WO1999054489A1 - Vegetaux tolerants aux agressions - Google Patents

Vegetaux tolerants aux agressions Download PDF

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Publication number
WO1999054489A1
WO1999054489A1 PCT/EP1999/002696 EP9902696W WO9954489A1 WO 1999054489 A1 WO1999054489 A1 WO 1999054489A1 EP 9902696 W EP9902696 W EP 9902696W WO 9954489 A1 WO9954489 A1 WO 9954489A1
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plant
cdk
stress
nucleic acid
plants
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PCT/EP1999/002696
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English (en)
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Sylvia Burssens
Nathalie Verbruggen
Dirk INZÉ
Tom Beeckman
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Cropdesign N.V.
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Priority to CA002326689A priority Critical patent/CA2326689A1/fr
Priority to EP99922120A priority patent/EP1071803A1/fr
Priority to JP2000544818A priority patent/JP2002512040A/ja
Priority to AU39283/99A priority patent/AU758559B2/en
Publication of WO1999054489A1 publication Critical patent/WO1999054489A1/fr

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    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a method for obtaining stress tolerant plants, for example tolerant to salinity, to vectors comprising genetic information capable of conferring said tolerance to the plants, to muteins encoded by the said genetic information and to plants and plant materials obtainable by the said method.
  • Abiotic stress conditions such as shortage or excess of solar energy, water and nutrients, salinity, high and low temperature and pollution (e.g., heavy metals), can have a major impact on plant growth and can significantly reduce the yield of, e.g., cultivars.
  • the growth of plant cells is inhibited by arresting the cell cycle in late G ⁇ , before DNA synthesis, and/or at the G 2 /M boundary; see reviews of Dudits, 1997, Plant Cell Division, Portland Press Research, Monograph, Francis, D., Dudits, D. and Inze, D. eds, ch 2, pp 21, and Bergounioux, Protoplasma 142 (1988), 127-136.
  • WO 92/09685 generally describes a method for controlling plant cell growth comprising modulating the level of cell cycle control proteins in said plant cells.
  • the method disclosed in WO92/09685 is described to be applicable for improvements of plant growth behavior in the presence of one or more environmental conditions.
  • WO 92/09685 describes the presence of a p34 cdc2 protein in plants, a protein which is known to play a key role in the cell cycle of yeasts and vertebrates (see, e.g., the review by Lew and Kornbluth, in Curr. Op. Cell Biol.
  • WO 97/13843 describes the production of water stress or salt stress tolerant transgenic cereal plants by transforming the cereal plant cell or protoplast with a nucleic acid encoding a late embryogenesis abundant protein.
  • the technical problem underlying the present invention is to provide means and methods for conferring or enhancing stress tolerance to plants which are particularly useful in agriculture.
  • the present invention relates to a method for obtaining plants, tolerant to abiotic stress conditions, comprising introducing into a plant cell, plant tissue or plant a nucleic acid molecule and/or regulatory sequence, wherein the introduction of said nucleic acid molecule or regulatory sequence results in the presence of a Cyclin Dependent Kinase (CDK) protein that is not susceptible to inhibitory phosphorylation under abiotic stress conditions.
  • CDK Cyclin Dependent Kinase
  • Control of CDK activity can be achieved by cyclin association and phosphorylation.
  • the phosphorylation of CDK can either have an inhibitory effect or an activating effect on its activity depending on the position of the phosphorylation site.
  • p34 cdc2 is regulated by an activating phosphorylation during G2 at Thr 167 and by inhibitory phosphorylations at Thr-14 and/or Tyr-15 (Jacobs, Annu. Rev. Plant Physiol. Plant Mol Biol. 46 (1995), 317-339).
  • not susceptible to inhibitory phosphorylation under abiotic stress conditions means that the CDK protein that is usually phosphorylated under stress conditions in the plant cell, is under-phosphorylated, i.e., non- phosphorylated at certain inhibitory phosphorylation sites which are otherwise phosphorylated under stress conditions.
  • the CDK protein comprises four inhibitory phosphorylation sites which are usually phosphorylated in the plant cell under stress conditions, said CDK is present in a non-phosphorylated form in accordance with the present invention if at least one of said phosphorylation sites is unphosphorylated.
