WO2005049843A2 - Cellules vegetales et vegetaux presentant une meilleure resistance a la contrainte - Google Patents

Cellules vegetales et vegetaux presentant une meilleure resistance a la contrainte Download PDF

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WO2005049843A2
WO2005049843A2 PCT/EP2004/053093 EP2004053093W WO2005049843A2 WO 2005049843 A2 WO2005049843 A2 WO 2005049843A2 EP 2004053093 W EP2004053093 W EP 2004053093W WO 2005049843 A2 WO2005049843 A2 WO 2005049843A2
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nucleic acid
acid molecule
plant
protein
plants
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WO2005049843A3 (fr
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Klaus Apel
Daniela Wagner
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Eidgenoessische Technische Hochschule Zürich
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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
    • C12N15/8274—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 herbicide 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
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants

Definitions

  • This invention relates generally to transformed plant cells and plants comprising an inactivated or down -regulated gene resulting in increased tolerance and/or resistance to environmental stress as compared to non-transformed wild type cells and methods of producing such plant cells or plants.
  • This invention further relates generally to transformed plant cells with increased tolerance and/or resistance to environmental stress as compared to a corresponding non-transformed wild type plant cell, wherein the increased tolerance and/or resistance to environmental stress as compared to a corresponding non- transformed wild type plant cell is altered by an inactivated or down-regulated gene, methods of producing, screening for and breeding such plant cells or plants and method of detecting stress in plants cells or plants.
  • this invention relates to transformed plant cells and plants comprising an altered gene expression resulting in increased tolerance and/or resistance to environmental stress, preferably by an inactivation or down-regulation of the expression of certain gene(s) , as compared to non-transformed wild type cells and methods of producing such plant cells or plants.
  • Abiotic environmental stress such as drought stress, salinity stress, heat stress, and cold stress, is a major limiting factor of plant growth and productivity (Boyer. 1982. Science 218, 443-448). Crop losses and crop yield losses of major crops such as rice, maize (corn) and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped and third-world countries.
  • Another abiotic stress according to the present invention is the stress induced by contamination with toxic compounds which preferably are used as herbicides. Further stress conditions are high light, UVA light and a limitation in C02.
  • Plants are typically exposed during their life cycle to stress conditions like reduced environmental water content or herbicides activity. Most plants have evolved strategies to protect themselves against these conditions for short period of time. However, if the severity and duration of the stress conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Continuous exposure to stress conditions major alterations in the plant metabolism. These great changes in metabolism ultimately lead to cell death and consequently yield losses. Developing stress-tolerant and/or resistant plants is a strategy that has the potential to solve or mediate at least some of these problems (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers).
  • ROS reactive oxygen radicals
  • Transgenic plants that overproduce osmolytes such as mannitol, fructans, proline or glycine-betaine also show increased resistance to some forms of abiotic stress and it is proposed that the synthesized osmolytes act as ROS scavengers (Tarczynski. et al. 1993 Science 259, 508-510; Sheveleva,. et al. 1997. Plant Physiol.115, 1211-1219).
  • Abiotic stress conditions limit the ability of plants to use light energy for photosynthesis, often reducing their growth and productivity and causing photooxidative damages (Barber et al. compliment Trends Biol. Sci. 17, 61 , (1992); . Apel et al., Annu. Rev. Plant Biol. 55, 373, (2004), Niyogi et al., Annu. Rev. Plant Physiol. Plant Mol. Biol., 50, 333 (1999)).
  • ROS reactive oxygen species
  • Another object of the invention is to produce transgenic cells and plants which are more resistant to stress conditions, such as drought, limitation in C02, high light, UV, high and low temperatures as compared to non-transformed wild type cells or plants, giving rise to higher crop yields under adverse conditions.
  • stress conditions such as drought, limitation in C02, high light, UV, high and low temperatures
  • transgenic plants should be resistant or tolerant to high ROS concentrations and/or herbicide treatments, allowing efficient weed control using such herbicides, even in high concentrations.
  • the present invention provides a transformed plant cell, preferably with altered gene expression level compared to a corresponding non transformed wild type plant cell, wherein the increased tolerance and/or resistance to an environmental stress as compared to a corresponding non-transformed wild type plant cell is altered by alteration of the expression of at least one gene.
  • altered gene expression or gene expression level refers to the change (increase or decrease) of the amount, concentration or activity (meaning here the effective concentration for the purposes of chemical reactions and other mass action) of a peptide expressed by said gene, meaning the gene product, in a specific volume relative to a corresponding volume (e.g. in an organism, a tissue, a cell or a cell compartment) of a control, reference or wild type, measured for example by one of the methods described herein below or being common knowledge of a person skilled in the art, which is changed (increased or decreased) as compared to a corresponding non transformed wild type plant cell.
  • the present invention provides a transformed plant cell with increased tolerance and/or resistance to an environmental stress as compared to a corresponding non-transformed wild type plant cell, wherein the increased tolerance and/or resistance to an environmental stress is altered by an inactivated or downregulated gene and a process for the production of such plants and plant cells.
  • the term "inactivated or down-regulated gene” means the transgenic reduction or deletion of the expression of nucleic acid sequence as shown in Fig. 7, 9, 11, 12, 13 and/or 16 leading to an increased tolerance and/or resistance to an environmental stress as compared to a corresponding non- transformed wild type plant cell.
  • the reduction or deletion of the expression of said nucleic acid results in increased tolerance to an environmental stress as compared to a corresponding non-transformed wild type plant cell.
  • the environmental stress is selected from the group consisting of salinity, drought, high and low temperature, chemical such as metal or herbicide treatments, pathogenic, light such as brightness, darkness or UV radiation, as compared to non-transformed wild type cells or plants, giving rise to higher crop yields under adverse conditions.
  • such transgenic plants should be preferably resistant or tolerant to oxidative stresses induced by ROSs, or combinations thereof, preferably light and/or chemical treatment, more preferably light and/or herbicide treatment.
  • the plant cells and plant of the present invention are preferably resistant or tolerant to stress induced by high ROS concentrations and/or high herbicides concentrations which induce ROS.
  • expression refers to the transcription and/or translation of a codogenic gene segment or gene.
  • the resulting product is an mRNA or a protein.
  • expression products can also include functional RNAs such as, for example, antisense, nucleic acids, tRNAs, snRNAs, rRNAs, RNAi, siRNA, ribozymes etc. Expression may be systemic.local or temporal, for example limited to certain cell types, tissuesorgans or time periods.
  • polynucleotides “nucleic acid” and “nucleic acid molecule” are interchangeably in the present context.
  • sequence may relate to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context in which the term “sequence” is used.
  • sequence may relate to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context in which the term “sequence” is used.
  • gene(s) polynucleotide
  • nucleic acid sequence nucleotide sequence
  • nucleic acid mo!ecule(s) refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The terms refer only to the primary structure of the molecule. [022.3] Thus, the terms "gene(s)", “polynucleotide”, “nucleic acid sequence”,
  • nucleotide sequence or “nucleic acid molecule(s)” as used herein include double- and single-stranded DNA and RNA. They also include known types of modifications, for example, methylation, "caps", substitutions of one or more of the naturally occurring nucleotides with an analog.
  • the DNA or RNA sequence of the invention comprises a coding sequence encoding the herein defined polypeptide.
  • a "coding sequence” is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3' -terminus.
  • a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
  • amplified relate to a corresponding change of a property in an organism, a part of an organism such as a tissue, seed, root, leave, flower etc. or in a cell and are interchangeable.
  • the overall activity in the volume is increased or enhanced in cases if the increase or enhancement is related to the increase or enhancement of an activity of a gene product, independent whether the amount of gene product or the specific activity of the gene product or both is increased or enhanced or whether the amount, stability or translation efficacy of the nucleic acid sequence or gene encoding for the gene product is increased or enhanced.
  • the terms “reduction”, “decrease” or “deletion” relate to a corresponding change of a property in an organism, a part of an organism such as a tissue, seed, root, leave, flower etc. or in a cell.
  • the overall activity in the volume is reduced, decreased or deleted in cases if the reduction, decrease or deletion is related to the reduction, decrease or deletion of an activity of a gene product, independent whether the amount of gene product or the specific activity of the gene product or both is reduced, decreased or deleted or Whether the amount, stability or translation efficacy of the nucleic acid sequence or gene encoding for the gene product is reduced, decreased or deleted.
  • the terms "increase” or “decrease” relate to a corresponding change of a property an organism or in a part of an organism, such as a tissue, seed, root, leave, flower etc. or in a cell.
  • the overall activity in the volume is increased in cases the increase relates to the increase of an activity of a gene product, independent whether the amount of gene product or the specific activity of the gene product or both is increased or generated or whether the amount, stability or translation efficacy of the nucleic acid sequence or gene encoding for the gene product is increased.
  • the term “increase” or “decrease” means that the specific activity of an enzyme as well as the amount of a compound or peptide, e.g. of a polypeptide or a nucleic acid molelcule or an encoding mRNA or DNA, can be increased or decreased in a volume.
  • the terms “reduction”, “decrease” or “deletion” relate to a corresponding change of a property in an organism, a part of an organism such as a tissue, seed, root, leave, flower etc. or in a cell.
  • change of a property it is understood that the activity, expression level or amount of a gene product or peptide, e.g. a polypeptide content is changed in a specific volume or in a specific amount of protein relative to a corresponding volume or amount of protein of a control, reference or wild type.
  • the overall activity in the volume is reduced, decreased or deleted in cases if the reduction, decrease or deletion is related to the reduction, decrease or deletion of an activity of a gene product, independent whether the amount of gene product or the specifip activity of the gene product or both is reduced, decreased or delated or whether the amount, stability or translation efficacy of the nucleic acid sequence or gene encoding for the gene product is reduced, decreased or deleted.
  • the terms “reduction”, “decrease” or “deletion” include the change of said property in only parts of the subject of the present invention, for example, the modification can be found in compartment of a cell, like an organelle, or in a part of a plant, like tissue, seed, root, leave, flower etc. but is not detectable if the overall subject, i.e. complete cell or plant, is tested.
  • the "reduction”, “decrease” or “deletion” is found cellular, thus the term “reduction, decrease or deletion of an acitivity” or “reduction, decrease or deletion of a metabolite content” relates to the cellular reduction, decrease or deletion compared to the wild typ cell.
  • the terms “reduction”, “decrease” or “deletion” include the change of said property only during different growth phases of the organism used in the inventive process, for example the reduction, decrease or deletion takes place only during the seed growth or during blooming. Furtheremore the terms include a transitional reduction, decrease or deletion for example because the used RNAi is not stable integrated in the genom of the organism and has therefore only a transient effect.
  • the term “reduction”, “decrease” or “deletion” means that the specific activity of an enzyme or other protein or regulatory RNA as well as the amount of a compound or metabolite, e.g. of a polypeptide, a nucleic acid molelcule or the fine chemical of the invention or an encoding mRNA or DNA, can be reduced, decreased or deleted in a volume.
  • wild type can be a cell or a part of organisms such as an organelle or tissue, or an organism, in particular a microorganism or a plant, which was not modified or treated according to the herein described process according to the invention. Accordingly, the cell or a part of organisms such as an organelle or a tissue, or an organism, in particular a microorganism or a plant used as wild type, control or reference corresponds to the cell, organism or part thereof as much as possible and is in any other property but in the result of the process of the invention as identical to the subject matter of the invention as possible. Thus, the wild type, control or reference is treated identically or as identical as possible, saying that only conditions or properties might be different which do not influence the quality of the tested property.
  • analogous conditions means that all conditions such as, for example, culture or growing conditions, assaytxinditions (such as buffer composition, temperature, substrates, pathogen strain, concentrations and the like) are kept identical between the experiments to be compared.
  • the "reference”, "control”, or “wild type” is preferably a subject, e.g. an organelle, a cell, a tissue, an organism, in particular a plant or a microorganism, which was not modified or treated according to the herein described process of the invention and is in any other property as similar to the subject matter of the invention as possible.
  • the reference, control or wild type is in its genome, transcriptome, proteome or metabolome as similar as possible to the subject of the present invention.
  • the term "reference-" "control-” or “wild type-”-organelle, -cell, -tissue or -organism, in particular plant or microorganism relates to an organelle, cell, tissue or organism, in particular plant or microorganism, which is nearly genetically identical to the organelle, cell, tissue or organism, in particular microorganism or plant, of the present invention or a part thereof preferably 95%, more preferred are 98%, even more preferred are 99,00%, in particular 99,10%, 99,30%, 99,50%, 99,70%, 99,90%, 99,99%, 99, 999% or more.
  • the "reference", "control”, or “wild type” is preferably a subject, e.g.
  • the reference, control or wild type differs form the subject of the present invention only in the cellular activity of the polypeptide or RNA of the invention, e.g. as result of a reduction, decrease or deletion in the level of the nucleic acid molecule of the present invention or a reduction, decrease or deletion of the specific activity of the polypeptide or RNA of the invention, e.g. by or in the expression level or activity of protein or RNA that means its biological activity and/or its biochemical or genetical causes.
  • RNA rRNA, tRNA, miRNA
  • mRNA messenger RNA
  • expression can be detected by e.g. Northern, qRT PCR, transcriptional run-on assays or Western blotting and other immuno assays.
  • decrease or deletion of the expression that means as consequence of the reduced, decreased or deleted transcription of a gene a related phenotypic trait appears such as the enhanced or increased stress tolerance.
  • preferred reference subject is the starting subject of the ⁇ resent process of the invention.
  • the reference and the subject matter of the invention are compared after standardization and normalization, e.g.
  • RNA, DNA, or Protein or activity or expression of reference genes like housekeeping genes, such as ubiquitin.
  • the molecule number or the specific activity of the polypeptide of the invention or the number of expression of the nucleic acid molecule of the invention may be reduced, decreased or deleted.
  • it is also possible to reduce, decrease or delete the expression of the gene which is naturally present in the organisms for example by modifying the regulation of the gene, or by reducing or decreasing the stability of the mRNA or of the gene product encoded by the nucleic acid molecule of the invention.
  • the reduction, decrease, deletion or modulation according to this invention can be constitutive, e.g. due to a stable permanent transgenic expression or to a stable mutation in the corresponding endogenous gene encoding the nucleic acid molecule of the invention or to a modulation of the expression or of the behaviour of a gene conferring the expression of the polypeptide of the invention, or transient, e.g. due to an transient transformation, a transiently active promotor or temporary addition of a modulator such as an antagonist or inductor, e.g. after transformation with a inducible construct carrying a double-stranded RNA nucleic acid molecule, an antisense nucleic acid molecule, a ribozyme of the invention etc. under control of a inducible promoter and adding the inducer, e.g. tetracycline or as described herein below.
  • a modulator such as an antagonist or inductor
  • the reduction, decrease or deletion in activity amounts preferably by at least 10%, preferably by at least 30% or at least 60%, especially preferably by at least 70%, 80%, 85%, 90% or more, very especially preferably are at least 95%, more preferably are at least 99% or more in comparison to the control, reference or wild type. Most preferably the reduction, decrease or deletion in activity amounts to 100%.
  • inactivation means that the enzymatic or biological activity of the polypeptides encoded is no longer detectable in the organism or in the cell such as, for example, within the plant or plant cell.
  • downregulation means that the enzymatic or biological activity of the polypeptides encoded is partly or essentially completely reduced in comparison with the activity of the untreated organism. This can be achieved by different cell- biological mechanisms.
  • the activity can be downregulated in the entire organism or, in the case of multi-celled organisms, in individual parts of the organism, in the case of plants for example in tissues such as the seed, the leaf, the root or other parts.
  • the enzymatic activity or biological activity is reduced by at least 10%, advantageously at least 20%, preferably at least 30%, especially preferably at least 40%, 50% or 60%, very especially preferably at least 70%, 80%, 85% or 90% or more, very especially preferably are at least 95%, more preferably are at least 99% or more in comparison to the control, reference or wild type. Most preferably the reduction, decrease or deletion in activity amounts to 100%.
  • biological activity means the biological function of the protein of the invention.
  • activity means the increase in the production of the compound produced by the inventive process.
  • biological activity preferably refers to the enzymatic function, transporter carrier function, DNA-packaging function, heat shock protein function, recombination protein function, beta-galactosidase function, Serine/threonine-protein kinase CTR1 function, lipase function, enoyl-CoA hydratase function, UDP-glucose glucosyltransferase function, cell division protein function, fiavonol synthase function, tracylglycerol lipase, MADS-box protein function, pectinesterase function, pectin metylesterase function, calcium transporting ATPase function, protein kinase function, lysophospholipase function, Chlorophyll A-B
  • Suitable substrates are low-molecular-weight compounds and also the protein interaction partners of a protein.
  • the term "reduction" of the biological function refers, for example, to the quantitative reduction in binding capacity or binding strength of a protein for at least one substrate in an organism, a tissue, a cell or a cell compartment - for example by one of the methods described herein below - in comparison with the wild type of the same genus and species to which this method has not been applied, under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like).
  • Reduction is also understood as meaning the modification of the substrate specificity as can be expressed for example, by the kcat/Km value.
  • a reduction of the function of at least 10%, advantageously of at least 20%, preferably at least 30%, especially preferably of at least 40%, 50% or 60%, very especially preferably of at least 70%, 80%, 90% or 95%, in comparison with the untreated organism is advantageous.
  • a particularly advantageous embodiment is the inactivation of the function. Binding partners for the protein can be identified in the manner with which the skilled worker is familiar, for example by the yeast 2-hybrid system.
  • a modification i.e. a decrease
  • a decrease in activity in an organism or a part thereof can be caused by adding a chemical compound such as an antagonist to the media, nutrition, soil of the plants or to the plants themselves.
  • the transformed plant cells are compared to the corresponding non- transformed wild type of the same genus and species under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like).
  • a change of at least 10%, advantageously of at least 20%, preferably at least 30%, especially preferably of at least 40%, 50% or 60%, very especially preferably of at least 70%, 80%, 90%, 95% or even 100% or more, in comparison with the non-transformed organism is advantageous.
  • Data significance can be determinated by all statistical methods known by a person skilled in the art, preferably by a t-test, more preferably by the student t- test.
  • inactivation or down-regulation of a gene in the plant cell results in altered metabolic activity as compared to a corresponding non -transformed wild type plant cell.
  • One preferred wild type plant cell is a non-transformed Arabidopsis plant cell.
  • An example here is the Arabidopsis wild type C24 (Nottingham Arabidopsis Stock Centre, UK; NASC Stock N906).
