 13800.1: MTTTTAEGGGGVAPFV YAFPWDSGY* 15520.1: MGSECWAHFEKKADNMAECRHCH
SEQ ID NO. 303: LOC_Os02g35560.
SEQ ID NO. 305: L
SEQ ID NO.306: LOC_
SEQ ID NO. 308: LOC_Os02g44790.1: M
SEQ ID NO. 309: LOC_Os02g4 SEQ ID NO. 310: LOC_Os02g46030.1 :
MCGGAILAEFIPAPSRAAAATKRVTAS
Figure imgf000049_0001
NTDMNAGVNLWSFDDFPIDGALP SEQ ID NO. 315 LOC_Os03g08470 3 MCGGAILAEFIPAPSRAAAATKRVTASHLWPAGSKNAARGKSKSKR
GALP SEQ ID NO. 316 LOC_Os03g08470 2 MCGGAILAEFIPAPSRAAAATKRVTASHLWPAGSKNAARGKSKSKRQQRSFADVDDFEAAFEQF
3.317 LOC _Os03g17150 1 MAYGKRPRQQAEEAAFSLFDSSDMARIMLLFSGAHGGGGGAAAASPPERMFECKTCNRQFPSFQALGGHRASHKKPRLADGD
GLAVEFPVWDFPC* SEQ ID NO. 318 LOC_θs03g18340 1 MATRRRAAAPPQPPAWTPEPWSDGETSALLDAWGPRHIRAAGGPLRTADWRAC
DTATAASSSW* SEQ ID NO. 319 LOC_Os03g 19630 1 MDMELSSSSTTVASSPASPPLGRCWRIRLPPAWTPEEDAVLRLAMENGSRHWRRMA PMRRSRRRVDELWMSARARWAHLIDDLLFQEWFLLVGH* SEQ ID NO. 320 LOC_Os03g20870 1 MEEEPSATCRYFCHMCSLIVRPEMGIEEV
RSSRRSSSS* SEQ ID NO. 321 LOC_Os03g21060 1 MVLSNPAMLPPGFRFHPTDEELIVHYLRNRAASSPCPVSIIADVDIYKFDPWDLPSKENYG
LOC_Os03g22170 1 MCGGAIPLISSRGPGGKRSLSAAD MEEMIKPAPAPASSEEEKEWATAATGERRHEEATAGR
ID SEQ ID NO 326 LOC_O
Figure imgf000050_0001
SG 3. 3
29 LOC_Os03g51690 1 MEEISHHFGWGASGVHGGHQHQHHHHPWGSSLSAIVAPPPPPQLQQQQTQAGGMAHTPLTLNTAAAAVGNPVLQLA
\YHEMLV (KGKLPK 3* SEQ I D NO. 330 LOC_Os03g51690 2 MEEISHHFGWGASGVHGGHQHQHHHHPWGSSLSAIVAPPPPPQLQQQQTQAGGMAHTPLTLNTAAAAVGNP
SEQ ID NO. 331 LOC_Os03g51690 3 MCRGGLQVGAPPEVAARLTAVAQDLELRQRTALGVLGAATEPELDQFMEAYHEMLVKYREELTRPLQEA HYKWPYPSESQKVALAESTGLDLKQINNWFINQRKRHWKPSDEMQFVMMDGYHPTNAAAFYMDGHFINDGGLYRLG* SEQ ID NO. 332 LOCJDs RLRAENRELAARLHAVARHGLAARCQNARLRAEAAALARRLLALQRLARGRHMMITASPPQFSRR'SEQ ID NO. 333 LOC_Os03g59460 1 MASS
1 MHCCMSLHPHRRHGDGDVDGSAS
SEQ ID NO. 337 LOC_Os04g27990 1 MPGDHAGAGAA
Figure imgf000050_0002
1 MMNFSSYFYSSSAAAAGGGGGGGEKKSSSSSAS
LOC_Os04g34970 1 MALSEPDLAAETEAIVSALTH 1 MTQWADLDALRPAVAADVQWTSDGKSIAA
Figure imgf000051_0001
VTRARAMSRLAAGREREWPEWAASWARYSSSGGAARLR* SEQ ID NO. 344 LOC_Os04g40630 4 MIERARRGWNAECTIRVLGVSSDAVFAF
SEQ ID NO 345 LOC_Os04g40630 2 MDFWFFSVQGTASPVLERMIERARRGWNAECTIRVLGVSSDAVFAFLQLLYASRVTPEDEEWTAHGPQL RPCRVPLCSHFKGKMRAEKADKTWRLLVKKVTRARAMSRLAAGREREWPEWAASWARYSSSGGAARLR* SEQ ID NO. 346 LOC_Os04g4063 0 3
RLLVKKVTRARAMSRLAAGREREWPEWAASWARYSSSGGAARLR* SEQ ID NO. 347 LOC_Os04g42950 1 MAAAEVQSAAGWGRQLQQDG
HQDAMDDLMMCPAMSTTSSSTAPPSPGIS* SEQ ID NO. 348 LOC_Os04g45340 1 MLHVQEEGWWCRVFKKRVAAVQRAAGDGGDSPFWFN WRDLDKLVASQFGHGDSTAKEPSYCNPVQVFQVEGKQEDSLDYVSTSASCGGEEDLWK'SEQ ID NO. 349 LOC_Os04g45810 1 MNGRTQLAS SEQ ID NO. 350 LO
SEQ ID NO. 352 LOC_Os04g47860 8 MASN
5 MASNSSAAAAAAFFGISRDGDQH
ID NO. 357 LOC_Os04g47860 6 MASNSSAAAAAAFF
Figure imgf000051_0002
SMPARADEQ* SEQ ID NO 358 LOC_Os04g47860 7 MASNSSAAAAAAFFGISRDGDQHDQIKPLISHQQHQHQQQQLAASLTGVATAAPTAASS QGAPPAAPPAKKKRNLPGNPSNQPKYPFTISAMHAYISVLRDLVSIDWSLIICFLTVKASYRSHRRA* SEQ ID NO 359 LOC_Os04g48350 1 MEW
MLIEPPPLAGQSTWAEEDYDCEVNLWSY* SEQ ID NO. 360 LOC_Os04g51400 1 MDDHMGRRTVGGLLFTKGGSILLFREDSARHKATNCCTRH
GEEVGKMVCKHYYHFSCIKNWLRQKNWCPICKSVALNTN* SEQ ID NO. 361 LOC_Os04g51400 2 MDDHMGRRTVGGLLFTKGGSILLFREDSA
RKCSICQEEYSDGEEVGKMVCKHYYHFSCIKNWLRQKNWCPICKSVALNTN* SEQ ID NO. 362 LOC_Os04g51400 3 MDDHMGRRTVGGLLFTK SEQ ID NO. 363 LOC_Os04g514004 MDDHM
5 MDDHMGRRTVGGLLFTKGGSILLFREDSARHK 365 LOC_Os04g56150 1 MTKYKGVRQRHWGSWV
367 LOC_Os04g56850 2 MASSQEKAKTGVLRNAAALL
1 MENHKDHTCKIATTTLVIVPLLLSLHQLYLFFLHL
LOC_Os05g28320 1 MLIHGEGSSSSAAAAGGKIKGS S 1 MCGGAIIADFVPPAGARRAAASDISDNAVLSAAG
Figure imgf000052_0001
AANHHHHHHQQESSGSSSASSLPPTPPPAAEHQLRECMSGLEAFLGLEEEEDDGGAGEPWDAVDMMLE* SEQ ID NO 373 LOC_Os05g34310
1
Dl
SI
MEQMLMDLMDQDFFGNDQPQE* SEQ ID NO. 374 LOC_Os05g34730 1 MGRVAASGGGGGGGEMMRYRGVRRRRWGKWVSEIRVPGTRERL