  • abiotic stress refers to any adverse effect on metabolism, growth or viability of the cell, tissue, organ or whole plant which is produced by an non-living or non-biological (i.e., not biotic: insect, bacteria, fungal, virus) environmental stressor, e.g., environmental factors such as water (flooding, drought, dehydration), anaerobic (low level of oxygen, CO 2 etc.), osmotic (salt), temperature (hot/heat, cold, freezing, frost), nutrients/pollutants, or by a hormone, second messenger or other molecule which is related to or induced by said stressor.
  • environmental stressor e.g., environmental factors such as water (flooding, drought, dehydration), anaerobic (low level of oxygen, CO 2 etc.), osmotic (salt), temperature (hot/heat, cold, freezing, frost), nutrients/pollutants, or by a hormone, second messenger or other molecule which is related to or
  • anaerobic stress means any reduction in oxygen levels sufficient to produce a stress as hereinbefore defined, including hypoxia and anoxia.
  • the term “flooding stress” refers to any stress which is associated with or induced by prolonged or transient immersion of a plant, plant part, tissue or isolated cell in a liquid medium such as occurs during monsoon, wet season, flash flooding or excessive irrigation of plants, etc.
  • Cold stress and heat stress are stresses induced by temperatures which are respectively, below or above, the optimum range of growth temperatures for a particular plant species. Such optimum growth temperature ranges are readily determined or known to those skilled in the art.
  • “Dehydration stress” is any stress which is associated with or induced by the loss of water, reduced turgor or reduced water content of a cell, tissue, organ or whole plant.
  • “Drought stress” refers to any stress which is induced by or associated with the deprivation of water or reduced supply of water to a cell, tissue, organ or organism.
  • the terms "salinity-induced stress”, “salt-stress” or similar term refer to any stress which is associated with or induced by a perturbation in the osmotic potential of the intracellular or extracellular environment of a cell.
  • the transgenic plant obtained in accordance with the method of the present invention upon the presence of the nucleic acid molecule and/or regulatory sequence introduced into said plant, attains tolerance or improved tolerance against abiotic stress which the corresponding wild-type plant was susceptible to.
  • the terms “tolerance” and “tolerant” cover the range of protection from a delay to complete inhibition of alteration in cellular metabolism, reduced cell growth and/or cell death caused by the abiotic stress defined hereinbefore.
  • the transgenic plant obtained in accordance with the method of the present invention is tolerant to abiotic stress in the sense that said plant is capable of growing substantially normal under environmental conditions where the corresponding wild-type plant shows reduced growth, metabolism, viability and/or male or female sterility.
  • CDKs cyclin dependent kinases
  • Thr14 and Tyr15 phosphorylation sites are conserved in the protein kinase CDC2aAt (Mironov (1999)). No phenotypic changes were however detected in transgenic Arabidopsis lines overexpressing a dominant negative mutant form of CDC2aAt with substituted Thr14 and Tyr15, except for a tendency to loose apical dominance (Hemerly, EMBO J. 14 (1995), 3925-3936).
  • the plant CDK protein being functionally equivalent to the known CDC2a of Arabidopsis thaliana, was phosphorylated at a tyrosine and optionally also at a threonine residue, corresponding to the tyrosine of position 15 and the threonine of position 14 of said CDC2a respectively.
  • the expression of non-phosphorylatable mutants of CDKs results in abiotic stress tolerant plants.
  • the present invention is based on the finding that the transgenic plants overexpressing a mutant CDK, i.e., CDC2aAt with non- phosphorylatable Ala14 and Phe15 residues show increased tolerance to abiotic stress, in particular salt stress.
  • a mutant CDK i.e., CDC2aAt with non- phosphorylatable Ala14 and Phe15 residues
  • WT wild type plants
  • YF2 and YF5 Example 2
  • YF lines displayed an enhanced shoot growth after cultivation in the presence of NaCI.
  • the YF lines recovered faster upon release from salinity than the CDC2aWT and WT plants (Fig. 1).
  • the results obtained in accordance with the present invention strongly suggest that the said phosphorylation in particular of plant CDK proteins appears to be one of the key events in abiotic stress-induced cell cycle arrest.
  • CDK or "plant CDK' are meant to encompass all plant CDK proteins having a cell cycle regulatory function in plants or plant cells having the above- mentioned phosphorylatable tyrosine residue, and optionally in addition thereto, the said threonine residue.
  • Examples of these CDKs are the members of the CDC2 family, as identified in Arabidopsis thaliana, such as CDC2a and CDC2b. While the findings described above have been obtained with the CDK protein CDC2a of Arabidopsis thaliana, the present invention can be performed with any CDK protein that is functional in plants, i.e., plant CDK proteins, functionally equivalent to the known CDC2a of Arabidopsis thaliana.