  • Other preferred wild type plant cells are a non-transformed from plants selected from the group consisting of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass and forage crops, preferably dicotyledonean plants such as A.t., Arabidopsis thaliana; S.t, Solanum tuberosum; N.s., Nicotiana sylvestris; B.o., Brassica oleracea; V.v., Vitis vinifera; L.e., Lycopersicum esculentum; C.s., Cit
  • More preferred wild type plant cells are a non-transformed Linum plant cell, preferably Linum usitatissimum, more preferably the variety Brig ' itta, Golda, Gold Merchant, Helle, Juliel, Olpina, Livia, Marlin, Maedgold, Sporpion, Serenade, Linus, Taunus, Lifax or Liviola, a non-transformed Heliantus plant cell, preferably Heliantus annuus, more preferably the variety Aurasol, Capella, Flavia, Flores, Jazzy, Palulo, Pegasol, PIR64A54, Rigasol, Sariuca, Sideral, Sunny, Alenka, Candisol or Floyd, or a non-transformed Brassica plant cell, preferably Brassica napus, more preferably the variety Dorothy, Evita, Heros, Hyola, Kimbar, Lambada, Licolly, Liconira, Licosmos, Lisonne, Mistral, Passat, Serator, Siapula, Sponsor
  • Inactivation or down-regulation of a gene is advantageous since no new gene must be introduced to achieve the altered gene expression activity resulting in increased tolerance and/or resistance to environmental stress. Only an endogenous gene is hindered in its expression.
  • the process of conferring stress tolerance and resistance of the present invention comprises one or more of the following steps a) destabilizing a protein enabling the reduced, decreased or deleted expression of a protein encoded by the nucleic acid molecule of the invention or of the polypeptid of the invention, e.g. of a polypeptide having the biological activity of a protein as depicted in Fig.
  • This can be achieved for example through the expression of the nucleic acids of the invention or parts of it in antisense orientation or by the expression of hairpin RNAi constructs or the simultaneous expression of sense and antisense RNA for the nucleic acids of the invention.
  • Advantogeously this can be achieved for example through the expression of the nucleic acids encoding a protein, which has lost its biological activity and which binds to another protein in a multimeric complex and thereby decreasing or delting the activity of said complex or which binds for example as a transcription factor to DNA and thereby decreasing or deleting the activity of the translated protein; or h) expression of an antibody, which binds to the nucleic acid molecule of the invention or the protein of the invention such as a protein as depicted in Fig.
  • said mRNA is one kind of the nucleic acid molecule of the present invention and/or the protein enabling the reduced or decreased expression of a protein encoded by the nucleic acid molecule of the present invention or the polypeptide having the herein mentioned biological activity of the polypeptide of the present invention, e.g. conferring increased tolerance and/or resistance to environmental stress as compared to a corresponding non-transformed wild type plant cell after decreasing the expression or activity of the encoded polypeptide or having the biological activity of a polypeptide having the biological activity of the protein of the invention.
  • the amount of mRNA, polynucleotide or nucleic acid molecule in a cell or a compartment of an organism correlates to the amount of encoded protein and thus with the overall activity of the encoded protein in said volume. Said correlation is not always linear, the activity in the volume is dependent on the stability of the molecules, the degradation of the molecules or the presence of activating or inhibiting co-factors. Further, product and educt inhibitions of enzymes are well known.
  • the activity of the abovementioned proteins and/or poylpeptide encoded by the nucleic acid molecule of the present invention can be reduced, decreased or deleted in various ways.
  • the activity in an organism or in a part thereof, like a cell is reduced or decreased via reducing or decreasing the gene product number, e.g. by reducing or decreasing the expression rate, like mutating the natural promoter to a lower activity, or by reducing or decreasing the stability of the mRNA expressed, thus reducing or decreasing the translation rate, and/or reducing or decreasing the stability of the gene product, thus increasing the proteins decayed.
  • the activity or turnover of enzymes or channels or carriers transcription factors, and similar activ proteins can be influenced in such a manner that a reduction of the reaction rate or a modification (reduction, decrease or deletion) of the affinity to the substrate results, is reached.
  • a mutation in the catalytic centre of an polypeptide of the invention e.g.as enzyme, can modulate the turn over rate of the enzyme, e.g. a knock out of an essential amino acid can lead to a reduced or completely knock out activity of the enzyme, or the deletion of regulator binding sites can reduce a negative regulation like a feedback inhibition (or a substrate inhibition, if the substrate level is also increased).
  • the specific activity of an enzyme of the present invention can be decreased such that the turn over rate is decreased or the binding of a co-factor is reduced. Reducing the stability of the encoding mRNA or the protein can also decrease the activity of a gene product. The reduction of the activity is also under the scope of the term "reduced, decreased or deleted activity”. Beside this, advantegously the reduction of the activity in cis, eg. mutating the promotor including other cis-regulatory elements, or the transcribed or coding parts of the gene, inhibition can be achieved in trans, eg.
  • transfactors like chimeric transcription factor, ribozymes, antisense RNAs, dsRNAs or dominant negative proteins versions, which interfere with various stages of expression, eg the transcription, the translation or the acitivity of the protein or protein complex itself.
  • the inventive process as mentioned above preferably the reduction, decrease or deletion of the biological activity represented by protein of the invention is achieved by reducing, decreasing or deleting the expression of at least one nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of: a) nucleic acid molecule encoding the polypeptide shown in Fig. 7, 9, 11 , 12, 13 and/or 16 and/or homologues thereof; b) nucleic acid molecule comprising the nucleic acid molecule shown in Fig.
  • nucleic acid molecule comprising a nucleic acid sequence, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence depicted in Fig. 7, 9, 11, 12, 13 and/or 16 and/or homologues thereof; d) nucleic acid molecule encoding a polypeptide having at least 25%, 27%, 28%, 30%, 40%, 45%, 46%, preferably 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and having the biological activity represented by protein as depicted in Fig.
  • nucleic acid molecule which comprises a polynucleotide which is obtained by amplifying a cDNA library or a genomic library using the primers depicted in Fig. 15 and having the biological activity represented by protein as depicted in Fig. 7, 9, 11 , 12, 13 and/or 16; f) nucleic acid molecule encoding a polypeptide which is isolated with the aid of monoclonal or polyclonal antibodies against a polypeptide encoded by one of the nucleic acid molecules of (a) to (d or e) and having the biological activity represented by the protein as depicted in Fig.
  • nucleic acid molecule encoding a polypeptide comprising the consensus sequence shown in Fig 17 or 19 and having the biological activity represented by the protein as depicted in Fig. 7, 9, 11, 12, 13 and/or 16; h) nucleic acid molecule encoding a polypeptide having the biological activity represented by the protein as depicted in Fig.
  • nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridisation conditions with a probe comprising one of the sequences of the nucleic acid molecule of (a) or (b) or with a fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized in (a) to (c) and encoding a polypeptide having the biological activity represented by protein as depicted in Fig. 7, 9, 11, 12, 13 and/or 16; or which comprises a sequence which is complementary thereto.
  • nucleic acid sequences may be modified so that gene expression is decreased.
  • This reduction, decrease or deletion (reduction, decrease, deletion, inactivation or down-regulation shall be used as synonyms throughout the specification) can be achieved as mentioned above by all methods known to the skilled person, preferably by double- stranded RNA interference (dsRNAi), introduction of an antisense nucleic acid, a ribozyme, an antisense nucleic acid combined with a ribozyme, a nucleic acid encoding a co-suppressor, a nucleic acid encoding a dominant negative protein, DNA- or protein- binding factors targeting said gene or -RNA or -proteins, RNA degradation inducing viral nucleic acids and expression systems, systems for inducing a homolog recombination of said genes, mutations in said genes or a combination of the above.
  • dsRNAi double- stranded RNA interference
  • an activity of a gene product in an organism or part thereof, in particular in a plant cell, a plant, or a plant tissue or a part thereof or in a microorganism can be decreased by decreasing the amount of the specific encoding mRNA or the corresponding protein in said organism or part thereof, "amount of protein or mRNA” is understood as meaning the molecule number of polypeptides or mRNA molecules in an organism, a tissue, a cell or a cell compartment.
  • Decrease in the amount of a protein means the quantitative decrease of the molecule number of said protein in an organism, a tissue, a cell or a cell compartment or part thereof - for example by one of the methods described herein below - in comparison to a wild type, control or reference.
  • inactivation means that the enzymatic or biological activity of the polypeptides encoded is no longer detectable in the organism or in the cell such as, for example, within the plant or plant cell.
  • downregulation means that the enzymatic or biological activity of the polypeptides encoded is partly or essentially completely reduced in comparison with the activity of the untreated organism. This can be achieved by different cell- biological mechanisms.
  • the activity can be downregulated in the entire organism or, in the case of multi-celled organisms, in individual parts of the organism, in the case of plants for example in tissues such as the seed, the leaf, the root or other parts.
  • the enzymatic activity or biological activity is reduced by at least 10%, advantageously at least 20%, preferably at least 30%, especially preferably at least 40%, 50% or 60%, very especially preferably at least 70%, 80%, 85% or 90% or more, , very especially preferably are at least 95%, more preferably are at least 99% or more in comparison to the control, reference or wild type. Most preferably the reduction, decrease or deletion in activity amounts to 100%.
  • a modification i.e. a decrease
  • a decrease in activity in an organism or a part thereof can be caused by adding a chemical compound such as an antagonist to the media, nutrition, soil of the plants or to the plants themselves.
  • the increase in tolerance and/or resistance to an environmental stress as compared to a corresponding non-transformed wild type plant cell can be achived by decreasing the endogenous level of the polypeptide of the invention.
  • the present invention relates to a process, wherein the gene copy number of a gene encoding the polynucleotide or nucleic acid molecule of the invention is decreased.
  • the endogenous level of the polypeptide of the invention can for example be decreased by mod ' rfiying the transcriptional or translational regulation of the polypeptide.
  • a further embodiment of the inventive process is a process, whereby the reduction or deletion of the biological activity represented by the protein used in the inventive process is achieved by a process comprising a step selected from the group consisting of: (a) introducing of a nucleic acid molecule encoding a ribonucleic acid sequence, which are able to form double-stranded ribonucleic acid molecule, whereby the sense strand of said double-stranded ribonucleic acid molecule has a homology of at least 25%, preferably 30% to a nucleic acid molecule conferring the expression of or encoding a protein having the biological activity of the protein as depicted in Fig.
  • nucleic acid sequences mentioned under point (a) to (j) above it is easy possible to isolate the 5'- and/or 3' -regions of said nucleic acid sequences and to use said 5'- and/or 3'- sequences for the reduction, decrease or deletion of the nucleic acid sequences used in the inventive process according to the different process steps (a) to (j) mentioned above.
  • RNA nucleic acid sequence a double-stranded RNA nucleic acid sequence (dsRNA) as described above or of an expression cassette, or more than one expression cassette, ensuring the expression of the latter; b) introduction of an antisense nucleic acid sequence or of an expression cassette ensuring the expression of the latter.
  • dsRNA double-stranded RNA nucleic acid sequence
  • an antisense nucleic acid sequence or of an expression cassette ensuring the expression of the latter.
  • the antisense nucleic acid sequence is directed against a gene (i.e. genomic DNA sequences) or a gene transcript (i.e. RNA sequences) including the 5 ' and 3 ' non- translated regions.
  • a-anomeric nucleic acid sequences c) introduction of an antisense nucleic acid sequence in combination with a ribozyme or of an expression cassette ensuring the expression of the former; d) introduction of sense nucleic acid sequences for inducing cosuppression or of an expression cassette ensuring the expression of the former; e) introduction of a nucleic acid sequence encoding dominant-negative protein or of an expression cassette ensuring the expression of the latter; f) introduction of DNA-, RNA- or protein-binding factors against genes, RNA's or proteins or of an expression cassette ensuring the expression of the latter; g) introduction of viral nucleic acid sequences and expression constructs which bring about the degradation of RNA, or of an expression cassette ensuring the expression of the former; h) introduction of constructs for inducing homologous recombination on endogenous genes, for example for generating knockout mutants; i) introduction of mutations into endogenous genes for generating a loss of function (e.
  • Each of these methods may bring about a reduction in the expression, the activity or the function for the purposes of the invention. A combined use is also feasible. Further methods are known to the skilled worker and may encompass hindering or preventing processing of the protein, transport of the protein or its mRNA, inhibition of ribosomal attachment, inhibition of RNA splicing, induction of an enzyme which degrades RNA or the protein of the invention and/or inhibition of translational elongation or termination, organism.
  • protein quantity refers to the amount of a polypeptide in an organism, a tissue, a cell or cell compartment.
  • reduction of the protein quantity refers to the quantitative reduction of the amount of a protein in an organism, a tissue, a cell or a cell compartment - for example by one of the methods described herein below - in comparison with the wild type of the same genus and species to which this method has not been applied under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like).
  • a reduction of at least 10%, advantageously of at least 20%, preferably at least 30%, especially preferably of at least 40%, 50% or 60%, very especially preferably of at least 70%, 80%, 90% or 95%, 99% or even 100% in comparison with the untreated organism is advantageous.
  • An especially advantageous embodiment is the inactivation of the nucleic acids, or of the polypeptides encoded by them. organism.
  • activity preferably refers to the activity of a polypeptide in an organism, a tissue, a cell or a cell compartment.
  • reduction in the activity refers to the reduction in the overall activity of a protein in an organism, a tissue, a cell or a cell compartment - for example by one of the methods described herein below - in comparison with the wild type of the same genus and species, to which this method has not been applied, under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like).
  • a reduction in activity of at least 10%, advantageously of at least 20%, preferably at least 30%, especially preferably of at least 40%, 50% or 60%, very especially preferably of at least 70%, 80%, 90% or 95%, 99% or even 100% in comparison with the untreated organism is advantageous.
  • a particularly advantageous embodiment is the inactivation of the nucleic acids or of the polypeptides encoded by them.
  • the term "function" preferably refers to the enzymatic or regulatory function of a peptide in an organism, a tissue, a cell or a cell compartment.
  • Suitable substrates are low-molecular-weight compounds and also the protein interaction partners of a protein.
  • reduction of the function refers, for example, to the quantitative reduction in binding capacity or binding strength of a protein for at least one substrate in an organism, a tissue, a cell or a cell compartment - for example by one of the methods described herein below - in comparison with the wild type of the same genus and species to which this method has not been applied, under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like).
  • Reduction is also understood as meaning the modification of the substrate specificity as can be expressed for example, by the kcat/Km value.
  • a reduction of the function of at least 10%, advantageously of at least 20%, preferably at least 30%, especially preferably of at least 40%, 50% or 60%, very especially preferably of at least 70%, 80%, 90% or 95%, 99% or even 100% in comparison with the untreated organism is advantageous.
  • a particularly advantageous embodiment is the inactivation of the function. Binding partners for the protein can be identified in the manner with which the skilled worker is familiar, for example by the yeast 2-hybrid system. [072.3] What follows is a brief description of the individual preferred methods:
  • dsRNAi double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • RNAi is also documented as an advantageously tool for the repression of genes in bacteria such as E. coli for example by Tchurikov et al. [J. Biol. Chem., 2000, 275 (34): 26523 - 26529].
  • Fire et al. named the phenomenon RNAi for RNA interference.
  • the techniques and methods described in the above references are expressly referred to.
  • Efficient gene suppression can also be observed in the case of transient expression or following transient transformation, for example as the consequence of a biolistic transformation (Schweizer P et al. (2000) Plant J 2000 24: 895-903).
  • dsRNAi methods are based on the phenomenon that the simultaneous introduction of complementary strand and counterstrand of a gene transcript brings about highly effective suppression of the expression of the gene in question.
  • the resulting phenotype is very similar to that of an analogous knock-out mutant (Waterhouse PM et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-64).
  • Tuschl et al. [Gens Dev., 1999, 13 (24): 3191 - 3197] was able to show that the efficiency of the RNAi method is a function of the length of the duplex, the length of the 3'-end overhangs, and the sequence in these overhangs. Based on the work of Tuschl et al.
  • dsRNAi has proved to be particularly effective and advantageous for reducing the expression of the nucleic acid sequences of the sequences as shown in Fig. 11 , 12, 14 and/or 16 and/or homologs thereof.
  • dsRNAi approaches are clearly superior to traditional antisense approaches.
  • the invention therefore furthermore relates to double-stranded RNA molecules (dsRNA molecules) which, when introduced into an organism, advantageously into a plant (or a cell, tissue, organ or seed derived therefrom), bring about altered metabolic activity by the reduction in the expression of the nucleic acid sequences of the figures 11, 12, 14 and/or 16 and/or homologs thereof.
  • dsRNA molecules double-stranded RNA molecules
  • one of the two RNA strands is essentially identical to at least part of a nucleic acid sequence
  • the respective other RNA strand is essentially identical to at least part of the complementary strand of a nucleic acid sequence.
  • the term "essentially identical" refers to the fact that the dsRNA sequence may also include insertions, deletions and individual point mutations in comparison to the target sequence while still bringing about an effective reduction in expression.
  • the homology as defined above amounts to at least 30%, preferably at least 40%, 50%, 60%, 70% or 80%, very especially preferably at least 90%, most preferably 100%, between the "sense" strand of an inhibitory dsRNA and a part-segment of a nucleic acid sequence of the invention including in a preferred embodiment of the invention their endogenous 5 ' - and 3 ' untranslated regions (or between the "antisense" strand and the complementary strand of a nucleic acid sequence, respectively).
  • the part-segment amounts to at least 10 bases, preferably at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases, especially preferably at least 40, 50, 60, 70, 80 or 90 bases, very especially preferably at least 100, 200, 300 or 400 bases, most preferably at least 500, 600, 700, 800, 900 or more bases or at least 1000 or 2000 bases or more in length.
  • the part-segment amounts to 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 bases, preferably to 20, 21, 22, 23, 24 or 25 bases. These short sequences are preferred in animals and plants.
  • RNAs preferably between 200 and 800 bases are preferred in nonmammalian animals, preferably in invertebrates, in yeast, fungi or bacteria, but they are also useable in plants.
  • siRNAs small/short interfering RNAs
  • Dicer which is a ds-specific Rnase III enzyme.
  • an "essentially identical" dsRNA may also be defined as a nucleic acid sequence, which is capable of hybridizing with part of a gene transcript (for example in 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12 to 16 h).