ASSSGSGVAGDGARKQGTRGEVSDTYWCRNGEDGSRSRSSGSEELIVYEGLSVDDMEILM* SEQ ID NO. 375 LOC_Os05g34830 1 MSGGGE
GPILGQLDPAAAVAGGGDPLLQDILMYWGKPF* SEQ ID NO. 376 LOC_Os05g34830 2 MALYGEKEWYFFSPRDRKYPNGSRPNRAAGSGYWK GPILGQLDPAAAVAGGGDPLLQDILMYWGKPF* SEQ ID NO. 377 LOC_Os05g34830 3 MALYGEKEWYFFSPRDRKYPNGSRPNRAAGSGYWK GPILGQLDPAAAVAGGGDPLLQDILMYWGKPP SEQ ID NO. 378 LOC_Os05g36100 1 MADQRRRFRGGGDWQASVDDWDDGGELEAAAAAA
SSSGYGAPAEHGEAVQWTSWPDGGGWTYPATTSSWSGSSQYPPPPRPPQQ* SEQ ID NO. 379 LOC_Os05g45020 1 MASREHLLLDPAALAV ADAEMEELMFAMRELGLRKVRPSASSVTPVLPPVTDEDGPDFGWVSELVM* SEQ ID NO 380 LOC_Os05g45060 1 MASFLDSFSSLGVGYAVA
Q ID NO. 381 LOC_Os05g49310 1 MQYTVEGSGGGGVQTVEAAVRKGPWTMEEDLSLVNYIAANGEGAWNTLARAAGLNRTGKSCRLRWLNYL
ATVAVTQEYATEAPPPSGMSSGSYLDQLQPGYASSIHGGQDGGAAAAAAGDVWSDEFLAASSDNFWALEDLWPTVQSLHGNC* SEQ ID NO. 38 2 LOC_Os05g49620 1 MVELCGGEGEGQIMLATELAQLRAMARELEAKMDPDRVAARELCRALASSVDRSIRLAASCFPPPEHPPPAAGNAGRDA
FFQSEIQNL* SEQ ID NO. 383 LOC_Os05g50350 1 MASAAGSKQQQAMMSLPSSRGGGGGGWTQRQNKQFECALAVYDKETPDRWHNIARYM GGAKSADEVRRHFDHLVEDVSRIESGRVPFPRYSSSSSSRGADDGNRSRYLKYQ* SEQ ID NO. 384 LOC_Os05g50700 1 MTITHASSLSRFHPL
MDMNDFFGPFDDDLDHFLDDDAVLGRRLSL* SEQ ID NO. 385 LOC_Os05g51040 1 MANNQERPKCSCVQSSCTQLYCRCFRSRYFCSDNCNC CKCLECSNSFGVKNSESSNKPDPDDKSATDGLTHEEΓΓTTENITLPGETWNSDPNKRPRYP SEQ ID NO. 386 LOC_Os05g51820 1 MSHIAVERN
VHQSFMPPPAAHPDNHLHS* SEQ ID NO. 387 LOC_Os06g03670 1 MEYYEQEEYATVTSAPPKRPAGRTKFRETRHPVYRGVRRRGPAGRWV PAMMMQYQDDMAATPSSYDYAYYGNMDFDQPSYYYDGMGGGGEYQSWQMDGDDDGGAGGYGGGDVTLWSY* SEQ ID NO. 388 LOC_θs06
GPSGHASEANQEFLHHAICDPSLHIGYQAYMDHLNQ* SEQ ID NO. 389 LOC_Os06g07030 1 MDRREATVFLPPPPPPQPTQPQQQPAAAAVRA
AAVIRKKAAEVGARVDAQHSWGAAAPVPLQPPQPPPPQRRRTKNPDLNREPTPDTSDDE* SEQ ID NO. 390 LOC_Os06g07440 1 MDDDPTSV NYELRTMGMRGDDDDDVEEDRVEVFGNTSDIPIHVNVDDDDPPVDDSGNGTPTGSSATCTNKKTKTSKVWDDFEELYETTNGNRVRVSAKCNYC
SEQ ID NO. 391 LOC_Os06g 10780 1 ME NO.
Figure imgf000053_0001
MELEAEVAKLKEQKAELQKKQVEMIQKQNDEVMERITQQLGPKAKRFCLRRTLTGPC* SEQ ID NO. 393 LOC_Os06g 10880 3 MELPADGSALA
PKAKRFCLRRTLTGPC* SEQ ID NO. 394 LOC_Os06g 10880 2 MELPADGSALARQGSIYSLTFDEFQSALGSAEKDFGSMNMDELLRNIWTAEES kGWRED rVEKWE 3. 395 LO
C_Os06g15330 1 MSSTAKAAAAGAVGAKSARACDGCLRRRARWYCAADDAFLCQGCDTSVHSANPLARRHERLRLRPSSPPPLVPPSGSGRRD
AAAGVFGHGGEEQALTPRLGMDGGREARVSRYREKRRTRLFSKKIRYEVRKLNAEKRPRMKGRFVKRAAAAATAAVATACVA* SEQ ID NO. 396
VQHCVTALPNQQFLPSSEDVKPMIHHVPSSSYGWNTSIDSKPNSSDAHWGARRYFPK* SEQ ID NO. 397 LOC_Os06g28630 1 MWRGRWAVA RQRMMRRGTIRSWKMTSAALIQQVPAAATPMAVAAVRGRGTSSPSDGWCSPRASLKLQLAPPTSISSIQQVPYIH* SEQ ID NO. 398 LOC_Os06g
DMWLGSHATCPVCRRRVERKHKGGVLPPMPPEPETEPPV* SEQ ID NO. 399 LOC_Os06g34860 1 MSMPPPVPQENQSVGKGTAIFSYTCVGL IDMWLASHATCPLCRTEVEPPPGDGGGRPAPAADFSςPTFΔLPP"* SEQ !D NO 400 LOC_θ308g410G0 1 MELLKELvPGCSlwSGTALVLDEII 1 MAATQATAARKFPEGLRVLAVDDSPVCLMLLEA
Figure imgf000053_0002
2 MNTKTEASKEGSGNIFGNELDEEFDAEKHNK
3 MNTKTEASKEGSGNIFGNELDEEFDAEKHNKEATT
MNTKTEASKEGSGNIFGNELDEEFDAEKHNKEATTRTG
Figure imgf000053_0003
ID NO. 411 LOC_θs07g32170 1 MDRKDKARKNFSSSS SEQ ID NO. 413 LOC_Os07g37210 1
Figure imgf000054_0001
Figure imgf000054_0002
SEQ ID NO. 415 LOC_Os
Figure imgf000054_0003
RYLLKRWRRTAKSANEENQGYVANGNGSSLNSIVPPANHHGLQGFSAMIQDTPVSNMHENSFRRSS* SEQ ID NO.416 LOC_Os07g42610 1 M 3 MGRSFRDSLKVLEADIQHANSLAAEFRREYDGACL
2 MGRSFRDSLKVLEADIQHANSLAAEFRR
SEQ ID NO 422 LOC_Os MRGSDHHQDWAAPRGGGGGGDDGQAHDMVMPGF
Figure imgf000054_0004
SEQ ID NO. 428 LOC_Os08g24
LOC_Os08g34360 1 MAKRRSNGETAAASSDDSSSGV
SEQ ID NO.432 LOC_Os08g41030 1 M
LOC_Os08g41950 2 MGRGRVELKRIENKINRQVTFAKR 1 MEAQAQDKAEEGEEEGTRQQHAQAGPVGAAGGG
ID NO 436 LOC_Os08g43210 1 MCTSKLEEITGEWP LOC_Os08g43600 1 MERAAGTGSGGDDDELVLPPA
Figure imgf000055_0001
MLTTKLVQHDGPTARSAKHKRKNQYRGIRQRPWGK SEQ ID NO. 446 LOC_Os09g26420 2 MCGGAIIS