  • plant CDK protein functionally equiva- lent to the known CDC2a of Arabidopsis thaliana
  • each CDK protein having a similar regulatory function as CDC2a of Arabidopsis thaliana in plants or plant cells respectively, e.g., having the PSTAIRE conserved cyclin binding motif, and the above-mentioned phosphorylatable tyrosine and threonine residues.
  • Intensive cloning efforts have identified a large number of CDK proteins in diverse plant species, among which at least five types can be distinguished on the basis of their sequences (for a compilation see Segers, In: Plant ceil proliferation and its regulation in growth and development. Bryant JA, Chiatante D, editors.
  • CDC2bAt contains a PPTALRE motif and its mRNA levels are preferentially present during S and G2 phase (Segers, 1996 and references cited therein).
  • the protein follows the transcriptional level but the CDC2bAf kinase activity becomes only maximal during mitosis, implying a role during the M phase.
  • CDKs or mutants thereof that can be employed in accordance with the present invention can be tested for their ability to confer abiotic stress tolerance to plants according to methods well-known in the art, see, e.g., Physical Stresses in Plants: Genes and Their Products for Tolerance. S. Grillo (Editor), A.
  • Determination of phosphorylation sites in CDKs corresponding to tyrosine at position 15 and the threonine of position 14 of CDC2a can be done, for example, by computer-assisted identification of such sites in the amino acid sequence of a given CDK using, e.g., BLAST2, which stands for Basic Local Alignment Search Tool (Altschul, 1997; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), which can be used to search for local sequence alignments.
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity.
  • BLAST is especially useful in determining exact matches or in identifying homologues.
  • Phosphorylating sites can also be determined using anti- phospho-tyrosine and anti-phospho-threonine antibodies as described by Zhang, Planta 200 (1996), 2-12.
  • nucleic acid molecule in the method of the present invention enhances the amount or results in de novo production of said non- phosphorylated form of CDK protein.
  • said nucleic acid molecule comprises a coding sequence of the mentioned protein or of a regulatory protein, e.g., a transcription factor, capable of inducing the expression of said CDK protein in its non-phosphorylated form or, e.g., of a CDK dephosphorylating enzyme or for example antisense to enzymes phosphorylating CDKs.
  • 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.
  • nucleic acid molecule(s) refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog.
  • regulatory sequence denotes a nucleic acid molecule increasing the expression of the said protein, e.g., the above-mentioned protein, due to its integration into the genome of a plant cell in close proximity to the gene, e.g., encoding inhibitors of phosphorylation.
  • regulatory sequences comprise promoters, enhancers, inactivated silencer intron sequences, 3'UTR and/or 5'UTR coding regions, protein and/or RNA stabilizing elements or other gene expression control elements which are known to activate gene expression and/or increase the amount of the gene products.
  • the present invention is aiming at providing de novo and/or increased expression of non-phosphorylated CDKs.
  • said CDK is a PSTAIRE type CDK, preferably said CDK is CDC2a.
  • CDKs Cyclin Dependent Kinases
  • tyrosine at position 15 and threonine residue at position 14 are therefor meant to encompass the positions 14 and 15 of the respective CDK, as well as such positional changes of the said tyrosine and threonine residues within the plant CDK protein, wherein the characteristic of these residues being phosphorylated at the onset of the stress-induced cell cycle arrest is retained.
  • the said positions as defined herein correspond to the tyrosine at position 15 and the threonine at position 14 of CDC2a of Arabidopsis thaliana, respectively.
  • the CDK is free of phosphate at the tyrosine at a position that corresponds to position 15 in the amino acid sequence of CDC2a of Arabidopsis thaliana.
  • the CDK protein is free of phosphate groups at both the tyrosine and the threonine, corresponding to the tyrosine at position 15 and the threonine at position 14, respectively, in the amino acid sequence of CDC2a of Arabidopsis thaliana.
  • said non- phosphorylated CDK protein is a non-phosphorylatable CDK mutein.
  • a preferred embodiment of the present invention is by conferring to the plant the capacity to produce, under stress conditions, a CDK mutein, of which Y-15 is substituted to a non-phosphorylatable residue.
  • said mutein will substantially not be sensitive for the phosphorylation system, triggering the stress-induced cell cycle arrest. In this way, the plant circumvents the downregulation of the cell cycle, being more tolerant to said stress conditions.