  • the dsRNA may consist of one or more strands of polymerized ribonucleotides. Modification of both the sugar-phosphate backbone and of the nucleosides may furthermore be present. For example, the phosphodiester bonds of the natural RNA can be modified in such a way that they encompass at least one nitrogen or sulfur hetero atom. Bases may undergo modification in such a way that the activity of, for example, adenosine deaminase is restricted. These and other modifications are described herein below in the methods for stabilizing antisense RNA.
  • the dsRNA can be prepared enzymatically; it may also be synthesized chemically, either in full or in part. Short dsRNA up to 30 bp, which effectively mediate RNA interference, can be for example efficiently generated by partial digestion of long dsRNA templates using E. coli ribonuclease III (RNase III). (Yang, D., et al. (2002) Proc. Natl. Acad. Sci. USA 99, 9942.)
  • the double-stranded structure can be formed starting from a single, self-complementary strand or starting from two complementary strands.
  • "sense" and “antisense” sequence can be linked by a linking sequence ("linker") and form for example a hairpin structure.
  • the linking sequence may take the form of an intron, which is spliced out following dsRNA synthesis.
  • the nucleic acid sequence encoding a dsRNA may contain further elements such as, for example, transcription termination signals or polyadenylation signals.
  • the two strands of the dsRNA are to be combined in a cell or an organism advantageously in a plant, this can be brought about in a variety of ways: a) transformation of the cell or of the organism, advantageously of a plant, with a vector encompassing the two expression cassettes; b) cotransformation of the cell or of the organism, advantageously of a plant, with two vectors, one of which encompasses the expression cassettes with the "sense" strand while the other encompasses the expression cassettes with the "antisense” strand; c) supertransformation of the cell or of the organism, advantageously of a plant, with a vector encompassing the expression cassettes with the "sense” strand, after the cell or the organism had already been transformed with a vector encompassing the expression cassettes with the "antisense” strand; d) hybridization e.g.
  • RNA duplex can be initiated either outside the cell or within the cell.
  • the dsRNA is synthesized outside the target cell or organism it can be introduced into the organism or a cell of the organism by injection, microinjection, electroporation, high velocity particles, by laser beam or mediated by chemical compounds (DEAE-dextran, calciumphosphate, liposomes) or in case of animals it is also possible to feed bacteria such as E. coli strains engineered to express double- stranded RNAi to the animals.
  • chemical compounds DEAE-dextran, calciumphosphate, liposomes
  • the dsRNA may also encompass a hairpin structure, by linking the "sense” and “antisense” strands by a "linker” (for example an intron).
  • a "linker” for example an intron.
  • the self-complementary dsRNA structures are preferred since they merely require the expression of a construct and always encompass the complementary strands in an equimolar ratio.
  • the expression cassettes encoding the "antisense” or the "sense” strand of the dsRNA or the self-complementary strand of the dsRNA are preferably inserted into a vector and stably inserted into the genome of a plant, using the methods described herein below (for example using selection markers), in order to ensure permanent expression of the dsRNA.
  • Transient expression with bacterial or viral vectors are similar useful.
  • the dsRNA can be introduced using an amount which makes possible at least one copy per cell.
  • a larger amount for example at least 5, 10, 100, 500 or 1 000 copies per cell) may bring about more efficient reduction.
  • dsRNA can be synthesized either in vivo or in vitro. To this end, a
  • DNA sequence encoding a dsRNA can be introduced into an expression cassette under the control of at least one genetic control element (such as, for example, promoter, enhancer, silencer, splice donor or splice acceptor or polyadenylation signal).
  • at least one genetic control element such as, for example, promoter, enhancer, silencer, splice donor or splice acceptor or polyadenylation signal.
  • Suitable advantageous constructs are described herein below. Polyadenylation is not required, nor do elements for initiating translation have to be present.
  • a dsRNA can be synthesized chemically or enzymatically.
  • RNA polymerases or bacteriophage RNA polymerases can be used for this purpose. Suitable methods for the in-vitro expression of RNA are described (WO 97/32016; US 5,593,874; US 5,698,425, US 5,712,135, US 5,789,214, US 5,804,693).
  • a dsRNA which has been synthesized in vitro either chemically or enzymatically can be isolated to a higher or lesser degree from the reaction mixture, for example by extraction, precipitation, electrophoresis, chromatography or combinations of these methods.
  • the dsRNAcan be introduced directly into the cell or else be applied extracellularly (for example into the interstitial space).
  • the RNAi method leads to only a partial loss of gene function and therefore enables the skilled worker to study a gene dose effect in the disered organism and to fine tune the process of the invention.
  • it leads to a total loss of function and therefore increases the production of the fine chemical. Futhermore it enables a person skilled in the art to study multiple functions of a gene.
  • the antisense nucleic acid molecule hybridizes with, or binds to, the cellular mRNA and/or the genomic DNA encoding the target protein to be suppressed. This process suppresses the transcription and/or translation of the target protein. Hybridization can be brought about in the conventional manner via the formation of a stable duplex or, in the case of genomic DNA, by the antisense nucleic acid molecule binding to the duplex of the genomic DNA by specific interaction in the large groove of the DNA helix.
  • An "antisense" nucleic acid molecule comprises a nucleotide sequence, which is at least in part complementary to a "sense" nucleic acid molecule encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an encoding mRNA sequence. Accordingly, an antisense nucleic acid molecule can bind via hydrogen bonds to a sense nucleic acid molecule.
  • the antisense nucleic acid molecule can be complementary to an entire coding strand of a nucleic acid molecule conferring the expression of the polypeptide of the invention or to only a portion thereof.
  • an antisense nucleic acid molecule can be antisense to a "coding region" of the coding strand of a nucleotide sequence of a nucleic acid molecule of the present invention.
  • the noncoding region is in the area of 50 bp, 100 bp, 200bp or 300 bp, pererably 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp or 1000 bp up- and/or downstream from the coding region.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons, which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the mRNA flanking the coding region of a nucleotide sequence.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into a polypeptide, i.e., also referred to as 5' and 3' untranslated regions (5'-UTR or 3 - UTR).
  • antisense nucleic acid molecules of the invention can be designed according to the rules of Watson and Crick base pairing.
  • An antisense nucleic acid sequence which is suitable for reducing the activity of a protein can be deduced using the nucleic acid sequence encoding this protein, for example the nucleic acid sequence as shown in fig. 11, 12, 14 and/or 16 (or homologs, analogs, paralogs, orthologs thereof), by applying the base-pair rules of Watson and Crick.
  • the antisense nucleic acid sequence can be complementary to all of the transcribed mRNA of the protein; it may be limited to the coding region, or it may only consist of one oligonucleotide, which is complementary to part of the coding or noncoding sequence of the mRNA.
  • the oligonucleotide can be complementary to the nucleic acid region, which encompasses the translation start for the protein.
  • Antisense nucleic acid sequences may have an advantageous length of, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides but they may also be longer and encompass at least 100, 200, 500, 1000, 2000 or 5000 nucleotides. A particular preferred length is between 15 and 30 nucleotides such as 15, 20, 25 or 30 nucleotides.
  • Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically using methods known to the skilled worker. .
  • an antisense nucleic acid molecule e.g., an antisense oligonucleotide
  • an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • substances which can be used are phosphorothioate derivatives and acridine-substituted nucleotides such as 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthin, xanthin, 4-acetylcytosine, 5- (carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxy- methylaminomethyluracil, dihydrouracil, b-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, b-D
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid molecule of interest, described further in the following subsection).
  • the expression of a protein encoded by one of the polypeptodes of fig. 7, 9, 11, 12, 13 and/or 16 or homologs, analogs, paralogs, orthologs thereof can be inhibited by nucleotide sequences which are complementary to the regulatory region of a gene (for example a promoter and/or enhancer) and which may form triplex structures with the DNA double helix in this region so that the transcription of the gene is reduced.
  • nucleotide sequences which are complementary to the regulatory region of a gene (for example a promoter and/or enhancer) and which may form triplex structures with the DNA double helix in this region so that the transcription of the gene is reduced.
  • the antisense nucleic acid molecule can be an a-anomeric nucleic acid.
  • a-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA in which - as opposed to the conventional b-nucleic acids - the two strands run in parallel with one another (Gautier C et al. (1987) Nucleic Acids Res. 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise 2'-0-methylribonucleotides [Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148] or chimeric RNA-DNA analogs [Inoue et al. (1987) FEBS Lett 215: 327-330].
  • the antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide having the biological activity of protein of the invention thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation and leading to the aforementioned compound X increasing activity.
  • the antisense molecule of the present invention comprises also a nucleic acid molecule comprising a nucleotide sequences complementary to the regulatory region of an nucleotide sequence encoding the natural occurring polypeptide of the invention, e.g. the polypeptide sequences shown in the sequence listing, or identified according to the methods described herein, e.g., its promoter and/or enhancers, e.g. to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, LJ. (1992) Bioassays 14(12):807-15.
  • ribozyme sequences into "antisense” RNAs imparts this enzyme-like RNA-cleaving property to precisely these "antisense” RNAs and thus increases their efficiency when inactivating the target RNA.
  • the preparation and the use of suitable ribozyme "antisense” RNA molecules is described, for example, by Haseloff et al. (1988) Nature 33410: 585-591.
  • the antisense nucleic acid molecule of the invention can be also a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity, which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes for example "Hammerhead” ribozymes; Haselhoff and Gerlach (1988) Nature 33410: 585-591] can be used to catalytically cleave the mRNA of an enzyme to be suppressed and to prevent translation.
  • the ribozyme technology can increase the efficacy of an antisense strategy.
  • the function or activity of a protein can efficiently also be reduced by expressing a dominant-negative variant of said protein.
  • the skilled worker is familiar with methods for reducing the function or activity of a protein by means of coexpression of its dominant-negative form [Lagna G and Hemmati-Brivanlou A (1998) Current Topics in Developmental Biology 36: 75-98; Perlmutter RM and Alberola-lla J (1996) Current Opinion in Immunology 8(2): 285-90; Sheppard D (1994) American Journal of Respiratory Cell & Molecular Biology 11(1): 1-6; Herskowitz I (1987) Nature 329 (6136): 219-22].
  • a dominant-negative variant can be realized for example by changing of an amino acid in the proteins encoded by one of the sequences shown in fig. 7, 9, 11 , 12, 13 and/or 16 or homologs thereof. This change can be determined for example by computer-aided comparison ("alignment").
  • These mutations for achieving a dominant- negative variant are preferably carried out at the level of the nucleic acid sequences.
  • a corresponding mutation can be performed for example by PCR-mediated in-vitro mutagenesis using suitable oligonucleotide primers by means of which the desired mutation is introduced. To this end, methods are used with which the skilled worker is familiar.
  • the "LA PCR in vitro Mutagenesis Kit” (Takara Shuzo, Kyoto) can be used for this purpose. It is also possible and known to those skilled in the art that deleting or changing of functional domains, e. g. TF or other signaling components which can bind but not activate may achieve the reduction of protein activity.
  • a reduction in the expression of a gene encoded by one of the sequences shown in fig. 7, 9, 11 , 12, 13 and/or 16 or homologs thereof according to the invention can also be achieved with specific DNA-binding factors, for example factors of the zinc finger transcription factorlype. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory regions, and bring about repression of the endogenous gene.
  • the use of such a method makes possible the reduction in the expression of an endogenous gene without it being necessary to recombinantly manipulate the sequence of the latter.
  • Such methods for the preparation of relevant factors are described in Dreier B et al. [(2001 ) J. Biol. Chem. 276(31): 29466-78 and (2000) J. Mol.
  • genes can be selected using any portion of a gene.
  • This segment is preferably located in the promoter region.
  • it may also be located in the region of the coding exons or introns.
  • the skilled worker can obtain the relevant segments from Genbank by database search or starting from a cDNA whose gene is not present in Genbank by screening a genomic library for corresponding genomic clones.
  • proteins which are introduced into a cell may also be those which themselves inhibit the target protein.
  • the protein-binding factors can, for example, be aptamers [Famulok M and Mayer G (1999) Curr. Top Microbiol. Immunol. 243: 123-36] or antibodies or antibody fragments or single-chain antibodies. Obtaining these factors has been described, and the skilled worker is familiar therewith.
  • a cytoplasmic scFv antibody has been employed for modulating activity of the phytochrome A protein in genetically modified tobacco plants [Owen M et al. (1992) Biotechnology (NY) 10(7): 790-794; Franken E et al. (1997) Curr. Opin. Biotechnol. 8(4): 411-416; Whitelam (1996) Trend Plant Sci. 1: 286-272].
  • Gene expression may also be suppressed by tailor-made low- molecular-weight synthetic compounds, for example of the polyamide type [Dervan PB and B ⁇ rli RW (1999) Current Opinion in Chemical Biology 3: 688-693; Gottesfeld JM et al. (2000) Gene Expr. 9(1-2): 77-91].
  • These oligomers consist of the units 3- (dimethylamino)propylamine, N-methyl-3-hydroxypyrrole, N-methylimidazole and N-methylpyrroles; they can be adapted to each portion of double-stranded DNA in such a way that they bind sequence-specifically to the large groove and block the expression of the gene sequences located in this position.
  • a nucleic acid construct which, for example, comprises at least part of an endogenous gene which is modified by a deletion, addition or substitution of at least one nucleotide in such a way that the functionality is reduced or completely eliminated.
  • T e modification may also affect the regulatory elements (for example the promoter) of the gene so that the coding sequence remains unmodified, but expression (transcription and/or translation) does not take place and is reduced.
  • the modified region is flanked at its 5' and 3' end by further nucleic acid sequences, which must be sufficiently long for allowing recombination.
  • Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate.
  • One possibility of removing the randomly integrated sequences and thus increasing the number of cell clones with a correct homologous recombination is the use of a sequence-specific recombination system as described in US 6,110,736, by means of which unspecifically integrated sequences can be deleted again, which simplifies the selection of events which have integrated successfully via homologous recombination.
  • a multiplicity of sequence-specific recombination systems may be used, examples which may be mentioned being Cre/lox system of bacteriophage P1, the FLP/FRT system from yeast, the Gin recombinase of phage Mu, the Pin recombinase from E. coli and the R/RS system of the pSR1 plasmid.
  • the bacteriophage P1 Cre/lox system and the yeast FLP/FRT system are preferred.
  • the FLP/FRT and the cre/lox recombinase system have already been applied to plant systems [Odell et al. (1990) Mol. Gen. Genet. 223: 369-378].
  • RNA/DNA oligonucleotides into the plant [Zhu et al. (2000) Nat. Biotechnol. 18(5): 555-558], and the generation of knock-out mutants with the aid of, for example, T-DNA mutagenesis [Koncz et al. (1992) Plant Mol. Biol.
  • the mutation sites may be specifically targeted or randomly selected. If the mutations have been created randomly e.g. by Transposon-Tagging or chemical mutagenesis, the skilled worked is able to specifically enrich selected muation events in the inventive nucleic acids.
  • Nucleic acid sequences as described in item B) to I) are expressed in the cell or organism by transformation/transfection of the cell or organism or are introduced in the cell or organism by known methods, for example as disclosed in item A).
  • Screening is well known to those skilled in the art and generally refers to the search for a particular attribute or trait.
  • this trait in a plant or plant cell is preferably the concentration of a metabolite or estimate the general appearance.
  • the methods and devices for screening are familiar to those skilled in the art and include GC (gas chromatography), LC (liquid chromatography), HPLC (high performance (pressure) liquid chromatography), MS (mass spectrometry), NMR (nuclear magnetic resonance) spectroscopy, IR (infra red) spectroscopy, photometric methods etc and combinations of these methods.
  • the screening for plants or plant cells with an increased tolerance and/or resistance to environmental stress as compared to non-transformed wild type cells is practiced with flu-mutants (op den Camp et al., 2003), as described below.
  • flu-mutants op den Camp et al., 2003
  • a preferred metabolite in the above described screening are Pchlide, protochlorophyllide. Further preferred metabolites are the oxygenation products of linolenic acid, hydroperoxy octadecatrieonic acid (HPOTE) and hydroxy octadecatrieonic acid (HOTE) and 2-Oxo-phytodienoic acid (OPDA) and jasmonic acid.
  • HPOTE hydroperoxy octadecatrieonic acid
  • HOTE hydroxy octadecatrieonic acid
  • OPDA 2-Oxo-phytodienoic acid
  • One of the advantages of the stress resistant plants or plant cells of the present invention is their increased tolerance and/or resistance to ROS induced stress to a concentration of protochlorophyllide 9-10 times higher as compared to the wild type.
  • herbicides which induce high ROS concentrations are: a) inhibitors of photosynthesis such as Atraton, Atrazine, Ametryne, Aziprotryne, Cyanazine, Cyanatryn, Chlorazine, Cyprazine, Desmetryne, Dimethametryne, Dipropetryn, Eglinazine, Ipazine, Mesoprazine, Methometon, Methoprotryne, Procyazine, Proglinazine, Prometon, Prometryne, Propazine, Sebuthylazine, Secbumeton, Simazine, Simeton, Simetryne, Terbumeton, Terbuthylazine, Terbutryne, Trietazine, Ametridione, Amibuzin, Hexazinone, Isomethiozin, Metamitron, Metribuzin, Bromacil, Isocil, Lenacil, Terbacil, Brompyrazon, Chloridazon, Dioxide, a
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.
  • Different breeding measures can be taken, depending on the desired properties. All the techniques are well known by a person skilled in the art and include for example, but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also can include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both of the parental lines.
  • transgenic seeds and plants according to the invention can therefor be used for the breeding of improved plant lines, which can increase the effectiveness of conventional methods such as herbicide or pesticide treatment or which allow one to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance preferably drought and temperature
  • Environmental stress includes but is not limited to salinity, drought, temperature, metal, chemical, pathogenic and oxidative stress, or combinations thereof, preferably ROS induced stress and/or herbicides.
  • environmental stress refers to any sub- optimal growing condition and includes, but is not limited to, sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof.
  • environmental stress may be salinity, drought, heat, or low temperature, or combinations thereof, and in particular, may be low water content or low temperature.
  • drought stress means any environmental stress which leads to a lack of water in plants or reduction of water supply to plants, wherein low temperature stress means freezing of plants below + 4°C as well as chilling of plants below 15°C and wherein high temperature stress means for example a temperature above 35°C.
  • ROS induced stress refers to any sub-optimal growing condition which induce a higher ROS concentration in plants or plant cells as compared to the wild type and leads to apoptotic cells, cell suicide or growth inhibition.
  • chemical stress refers to any sub-optimal growing condition associated with chemical compounds, preferably herbicides.