NO. 448 LOC_Os09g28210 1 MDFDLFNSYPESQLDL
Figure imgf000055_0002
TTPVDWQMGADEAGSNQLWDGLQDLMKLDEADTWFPPFSGAASSF* SEQ ID NO. 450 LOC_Os09g29130 1 MDFDDHDDGDEEMPPMPVSS PSGSGSGKKRFRTKFTQEQKDKMLAFAERVGWRIQKHDEAAVQQFCDEVGVKRHVLKVWMHNNKHTLGKKLP* SEQ ID NO.451 LOC_Os09g2 99302 SEQ ID NO.452 LOC_Os09g29930 3 MDSGSRS 1 MGIQGNKATTREHDFLSLYTTAAKDPSLQLH
2 MSFGGMFDGAGSGVFSYDAGGGGG
1 MPRALQRSGSNSLAALLRADPPPNAAIAD
SEQ ID NO. 463 LOC_Os10g230
Figure imgf000056_0001
W SEQ lD NO 464 LOC_Os10g23090 1 MKRPSCRGSSMAIIHDTSDQQEDNMRSYMDGGGAAAYEEEEEEVEDDDGGGGGGGGGGGGGLGE NO.465 LOC_Os10g26270 1 MACAETYIENMEDRCIYF
Figure imgf000056_0002
SEQ ID NO. 469 L
1 MKKGKWSKEEDDLIKNHMEKYGIG SEQ ID NO.471 LOC_Os10g3
LOC_Os10g42490 1 MRRMFGDCQVLSSMAAMAGAAS
1 MKNSSNKRSLVADQWHPSSVCCDHRA
Figure imgf000057_0001
478 LOC_Os11g08410 1 MPKPTPSSSSFLDFTGGV
1 MWREKQGGRMGKGKGKGKEKECDINCFGRSF
SEQ ID NO. 480
ID NO. 482 LOC_Os12g03150 1 MGRPPCCDKEGIKK
Figure imgf000057_0002
GGGGDGDGGPPAEEGEEKRRKGPSLPALSEIRWGELLTPEPANAAAVALSAALAWAGASLLLQLALISFAIFTAAVKYSFVAALLLFVLIALL' SEQ ID NO. 485 LOC_Os12g13130 1 MPRREGGRSRSAAYLVLFASCLLAVAAASHQEFHEAAGSRTLLMSHEHTNQVHCSRERSRAAWKAIDEYLMPFV
WQKKKPS* SEQ ID NO. 486 LOC_Os12g131302 MPRREGGRSRSAAYLVLFASCLLAVAAASHQEFHEAAGSRTLLMSHEHTNQVHCSRERSR EMKKGGQNLRRISKWQKKKPS* SEQ ID NO. 487 LOC_Os12g13130 3 MPRREGGRSRSAAYLVLFASCLLAVAAASHQEFHEAAGSRTLLMSH 4 MPRREGGRSRSAAYLVLFASCLLAVAAASHQEF LOC_Os12g18120 1 MADADARAPPKSDPGATPI
2 MADADARAPPKSDPGATPIGSISPSSAAPAAG
SEQ ID NO. 492 LOC_Os12g181203 MADADAR
1 MASLDLLLKLGFRFNPSQEEVITYYLPR StQ l
Figure imgf000058_0001
PCY* SEQ ID NO. 498 LOC_Os12g38200 1 MVFSSLPIFLDPPNWTQMQQQPLQCLIGGGGSDHHHLMPPPSGLAPLPSAAGAADTAASAPAAAA
VL* SEQ ID NO 499 LOC_Os12g38950 1 MASDVPQDDVQCHFCGTYLRPRSFRKHQQRCKYNPDALTRQNLSASSIPTSATRATHSEMASDVPQ
1 MSVLAGLLAQDQDGDDQEVTSHAAAASTQGDANFTG 1 MGDQKRSFINVMIGDFVAVPTKFANFIRGQISEWKL
Figure imgf000058_0002
KGWGKFVNDNKLEIHDICLFQLMKNKKKLTMTVHIIRKGECS* SEQ ID NO 502 LOC_Os12g40120 1 MGEKGCESCREWQEHCYREHMDVSRI
FNCQRWIKFIRENRLREGYICIFELMKGARRVTMTVHVIGKVDDRFVLLG* SEQ ID NO. 503 LOC_Os12g42400 2 MMSFNKSQEGFGQVAAVAT
LLKA* SEQ ID NO 504 LOC_Os12g42400 3 MMSFNKSQEGFGQVAAVATLASNGGGSLPWLLYGEPLGQGKPAMSPEGWPRAQTPLDPPQVP SIYEHEDMDHFHSFDHLRTHFFTPLPSLMDVEHGAGNPFKWTAASDGCCDLLKA* SEQ ID NO. 505 LOC_Os12g42400 1 MMSFNKSQEGFGQ
SDGCCDLLKA* SEQ ID NO. 506 LOC_Os12g43930 1 MAANVGESTSGSSSGGADSGGSFECNICFELPQEPIVTLCGHLFCWPCLYKWLHIHSHS LLSFQVHGFPDATAYGQPAGFPYGYGHGHGHGHGHAFHGGHAHAAAAPRHGPPGQQQQADVYLKALLILVGFLVIASLITF* Especially preferred is an isolated polypeptide selected from the group comprising:
(a) a polypeptide consisting of a sequence selected from the group comprising SEQ ID NO. 254, SEQ ID NO. 255, SEQ ID NO. 267 to SEQ ID NO. 270, SEQ ID NO. 273 to SEQ ID NO. 276, SEQ ID NO. 278, SEQ ID NO. 280,SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 297 to SEQ ID NO. 299, SEQ ID NO. 301 , SEQ ID NO. 305, SEQ ID NO. 309 to SEQ ID
NO. 311, SEQ ID NO. 314 to SEQ ID NO. 320, SEQ ID NO. 322, SEQ ID NO. 324, SEQ ID NO. 328, SEQ ID NO. 331, SEQ ID NO. 332, SEQ ID NO. 334, SEQ ID NO. 337, SEQ ID NO. 338, SEQ ID NO. 340, SEQ ID NO. 341, SEQ ID NO. 348, SEQ ID NO. 351 to SEQ ID NO. 364, SEQ ID NO. 370, SEQ ID NO. 373, SEQ ID NO. 375 to SEQ E) NO. 377, SEQ ID NO. 384 to SEQ ID NO. 386, SEQ ID NO. 388, SEQ ID NO. 389, SEQ ID NO. 392 to SEQ
ID NO. 394, SEQ ID NO. 397, SEQ ID NO. 399 to SEQ ID NO. 401, SEQ ID NO. 405, SEQ ID NO. 410 to SEQ ID NO. 412, SEQ ID NO. 415, SEQ ID NO. 418 to SEQ ID NO. 420, SEQ ID NO. 423, SEQ ID NO. 424, SEQ ID NO. 433, SEQ ID NO. 436 to SEQ ID NO. 439, SEQ ID NO. 443, SEQ ID NO. 444 to SEQ ID NO. 447, SEQ ID NO. 450 to SEQ ID NO. 453, SEQ ID NO. 458, SEQ ID NO. 461, SEQ ID NO. 460, SEQ ID NO. 467, SEQ ID NO.