  • the invention also provides a transformed plant cell with one or more nucleic acid sequences homologous to one or more of sequences shown in Fig. 11 , 12, 13 and/or 14 , wherein the plant is selected from the group comprised of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa, more preferably dicotyledonean plants, such as A.t, Arabidopsis thaliana; S
  • the present invention further provides a transgenic plant cell with an inactivated or down-regulated gene selected from the group comprising sequences as shown in Fig. 11 , 12, 13 and/or 14 and/or homologs thereof, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa, more preferably dicotyledonean plants, such as A.t., Arabidopsis thaliana; S.t., Solanum tuberosum; N.s., Nicotiana sylvestris; B.o., Brassica oleracea; V.v., Vitis vinifera; L.e., Lycopersicum esculentum; C.s., Citrus sinensis; and P.b., Populus balsamifera and monocotyledonean plants, such as O.s., Oryza sativa; H.v., Hordeum vulgare; T.a., Triticum aestivum; S.o., Sac
  • nucleic acid sequence selected from the group consisting of sequences as shown in Fig. 11, 12, 13 and/or 14 and/or homologs thereof in target plants, especially crop plants, and then inactivate or down-regulate the corresponding gene to achieve increased tolerance and/or resistance to environmental stress. Consequently the invention, is not limited to a specific plant.
  • the invention also provides a transformed plant cell with a nucleic acid sequence homologous to one of sequences as shown in Fig. 11 , 12, 13 and/or 14 , wherein the plant is selected from the group comprised of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa, more preferably At., Arabidopsis thaliana; S.t., Solanum tuberosum; N.s
  • the transformed plant cell may be derived from a monocotyledonous or a dicotyledonous plant.
  • the monocotyledonous or a dicotyledonous plant may be selected from the group comprised of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa, more preferably dicotyledonean plants, such as A.t., Arabidopsis thaliana; S.t., Solanum tuberosum; N.s., Nicotiana sylvestris; B.o.
  • the transformed plant cell may be derived from a gymnosperm plant and can preferably be selected from the group of spruce, pine and fir.
  • the invention also provides a transformed plant generated from said plant cell and which is a monocot or dicot plant.
  • the transformed plant may be selected from the group comprised of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa, more preferably dicotyledonean plants, such as A.t., Arabidopsis thaliana; S.t., Solanum tuberosum; N.s., Nicotiana sylvestris; B.o., Brassica oleracea; V.v.,
  • the invention not only deals with plants but also with an agricultural product produced by any of the described transformed plants, plant parts such as leafs, petal, anther, roots, tubers, stems, buds, flowers or especially seeds produced by said transformed plant, which are at least genetically heterozygous, preferably homozygous for a gene or its homolog, that when inactivated or down-regulated confers an increased tolerance and/or resistance to environmental stress as compared to a wild type plant.
  • Homologs of the aforementioned sequences can be isolated advantageously from yeast, fungi, viruses, algae bacteria, such as Acetobacter
  • Actinobacillus sp Aeromonas salmonicida; Agrobacterium tumefaciens; Aquifex aeolicus; Arcanobacterium pyogenes; Aster yellows phytoplasma; Bacillus sp.;
  • Bifidobacterium sp. Borrelia burgdorferi; Brevibacterium linens; Brucella melitensis;
  • Klebsiella pneumoniae Lactobacillus sp.; Lactococcus lactis; Listeria sp.; Mannheimia haemolytica; Mesorhizobium loti; Methylophaga thalassica; Microcystis aeruginosa;
  • Neisseria sp. Nitrosomonas sp.; Nostoc sp. PCC 7120; Novosphingobium aromaticivorans; Oenococcus oeni; Pantoea citrea; Pasteurella multocida;
  • Pediococcus pentosaceus Phormidium foveolarum; Phytoplasma sp.; Plectonema boryanum; Prevotella ruminicola; Propionibacterium sp.; Proteus vulgaris;
  • Salmonella sp. Salmonella sp.; Selenomonas ruminantium; Serratia entomophila; Shigella sp.;
  • yeasts such as from the genera Saccharomyces, Pichia, Candida, Hansenula, Torulopsis or Schizosaccharomyces, or even more preferred from plants such as Arabidopsis thaliana, maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, borage, safflower, linseed, primrose, rapeseed, canola and turnip rape, manihot, pepper, sunflower, tagetes, solanaceous plant such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bushy plants such as coffee, cacao, tea, Salix species, trees such as oil palm, coconut, perennial grass, such as ryegrass and fescue, and forage crops, such as alfalfa and clover and from spruce, pine or fir for example, more preferably from Saccharomyces cerevisiae or plants
  • Homologs are defined herein as two nucleic acids or proteins that have similar, or “homologous", nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists and antagonists of stress related proteins of the present invention (SRP) as defined hereafter.
  • SRP stress related proteins of the present invention
  • the term “homolog” further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in Fig. 11 , 12, 13 and/or 14 (and portions thereof) due to degeneracy of the genetic code and thus encode the same SRP as that encoded by the amino acid sequences shown in Fig. 7, 9, 11 , 12, 13 and/or 16.
  • a "naturally occurring" SRP refers to a SRP amino acid sequence that occurs in nature.
  • the term "homology” means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent.
  • the nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other plant varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques.
  • the allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants.
  • Structurally equivalents can, for example, be identified by testing the binding of said polypeptide to antibodies or computer based predictions. Structurally equivalent have the similar immunological characteristic, e.g. comprise similar epitopes. [149.5] Functional equivalents derived from one of the polypeptides as shown in
  • Fig. 7, 9, 11, 12, 13 and/or 16 according to the invention by substitution, insertion or deletion have at least 20%, 27%, 28%, 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with one of the polypeptides as shown in in Fig.
  • 11, 12, 13 and/or 14 according to the invention by substitution, insertion or deletion have at least 20%, 27%, 28%, 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%,-97%, 98% or 99% homology with one of the polypeptides as shown in Fig.
  • hybridizing it is meant that such nucleic acid molecules hybridize under conventional hybridization conditions, preferably under stringent conditions such as described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) or in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • DNA as well as RNA molecules of the nucleic acid of the invention can be used as probes. Further, as template for the identification of functional homologues Northern blot assays as well as Southern blot assays can be performed.
  • the Northern blot assay advantageously provides further informations about the expressed gene product: e.g. expression pattern, occurance of processing steps, like splicing and capping, etc.
  • the Southern blot assay provides additional information about the chromosomal localization and organization of the gene encoding the nucleic acid molecule of the invention.
  • SSC sodium chloride/sodium citrate
  • 0.1% SDS 0.1% SDS at 50 to 65°C, for example at 50°C, 55°C or 60°C.
  • these hybridization conditions differ as a function of the type of the nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer.
  • the temperature under "standard hybridization conditions” differs for example as a function of the type of the nucleic acid between 42°C and 58°C, preferably between 45°C and 50°C in an aqueous buffer with a concentration of 0.1 x 0.5 x, 1 x, 2x, 3x, 4x or 5 x SSC (pH 7.2). If organic solvents) is/are present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 40°C, 42°C or45°C.
  • the hybridization conditions for DNADNA hybrids are preferably for example 0.1 x SSC and 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, preferably between 30°C and 45°C.
  • the hybridization conditions for DNA:RNA hybrids are preferably for example 0.1 x SSC and 30°C, 35°C, 40°C, 45°C, 50°C or 55°C, preferably between 45°C and 55°C.
  • a further example of one such stringent hybridization condition is hybridization at 4XSSC at 65°C, followed by a washing in 0.1XSSC at 65 ⁇ C for one hour.
  • an exemplary stringent hybridization condition is in 50 % formamide, 4XSSC at 42°C.
  • the conditions during the wash step can be selected from the range of conditions delimited by low-stringency conditions
  • the temperature during the wash step can be raised from low-stringency conditions at room temperature, approximately 22°C, to higher-stringency conditions at approximately 65°C.
  • Both of the parameters salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant while only the other is varied.
  • Denaturants for example formamide or SDS, may also be employed during the hybridization. In the presence of 50% formamide, hybridization is preferably effected at 42°C. Relevant factors like i) length of treatment, ii) salt conditions, iii) detergent conditions, iv) competitor DNAs, v) temperature and vi) probe selection can be combined case by case so that not all possibilities can be mentioned herein.
  • Northern blots are prehybridized with
  • Hybri-Quick buffer (Roth, Düsseldorf) at 68°C for 2h.
  • the hybridzation with radioactive labelled probe is conducted over night at 68°C. Subsequently the hybridization buffer is discarded and the filter shortly washed using 2xSSC; 0,1% SDS. After discarding the washing buffer new 2xSSC; 0,1% SDS buffer is added and incubated at 68°C for 15 minutes. This washing step is performed twice followed by an additional washing step using 1xSSC; 0,1% SDS at 68°C for 10 min.
  • Hybridization conditions can be selected, for example, from the following conditions: a) 4X SSC at 65°C, b) 6X SSC at 45°C, c)6X SSC, 100 mg/ml denatured fragmented fish sperm DNA at 68°C, d) 6X SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 68°C, e) 6X SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm DNA, 50% formamide at 42°C, f) 50% formamide, 4X SSC at 42°C, g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCI, 75 mM
  • Wash steps can be selected, for example, from the following conditions: a) 0.015 M NaCI/0.0015 M sodium citrate/0.1 % SDS at 50°C. b) 0.1X SSC at 65°C. c) 0.1X SSC, 0.5 % SDS at 68°C. d) 0.1X SSC, 0.5% SDS, 50% formamide at 42°C. e) 0.2X SSC, 0.1% SDS at 42 ⁇ C. f) 2X SSC at 65°C (low-stringency condition).
  • transformed means all those plants or parts thereof which have been brought about and/or modified by manipulation methods and in which either a) one or more genes, preferably encoded by one or more nucleic acid sequence as depicted in Fig. 11, 12, 13 and/or 14 or a homolog thereof, or b) .. c . a genetic regulatory element or elements, for example promoters, which are functionally linked e.g. to a nucleic acid sequence of sequences as shown in Fig. 11 , 12, 13 and/or 14 or a homolog thereof, or c) (a) and (b) is/are not present in its/their natural genetic environment and/or has/have been modified by means of manipulation methods.
  • the modification is possible for the modification to be, by way of example, a substitution, addition, deletion, inversion or insertion of one or more nucleotides.
  • Manipulation in the present invention is also meant to encompass all changes in the plant cell, including induced or non-induced (spontaneous) mutagenesis, directed or non-directed genetic manipulation by conventional breeding or by modern genetic manipulation methods, e. g.
  • dsRNAi double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • Natural genetic environment means the natural chromosomal locus in the organism of origin or the presence in a genomic library.
  • the natural, genetic environment of the nucleic acid sequence is preferably at least partially still preserved.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particulariy preferably at least 1000 bp, very particularly preferably at least 5000 bp.
  • a plant or plant cell is considered "true breeding" for a particular attribute if it is genetically homozygous for that attribute to the extent that, when the true- breeding plant is self-pollinated, a significant amount of independent segregation of the attribute among the progeny is not observed.
  • nucleic acid and “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an "isolated" nucleic acid molecule is one that is substantially separated from other nucleic acid molecules, which are present in the natural source of the nucleic acid. That means other nucleic acid molecules are present in an amount less than 5% based on weight of the amount of the desired nucleic acid, preferably less than 2% by weight, more preferably less than 1 % by weight, most preferably less than 0.5% by weight.
  • an "isolated" nucleic acid is free of some of the sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated gene encoding nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule encoding a gene or a portion thereof or a homolog thereof which confers tolerance and/or resistance to environmental stress in plants, when inactivated or down-regulated, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • an Arabidopsis thaliana gene encoding cDNA can be isolated from an A. thaliana library using all or portion of one of sequences of the nucleic acid according to at least one of the sequences as shown in Fig. 11, 12, 13 and/or 14 .
  • a nucleic acid molecule encompassing all or a portion of one of the sequences of sequences as shown in Fig.
  • RNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979 Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences as shown in Fig. 11, 12, 13 and/or 14 .
  • a nucleic acid molecule of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid molecule so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to a gene encoding nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in Fig. 11, 12, 13 and/or 14 or homologs thereof encoding a gene (i.e., the "coding region"), as well as 5' untranslated sequences and 3' untranslated sequences.
  • the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of sequences as shown in Fig. 11, 12, 13 and/or 14 or homologs thereof, for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a gene.
  • portions of genes or proteins encoded by said gene encoding nucleic acid molecules of the invention are preferably biologically active portions of genes or proteins described herein.
  • biologically active portion of a gene or protein encoded by said gene is intended to include a portion, e.g., a domain/motif, of the gene or protein that participates in stress tolerance and/or resistance response in a plant, which is preferably achieved by altering metabolic activity, and finally resulting in increased tolerance and/or resistance to environmental stress.
  • a stress analysis of a plant comprising the protein may be performed for example by the above screening method or by estimating the general appearance and health. More specifically, nucleic acid fragments encoding biologically active portions of a gene or protein encoded by said gene can be prepared by isolating a portion of one of sequences of the nucleic acid as shown in Fig.
  • conserved regions are those, which show a very little variation in the amino acid in one particular position of several homologs from different origin.
  • portions of a protein encompassed by the present invention include peptides comprising amino acid sequences derived from the amino acid sequence of the protein encoded by one of sequences as shown in Fig. 7, 9, 11 , 12, 13 and/or 16, or the amino acid sequence of a protein homologous to the protein, which include fewer amino acids than the full length protein or a full length protein which is homologous to the protein, and exhibits at least some activity of the protein.
  • Preferred portions according to the present invention e.g., peptides or proteins which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length
  • biologically active portions in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the activities described herein.
  • the biologically active portions of the protein include one or more selected domains/motifs or portions thereof having biological activity.
  • the present invention especially includes homologs and analogs of naturally occurring proteins and protein encoding nucleic acids in a plant.
  • Homologs are defined herein as two nucleic acids or proteins that have similar, or “homologous", nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists and antagonists of the protein as defined hereafter.
  • the term “homolog” further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in Fig. 11, 12, 13 and/or 14 (and portions thereof) due to degeneracy of the genetic code and thus encode the same protein as that encoded by the amino acid sequences.
  • a "naturally occurring" refers to an amino acid sequence that occurs in nature.
  • the present invention includes homologs and analogs of naturally occurring proteins and protein encoding nucleic acids of the invenion in a plant.
  • "Homologs” are defined herein as two nucleic acids or polypeptides that have similar, or substantially identical, nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists and antagonists of SRPs as defined hereafter.
  • the term “homolog” further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in Fig.
  • a naturally occurring protein refers to amino acid sequence that occurs in nature.
  • a naturally occurring protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress comprises an amino acid sequence selected from the group consisting of ones shown in Fig. 11, 12, 13 and/or 14.
  • An agonist of the protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress can retain substantially the same, or a subset, of the biological activities of the said protein.
  • An antagonist of the said protein can inhibit one or more of the activities of the naturally occurring form of the protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress.
  • an antagonist can competitively bind to a downstream or upstream member of the cell membrane O
  • component metabolic cascade that includes said protein, or bind to the protein of the invention that mediates transport of compounds across such membranes, thereby preventing translocation from taking place.
  • Nucleic acid molecules corresponding to natural allelic variants and analogs, orthologs and paralogs of a protein of the invention cDNA can be isolated based on their identity to the A.t., Arabidopsis thaliana; S.t., Solanum tuberosum; N.s., Nicotiana sylvestris; B.o., Brassica oleracea; V.v., Vitis vinifera; L.e., Lycopersicum esculentum; C.s., Citrus sinensis; and P.b., Populus balsamifera, O.s., Oryza sativa; H.v., Hordeum vulgare; T.a., Triticum aestivum; S.o., Saccharum officinarum; Z.m., Zea mays; and S.b., Sorghum bicolor protein nucleic acids described herein using said proteins cDNAs, or a portion thereof, as a hybridization probe
  • homologs of the protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of said protein for their agonist or antagonist activity.
  • a variegated library of protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of SRP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SRP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion polypeptides (e.g., for phage display) containing the set of protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress sequences therein.
  • a set of larger fusion polypeptides e.g., for phage display
  • Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector.
  • Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential protein of the invention sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art. See, e.g., Narang, S.A., 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic Acid Res.
  • libraries of fragments of the protein of the invention coding regions can be used to generate a variegated population of protein fragments for screening and subsequent selection of homologs of a said proteins.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a protein of the invention coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA, which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal, and internal fragments of various sizes of the protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify SRP homologs (Arkin and Yourvan, 1992, PNAS 89:7811-7815; Delgrave et al., 1993, Polypeptide Engineering 6(3):327-331).
  • cell based assays can be exploited to analyze a variegated protein (of the invention) library, using methods well known in the art.
  • the present invention further provides a method of identifying a novel protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress, comprising (a) raising a specific antibody response to said protein, or a fragment thereof, as described herein; (b) screening putative SRP material with the antibody, wherein specific binding of the antibody to the material indicates the presence of a potentially novel protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress; and (c) analyzing the bound material in comparison to known proteins, to determine its novelty.
  • the present invention includes protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress and homologs thereof.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid).
  • the amino acid residues at corresponding amino acid positions are then compared.
  • a position in one sequence e.g., one of the sequences as shown in Fig. 7, 9, 11, 12, 13 and/or 16
  • the molecules are identical at that position.
  • the same type of comparison can be made between two nucleic acid sequences.
  • the isolated amino acid homologs included in the present invention are at least about 20%, 27%, 28%, 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical to an entire
  • the isolated amino acid homologs included in the present invention are at least about 20%, 27%, 28%. 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical to an entire amino acid sequence encoded by a nucleic acid sequence as shown in Fig.
  • amino acid homologs of the proteins of the invention have sequence identity over at least 15 contiguous amino acid residues, more preferably at least 25 contiguous amino acid residues, and most preferably at least 35 contiguous amino acid residues of a polypeptide as shown in Fig. 7, 9, 11, 12, 13 and/or 16.
  • an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which is at least about 20%, 27%, 28%, 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical to a nucleotide sequence as shown in Fig.
  • nucleic acids are at least 75 nucleotides, more preferably at least 100 nucleotides and most preferably the entire length of the coding region.
  • the isolated nucleic acid homolog of the invention encodes a SRP, or portion thereof, that is at least 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence as shown in Fig. 7, 9, 11, 12, 13 and/or 16 and that functions as a modulator of an environmental stress response in a plant.
  • overexpression of the nucleic acid homolog in a plant increases the tolerance of the plant to an environmental stress.