469, SEQ ID NO. 470, SEQ ID NO. 472, SEQ ID NO. 478, SEQ ID NO. 481, SEQ E) NO. 484 to SEQ ID NO. 492, SEQ ID NO. 495 to SEQ ID NO. 502,
(b) a polypeptide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a sequence selected from the group comprising SFQ ID NO. 254, SEQ ID NO. 255, SEQ ID NO. 267 io SEQ ID NO. 270, SEQ
ID NO. 273 to SEQ ID NO. 276, SEQ ID NO. 278, SEQ ID NO. 280,SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 297 to SEQ ID NO. 299, SEQ ID NO. 301, SEQ ID NO. 305, SEQ ID NO. 309 to SEQ ID NO. 311, SEQ ID NO. 314 to SEQ ID NO. 320, SEQ ID NO. 322, SEQ ID NO. 324, SEQ ID NO. 328, SEQ ID NO. 331, SEQ ID NO. 332, SEQ ID NO. 334, SEQ ID NO. 337, SEQ ID NO. 338, SEQ E) NO. 340, SEQ E) NO. 341 , SEQ E) NO. 348,
SEQ E) NO. 351 to SEQ E) NO. 364, SEQ E) NO. 370, SEQ E) NO. 373, SEQ E) NO. 375 to SEQ E) NO. 377, SEQ E) NO. 384 to SEQ E) NO. 386, SEQ E) NO. 388, SEQ E) NO. 389, SEQ E) NO. 392 to SEQ E) NO. 394, SEQ E) NO. 397, SEQ E)-NO. 399 to SEQ E) NO. 401, SEQ E) NO. 405, SEQ E) NO. 410 to SEQ E) NO. 412, SEQ E) NO. 415, SEQ E) NO. 418 to SEQ E) NO. 420, SEQ E) NO. 423, SEQ E) NO. 424, SEQ E) NO. 433, SEQ E)
NO. 436 to SEQ E) NO. 439, SEQ E) NO. 443, SEQ E) NO. 444 to SEQ E) NO. 447, SEQ E) NO. 450 to SEQ E) NO. 453, SEQ E) NO. 458, SEQ E) NO. 461, SEQ E) NO. 460, SEQ E) NO. 467, SEQ E) NO. 469, SEQ E) NO. 470, SEQ E) NO. 472, SEQ E) NO. 478, SEQ E) NO. 481, SEQ E) NO. 484 to SEQ JD NO. 492, SEQ E) NO. 495 to SEQ E) NO. 502 (c) a polypeptide of (a) and/or (b), wherein said sequence is modified, to alter an abiotic stress tolerance of a plant, preferred osmotic tolerance and/or salt tolerance. As used herein, the term "polypeptide" means an unbranched chain of amino acid residues that are covalently linked by an amide linkage between the carboxyl group of one amino acid and the amino group of another. The term polypeptide can encompass whole proteins (i.e. a functional protein encoded by a particular gene), as well as fragments of proteins. Of particular interest are polypeptides of the present invention which represent whole proteins or a sufficient portion of the entire protein to impart the relevant biological activity of the protein. The term "protein" also includes molecules consisting of one or more polypeptide chains. Thus, a polypeptide of the present invention may also constitute an entire gene product, but only a portion of a functional oligomeric protein having multiple polypeptide chains.
Of particular interest in the present invention are polypeptides involved in one or more important biological properties in plants. Such polypeptides may be produced in transgenic plants to provide plants having improved phenotypic properties and/or improved response to stressful environmental conditions. In some cases, decreased expression of such polypeptides may be desired, such decreased expression being obtained by use of the polynucleotide sequences provided herein, for example in antisense or cosuppression methods.
Polypeptides of the present invention that are variants of the polypeptides provided herein will generally demonstrate significant identity with the polypeptides provided herein. Of particular interest are polypeptides having ai least about 35% sequence identity, at least about 50% sequence identity, at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, and more preferably at least about 85%, 90%, 95% or even greater, sequence identity with polypeptide sequences described herein. Of particular interest in the present invention are polypeptides having amino acid sequences provided herein (reference polypeptides) and functional homologs of such reference polypeptides, wherein such functional homologs comprises at least 50 consecutive amino acids having at least 90% identity to a 50 amino acid polypeptide fragment of said reference polypeptide.
The terms "protein(s)", "peptide(s)" or "oligopeptide(s)", when used herein refer to amino acids in a polymeric form of any length. Said terms also include known amino acid modifications such as disulphide bond formation, cysteinylation, oxidation, glutathionylation, methylation, acetylation, farnesylation, biotinylation, stearoylation, formylation, lipoic acid addition, phosphorylation, sulphation, ubiquitination, myristoylation, palmitoylation, geranylgeranylation, cyclization (e.g. pyroglutamic acid formation), oxidation, deamidation, dehydration, glycosylation (e.g. pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses, sialic acid etc.), acylation and radiolabels
(e.g. S35, C14, P32, P3^, 3HJ) as well as non-naturally occurring amino acid residues, L-amino acid residues and D-amino acid residues. Helping plants to tolerate stressful growth conditions results in a yield improvement resulting from improved plant growth and development. Polypeptides useful for improved stress tolerance under a variety of stress conditions include polypeptides involved in gene regulation, such as ion antiporters, ion transporters, H+ pyrophosphatases, H+ ATPases, aquaporines, CNGCs, glutamate receptors, Ca2+- ATPases, transcription factors, serine/threonine-protein kinases, MAP kinases, MAP kinase kinases, and MAP kinase kinase kinases; polypeptides that act as receptors for signal transduction and regulation, such as receptor protein kinases; intracellular signaling proteins, such as protein phosphatases, GTP binding proteins, and phospholipid signaling proteins; polypeptides involved in arginine biosynthesis; polypeptides involved in ATP metabolism, including for example ATPase, adenylate transporters, and polypeptides involved in ATP synthesis and transport; polypeptides involved in glycine betaine, proline, jasmonic acid, flavonoid or steroid biosynthesis; and hemoglobin. Enhanced or reduced activity of such polypeptides in transgenic plants will provide changes in the ability of a plant to respond to a variety of environmental stresses, such as chemical stress, osmotic stress and/or salt stress.
Also preferred is the polypeptide, wherein said polypeptide is a transcription factor.
Transcription factors play a key role in plant growth and development by controlling the expression of one or more genes in spatial, temporal and physiological specific patterns. Enhanced or reduced activity of such polypeptides in transgenic plants will provide significant changes in gene transcription patterns and provide a variety of beneficial effects in plant growth, development and response to environmental conditions. Transcription factors of interest include, but are not limited to ABBVPl, C3H, HRT, SBP, Alfin-like, CAMTA, HSF, Sigma70-like, AP2-EREBP, CCAAT, LFY, SRS, ARF, CPP, LM, TAZ, ARR-B, CSD, MADS, TCP, BBR/BPC, DBP, MYB, Trihelix, BESl, E2F-DP, MYB-related, TUB, bHLH, EIL, NAC, ULT, bZIP, FHA, Orphans, VOZ, C2C2-CO-like, G2-like, PBF-2-like, WRKY, C2C2-Dof, GeBP, PLATZ, zf-HD, C2C2-GATA, GRAS, Pseudo ARR-B, ZIM, C2C2-YABBY, GRF, RWP-RK, C2H2, HB, Sl Fa-like, ARID, HMG, MBFl, SET, AUX/IAA, Jumonji, PHD, SNF2, DDT, LUG, RB.
It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homologue polypeptides of the invention. Due to the degeneracy of the genetic code, many diffident polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (i.e., peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code, are also within the scope of the invention. As well-known to those skilled in the art, some amino acids have analogous physicochemical properties so that these amino acids advantageously can be replaced by each other. For example, these include the group of nonpolar (hydrophobic) amino acids (a) glycine, alanine, valine, leucine and/or isoleucine; or the hydroxy amino acids (b) serine, threonine and/or tyrosine, the amides of amino dicarboxylic acids (c) asparagine and glutamine, the amino dicarboxylic acids (d) aspartic acid and glutamic acid; the basic amino acids (e) lysine, arginine and/or ornithine as well as the group of aromatic amino acids (f) phenylalanine, tyrosine and/or tryptophan. The replacement of amino acids by structural similar amino acids is also possible. For example this is the case in the group with a β- functional group (g) cysteine, methionine, serine, α- aminobutyric acid and selenocysteine as well as the turn-inducing group (h) proline, 1 -amino-2-carboxy cyclohexane, pipecolic acid and an ortho- aminobenzoic acid. The skilled artisan knows that amino acids within one and the same group (a-h) can be replaced with one another. In all cases, peptide sequences will have a sufficient homology to be an analogous to an amino acid sequence of the peptides of the invention. Furthermore, the amino acids can be replaced by modified amino acids or specific enantiomers.
hi another preferred embodiment the invention relates to a vector comprising said polynucleotide and/or said nucleotide construct.
Also preferred is the vector, wherein the vector is a viral expression vector, a phage display vector, a bacterial expression vector, a yeast expression vector, a vector for expression in insects cells, a vector for in-vitro expression, a mammalian expression vector, a fungus expression vector, an algae expression vector or a plant expression vector.
With "vector" is meant a DNA sequence, which can be introduced in an organism by transformation and can be stably maintained in said organism. Vector maintenance is possible in e.g. cultures of Escherichia coli, Agrobacterium tumefaciens, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Other vectors such as phagemids and cosmid vectors can be maintained and multiplied in bacteria and/or viruses. Vector sequences generally comprise a set of unique sites recognised by restriction enzymes, the multiple cloning site (MCS), wherein one or more non-vector sequence(s) can be inserted.
"Expression vectors" form a subset of vectors which, by virtue of comprising the appropriate regulatory sequences enabling the creation of an expressible format for the inserted non- vector sequence(s), thus allowing expression of the protein encoded by said non-vector sequence(s).
Expression vectors are known in the art enabling protein- (gene-) expression in organisms including bacteria (e.g. Escherichia coli), fungi (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris), insect cells (e.g. baculoviral expression vectors), animal cells (e.g. COS or CHO cells) and plant cells (e.g. potato virus X-based expression vectors, see e.g. Vance et al. 1998— WO9844097). The current invention clearly includes any vector or expression vector comprising a non- vector DNA sequence encoding a polypeptide of the invention, homologue and/or derivative.
Vectors may also include a screenable marker. Screenable markers may be used to monitor transformation. Exemplary screenable markers include antibiotic resistant genes or genes expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP), a beta- glucuronidase or uidA gene (GUS), which encodes an enzyme for which various chromogenic substrates are known or an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues. Other possible selectable and/or screenable marker genes will be apparent to those of skill in the art.
In another preferred embodiment the invention relates to a host cell comprising said polynucleotide, said nucleotide construct, said polypeptide and/or said vector.
Also preferred is the host cell, wherein the host cell is selected from the group comprising a bacterial cell, a yeast cell, a fungus cell, a mammalian cell, an insect cell, an algae cell and/or a plant cell.
In another preferred embodiment the invention relates to a transgenic plant cell having stably incorporated into its genome at least one nucleotide construct comprising said polynucleotide, operably linked to a promoter that drives expression in said cell.
As used herein a "transgenic" organism is one whose genome has been altered by the incorporation of foreign genetic material or additional copies of native genetic material, e.g. by transformation or recombination.
A "transgenic plant" refers to a plant that contains genetic material not found in a wild type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
"Transgenic plant" in terms of the invention does also relate to cisgenic plants.
In terms of the invention "Plant" or "Plants" comprise all plant species which belong to the superfamily Viridiplantae. The present invention is applicable to any plant, in particular monocotyledonous plants and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp., Camellia sinensis, Carina indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis snn , Erythrina spp., Eucalyptus spp., Euclca scnirnperi, Eulalia viliosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp., Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., ■ «■ Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburusi alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidatai, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, amongst others, or the seeds of any plant specifically named above or a tissue, cell or organ culture of any of the above species.
Polynucleotides or DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).
Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
A person skilled in the art will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Preferred is a transgenic plant having an altered tolerance to abiotic stress, preferred osmotic stress and/or salt stress compared to a wild-type plant, wherein the transgenic plant comprises at least one modified polynucleotide, wherein the modified polynucleotide is selected from the group comprising an overexpressed polynucleotide, a suppressed polynucleotide and/or a knocked out polynucleotide. Also preferred is a transgenic plant having an altered tolerance to abiotic stress preferred osmotic stress and/or salt stress compared to a control plant, wherein the transgenic plant comprises at least one of said polynucleotides, and the control plant does not overexpress a polypeptide encoded by the polynucleotide.
A transgenic plant having an altered tolerance to abiotic stress preferred osmotic stress and/or salt stress compared to a control plant, wherein the transgenic plant comprises at least one of said polynucleotides foreign DNA, and the gene including the polynucleotide is not knocked out in the control plant.
A transgenic plant having an altered tolerance to abiotic stress preferred osmotic stress and/or salt stress compared to a control plant, wherein at least one of said polynucleotides is suppressed and the gene including the polynucleotide is not suppressed in the control plant.
Further preferred is a transgenic plant comprising at least one said polypeptides.
Salt stress tolerance can be assayed according to any of a number of well-know techniques. For example, plants can be grown under conditions in which more than optimum salt concentration is provided to the plant. Salt stress tolerance can be determined by any of a number of standard measures including turgor pressure, growth, yield and the like, yield of photosynthesis, stomatal conductivity, transpiration rate, osmotic potential etc.
Further preferred is the transgenic plant, wherein the transgene comprises a polynucleotide sequence that hybridizes under stringent conditions to the said complement polynucleotide.