  • the percent sequence identity between two nucleic acid or polypeptide sequences may be determined using the Vector NTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814).
  • a gap opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids.
  • a gap opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings.
  • the gap opening penalty is 10
  • the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the pu ⁇ oses of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide.
  • the invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes to the polynucleotide with a sequence as shown in Fig. 11, 12, 13 and/or 14 under stringent conditions. More particularly, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence as shown in Fig. 11 , 12, 13 and/or 14. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length.
  • an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which hybridizes under highly stringent conditions to the nucleotide seq uence shown in in Fig. 11 , 12, 13 and/or 14 , and functions as a modulator of stress tolerance in a plant.
  • overexpression of the isolated nucleic acid homolog in a plant increases a plant's tolerance to an environmental stress.
  • stringent conditions refers in one embodiment to hybridization overnight at 601 C in 10X Denharts solution, 6X SSC, 0.5% SDS and 100 g/ml denatured salmon sperm DNA. Blots are washed sequentially at 621C for 30 minutes each time in 3X SSC/0.1 % SDS, followed by 1X SSC/0.1 % SDS and finally 0.1X SSC/0.1 % SDS.
  • highly stringent conditions refers to hybridization overnight at 651 C in 10X Denharts solution, 6X SSC, 0.5% SDS and 100 g/ml denatured salmon sperm DNA.
  • Blots are washed sequentially at 651 C for 30 minutes each time in 3X SSC/0.1 % SDS, followed by 1X SSC/0.1% SDS and finally 0.1X SSC/0.1% SDS.
  • Methods for nucleic acid hybridizations are described in Meinkoth and Wahl, 1984, Anal. Biochem. 138:267-284; Ausubel et al. eds, 1995, Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-lnterscience, New York; and Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, New York.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent or highly stringent conditions to a sequence as shown in Fig. 11 , 12, 13 and/or 14 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural polypeptide).
  • the nucleic acid encodes a naturally occurring, .t, Arabidopsis thaliana; S.t., Solanum tuberosum; N.s., Nicotiana sylvestris; B.o., Brassica oleracea; V.v., Vitis vinifera; L.e., Lycopersicum esculentum; C.s., Citrus sinensis; and P.b., Populus balsamifera, a O.s., Oryza sativa; H.v., Hordeum vulgare; T.a., Triticum aestivum; S.o., Saccharum officinarum; Z.m., Zea mays; and S.b., Sorghum bicolor protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress.
  • .t Arabidopsis thaliana
  • S.t. Solanum tuberosum
  • N.s. Nicotiana sylvestris
  • allelic variants refers to a nucleotide sequence containing polymorphisms that lead to changes in the amino acid sequences of said protein and that exist within a natural population (e.g., a plant species or variety). Such natural allelic variations can typically result in 1-5% variance in a nucleic acid of the invention. Allelic variants can be O
  • nucleic acid sequence of interest identified by sequencing the nucleic acid sequence of interest in a number of different plants, which can be readily carried out by using hybridization probes to identify the same SRP genetic locus in those plants.
  • Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations in a protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress that are the result of natural allelic variation and that do not alter the functional activity of said protein, are intended to be within the scope of the invention.
  • An isolated nucleic acid molecule encoding a protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress having sequence identity with a polypeptide sequence as shown in Fig. 7, 9, 11 , 12, 13 and/or 16 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence as shown in Fig. 11, 12, 13 and/or 14, respectively, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced into one of the sequences as shown in Fig. 11, 12, 13 and/or 14 by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. [189.5] To knock the mutation is earned out preferably at essential positions.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in a protein of the invention is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a protein of the invention coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a SRP activity described herein to identify mutants that retain protein activity.
  • the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined by analyzing the stress tolerance of a plant expressing the polypeptide as described herein.
  • nucleic acid molecules encoding the protein whose reduction or deletion results in increased tolerance and/or resistance to an environmental stress described above another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto.
  • Antisense polynucleotides are thought to inhibit gene expression of a target polynucleotide by specifically binding the target polynucleotide and interfering with transcription, splicing, transport, translation, and/or stability of the target polynucleotide. Methods are described in the prior art for targeting the antisense polynucleotide to the chromosomal DNA, to a primary RNA transcript, or to a processed mRNA.
  • the target regions include splice sites, translation initiation codons, translation termination codons, and other sequences within the open reading frame.
  • antisense refers to a nucleic acid comprising a polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA, so as to interfere with expression of the endogenous gene.
  • “Complementary" polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules, specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • antisense nucleic acid includes single stranded RNA as well as double-stranded DNA expression cassettes that can be transcribed to produce an antisense RNA.
  • Active antisense nucleic acids are antisense RNA molecules that are capable of selectively hybridizing with a primary transcript or mRNA encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the polypeptide as shown in Fig. 7, 9, 11 , 12, 13 and/or 16.
  • the antisense nucleic acid can be complementary to an entire SRP coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a SRP.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding a SRP.
  • noncoding region refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • the antisense nucleic acid molecule can be complementary to the entire coding region of SRP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of SRP mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SRP mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • the antisense molecules of the present invention comprise an RNA having 60-100% sequence identity with at least 14 consecutive nucleotides of at least one of the sequences as shown in Fig. 11, 12, 13 and/or 14 .
  • the sequence identity will be at least 70%, more preferably at least 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecule of the invention is an 9-anomeric nucleic acid molecule.
  • An 9-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual 9-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids. Res.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
  • the antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a SRP to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • the antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic (including plant) promoter are preferred.
  • ribozymes As an alternative to antisense polynucleotides, ribozymes, sense polynucleotides, or double stranded RNA (dsRNA) can be used to reduce expression of a SRP polypeptide.
  • ribozyme is meant a catalytic RNA-based enzyme with ribonuclease activity which is capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which it has a complementary region.
  • Ribozymes e.g., hammerhead ribozymes described in Haselhoff and Gerlach, 1988, Nature 334:585-591
  • a ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a cDNA, as disclosed herein (i.e., sequences as shown in Fig. 11 , 12, 13 and/or 14) or on the basis of a heterologous sequence to be isolated according to methods taught in this invention.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a SRP-encoding mRNA.
  • SRP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W., 1993, Science 261 :1411 -1418.
  • the ribozyme will contain a portion having at least 7, 8, 9, 10, 12, 14, 16, 18 or 20 nucleotides, and more preferably 7 or 8 nucleotides, that have 100% complementarity to a portion of the target RNA.
  • Methods for making ribozymes are known to those skilled in the art. See, e.g., U.S. Patent Nos. 6,025,167; 5,773,260; and 5,496,698.
  • dsRNA refers to RNA hybrids comprising two strands of RNA.
  • the dsRNAs can be linear or circular in structure.
  • dsRNA is specific for a polynucleotide encoding either the polypeptide as shown in Fig.
  • RNAs may be substantially or completely complementary.
  • the hybridizing portions are at least 95% complementary.
  • the dsRNA will be at least 100 base pairs in length.
  • the hybridizing RNAs will be of identical length with no over hanging 5' or 3' ends and no gaps.
  • dsRNAs having 5' or 3' overhangs of up to 100 nucleotides may be used in the methods of the invention.
  • the dsRNA may comprise ribonucleotides or ribonucleotide analogs, such as 2'-0-methyl ribosyl residues, or combinations thereof. See, e.g., U.S. Patent Nos. 4,130,641 and 4,024,222.
  • a dsRNA polyriboinosinic acid:polyribocytidylic acid is described in U.S. patent 4,283,393.
  • Methods for making and using dsRNA are known in the art.
  • One method comprises the simultaneous transcription of two complementary DNA strands, either in vivo, or in a single in vitro reaction mixture. See, e.g., U.S. Patent No. 5,795,715.
  • dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures.
  • dsRNA can be expressed in a plant cell by transcribing two complementary RNAs.
  • Other methods for the inhibition of endogenous gene expression such as triple helix formation (Moser et al., 1987, Science 238:645-650 and Cooney et al., 1988, Science 241:456-459) and cosuppression (Napoli et al., 1990, The Plant Cell 2:279-289) are known in the art. Partial and full-length cDNAs have been used for the cosuppression of endogenous plant genes. See, e.g., U.S. Patent Nos.
  • a sense polynucleotide blocks transcription of the corresponding target gene.
  • the sense polynucleotide will have at least 65% sequence identity with the target plant gene or RNA.
  • the percent identity is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.
  • the introduced sense polynucleotide need not be full length relative to the target gene or transcript.
  • the sense polynucleotide will have at least 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with at least 100 consecutive nucleotides of one of the sequences as shown in Fig. 11, 12, 13 and/or 14.
  • the regions of identity can comprise introns and and/or exons and untranslated regions.
  • the introduced sense polynucleotide may be present in the plant cell transiently, or may be stably integrated into a plant chromosome or extrachromosomal replicon.
  • nucleic acid molecules encoding proteins from the same or other species such as protein analogs, orthologs and paralogs, are intended to be within the scope of the present invention.
  • analogs refers to two nucleic acids that have the same or similar function, but that have evolved separately in unrelated organisms.
  • orthologs refers to two nucleic acids from different species that have evolved from a common ancestral gene by speciation. Normally, orthologs encode proteins having the same or similar functions.
  • paralogs refers to two nucleic acids that are related by duplication within a genome.
  • Paralogs usually have different functions, but these functions may be related (Tatusov, R.L. et al. 1997 Science 278(5338):631-637).
  • Analogs, orthologs and paralogs of naturally occurring proteins can differ from the naturally occurring proteins by post-translational modifications, by amino acid sequence differences, or by both.
  • Post-translational modifications include in vivo and in vitro chemical derivatisation of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation, and such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.
  • orthologs of the invention will generally exhibit at least 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity or homology with all or part of a naturally occurring protein amino acid sequence and will exhibit a function similar to a protein.
  • homologs Such homologs, analogs, orthologs and paralogs will be referred to in general as homologs or being homologous throughout the present application.
  • the homology ( identity) was calculated over the entire amino acid range.
  • the program used was PileUp (J. Mol. Evolution., 25 (1987), 351 - 360, Higgens et al., CABIOS, 5 1989: 151 - 153) or the program Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443 -453 (1970) and Smith and Waterman respectively (Adv. Appl. Math. 2; 482 - 489 (1981)] which are part of the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)].
  • the invention provides a method of producing a transformed plant, wherein inactivation or down-regulation of a gene in the transformed plant results in increased tolerance and/or resistance to environmental stress as compared to a corresponding non-transformed wild type plant, comprising (a) transforming a plant cell by inactivation or down-regulation of one or more genes, preferably encoded by one or more nucleic acids selected from a group consisting of the nucleic acids as shown in Fig. 11, 12, 13 and/or 14 and/or homologs thereof and (b) generating from the plant cell a transformed plant with an increased tolerance and/or resistance to environmental stress as compared to a corresponding wild type plant.
  • the invention also incorporates a method of inducing increased tolerance and/or resistance to environmental stress as compared to a corresponding non-transformed wild type plant in said plant cell or said plant by inactivation or down- regulation of one or more genes encoded by one or more nucleic acids selected from a group consisting of the nucleic acids as shown in Fig. 11, 12, 13 and/or 14 and/or homologs thereof.
  • the nucleic acid is at least about 20%, 27%, 28%, 30%, 40%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or preferably of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% more preferably of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% most preferably of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to said sequence (see above).
  • the homolog sequence stems from a plant selected from the group comprised of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed, turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops and Arabidopsis thaliana, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa , more preferably dicotyledonean plants, such as A.t, Arabidopsis thaliana; S.t, Solanum tuberosum; N.s., Nicotiana sylvestris; B.o., Brassica oleracea;
  • Inactivation or down-regulation of said gene or genes may be achieved by all methods known to one skilled in the art, preferably by double-stranded RNA interference (dsRNAi), introduction of an antisense nucleic acid, a ribozyme, an antisense nucleic acid combined with a ribozyme, a nucleic acid encoding a co- suppressor, a nucleic acid encoding a dominant negative protein, DNA- or protein- binding factors targeting said gene or -RNA or -proteins, RNA degradation inducing viral nucleic acids and expression systems, systems for inducing a homolog recombination of said genes, mutations in said genes or a combination of the above.
  • dsRNAi double-stranded RNA interference
  • nucleic acid sequences of the invention or their homologs are isolated nucleic acid sequences which encode polypeptides. These nucleic acids or the polypeptides encoded by them and their biological and enzymatic activity are inactivated or downregulated in the method according to the invention which leads to increased resistance and/or tolerance to environmental stress.
  • inactivation means that the enzymatic or biological activity of the polypeptides encoded is no longer detectable in the organism or in the cell such as, for example, within the plant or plant cell.
  • downregulation means that the enzymatic or biological activity of the polypeptides encoded is partly or essentially completely reduced in comparison with the activity of the untreated organism.
  • the activity can be downregulated in the entire organism or, in the case of multi-celled organisms, in individual parts of the organism, in the case of plants for example in tissues such as the seed, the leaf, the root or other parts.
  • the enzymatic activity or biological activity is reduced by at least 10%, advantageously at least 20%, preferably at least 30%, especially preferably at least 40%, 50% or 60% contour.very especially preferably at least 70%, 80%, 90% or 95%, 99% or even 100% in comparison with the untreated organism.
  • a particularly advantageous embodiment is the inactivation of the nucleic acids or of the polypeptides encoded by them.
  • the invention provides a method of producing a transformed plant with a gene encoding nucleic acid, wherein inactivation or down-regulation of said gene(s) in the plant results in increased tolerance to environmental stress as compared to a wild type plant, comprising the inactivation or down-regulation by mutation of a nucleic acid sequence as shown in Fig. 11, 12, 13 and/or 14 or homologs thereof.
  • binary vectors such as pBinAR can be used (H ⁇ fgen and Willmitzer, 1990 Plant Science 66:221-230).
  • suitable binary vectors are such as pBIN19, pBI101, pGPTV or pPZP (Hajukiewicz, P.
  • Construction of the binary vectors can be performed by ligation of the cDNA in sense or antisense orientation into the T-DNA. 5-prime to the cDNA a plant promoter activates transcription of the cDNA. A polyadenylation sequence is located 3- prime to the cDNA. Tissue-specific expression can be achieved by using a tissue specific promoter as listed below. Also, any other promoter element can be used. For constitutive expression within the whole plant, the CaMV 35S promoter can be used.
  • the expressed protein can be targeted to a cellular compartment using a signal peptide, for example for plastids, mitochondria or endoplasmic reticulum (Kermode, 1996 Crit Rev. Plant Sci. 4(15):285-423).
  • the signal peptide is cloned 5-prime in frame to the cDNA to archive subcellular localization of the fusion protein.
  • promoters that are responsive to abiotic stresses can be used with, such as the Arabidopsis promoter RD29A.
  • the promoter used should be operatively linked to the nucleic acid such that the promoter causes transcription of the nucleic acid which results in the synthesis of a mRNA which encodes a polypeptide.
  • the RNA can be an antisense RNA for use in affecting subsequent expression of the same or another gene or genes.
  • Alternate methods of transfection include the direct transfer of DNA into developing flowers via electroporation or Agrobacterium mediated gene transfer.
  • Agrobacterium mediated plant transformation can be performed using for example the GV3101 (pMP90) (Koncz and Schell, 1986 Mol. Gen. Genet. 204:383-396) or LBA4404 (Ooms et al., Plasmid, 1982, 7: 15-29; Hoekema et al., Nature, 1983, 303: 179-180) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994 Nucl. Acids. Res.
  • rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989 Plant Cell Reports 8:238-242; De Block et al., 1989 Plant Physiol. 91 :694-701).
  • Agrobacterium and plant selection depend on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker.
  • Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994 Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. o
  • a useful method to ascertain the level of transcription or activity of the gene is to perform a Northern blot (for reference see, for example, Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: New York).
  • This information at least partially demonstrates the degree of transcription of the transformed gene.
  • Total cellular RNA can be prepared from cells, tissues or organs by several methods, all well-known in the art, such as that described in Bormann, E.R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed.
  • the invention may further be combined with an isolated recombinant expression vector comprising a stress related protein encoding nucleic acid, wherein expression of the vector or stress related protein encoding nucleic acid, respectively in a host cell results in increased tolerance and/or resistance to environmental stress as compared to the wild type of the host cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • expression vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses
  • a plant expression cassette comprising a nucleic acid construct, which when expressed allows inactivation or down-regulation of a gene encoded by a nucleic acid selected from the group consisting of sequences as shown in Fig. 11, 12, 13 and/or 14 and/or homologs thereof and/or parts thereof by a method mentioned above leading to increased stress tolerance and/or resistance is also included in the scope of the present invention.
  • the plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells and operably linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals.
  • Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens T-DNA such as the gene 3 known as octopine synthase of the Ti-plas id pTiACH ⁇ (Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.
  • Plant gene expression must be operably linked to an appropriate promoter conferring gene expression in a time, cell or tissue specific manner.
  • Preferred promoters are such that drive constitutive expression (Benfey et al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CaMV (Franck et al., 1980 Cell 21 :285-294), the 19S CaMV (see also U.S. Patent No. 5352605 and PCT Application No. WO 8402913) or plant promoters like those from Rubisco small subunit described in U.S. Patent No. 4,962,028.
  • Additional advantageous regulatory sequences are, for example, included in the plant promoters such as CaMV/35S [Franck et al., Cell 21 (1980) 285 - 294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, LEB4, nos or in the ubiquitin, napin or phaseolin promoter.
  • Additional useful plant promoters are the cytosolic FBPase promoter or ST-LSI promoter of the potato (Stockhaus et al., EMBO J.
  • phosphorybosyl phyrophoshate amido transferase promoter of Glycine max gene bank accession No. U87999
  • noden specific promoter described in EP-A-0 249 676 are seed specific promoters which can be used for monocotyledons or dicotyledons.
  • the construct may also comprise further genes which are to be inserted into the organisms and which are for example involved in stress resistance, i.e. next to inactivating certain genes or incorporating inactivated genes at their place, it is possible to introduce favorable genes that are related to production of proteins which actively increase stress tolerance or resistance. It is therefore feasible and advantageous to insert and express in host organisms regulatory genes such as genes for inducers, repressors or enzymes which intervene by their enzymatic activity in the regulation of one or more or all genes of a biosynthetic pathway. These genes can be heterologous or homologous in origin.
  • the inserted genes may have their own promoter or else be under the control of same promoter as sequences shown in Fig. 11, 12, 13 and/or 14 or their homologs.