The term "hybridize" refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (in other words, the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tn, of the formed hybrid, and the G:C ratio within the nucleic acids.
The term "Tn," refers to the "melting temperature" of a nucleic acid. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art.
As used herein the term "stringent conditions" refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Also preferred is the transgenic plant, wherein the plant is selected from the group consisting of poales, preferred poaceae, more preferred ehrhartoideae and/or panicideae, especially preferred rice and/or maize.
Further preferred is the transgenic plant, wherein said plant is a crop plant or a monocot or a cereal, such as maize, wheat, barley, millet, rye, sorghum, oats preferred rice; or a plant cell derived from said transgenic plant.
Also preferred is the transgenic plant, wherein the abiotic stress is osmotic stress, preferred salt stress.
Rice is one of the most important alimentary corps. The tolerance to abiotic stress, preferred osmotic stress and/or salt stress is particularly important for rice. Thus, it was meaningful and important to find out transcription factor associated with tolerance to high or low salinity for culturing a rice plant with tolerance to high or low salinity and thereby increasing rich production.
Also preferred is the transgenic plant, wherein the transgenic plant is a cultured host cell.
The invention also relates to a seed produced from the transgenic plant, preferred a transformed seed.
In another preferred embodiment the invention relates to a seed produced fiυrn the transgenic plant.
The invention also relates to a method for producing one of said plants, said method comprising the steps of transforming a target plant with an expression vector comprising a polynucleotide, encoding a transcription factor polypeptide.
Also preferred is a method for producing one of said plants, said method comprising the step of suppressing the expression level of at least one of said polynucleotides or at least one of said polypeptides.
Also preferred is the method of the preceding claim, wherein the transformed plant has a morphology that is substantially similar to a control plant.
Further preferred is a method for altering a plant stress response, said method comprising stably introducing into the genome of a plant at least one nucleotide construct comprising a polynucleotide operably linked to a promoter that drives expression in a plant cell, wherein said polynucleotide is selected from the group comprising:
(a) polynucleotide consisting of a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ED NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ ID NO. 111, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ DD NO. 133, SEQ DD NO. 135, SEQ DD NO. 136, SEQ DD NO. 139 to SEQ DD NO. 141, SEQ DD NO. 144, SEQ DD NO. 146 to SEQ DD NO. 148, SEQ DD NO. 152, SEQ DD NO. 157 to SEQ DD NO. 159, SEQ DD NO. 162, SEQ DD NO. 165 to SEQ DD NO167, SEQ ID NO. 170, SEQ DD NO. 171, SEQ DD
NO. 180, SEQ DD NO. 183 to SEQ DD NO. 186, SEQ DD NO. 190 to SEQ DD NO. 194, SEQ DD NO. 197 to SEQ DD NO. 200, SEQ DD NO. 205, SEQ DD NO. 208, SEQ DD NO. 207, SEQ ID NO. 214, SEQ DD NO. 216, SEQ DD NO. 217, SEQ DD NO. 219, SEQ DD NO. 225, SEQ ID NO. 228, SEQ DD NO. 231 to SEQ DD NO. 239, SEQ DD NO. 242 to SEQ DD NO. SEQ DD NO. 249,
(b) a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a nucleotide sequence of
(a),
(c) a polynucleotide of (a) and/or (b), wherein said sequence is modified.
Also preferred is the method, wherein the method improves the plant stress tolerance.
Further preferred is a method for improving the yield of a plant, comprising stably incorporating into the genome of said plant at least one nucleotide construct comprising a polynucleotide operably linked to a promoter that drives expression in a plant cell, wherein said polynucleotide is selected from the group comprising:
(a) polynucleotide consisting of a sequence selected from the group comprising SEQ ID • NO. 1, SEQ DD NO. 2, SEQ DD NO. 14 to SEQ DD NO. 17, SEQ DD NO. 20 to SEQ DD NO. 23, SEQ ID NO. 25, SEQ DD NO. 27, SEQ DD NO. 40, SEQ DD NO. 41, SEQ DD NO. 44 to SEQ DD NO. 46, SEQ DD NO. 48, SEQ DD NO. 52, SEQ DD NO. 56 to SEQ
DD NO. 58, SEQ DD NO. 61 to SEQ DD NO. 67, SEQ DD NO. 69, SEQ DD NO. 71, SEQ DD NO. 75, SEQ DD NO. 78, SEQ DD NO. 79, SEQ DD NO. 81, SEQ DD NO. 84, SEQ DD NO. 85, SEQ DD NO. 87, SEQ DD NO. 88, SEQ DD NO. 95, SEQ DD NO. 98 to SEQ DD NO. 11 1, SEQ DD NO. 117, SEQ DD NO. 120, SEQ DD NO. 122 to SEQ DD NO. 124, SEQ DD NO. 131 to SEQ DD NO. 133, SEQ DD NO. 135, SEQ DD NO. 136,
SEQ DD NO. 139 to SEQ DD NO. 141, SEQ DD NO. 144, SEQ DD NO. 146 to SEQ DD NO. 148, SEQ DD NO. 152, SEQ DD NO. 157 to SEQ DD NO. 159, SEQ DD NO. 162, SEQ DD NO. 165 to SEQ DD NO 167, SEQ DD NO. 170, SEQ DD NO. 171, SEQ DD NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ ID NO. 217, SEQ ID NO. 219, SEQ ID NO. 225, SEQ ID NO. 228, SEQ ID NO. 231 to SEQ ID NO. 239, SEQ ID NO. 242 to SEQ ID NO.SEQ ID NO. 249,
(b) a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a sequence of (a),
(c) a polynucleotide of (a) and/or (b), wherein said sequence is modified.
"Yield" refers to increased plant growth, increased crop growth, increased biomass, and/or increased plant product production, and is dependent to some extent on temperature, plant size, organ size, planting density, light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature acclimation and water or nutrient use efficiency.
The teachings of the present invention are characterised by the following features:
departure from the beaten track a new perception of the problem satisfaction of a long-felt need or want - hitherto all efforts of experts were in vain the simplicity of the solution, which proves inventive action, especially since it replaces a more complex doctrine the development of scientific technology followed another direction the achievement forwards the development - misconceptions among experts about the solution of the according problem (prejudice) technical progress, such as: improvement, increased performance, price-reduction, saving of time, material, work steps, costs or resources that are difficult to obtain, improved reliability, remedy of defects, improved quality, no maintenance, increased efficiency, better yield, augmentation of technical possibilities, provision of another product, opening of a second way, opening of a new field, first solution for a task, spare product, alternatives, possibility of rationalisation, automation or miniaturisation or enrichment of the pharmaceutical fund special choice; since a certain possibility, the result of which was unforeseeable, was chosen among a great number of possibilities, it is a patentable lucky choice error in citations - young field of technology combined invention; a combination of a number of known elements, with a surprising effect licensing praise of experts and commercial success
Said advantages are shown especially in the preferential embodiments of the invention.