  • the expression cassettes according to the invention are also understood as meaning analogs which can be brought about, for example by a combination of the individual nucleic acid sequences on a polynucleotide (multiple constructs), on a plurality of polynucleotides in a cell (cotransformation) or by sequential transformation.
  • Advantageous genetic control sequences of expression cassettes according to the invention or for vectors comprising expression cassettes according to the invention are, for example, promoters such as the cos, tac, trp, tet, Ipp, lac, laclq, T7, T5, T3, gal, trc, ara, SP6, l-PR or the l-PL promoter, all of which can be used for expressing SRP in Gram-negative bacterial strains.
  • promoters such as the cos, tac, trp, tet, Ipp, lac, laclq, T7, T5, T3, gal, trc, ara, SP6, l-PR or the l-PL promoter, all of which can be used for expressing SRP in Gram-negative bacterial strains.
  • promoters a y and SP02 both of which can be used for expressing SSP in Gram- positive bacterial strains, and in the yeast or fungal promoters AUG1, GPD-1, PX6, TEF, CUP1, PGK, GAP1, TPI, PH05, AOX1, GAL10/CYC1, CYC1, OliC, ADH, TDH, Kex2, MFa or NMT or combinations of the abovementioned promoters (Degryse et al., Yeast 1995 Jun 15; 11(7):629-40; Romanos et al. Yeast 1992 Jun;8(6):423-88; Benito et al. Eur. J.
  • Plant Pathol. 104, 207-220 (1998); Cregg et al. Biotechnology (N Y) 1993 Aug;11(8):905-10; Luo X., Gene 1995 Sep 22;163(1):127-31 ; Nacken etal., Gene 1996 Oct 10;175(1-2): 253-60; Turgeon et al., Mol Cell Biol 1987 Sep;7(9):3297-305) or the transcription terminators NMT, Gcy1, TrpC, AOX1, nos, PGK or CYC1 (Degryse et al., Yeast 1995 Jun 15; 11 (7):629-40; Brunelli et al. Yeast 1993 Dec9(12): 1309-18; Frisch et al., Plant Mol.
  • genetic control sequences which are suitable for expression in insect cells are the polyhedrin promoter and the p10 promoter (Luckow, V.A. and Summers, M.D. (1988) BioTechn. 6, 47-55).
  • Advantageous genetic control sequences for expressing SRP in cell culture are, in addition to polyadenylation sequences such as, for example, from simian virus 40, eukaryotic promoters of viral origin such as, for example, promoters of the polyoma virus, adenovirus 2, cytomegalovirus or simian virus 40.
  • polyadenylation sequences such as, for example, from simian virus 40, eukaryotic promoters of viral origin such as, for example, promoters of the polyoma virus, adenovirus 2, cytomegalovirus or simian virus 40.
  • promoters of viral origin such as the promoter of the cauliflower mosaic virus 35S transcript (Franck et al., Cell 21 (1980), 285-294; Odell et al., Nature 313 (1985), 810-812).
  • constitutive promoters are, for example, the Agrobacterium nopaline synthase promoter, the TR dual promoter, the Agrobacterium OCS (octopine synthase) promoter, the ubiquitin promoter (Holtorf S et al., Plant Mol Biol 1995, 29:637-649), the promoters of the vacuolar ATPase subunits, or the promoter of a proline-rich protein from wheat (WO 91/13991).
  • the expression cassettes may also comprise, as genetic control sequence, a chemically inducible promoter, by means of which the expression of the exogenous gene in the plant can be controlled at a specific point in time.
  • a chemically inducible promoter such as, for example, the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic-acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP-A-0388186), a tetracyclin-inducible promoter (Gatz et al., (1992) Plant J.
  • an abscisic-acid-inducible promoter EP-A 335528
  • an ethanol- or cyclohexanone-inducible promoter WO 93/213344
  • suitable promoters are those which confer tissue- or organ-specific expression in, for example, anthers, ovaries, influorescences and floral organs, leaves, stomata, trichomes, stems, vascular tissues, roots and seeds.
  • Others which are suitable in addition to the abovementioned constitutive promoters are, in particular, those promoters which ensure leaf-specific expression.
  • Promoters which must be mentioned are the potato cytosolic FBPase promoter (WO 97/05900), the RuBisCO (ribulose-1,5-bisphosphate carboxylase) SSU promoter (small subunit) or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989), 2445 - 245). Promoters which are furthermore preferred are those which control expression in seeds and plant embryos. Examples of seed-specific promoters are the phaseolin promoter (US 5,504,200, Bustos MM et al., Plant Cell.
  • sucrose binding protein promoter WO 00/26388
  • LeB4 promoter Bact al., Mol Gen Genet 1991, 225: 121-128; Fiedler, U. et al., Biotechnology (NY) (1995), 13 (10) 1090.
  • promoters which are suitable as genetic control sequences are, for example, specific promoters for tubers, storage roots or roots, such as, for example, the class I patatin promoter (B33), the potato cathepsin D inhibitor promoter, the starch synthase (GBSS1) promoter or the sporamin promoter, fruit-specific promoters such as, for example, the fruit-specific promoter from tomato (EP-A 409625), fruit-maturation- specific promoters such as, for example, the fruit-maturation-specific promoter from tomato (WO 94/21794), influoresence-specific promoters such as, for example, the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593), or plastid- or chromoplast-specific promoters such as, for example, the RNA polymerase promoter (WO 97/06250), or else the Glycine max phosphoribosyl pyrophosphate
  • Additional functional elements arew possible and are understood as meaning by way of example but not by limitation reporter genes, replication origins, selection markers and what are known as affinity tags, in direct fusion with SRP or in fusion by means of a linker optionally comprising a protease cleavage site.
  • Further suitable additional functional elements are sequences which ensure targeting into the apoplast, into plastids, into the vacuole, the mitochondrion, the peroxisome, the endoplasmic reticulum (ER), or, owing to the absence of such operative sequences, the remaining of the product in the compartment where it is formed, the cytosol (Kenmode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).
  • vectors comprising at least one copy of the nucleic acid sequences according to the invention and/or the expression cassettes according to the invention.
  • vectors are furthermore also understood as meaning all of the other known vectors with which the skilled worker is familiar, such as, for example, phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear DNA or circular DNA.
  • phages viruses
  • viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear DNA or circular DNA.
  • the nucleic acid construct according to the invention can advantageously also be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA may consist of a linearized plasmid or only of the nucleic acid construct as vector, or the nucleic acid sequences used.
  • the expression cassette according to the invention and vectors derived therefrom can be used for transforming bacteria, cyanobacteria (for example of the genus Synechocystis, Anabaena, Calothrix, Scytonema, Oscillatoria, Plectonema and Nostoc), proteobacteria such as, for example, Magnetococcus sp. MC1 , yeasts, filamentous fungi and algae and eukaryotic nonhuman cells (for example insect cells) with the aim of producing SSP recombinantly, the generation of a suitable expression cassette depending on the organism in which the gene is to be expressed.
  • cyanobacteria for example of the genus Synechocystis, Anabaena, Calothrix, Scytonema, Oscillatoria, Plectonema and Nostoc
  • proteobacteria such as, for example, Magnetococcus sp. MC1 , yeasts, filamentous fungi
  • the gene construct advantageously comprises, for expression of the other genes present, additionally 3' and/or 5' terminal regulatory sequences to enhance expression, which are selected for optimal expression depending on the selected host organism and gene or genes.
  • These regulatory sequences are intended to make specific expression of the genes and protein expression possible as mentioned above. This may mean, depending on the host organism, for example that the gene is expressed or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.
  • the regulatory sequences or factors may moreover preferably have a beneficial effect on expression of the introduced genes, and thus increase it. It is possible in this way for the regulatory elements to be enhanced advantageously at the transcription level by using strong transcription signals such as promoters and/or enhancers. However, in addition, it is also possible to enhance translation by, for example, improving the stability of the mRNA.
  • the intermediary vectors can be integrated into the agrobacterial Ti or Ri plasmid by means of homologous recombination owing to sequences which are homologous to sequences in the T-DNA.
  • This plasmid additionally contains the vir region, which is required for the transfer of the T-DNA.
  • Intermediary vectors are not capable of replicating in agrobacteria.
  • the intermediary vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
  • Binary vectors are capable of replication both in E.coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border region.
  • Agrobacteria which have been transformed with a vector according to the invention can likewise be used in a known manner for the transformation of plants, such as test plants such as Arabidopsis or crop plants like cereals, maize, oats, rye, barley, wheat, soya, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot, capsicum, oilseed rape, tapioca, cassava, arrowroot, Tagetes, alfalfa, lettuce and the various tree, nut and grapevine species, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently growing them in suitable media.
  • test plants such as Arabidopsis or crop plants like cereals, maize, oats, rye, barley, wheat, soya, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot, capsicum, oilseed rape, tapioca,
  • the genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Such methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or H ⁇ fgen and Willmitzer.
  • the transgenic organisms generated by transformation with one of the above- described embodiments of an expression cassette comprising a nucleic acid sequence according to the invention or a vector comprising the abovementioned expression cassette, and the recombinant SSP which can be obtained from the transgenic organism by means of expression are subject matter of the present invention.
  • the use of transgenic organisms comprising an expression cassette according to the invention, for example for providing recombinant proteins, and/or the use of these organisms in in vivo assay systems are likewise subject matter of the present invention.
  • nucleotide acid sequences of the invention as shown in Fig. 11 , 12, 13 and/or 14 or the nucleotide acid sequences of the invention coding for polypeptides as shown in Fig. 7, 9, 11 , 12, 13 and/or 16 may be overexpressed or the above mentioned cassettes, plasmids, vectors or gene constructs may be used for overexpression of the above mentioned nucleic acid or their homologs in a plant increases the tolerance of the plant to an environmental stress.
  • Selection marker systems like the AHAS marker or other promoters, e.g. superpromotor (Ni et al,., Plant Journal 7, 1995: 661-676), Ubiquitin promotor (Callis et al., J. Biol. Chem., 1990, 265: 12486-12493; US 5,510,474; US 6,020,190; Kawalleck et al., Plant. Molecular Biology, 1993, 21 : 673-684) or 10S promotor (GenBank Accession numbers M59930 and X16673) may be similar useful for the combination with the present invention and are known to a person skilled in the art.
  • superpromotor Ne et al,., Plant Journal 7, 1995: 661-676
  • Ubiquitin promotor Callis et al., J. Biol. Chem., 1990, 265: 12486-12493; US 5,510,474; US 6,020,190; Kawalleck et al., Plant. Molecular Biology,
  • the present invention describes the use of comparing particular attributes or traits, estimating the general appearance or comparing the altered metabolic activity by inactivation or down -regulation of genes to engineer stress-tolerant and/or resistant, i.e. drought-, salt- and/or cold-tolerant and/or herbicide tolerant and/or ROS tolerant and/or resistant plants.
  • stress-tolerant and/or resistant i.e. drought-, salt- and/or cold-tolerant and/or herbicide tolerant and/or ROS tolerant and/or resistant plants.
  • This strategy has herein been demonstrated for Arabidopsis thaliana, but its application is not restricted to these plants.
  • the invention provides a transformed plant containing one or more (stress related protein encoding) genes selected from sequences as shown in Fig.
  • the environmental stress is herbicide induces and/or ROS induced.
  • the methods of the invention may be used to detect environmental stress in plant cells or plants by screening the plant cells for particular attributes or traits, estimating the general appearance, analyzing the growth characteristics or screening for altered metabolic activity as compared to non-stress conditions, which allows for selection of resistant or tolerant plants or plant cells and also provides detection of stress in plants or plant cells before symptoms are visable and damage is high.
  • the methods of the invention also allow breeding of plant cells or plants towards increased tolerance and/or resistance to environmental stress by screening the plant cells under stress conditions for particular attributes or traits, estimating the general appearance, analyzing the growth characteristics or screening for altered metabolic activity as compared to non-stress conditions and selecting those with increased tolerance and/or resistance to environmental stress for further replication.
  • the engineering of one or more stress related genes of the invention may also result in stress related proteins having altered activities which indirectly impact the stress response and/or stress tolerance of plants.
  • the normal biochemical processes of metabolism result in the production of a variety of products (e.g., hydrogen peroxide and other reactive oxygen species) which may actively interfere with these same metabolic processes (for example, peroxynitrite is known to react with tyrosine side chains, thereby inactivating some enzymes having tyrosine in the active site (Groves, J.T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235).
  • the sequences disclosed herein, or fragments thereof can be targeted to generate knockout mutations in the genomes of various other plant cells (Girke, T., 1998 The Plant Journal 15:39-48).
  • the resultant knockout cells can then be evaluated for their ability or capacity to tolerate various stress conditions, their response to various stress conditions, and the effect on the phenotype and/or genotype of the mutation.
  • U.S. Patent No. 6004804 Non-Chimeric Mutational Vectors
  • Puttaraju et al. 1999 Spliceosome -mediated RNA trans-splicing as a tool for gene therapy Nature Biotechnology 17:246-252.
  • the present invention relates to the use of a polypeptide with the biological activity of a polypeptide as shown in Fig. 7, 9, 11 , 12, 13 and/or 16 which, if not present, is charactarized in increased tolerance and/or resistance to environmental stress as compared to non-transformed wild type cells as target for herbicides in order to create herbicide-resistant plants.
  • the present invention relates to the use of the abovementioned polypeptides in a method for identifying herbicidal or growth - regulatory compounds which inhibit the polypeptides of the invention.
  • the invention relates to the use of these compounds which have been identified via the method as herbicides or growtlrregulators.
  • the present invention furthermore relates to the use of SRP of the invention and disclosed in Fig. 7, 9, 11, 12, 13 and/or 16 in a method for identifying active test compounds which confer increased tolerance and/or resistance to environmental stress as compared to non-transformed wild type cells, further named stress resistance conferring (SRC) compound.
  • SRC stress resistance conferring
  • the method according to the invention for identifying stress resistance conferring (SRC) active compounds comprises the following steps: i) bringing SRP into contact with one or more test compounds under conditions which permit binding of the test compound(s) to SRP; and ii) detecting whether the test compound binds to the SRP of i); or iii) detecting whether the test compound reduces or blocks the activity of the S RP of i); or iv) detecting whether the test compound reduces or blocks the transcription, translation or expression of SRP.
  • Detection in accordance with step (ii) of the above method can be effected using techniques which identify the interaction between protein and ligand.
  • either the test compound or the enzyme may contain the detectable label such as, for example, a fluorescent label, a radioisotope label, a chemiluminescent label or an enzyme label.
  • detectable label such as, for example, a fluorescent label, a radioisotope label, a chemiluminescent label or an enzyme label.
  • enzyme labels are horseradish peroxidase, alkaline phosphatase or luciferase. The detection which follows depends on the label and is known to the skilled worker.
  • HTS high-throughput methods
  • FCS fluorescence correlation spectroscopy
  • Fluorescence polarization exploits the characteristic of a quiescent fluorophore which is excited with polarized light to likewise emit polarized light. If, however, the fluorophore is allowed to rotate during the excited state, the polarization of the fluorescent light which is emitted is more or less lost. Under otherwise identical conditions (for example temperature, viscosity, solvent), rotation is a function of molecule size, whereby findings regarding the size of the fluorophore-bound residue can be obtained via the reading (Methods in Enzymology 246 (1995), pp. 283-300).
  • a method according to the invention can be designed directly for measuring the binding, to SRP, of a test compound labeled with a fluroescent molecule.
  • the method according to the invention may also take the form of the "displacement assay” described under 1.
  • the compounds identified thus may be suitable as inhibitors.
  • "Fluorescent resonant energy tranfer” (FRET) is based on irradiation -free energy transfer between two spatially adjacent fluroescent molecules under suitable conditions. A prerequisite is that the emission spectrum of the donor molecule overlaps with the excitation spectrum of the acceptor molecule. Using the fluorescent label of SRP and the on binding test compound, the binding can be measured by means of FRET (Cytometry 34, 1998, pp. 159-179).
  • the method according to the invention may also take the form of the "displacement assay” described under 1.
  • An especially suitable embodiment of FRET technology is "Homogenous Time Resolved Fluorescence” (HTRF) as can be obtained from Packard BioScience. The compounds identified thus may be suitable as inhibitors.
  • HTRF Homogenous Time Resolved Fluorescence
  • SRP Surface-enhanced laser desorption/ ionization
  • MALDI-TOF Time of Flight mass spectrometer
  • the measurement of surface plasmon resonance is based on the change in the refractive index at a surface when a test compound binds to a protein which is immobilized to said surface. Since the change in the refractive index is identical for virtually all proteins and polypeptides for a defined change in the mass concentration at the surface, this method can be applied to any protein in principle (Lindberg et al. Sensor Actuators 4 (1983) 299-304; Malmquist Nature 361 (1993) 186-187).
  • the measurement can be carried out for example with the automatic analyzer based on surface plasmon resonance which is available from Biacore (Freiburg) at a throughput of, currently, up to 384 samples per day.
  • a method according to the invention can be designed directly for measuring the binding of a test compound to SRP. As an alternative, the method according to the invention may also take the form of the "displacement assay" described under 1. The compounds identified thus may be suitable as inhibitors.
  • a preferred embodiment of the method according to the invention which is based on steps i) and ii), consists in i) expressing an SRP in a transgenic organism according to the invention, or growing an organism which naturally contains an SRP; ii) bringing the SRP of step i) in the cell digest of the transgenic or nontransgenic organism, in partially purified form or in homogeneously purified form, into contact with a test compound; and iii) selecting a compound which reduces or blocks the SRP activity, the activity of the SRP incubated with the test compound being determined with the activity of an SRP not incubated with a test compound.
  • the SRP-comprising solution may consist of the lysate of the original organism or of the transgenic organism which has beerrtransformed with an expression casette according to the invention. If appropriate, the SRP can be purified partially or fully via customary methods. A general overview over current protein purification techniques is described, for example, in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-lnterscience (1994); ISBN 0-87969-309-6. If SSP is obtained recombinantly, the protein, which is fused to an affinity tag, can be purified via affinity chromatography as is known to the skilled worker.
  • the SRP which is required for in vitro methods can thus be isolated either by means of heterologous expression from a transgenic organism according to the invention or from an organism with SRP activity, preferably from an desired plant, the term "desired plant” being understood as meaning the cultured species such as crops.