Examples Example 1
Photosynthetic activity in plants with reduced expression level of the gene Os02gl3800
Salt-stressed plants experience a decrease of their photosynthetic efficiency (Munns, 1993). This decrease seems to be associated with the photosystem II (PSII) complex. Salinity stress decreases the PSII activity and promotes the destruction of chlorophyll pigments via accumulation of ions that may inhibit the quantum yield of PSII electron transport (Sudhir and Murthy, 2004). The ratio of FVVFM- (i.e., Fv>: variable fluorescence from light-adapted material; FM-: the maximum fluorescence signal, when all PSII centres are in the closed state) is an excellent indicator of PSII quantum yield. To compare the damage of PSII after the onset of salt stress in the wild-type Nipponbare and plants with reduced expression level of the gene Os02gl3800 {os02gl 3800-1), the chlorophyll fluorescence was followed using pulse amplitude modulation (PAM). Photosynthesis yield was measured at the second most expanded leaf of plants exposed to 50 and 10OmM NaCl.
When plants were kept under normal growth conditions, i.e., in the absence of salt stress, wild-type and transgenic plants maintained high photosynthetic yield, regarded as 100%. However, in salt stress treated plants (5OmM and 10OmM NaCl), a reduction of the photosynthetic yield was observed already three days after onset of the stress in both wild-type and transgenic plants. Nevertheless, the decrease was more pronounced in the wild-type plants. Application of 5OmM and 10OmM NaCl led to a decline of the orginal photosynthetic yield to 71% and 41%, respectively, in Nipponbare, whereas the os02gl 3800-1 transgenic plants showed a reduction to 85% and 71%, respectively (Figure 1).
When the stress was prolonged (five days) in 10OmM NaCl, no significant differences in the photosynthetic yield were noticed between the wild-type and os02gl 3800-1 transgenic plants. In those plants, the second most expanded leaves were already strongly necrotic and curly five days after the onset of stress, not allowing measurements at the later time points. However, within the same period the transgenic plants growing at 5OmM NaCl maintained higher photosynthetic activity (i.e., 70% of the original rate) than the Nipponbare wild-type (around 25% of the initial yield), despite of a slight decrease in the chlorophyll fluorescence. These results indicated that PSII is less affected by high salinity in the os02gl 3800-1 transgenic plants than in the wild-type Nipponbare and consequently, the os02gl 3800-1 transgenic plants displayed enhanced salt tolerance. Although similar findings were made at both salt concentrations, differences between the os02gl3800- 1 transgenic plants and wild-type plants were more pronounced at 5OmM NaCl than at 10OmM NaCl.
Figure 1 shows the photosynthetic rate analysed in wild-type and os02gl 3800-1 plants grown at two different salt stress concentrations, i.e., 5OmM NaCl (left panel) and 10OmM NaCl (right panel). Data are mean ± SE of eight replicates plants eachs.
Example 2
Osmotic potential determination in plants with reduced expression level of the gene Os02g46030
High salt concentration represents a water deficit or osmotic stress because of decreased osmotic potential in the soil or hydroponic solution. To overcome an osmotic stress, solutes are accumulated as a response to falling water potential of the cell's environment. As a consequence of this net accumulation, the osmotic potential of the cell is lowered, which in turn attracts water into the cell helping to maintain turgor pressure water deficit (Babu et al., 1999). Here the effects of osmotic stress caused by high salinity were measured in leaves of plants with reduced expression level of the gene Os02g46030 (ps02g46030-\) and wild-type plants. Salt-treated plants (i.e., wild-type and transgenic plants) showed a slight decrease in the osmotic potential three days after the onset of the stress
(Figure 2). Nevertheless, wild-type plants showed no significant differences after 7 days of stress, when compared to plants grown under normal conditions. In contrast, salt stress led to a progressive decrease in the osmotic potential (from -1.5 to -2.5MPa) in the os02g46030-l plants indicating it can not accumulate easily osmolytes to restore the water content of the cells.
Example 3
Stomatal conductance and transpiration rate in plants with reduced expression level of the gene Os02gl3800
Generally, at high salinity plant water and osmotic potential become more negative and, as a primary response to minimize water loss, stomata close leading to a reduction of the transpiration rate. Such physiological responses changes can be observed within hours after the onset of salt stress. To identify possible differences between the plants with reduced expression level of the gene Os02gl3800 {os02gl 3800-1) and Nipponbare plants, gas exchange parameters were determined at days zero, one and three after imposition of the salt stress (5OmM NaCl). As shown in Figure 3A, application of salt stress led to a decline of the original transpiration rates to approximately 50% in both, os02gl 3800-1 transgenic and wild-type plants. However, in the third day after stress imposition, the os02gl 3800-1 plants exhibited a transpiration rate that was 25% higher than that of the wild-type.
A similar trend was observed for the stomatal conductance parameter (Figure 3B). Although, transgenic and wild-type plants displayed a decrease in stomatal conductance already after the first day of stress, no significant difference was detected between the two types of plants. However, three days after the onset of stress, the Nipponbare plants maintained their stomatal conductance rate (53% of the original value), whereas in the os02gl 3800-1 plants a slight increase to 70% of the original stomatal conductance rate was observed. The results suggest that the os02gl 3800-1 transgenic plants were less strongly affected by salt stress than the wild-type.
Figure 3 (A) shows the response of transpiration and (B) the stomatal conductance in os02gl 3800-1 transgenic (black bars) and Nipponbare wild-type (grey bars) seedlings upon application of salt stress (5OmM NaCl) for up to three days. Data are mean ± SE of four replicates.
Example 4
Stomatal conductance and transpiration rate in plants with increased expression level of the gene Os06g41060
Leaves of plants with increased expression level of the gene Os06g41060 (35S::Os06g41060) and Nipponbare wild-type plants were compared employing gas exchange as physiological parameter. Transpiration rate and stomatal conductance were analysed in plants 0 and 48h after start of the salt stress (5OmM NaCl; Figure 4). At 48h, leaves of Nipponbare showed a reduction by 35% of the original transpiration rate, whereas the 35S::Os06g41060 displayed a decline of only 21%. Stomatal conductance was less significantly different between the plants, with a reduction of 30 and 38%, respectively, of the initial rates detected in the mutant and the wild-type, respectively.
Figure 4 (A) shows the response of transpiration and (B) the stomatal conductance: 35S::Os06g41060 (black bars) and Nipponbare (grey bars) plants treated by salt stress (5OmM NaCl) for two days. Data are means ± SE of six replicates.

Claims

Claims
1. An isolated polynucleotide selected from the group comprising:
(a) polynucleotide consisting of a sequence selected from the group comprising SEQ DD NO. 1 , SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID
NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ ID NO. 111, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ ID NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 139 to SEQ ID NO. 141, SEQ ID NO. 144, SEQ ID NO. 146 to SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NOl 67, SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID
NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ ID NO. 217, SEQ ID NO. 219, SEQ ID NO. 225, SEQ ID NO. 228, SEQ ID NO. 231 to SEQ ID NO. 239, SEQ ED NO. 242 to SEO ID NO. SEQ ID NO. 249,
(b) a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ LO NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41 , SEQ ID NO. 44 to SEQ ED NO. 46, SEQ ED NO. 48, SEQ
ED NO. 52, SEQ ED NO. 56 to SEQ ED NO. 58, SEQ ID NO. 61 to SEQ ED NO. 67, SEQ ED NO. 69, SEQ ED NO. 71, SEQ ED NO. 75, SEQ ED NO. 78, SEQ LD NO. 79, SEQ LD NO. 81, SEQ ED NO. 84, SEQ ED NO. 85, SEQ LD NO. 87, SEQ LD NO. 88, SEQ LD NO. 95, SEQ ED NO. 98 to SEQ LD NO. 111, SEQ ED NO. 117, SEQ LD NO. 120, SEQ LD NO. 122 to SEQ LD NO. 124, SEQ LD NO. 131 to SEQ ED NO. 133,
SEQ LD NO. 135, SEQ ED NO. 136, SEQ LD NO. 139 to SEQ LD NO. 141, SEQ LD NO. 144, SEQ ED NO. 146 to SEQ LD NO. 148, SEQ ID NO. 152, SEQ LD NO. 157 to SEQ LD NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ LD NO 167, SEQ LD NO. 170, SEQ LD NO. 171, SEQ LD NO. 180, SEQ ED NO. 183 to SEQ LD NO. 186, SEQ LO NO. 190 to SEQ ED NO. 194, SEQ ED NO. 197 to SEQ ID NO. 200, SEQ ED
NO. 205, SEQ LD NO. 208, SEQ ED NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ LD NO. 217, SEQ LD NO. 219, SEQ ED NO. 225, SEQ ED NO. 228, SEQ LO NO. 231 to SEQ LO NO. 239, SEQ LD NO. 242 to SEQ LO NO.SEQ LO NO. 249, (c) a polynucleotide of (a) and/or (b), wherein said sequence is modified, to alter an abiotic stress tolerance of a plant, preferred osmotic tolerance and/or salt tolerance.
2. A nucleotide construct comprising a polynucleotide of claim 1 , wherein said polynucleotide is operably linked to a promoter that drives expression in a plant cell.
3. The nucleotide construct of the preceding claim, wherein said promoter is a constitutive promoter.
4. The nucleotide construct of at least one of the preceding claims, wherein said promoter is a tissue-preferred promoter.
5. The nucleotide construct of at least one of the preceding claims, wherein said promoter is an inducible promoter.
6. The nucleotide construct of at least one of the preceding claims, wherein said promoter is a stress-inducible promoter.
7. A polypeptide encoded by a polynucleotide and/or a nucleotide construct of at least one of the preceding claims.
8. The polypeptide of the preceding claim, wherein said polypeptide is a transcription factor.
9. A vector comprising the polynucleotide and/or the nucleotide construct of at least one of the preceding claims.
10. The vector of the preceding claim, wherein the vector is a viral expression vector, a phage display vector, a bacterial expression vector, a yeast expression vector, a mammalian vector, a vector for expression in insects cells, a vector for in-vitro expression, a fungus expression vector, an algae expression vector or a plant expression vector.
11. A host cell comprising a polynucleotide of claim 1 , a nucleotide construct of at least one of the claims 2 to 6, a polypeptide of the claim 7 or 8 and/or a vector of claim 9 or 10.
12. The host cell of the preceding claim, wherein the host cell is selected from the group comprising a bacterial cell, a yeast cell, a fungus cell, an algae cell, an mammalian cell, an insect cell and/or a plant cell.
13. A transgenic plant cell having stably incorporated into its genome at least one nucleotide construct comprising a polynucleotide according to claim 1 , operably linked to a promoter that drives expression in said cell.
5
14. A transgenic plant comprising at least one polypeptide of claim 7.
15. A transgenic plant having an altered tolerance to abiotic stress, preferred osmotic stress and/or salt stress, compared to a wild-type plant, wherein the transgenic plant comprises at least one 0 modified polynucleotide of claim 1, wherein the modified polynucleotide is selected from the group comprising an overexpressed polynucleotide, a suppressed polynucleotide and/or a knocked out polynucleotide.
16. The transgenic plant of at least one of the claims 13 to 15, wherein the transgene comprises a 5 polynucleotide sequence that hybridizes under stringent conditions to the complement polynucleotide of claim 1.
17. The transgenic plant according to any of the preceding claims, wherein said plant is a crop plant or a monocot or a cereal, such as maize, wheat, barley, millet, rye, sorghum, oats, 0 preferred rice.
18. The transgenic plant according to any of the preceding claims, wherein the transgenic plant is a cultured host cell. 5
19. A seed produced from the transgenic plant according to at least one of the preceding claims.
20. A transformed seed of the transgenic plant of at least one of the preceding claims.
21. A method for producing a plant according to at least one of the preceding claims, said method 50 comprising the steps of: transforming a target plant with an expression vector comprising a polynucleotide of claim 1, encoding a transcription factor polypeptide of claim 7 or 8.
22. A method for producing a plant according to any of the preceding claims, said method S 5 comprising the step of: suppressing the expression level of a polynucleotide of claim i or a polypeptide of claim 7 or
23. The method of the preceding claim, wherein the transformed plant has a morphology that is substantially similar to a control plant.
24. A method for altering a plant stress response, said method comprising stably introducing into the genome of a plant at least one nucleotide construct comprising a polynucleotide operably linked to a promoter that drives expression in a plant cell, wherein said polynucleotide is selected from the group comprising:
(a) polynucleotide consisting of a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID
NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ ID NO. 111, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID
NO. 124, SEQ ID NO. 131 to SEQ ID NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 139 to SEQ ID NO. 141, SEQ ID NO. 144, SEQ ID NO. 146 to
SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NO167, SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID
NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ ID NO. 217, SEQ ID NO. 219, SEQ ID NO. 225, SEQ ID NO. 228, SEQ ID NO. 231 to SEQ ID NO. 239, SEQ ID NO. 242 to SEQ ID NO.SEQ ID NO. 249, (b) a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a nucleotide sequence of
(a), (c) a polynucleotide of (a) and/or (b), wherein said sequence is modified.
25. A method of at least one of the preceding claims, wherein the method improves the plant salt stress tolerance and/or osmotic stress tolerance.
26. A method for improving the yield of a plant, comprising stably incorporating into the genome of said plant at least one nucleotide construct comprising a polynucleotide operably linked to a promoter that drives expression in a plant cell, wherein said polynucleotide is selected from the group comprising:
(a) polynucleotide consisting of a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 14 to SEQ ID NO. 17, SEQ ID NO. 20 to SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 44 to SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 52, SEQ ID NO. 56 to SEQ ID NO. 58, SEQ ID NO. 61 to SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 95, SEQ ID NO. 98 to SEQ ID NO. 111, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 122 to SEQ ID NO. 124, SEQ ID NO. 131 to SEQ ID NO. 133, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 139 to SEQ ID NO. 141, SEQ ID NO. 144, SEQ ID NO. 146 to
SEQ ID NO. 148, SEQ ID NO. 152, SEQ ID NO. 157 to SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 165 to SEQ ID NO167, SEQ ID NO. 170, SEQ ID NO. 171,
SEQ ID NO. 180, SEQ ID NO. 183 to SEQ ID NO. 186, SEQ ID NO. 190 to SEQ ID NO. 194, SEQ ID NO. 197 to SEQ ID NO. 200, SEQ ID NO. 205, SEQ ID NO. 208, SEQ ID NO. 207, SEQ ID NO. 214, SEQ ID NO. 216, SEQ ID NO. 217, SEQ ID NO. 219, SEQ ID NO. 225, SEQ ID NO. 228, SEQ ID NO. 231 to SEQ ID NO. 239, SEQ ID NO. 242 to SEQ ID NO.SEQ ID NO. 249
(b) a polynucleotide consisting of a sequence having at least 70%, preferred 80%, more preferred 90%, especially preferred 98% sequence identity to a sequence of (a),
(c) a polynucleotide of (a) and/or (b), wherein said sequence is modified.
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