  • SRP is incubated with a test compound. After a reaction time, the activity of the SRP incubated with a test compound is determined in comparison with the activity of an SRP which is not incubated with a test compound. If the SRP is inhibited, a significant decrease in activity is observed in comparison with the activity of the uninhibited polypeptide according to the invention, the result being a reduction of at least 10%, advantageously at least 20%, preferably at least 30%, especially preferably at least 50%, up to 100% reduction (blocking). Preferred is an inhibition of at least 50% at test compound concentrations of 10" 4 M, preferably at 10 5 M, especially preferably at 10" 6 M, based on enzyme concentration in the micromolar range.
  • the enzymatic activity of SRP can be determined for example by an activity assay in which the increase in the product, the decrease in the substrate (or starting material) or a combination of at least two of the abovementioned parameters are determined as a function of a defined period.
  • suitable substrates are, for example, sucrose-6-phosphate or nitrophenyl compounds such as, for example, p-nitrophenyl phosphate, preferably sucrose-6-phosphate, and examples of suitable cofactors are divalent metals such as magnesium or manganese, preferably magnesium. If appropriate, derivatives of the abovementioned compounds which contain a detectable label such as, for example, a fluorescent label, a radioisotope label or a chemiluminescent label, may also be used.
  • a detectable label such as, for example, a fluorescent label, a radioisotope label or a chemiluminescent label
  • the amounts of substrate to be employed in the activity assay can range between 0.5- 10 mM and the amounts of cofactor can range between 0.1-5 mM, based on 1-100 mg/ml enzyme.
  • the conversion of a substrate is determined via quantifying the phosphate formed during the reaction by means of ascorbate/ammonium molybdate reagent (Ames (1966), Methods Enzymol. 8, 115), following a method of Lunn et al. (2000, Pro . Natl. Acad. Sci. USA 97: 12914).
  • ascorbate/ammonium molybdate reagent Ames (1966), Methods Enzymol. 8, 115
  • Lunn et al. 2000, Pro . Natl. Acad. Sci. USA 97: 12914
  • modifications of Ames' method described for deteding phosphate may also be used, for example the method described by Chifflet et al. (1988) Analytical Biochemistry 168: 1), which is particularly suitable for unstable organic phosphates and in the presence of high protein concentrations, the method described by Lanzetta et al. (1979, Analytical Biochemistry 100: 95), which encompasses a method for detecting
  • PMB Phosphate assay
  • the adivity can be determined on the basis of the sucrose liberated from sucrose-6-P. Suitable for this purpose are, for example, optical-enzymatic methods, for example those described by Sonnewald (1992, Plant Journal 2: 571) or chromatographic methods using HPLC (B ⁇ rnke et al. 2001 , J Bacteriol 183: 2425). Moreover, methods for chemically deteding the sucrose formed can also be found by the skilled worker in the literature.
  • the abovementioned embodiment of the method according to the invention may also be used for identifying growth-regulatory substances.
  • the transgenic organism employed is a plant.
  • the method for identifying growth-regulatory substances thus encompasses the following steps: i) generation of a transgenic plant comprising a nucleic acid sequence according to the invention; ii) applying a test substance to the transgenic plant of i) and to a nontransgenic plant of the same cultivar; iii) determining the growth or the viability of the transgenic plant and the nontransgenic plants after application of the test substance; and iv) selection of test substances which bring about a modified growth of the nontransgenic plant in comparison with the growth of the transgenic plant.
  • test compounds are seleded in step iv) which bring about a modified growth of the nontransgenic organism in comparison with the growth of the transgenic organism.
  • Modified growth in this context is understood as meaning an inhibition of the vegetative growth of the plants, which may be manifested in particular in reduced longitudinal growth. Accordingly, the treated plants show stunted growth; moreover, the leaves are darker.
  • modified growth is also understood as meaning a change in the course of maturation over time, an inhibition or promotion of lateral branched growth of the plants, shortened or extended developmental stages, increased standing ability, the growth of larger amounts of buds, flowers, leaves, fruits, seed kernels, roots and tubers, an increased sugar content in plants such as sugar beet, sugar cane and citrus fruit, an increased protein content in plants such as cereals or soybean, or stimulation of the latex flow in rubber trees.
  • the skilled worker is familiar with the detection of such modified growth.
  • a group of test compounds affect the target, then it is either possible directly to isolate the individual test compounds or to divide the group of test compounds into a variety of subgroups, for example when it consists of a multiplicity of different components, in order to reduce the number of the different test compounds in the method according to the invention.
  • the method according to the invention is then repeated with the individual test compound or the relevant subgroup of test compounds.
  • the above-described steps can be carried out repeatedly, preferably until the subgroup identified in accordance with the method according to the invention only comprises a small number of test compounds, or indeed just one test compound.
  • supports which contain one or more of the nucleic acid molecules according to the invention, one or more of the vedors comprising the nucleic acid sequence according to the invention, one or more transgenic organisms comprising at least one of the nucleic acid sequences according to the invention, or one or more (poly)peptides encoded via the nucleic acid sequences according to the invention lends itself to carrying out an HTS method in practice.
  • the support used can be solid or liquid, it is preferably solid and especially preferably a microtiter plate.
  • the abovementioned supports are also subjed matter of the present invention.
  • microtiter plates which, as a rule, can comprise volumes of 200 ml, are used.
  • the other components of an HTS system which match the corresponding microtiter plates, such as a large number of instruments, materials, automatic pipetting devices, robots, automated plate readers and plate washers, are commercially available.
  • the invention furthermore relates to stress resistance conferring (SRC) adive compounds identified by the method according to the invention.
  • SRC stress resistance conferring
  • selected compounds These compounds are hereinbelow referred to as "selected compounds". They have a molecular weight of less than 1000 g/mol, advantageously less than 500 g/mol, preferably less than 400 g/mol, especially preferably less than 300 g/mol.
  • Herbicidally adive compounds have a Ki value of less than 1 mM, preferably less than 1 mM, especially preferably less than 0.1 mM, very especially preferably less than 0.01 mM.
  • the seleded compounds may also be present in the form of their agriculturally useful salts.
  • Agriculturally useful salts which are suitable are mainly the salts of those cations, or the acid addition salts of those acids, whose cations, or anions, respedively, do not adversely affect the tress resistance conferring (SRC) adion of the adive compounds identified by the methods according to the invention.
  • SRC tress resistance conferring
  • the selected compounds may furthermore either be present in the form of racemates, enantiomer mixtures, pure enantiomers or, if they have chiral substituents, also in the form of diastereomer mixtures.
  • the selected compounds may take the form of chemically synthesized substances or substances produced by microorganisms; they can be found, for example, in cell extrads of, for example, plants, animals or microorganisms?
  • the readion mixture can be a cell-free extrad or comprise a cell or cell culture. Suitable methods are known to the skilled worker and are described generally for example in Alberts, Molecular Biology the cell, 3 rd Edition (1994), for example chapter 17.
  • the seleded compounds may also originate from extensive substance libraries.
  • Candidate test compounds can be expression libraries such as, for example, cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein).
  • the selected compounds can be mixed with commen and well known herbicidal compositions and then used for controlling undesired vegetation, if appropriate also for the defoliation of, for example, potatoes, or the desiccation of, for example, cotton, and as growth regulators.
  • Herbicidal compositions comprising the selected compounds afford very good control of vegetation by conferring herbicidal oo
  • the seleded compounds can also be used in crops which tolerate the adion of herbicides owing to breeding, including recombinant methods. The generation of such crops is described hereinbelow.
  • the invention furthermore relates to a method of preparing the herbicidal composition which has already been mentioned above, which comprises formulating selected compounds with suitable adjuvants to give crop protedion products.
  • the seleded compounds can be formulated for example in the form of directly sprayable aqueous solutions, powders, suspensions, also highly concentrated aqueous, oily or other suspensions or suspoemulsions or dispersions, emulsifiable concentrates, emulsions, oil dispersions, pastes, dusts, materials for spreading or granules and be used by means of spraying, atomizing, dusting, spreading or pouring.
  • the use forms depend on the intended use and on the nature of the selected compounds; in any case, they should guarantee the finest possible distribution of the seleded compounds.
  • the herbicidal compositions comprise a herbicidally adive amount of at least one selected compound and adjuvants conventionally used in the formulation of herbicidal compositions. [280.5]
  • the selected compounds can be dissolved or dispersed in an oil or solvent, it being possible to add further formulation auxiliaries for homogenization purposes.
  • liquid or solid concentrates from selected compounds, if appropriate solvents or oil and, optionally, further adjuvants, and such concentrates are suitable for dilution with water.
  • emulsifiable concentrates EC, EW
  • suspensions SC
  • soluble concentrates SL
  • dispersible concentrates DC
  • pastes pills
  • wettable powders or granules it being possible for the solid formulations either to be soluble or dispersible (wettable) in water.
  • suitable powders or granules or tablets can additionally be provided with a solid coating which prevents abrasion or premature release of the active ingredient.
  • adjuvant is understood as meaning the following classes of compounds: antifoams, thickeners, wetters, stickers, dispersants, emulsifiers, bactericides and/or thixotropic agents.
  • antifoams thickeners, wetters, stickers, dispersants, emulsifiers, bactericides and/or thixotropic agents.
  • dispersants emulsifiers
  • bactericides bactericides and/or thixotropic agents
  • SLs, EWs and ECs can be -prepared by simply mixing the constituents in question; powders can be prepared by mixing or grinding in specific types of mills (for example hammer mills).
  • DC, SCs and SEs are usually prepared by wet milling, it being possible to prepare an SE from an SC by adding an organic phase which may comprise further adjuvants or selected compounds.
  • the preparation is known.
  • Powders, materials for spreading and dusts can advantageously be prepared by mixing or concomitantly grinding the adive substances together with a solid carrier.
  • Granules for example coated granules, impregnated granules and homogeneous granules, can be prepared by grinding the selected compounds to solid carriers.
  • inert liquid and/or solid carriers which are suitable for the formulations according to the invention, such as, for example, liquid additives such as mineral oil fractions of medium to high boiling point such as kerosene or diesel oil, furthermore coal tar oils and oils of vegetable or animal origin, or aliphatic, cyclic and aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene, alkylated naphthalenes or their derivatives, alkylated benzenes or their derivatives, alcohols such as methanol, ethanol, propanol, butanol, cyclohexanol, ketones such as cyclohexanone or strongly polar solvents, for example amines such as N-methylpyrrolidone or water.
  • liquid additives such as mineral oil fractions of medium to high boiling point such as kerosene or diesel oil, furthermore coal tar oils and oils of vegetable or animal origin, or aliphatic, cyclic and aromatic hydrocarbons, for example par
  • Examples of solid carriers are mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas and produds of vegetable origin such as cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders and other solid carriers.
  • mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate,
  • surfactants which are suitable for the formulations according to the invention such as, for example, alkali metal salts, alkaline earth metal salts or ammonium salts of aromatic sulfonic acids, for example ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid, and of fatty acids, of alkyl- and alkylarylsulfonates, of alkyl sulfates, lauryl ether sulfates and fatty alcohol sulfates, and also salts of sulfated hexa-, hepta- and octadecanols and of fatty alcohol glycol ethers, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenes
  • aromatic sulfonic acids for example ligninsulf
  • the herbicidal compositions, or the selected compounds can be applied pre- or post-emergence. If the selected compounds are less well tolerated by certain crop plants, application techniques may be used in which the seleded compounds are sprayed, with the aid of the spraying apparatus, in such a way that they come into as little contact, if any, with the leaves of the sensitive crop plants while the selected compounds reach the leaves of undesired plants which grow underneath, or the bare soil surface (post-direded, lay-by). [285.5] Depending on the intended aim of the control measures, the season, the target plants and the growth stage, the application rates of selected compounds amount to 0.001 to 3.0, preferably 0.01 to 1.0 kg/ha.
  • transgenic plants which are resistant to substances found by the methods according to the invention and/or to compositions comprising these substances can also be generated by transformation, followed by overexpression of a nucleic acid sequence according to the invention.
  • the invention therefore furthermore relates to a method for the generation of transgenic plants which are resistant to substances which have been found by a method according to the invention, wherein nucleic acids encoding an SRP variant are overexpressed in these plants.
  • a similar method is described for example in Lermantova et al., Plant Physiol., 122, 2000: 75 - 83.
  • the function of the target is taken over by another gene which is present in the plant, or its gene produd.
  • the skilled worker is familiar with alternative methods for identifying the homologous nucleic acids, for example in other plants with similar sequences such as, for example, using fransposons.
  • the invention therefore also relates to the use of alternative insertion mutagenesis methods for the insertion of foreign nucleic acids into the nucleic acid sequences as shown in Fig. 11, 12, 13 and/or 14 into sequences derived from these sequences on the basis of the genetic code, and/or their derivatives in other plants.
  • the transgenic plants are generated with one of the above-described embodiments of the expression cassette according to the invention by customary transformation methods, which have likewise been described above.
  • the expression efficacy of the recombinantly expressed SRP can be determined for example in vitro by shoot meristem propagation or by a germination test. Moreover, an expression of the SRP gene, which has been modified with regard to type and level, and its effect on the resistance to SRP inhibitors, can be tested on test plants in greenhouse experiments.
  • Cloning methods such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of Escherichia coli cells, growing bacteria and sequence analysis of recombinant DNA were carried out as described by Sambrook et al. (1989) (Cold Spring Harbor
  • the present invention is based on the identification of the Executerl gene of
  • the executerl mutation blocks a key branch point, from which various stress response pathways diverge and that is indispensable for the processing and transmission of the initial stress signal.
  • Such mutant plants are insensitive to certain environmental cues and are unable to activate their genetically controlled stress response program that normally would lead to the acclimation of the plants to more severe environmental stress. Instead, these plants continue to grow, as if not being affected by their environment. While most plants with such a genetic defed that lack the ability to acclimate would rapidly be eliminated, after environmental conditions have worsened, such a trait may be desirable for crop plants that are going to be harvested prior to such seasonal climatic changes.
  • the invention relates to the use of the Executerl and Executerl -like
  • antiapoptotic compounds that are interacting with the 30 products of such genes can be identified.
  • conditions for such assays in which such antiapoptotic compounds can be identified, will be established.
  • Such compounds should convert the phenotype of a plant under stress to that of a healthy plant that apparently is no longer affected by adverse environmental factors.
  • Assay conditions for the identifications of such compounds should favour the adivation of the genetically controlled stress response rather than triggering oxidative damage. These conditions can be adjusted by using the ex1 mutation to suppress stress responses. Oxidative damage will not be affeded by this mutation.
  • Such selected antiapoptotic compounds can then be used in suitable formulations for protective treatment of crop plants, in order to make these plants more resistant to stress conditions comprising drought, limitation in C02, high light, UV, high and low temperatures.
  • Such treatments may be applied at any growth stage of crop plants, from seed to the stage of harvest.
  • the second assay takes advantage of the singlet oxygen-induced suicide in flu mutants after a dark/light shift. Again, under such conditions, wildtype plants will suffer from the stress response. In a screen, antiapoptotic compounds can then be identified that proted the plants from the stress response. Compounds that seledively and specifically inactivate the same target in both plants should phenocopy the ex1 mutant under both assay conditions. Such a screening procedure with two different assays running in parallel would ensure a much higher selectivity and specificity of the selected compound. Furthermore, the invention relates to the use of the mutated forms of Executerl and other genes that have been identified as described in the previous paragraph.
  • Plants homozygous for these mutated genes are blocked in the perception and transmission of stress signals and thus display a greatly reduced susceptibility to external stress conditions. Stress resistance in such plants can be further enhanced by combining mutated alleles of two or more of the stress response genes characterized above. For instance, a double homozygous line with the ex1 and ex1-like mutations would be expected to show an enhanced resistance against a broader range of environmental stress conditions. Similar effects would be expected with combinations of other genes. Mutated alleles of these genes can be obtained also for crop plants of interest by using the genetic screen in the presence of Norflurazon that has been described above. Methods for the produdion of plant lines that are homozygous for mutations such as e.g. ex1 and ex1-like, even plant lines that carry more than one such mutated gene homozygously, are known to the experts in the field.
  • mutated forms of ex1 or other genes described above may be of particular interest for the seledion of herbicide-resistant plants.
  • Norflurazon for instance, is a powerful herbicide that has not yet been used in the field, simply because it does not discriminate between weed and crop plants and kills both.
  • bleaching caused by very low concentrations of the herbicide is primarily due to the adivation of genetic programs that induce growth retardation and collapse of seedlings. These stress responses are blocked in plants that carry mutations within the target genes mentioned above such as ex1.
  • these plants are expeded to show a significantly enhanced resistance against the herbicide and therefore such herbicides like Norflurazon will find an application as useful herbicides when herbicide formulations with low concentrations of the adive compound, such as concentrations in the range of 10-9 M, are used in combination with crop plant lines that are homozygous for at least one mutated ex1 or ex1-like gene, conferring resistance to such herbicides.
  • Such herbicides can then be used for weed control with crops that are then resistant and will survive such herbicide treatments. Since these resistance traits may be introduced into crop plants by mutations , such mutant plants can be grown and tested under field conditions.
  • transgenic plants may also be more resistant to stress conditions, such as drought, limitation in C02, high light, UV, high and low temperatures, giving rise to higher crop yields under adverse conditions.
  • transgenic plants may be useful in combination with herbicides such as Norflurazon, used in formulations with relatively low concentrations of the active compounds, such as in the range of 10-9M. These transgenic plants will be resistant or tolerant to such herbicide treatments, allowing efficient weed control using such herbicides.
  • the Executerl mutated gene alone or in combination with the flu gene or herbicides such as Norflurazon can be applied as a selectable marker for genetic screens. Illumination of the flu mutant or wild-type plants treated with low concentrations of e.g. Norflurazon triggers the release of singlet oxygen and provokes almost simultaneously rapid changes in gene expression that in the case of some genes may reach an up to 200-fold increase within the first 10 minutes of reillumination.
  • the specific association of such rapidly responding genes with singlet oxygen- mediated stress responses can be tested experimentally by using ex1 or other mutations mentioned above to suppress these rapid responses.
  • Promoters of such genes can be used as highly seledive and efficient tools to control and induce the expression of any gene that may confer resistance to stress conditions that lead to the generation of singlet oxygen.
  • stress conditions include drought, limitation in C02, high light, UV, high and low temperatures.
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • External conditions that adversely affed plants can be biotic, imposed by other organisms, or abiotic, arising from an excess or deficit in the physical or chemical environment (Apel and Hirt, 2004).
  • Apel and Hirt, 2004 As a result of these disturbances intracellular levels of ROS may rapidly rise.
  • plants differentially enhance the release of ROS that are either chemically distinct and/or generated within different cellular compartments (Neill et al., 2002).
  • ROS act as a signal that evokes these different stress responses their biological activities should exhibit a high degree of specificity that could be derived from their chemical identities and/or the intracellular locations at which they were generated. Because under natural stress conditions different ROS may be generated simultaneously at various intracellular sites it is difficult to test the proposed biological adivity of a given ROS (Hideg et al., 2002). We therefore developed an experimental strategy that allows to study the biological adivity of only one ROS at a given time.
  • M2 plants were grown on soil under continuous light until they reached the rosette leaf stage and were ready to bolt. Illumination conditions were then changed for 14 days from continuous light to an 8 hr dark/16 hr light regime. Most of the M2 plants, as well as the original flu plants, ceased to grow under these long-day conditions. Out of a total of 125000 plants that were used for this screen, 21 suppressor mutants were identified that continued to grow like wild-type plants. A second screen was done at the seedling stage. Approximately 2000 seeds from each of 60 different M2 seed batches were germinated separately on MS agar plates and kept for 7 days under a 16 hr light/8 hr dark regime.
  • Group I consisted of second-site mutants that ceased to grow when exposed as mature plants to a light/dark shift, whereas the seedling stage was unaffeded and developed similarly to those kept under continuous light ( Figure 1).
  • the second-site mutations in this group seem to affect genes that are normally required to control and/or to trigger a cell death program in Arabidopsis seedlings. These mutations were dubbed singlet oxygen linked death adivator (soldat).
  • the third group III contained 16 mutants, each of which had been isolated from a different batch of M1 plants. Allelism tests revealed that they were all allelic, and represent a single locus that has been named executerl (ex1). This mutation maps on chromosome IV between the SSLP marker CIW9 and the CAPS marker DHS1.
  • the ex1 flu mutants behaved like wild type in that the seedlings remained viable and the growth of mature plants was unaffected by shifts from the dark to the light ( Figure 1 , 2). However, in contrast to wild-type plants - but like flu - the ex 1 /flu mutant accumulated high levels of free Pchlide in the dark ( Figure 1) and upon reillumination generated similar amounts of singlet oxygen as flu (Figure 3).
  • the ex1 gene was identified by a map-based cloning strategy similar to the one used previously for the isolation of the FLU gene (Meskauskiene et al., 2001).
  • First the EX1 locus was genetically mapped in F2 plants from a cross of ex1 in ecotype Landsberg ereda (Ler) flu/flu with ecotype Columbia (Col-0) flu/flu plants. This latter line had been obtained by 6 backcrosses of flu in Ler with wild-type Col-0 plants.
  • EX1 was located on a 90 Kbp genomic fragment of chromosome IV ( Figure 4).
  • the genomic fragment that complemented the ex1 mutation contained three predided open reading frames. Their sequences of the mutant and of wild type were compared. Only one of them, ORF At4g33630, showed a difference between wild type and mutant, a guanine had been replaced by an adenine, leading to an amino acid exchange from glycine to aspartic acid. Once a EX1 candidate gene had been identified, the corresponding genomic sequences of 14 other allelic lines were analyzed in the same way. All these mutant lines could be complemented by the wild-type copy of the EX1 gene and in all of the mutants, except for two, single base changes could be found in the corresponding subunit sequences.
  • EX1 cDNA was synthesized from cDNA derived from total RNA of Arabidopsis seedlings by using gene-specific primers.
  • the ORF of the EX1 cDNA predids a protein of 684 amino acids that is unrelated to known proteins. It contains an N terminal extension that resembles import signal sequences of nuclear encoded plastid proteins.
  • the mature protein is predided to be hydrophilic, it is enriched in serine residues and the only known sequence motif that was recognized is a putative PEST sequence, a stretch of 20 amino acids starting at position 47 of the precursor protein (Figure 7). PEST sequences have been proposed to enhance the degradation of the protein by proteases.
  • EX1 was synthesized in a coupled transcription/translation system in the presence of 35S-methionine and then imported into chloroplasts isolated from 12-day-old pea seedlings. At the end of the incubation the plastid proteins were dissolved and separated eledrophoretically.
  • EX1 is not an integral membrane protein but, as predided from its amino acid composition, is a watersoluble protein part of which is recovered from the stroma, whereas the remaining part is loosely attached to the membrane surface.
  • EX1-like protein also contains a putative PESTmotif, which, however is found at a different position ( Figure 7).
  • a related EX1-like protein with a putative PEST-motif at the same position as in its Arabidopsis counterpart was also found in rice ( Figure 7).
  • the three amino acids of EX1 which were exchanged in three of the allelic mutants and that led to an inactivation of EX1 were also conserved among the EX1 - like proteins of Arabidopsis and rice. Thus, these amino acids of EX1 seem to be functionally important.
  • Linolenic acid has been shown to be a preferred target of ROS during lipid peroxidation. It is the most prominent polyunsaturated fatty acid in chloroplast membrane lipids, accounting for up to 80% of total fatty acids (Murakami et al., 2000).
  • Peroxidation of linolenic acid in flu plants after the release of singlet oxygen was monitored by measuring changes in the amount of the oxygenation products of linolenic acid, hydroperoxy odadecafrieonic acid (HPOTE) and hydroxy odadecafrieonic acid (HOTE) during the first 60 min of illumination.
  • Free hydroperoxides of linolenic acid represented only a minor fradion of less than 1 % of total oxidized fatty acids in both flu and wild-type plants prior to the release of singlet oxygen.
  • HOTE may be generated enzymatically, or non-enzymatically by dired interadion of linolenic acid with singlet oxygen.
  • Enzymatic peroxidation of linolenic acid is mainly catalyzed by lipoxygenases (LOX), leading to different hydroxyl derivatives by regio- and stereo-specific oxygenation.
  • LOX lipoxygenases
  • the origin of the HOTE formed in the flu cells could be deduced from its enantiomer composition (Berger et al., 2001 ).
  • the 13-HOTE that rapidly accumulated during the first 60 min of illumination showed a clear preponderance of the Sisomer over the R-isomer -the S-enantiomer making up more than 95% of the total (data not shown).
  • the composition of the positional isomers was also consistent with an enzymatic origin for most of the HOTE in the flu mutant.
  • 12- HOTE has been suggested to be a specific marker for non -enzymatic lipid peroxidation (Berger et al., 2001), since no lipoxygenase isoform is known to be able to catalyze the formation of this iso er.
  • 12-HOTE Within the fradion of esterified oxylipins only trace amounts of 12-HOTE were present in both wild-type and flu cells. Furthermore, its levels did not seledively increase during reillumination of flu.
  • Free 12- HOTE was barely detedable, its concentration changed only slightly during reillumination and was similar to levels in wild-type plants (op den Camp et al., 2003). Taken together, these data show that the rapid increase in free HOTE during reillumination of flu plants kept in the dark for 8 h is attributable almost exclusively to the enzymatic oxygenation of linolenic acid and thus cannot be explained by the cytotoxicity of singlet oxygen.
  • 13-HPOTE ads as a biosynthetic precursor of 12-Oxo-phytodienoic acid (OPDA) and jasmonic acid.
  • OPDA 12-Oxo-phytodienoic acid
  • jasmonic acid these two latter oxylipins are well known as biologically adive signaling compounds that are involved in the control of various stress- and development-related processes.
  • Figure 10 the accumulation of all three oxygenation derivatives of linolenic acid was suppressed ( Figure 10), even though similar amounts of singlet oxygen were released during a dark/light shift as in the original flu mutant.
  • Some claims may be directed to crop plants whose genes that are the fundional homologues of the Ex1 gene of Arabidopsis thaliana are inactivated (or the homologs of the Ex1-like genes, or any other gene in such stress signal pathway, or preferably more than one of such genes inadivated) and that thereby are resistant or tolerant to adverse environmental conditions.
  • Hideg E., Barta, C, Kalai, T., Vass, I., Hideg, K., and Asada, K. (2002). Detedion of singlet oxygen and superoxide with fluorescent sensors in leaves under stress by photo inhibition or UV radiation. Plant Cell Physiol. 43, 1154-1164.
  • FLU A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 98, 12826-12831.
  • Crop plant comprising intracellular genetic material, charaderized in that the genetic material of the crop plant comprises a mutation in the homologue of a gene that plays a key role in the perception and transmission of the genetically controlled stress response.
  • Crop plant as disclosed above charaderized in that the mutation is homozygous or combined homozygous of several genes from the group of Ex1 , Ex1 -like or other genes that play a key role in the perception and transmission of the genetically controlled stress response, wherein the mutations are induced by a treatment of the plant by at least one of a group of treatments comprising irradiation and treatment with a chemical substance.
  • Crop plant as disclosed above charaderized in that the mutant plant is insensitive to certain environmental cues and is unable to adivate its genetically controlled stress response program that normally would lead to the acclimation of the plants to more severe environmental stress.
  • Transgenic crop plant comprising intracellular genetic material, charaderized in that the transgenic material of the crop plant reduces or inhibits the activity of the homologues of the Ex1 gene, Ex1 -like genes, or of another gene that plays a key role in the perception and transmission of the genetically controlled stress response.
  • Gene promoter for controlling and inducing the expression of a gene, characterized in that the gene promoter is the promoter from a gene that plays a key role in the perception and transmission of the genetically controlled stress response of a plant mediated by singlet oxygen and that controls the expression of a gene of interest whose coding sequence is transcriptionally fused to such promoter and thus allows the adivation of such gene of interest under conditions that naturally induce the adivity of the natural gene.
  • Polynucleotide representing the coding sequence of a plant gene that plays a key role in the perception and transmission of the genetically controlled stress response, comprising a mutation in such coding sequence.
  • Polynucleotide as disclosed above charaderized in that the mutation is localized in a group of genes comprising the Ex1 and Ex1-like genes of Arabidopsis thaliana, and homologues of Ex1 and Ex1-like genes from other plants.
  • Polypeptide that plays a key role in the perception and transmission of the genetically controlled stress response comprising a mutation in such amino acid sequence.
  • Polypeptide as disclosed above charaderized in that the mutation is localized in the amino acid sequence of a group of proteins comprising EX1 and EX1 -like of Arabidopsis thaliana, and homologues of EX1 and EX1 -like from other plants.
  • Polypeptide as disclosed above charaderized in that the amino acid sequence of EX1 protein is as shown in Fig. 7.
  • the assay comprising the steps of: a) determining the concentration of a herbicide that causes bleaching to wild type plants, this bleaching being primarily due to the singlet oxygen-induced activation of the genetic suicide program of the plant; b) adding herbicide at the conditions as determined in step a) to wild type plants, c) adding a substance to be tested to these wild type plants; d) repeating steps a) to c) with different substances; and e) seleding the substances that prohibit bleaching of these wild type plants.
  • the assay comprising the steps of: a) transferring flu mutant plants from continuous light to the dark; b) moving such plants from a) to the light and adding a substance to be tested to such flu mutant plants; c) repeating steps a) to b) with different substances; and d) selecting the substances that prohibit bleaching of these flu mutant plants.
  • the present invention is based on the identification of the Executerl (Ex1) gene of Arabidopsis thaliana that plays a key role in the perception and transmission of singlet oxygen mediated stress signals.
  • Executerl Ex1
  • Exe- 5 cuterl genetic programs have been discovered that are being used by plants to cease growth and commit suicide, once generation of singlet oxygen within chloroplasts reaches a critical level.
  • Gene sequences can be used to identify identical or heterologous genes from cDNA or genomic libraries. Identical genes (e. g. full-length cDNA clones) can be isolated via nucleic acid hybridization using for example cDNA libraries. Depending on the abundance of the gene of interest, 100,000 up to 1 ,000,000 recombinant bacteriophages are plated and transferred to nylon membranes. After denaturation with alkali, DNA is immobilized on the membrane by e. g. UV cross linking. Hybridization is carried out at high stringency conditions. In aqueous solution, hybridization and washing is performed at an ionic strength of 1 M NaCI and a temperature of 68°C. Hybridization probes are generated by e.g. radioactive ( P) nick transcription labeling (High Prime, Roche, Mannheim, Germany). Signals are detected by autoradiography.
  • P radioactive
  • Partially identical or heterologous genes that are similar but not identical can be identified in a manner analogous to the above-described procedure using low stringency hybridization and washing conditions.
  • the ionic strength is normally kept at 1 M NaCI while the temperature is progressively loared from 68 to 42°C.
  • Radiolabeled oligonucleotides are prepared by phosphorylation of the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double stranded concatemers are then radiolabeled by, for example, nick transcription. Hybridization is normally performed at low stringency conditions using high oligonucleotide concentrations.
  • c-DNA clones can be used to produce recombinant polypeptide for example in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant polypeptides are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant polypeptides are then used to produce specific antibodies for example by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni- NTA column saturated with the recombinant antigen as described by Gu et al., 1994, BioTechniques 17:257-262.
  • the antibody can then be used to screen expression cDNA libraries to identify identical or heterologous genes via an immunological screening (Sambrook, J. et al., 1989, "Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al., 1994, “Current Protocols in Molecular Biology", John Wiley & Sons).
  • the genes of the invention and their homologous ORFs in other species may also be down regulated by introducing a synthetic spedfic repressor.
  • a gene for a chimeric zinc finger protein which binds to a specific region in the regulatory or coding region of the genes of interests or its homologs in other spezies is constructed.
  • the artificial zinc finger protein comprises a specific DNA-binding domain consting for example of zinc finger and optional an repression like the EAR domain (Hiratsu et al., 2003. Plant J. 34(5), 733-739 Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis.)
  • PCR readions are run on the DNA pools with specific combinations of T-DNA or transposon border primers and gene specific primers.
  • General rules for primer design can again be taken from Krysan et al., 1999 (Plant Cell 1999, 11 , 2283-2290).
  • Rescreening of lower levels DNA pools lead to the identification of individual plants in which the gene of interest is disrupted by the insertional mutagen.
  • These methods can as well be applied to crop populations that are mutated with insertional mutagenesis such as corn (Femandes J, Dong Q, Schneider B, Morrow DJ, Nan GL, Brendel V, Walbot V.Genome Biol. 2004;5(10):R82. Epub 2004 Sep 23 Genome-wide mutagenesis of Zea mays L.

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Abstract

L'invention porte sur des cellules végétales et des végétaux transformés contenant un gène inactivé ou régulé à la baisse, ce qui permet d'obtenir une meilleure tolérance et/ou résistance aux contraintes de l'environnement, par rapport aux cellules de type sauvage non transformées. Elle porte aussi sur des procédés de fabrication de ces cellules végétales ou de ces végétaux. L'invention concerne également des cellules végétales transformées présentant une meilleure tolérance et/ou résistance à une contrainte de l'environnement par rapport à une cellule végétale de type sauvage non transformée correspondante, des procédés de fabrication, de criblage et de culture de ces cellules végétales ou de ces végétaux, ainsi qu'un procédé de détection de contrainte dans des cellules végétales ou des végétaux.
PCT/EP2004/053093 2003-11-21 2004-11-22 Cellules vegetales et vegetaux presentant une meilleure resistance a la contrainte WO2005049843A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103122340A (zh) * 2011-11-18 2013-05-29 中国科学院上海生命科学研究院 一种改善木薯农艺性状的方法及其应用
AU2020267286B2 (en) * 2010-04-28 2022-12-08 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing plant yield and/or agricultural characteristics
CN116855517A (zh) * 2023-08-16 2023-10-10 青岛农业大学 一种葡萄VvFLS3基因及其在抗高温中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016655A2 (fr) * 2000-08-24 2002-02-28 The Scripps Research Institute Sequences nucleotidiques de plantes a stress regule, plantes transgeniques contenant ces sequences, et methodes d'utilisation stress-regulated nucleotide sequences of plants, transgenic plants containing same, and methods of use
WO2002097097A2 (fr) * 2001-05-31 2002-12-05 Performance Plants, Inc. Compositions et procedes d'augmentation de la resistance au stress des plantes
WO2003020015A2 (fr) * 2001-08-30 2003-03-13 Purdue Research Foundation Procedes de production de plantes transgeniques resistant aux contraintes osmotiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016655A2 (fr) * 2000-08-24 2002-02-28 The Scripps Research Institute Sequences nucleotidiques de plantes a stress regule, plantes transgeniques contenant ces sequences, et methodes d'utilisation stress-regulated nucleotide sequences of plants, transgenic plants containing same, and methods of use
WO2002097097A2 (fr) * 2001-05-31 2002-12-05 Performance Plants, Inc. Compositions et procedes d'augmentation de la resistance au stress des plantes
WO2003020015A2 (fr) * 2001-08-30 2003-03-13 Purdue Research Foundation Procedes de production de plantes transgeniques resistant aux contraintes osmotiques

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] 18 June 2002 (2002-06-18), "Arabidopsis thaliana unknown protein (At4g33630) mRNA, complete cds." XP002338210 retrieved from EBI accession no. EM_PRO:AY117173 Database accession no. AY117173 *
DATABASE EMBL [Online] 21 August 2000 (2000-08-21), "PI1_54_D12.b1_A002 Pathogen induced 1 (PI1) Sorghum bicolor cDNA, mRNA sequence." XP002338211 retrieved from EBI accession no. EM_PRO:BE595816 Database accession no. BE595816 *
WAGNER DANIELA ET AL: "The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana" SCIENCE (WASHINGTON D C), vol. 306, no. 5699, 12 November 2004 (2004-11-12), pages 1183-1185, XP002338197 ISSN: 0036-8075 *
WI SOO JIN ET AL: "Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants" MOLECULES AND CELLS, vol. 13, no. 2, 30 April 2002 (2002-04-30), pages 209-220, XP002338203 ISSN: 1016-8478 *
XIONG L ET AL: "Disease Resistance and Abiotic Stress Tolerance in Rice Are Inversely Modulated by an Abscisic Acid-Inducible Mitogen-Actived Protein Kinase" PLANT CELL, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 15, March 2003 (2003-03), pages 745-759, XP002264237 ISSN: 1040-4651 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020267286B2 (en) * 2010-04-28 2022-12-08 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing plant yield and/or agricultural characteristics
US11542522B2 (en) 2010-04-28 2023-01-03 Evogene Ltd. Isolated polynucleotides and polypeptides for increasing plant yield and/or agricultural characteristics
CN103122340A (zh) * 2011-11-18 2013-05-29 中国科学院上海生命科学研究院 一种改善木薯农艺性状的方法及其应用
CN116855517A (zh) * 2023-08-16 2023-10-10 青岛农业大学 一种葡萄VvFLS3基因及其在抗高温中的应用

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