  • CDK mutein used herein, is defined as a CDK fragment or CDK protein 10
  • the CDK mutein also comprises a non-phosphorylatable amino acid residue at position 14.
  • the said mutein is derived from endogenous CDK of the stress tolerant plant to be obtained.
  • endogenous CDK By starting from the endogenous CDK, the risk of malfunctioning muteins is minimized.
  • CDK of, e.g., yeast or vertebrate origin may, dependent on the homology with the endogenous plant CDK, as well be suitable in the present invention; the suitability can easily be determined by the skilled person.
  • the CDK mutein preferably comprises a Y-15 -> F-15 mutation, F being phenylalanine. In all plants investigated so far, the expression of said mutein led to enhanced stress tolerance.
  • the CDK mutein preferably comprises a T-14 -> A-14 mutation, A being alanine. Expression of such a mutein led to improved stress tolerance.
  • the method of the present invention can be performed in various ways.
  • a plant cell that already comprises in its genome a nucleic acid molecule encoding a non- phosphorylatable form of CDK as described above, but does not express the same in an appropriate manner due to, e.g., a weak promoter.
  • a regulatory sequence such as a strong promoter in close proximity to the endogenous nucleic acid molecule encoding said non-phosphorylatable form of CDK so as to induce expression of 11
  • nucleic acid molecule to be introduced into the plant cell or plant tissue or plant encodes said non-phosphorylatable form of CDK.
  • the method of the present invention can be performed wherein said non-phosphorylated form of CDK is due to dephosphorylation and/or inhibition of phosphorylation of CDK.
  • CDK in particular CDK, equivalent to CDC2a of Arabidopsis thaliana, being free of a phosphate at the Y-15 position.
  • a preferred embodiment of the present invention therefore relates to conferring to the plant the capacity to provide, at the stress conditions, CDK protein, being functionally equivalent to CDC2a of Arabidopsis thaliana, a substantial portion thereof being free of phosphate at the tyrosine, corresponding to the tyrosine of position 15 of said CDC2a.
  • a "substantial portion” in this respect is defined herein as the amount of CDK, being free of phosphate at the Y-15 position, that is sufficient to confer to the plant improved growth during abiotic stress conditions.
  • the person skilled in the art will understand that not all of the corresponding CDK present in the plant or plant cell has to be phosphate free at the Y-15 position to improve said stress tolerance.
  • the CDK protein is preferably free of phosphate groups at both the tyrosine and the threonine, corresponding to the tyrosine on position 15 and the threonine on position 14 of said CDC2a, respectively.
  • An attractive way to obtain stress tolerant plants according to the present invention is therefore by conferring to the plant the capacity to provide under stress conditions, CDC25 or a functional analogue thereof, capable of dephosphorylating at least the 12
  • tyrosine at position 15 of the endogenous CDK of the said plant.
  • the dephosphorylating activity of CDC25 is described in Lew and Kornbluth, supra.
  • Another attractive route to obtain stress tolerant plants according to the present invention is by conferring to the plant the capacity to inhibit, under stress conditions, the expression or activity of at least Wee-kinase, MIK1 or MYT or a functional equivalent thereof, thereby inhibiting or reducing the endogenous phosphorylation of CDK of the said plant at least the tyrosine at position 15.
  • Wee-kinase is reviewed in, e.g., Lew and Kornbluth, supra. This kinase phosphorylates the above-discussed Y- 15 of CDK and may also be responsible for the phosphorylation of the T-14.
  • wee-kinase any endogenous kinase of the plant having the function of known Wee-kinase in phosphorylating the respective tyrosine residue and optionally the threonine residue of the endogenous plant CDK.
  • the recently identified Myt1 kinase (Mueller, Science 270 (1995), pp 86) may therefore be regarded as such a functional equivalent.
  • engineering of transgenic plants in accordance with the present invention comprises the use of the animal or yeast CDC25, WEE1, MYT1 or MIK1 genes or more preferably their plant homologues such as Wee1 form maize; see Sun, supra.
  • the expression of the above-described phosphatases or inhibition of said protein kinases can be achieved in different ways.
  • the expression of the plant homologue of CDC25 can be induced by introduction of a regulatory sequence as defined above so as to induce the expression of the endogenous phosphatase gene.
  • said nucleic acid molecule introduced into the plant cell, plant tissue or plant encodes said CDC25 or its functional analog.
  • three phosphatases have been identified in : CDC25a, b, and c.
  • CDC25a plays a role at the G1/S transition while CDC25b and c are considered to be functional at the G2/M transition (Sahdu, Proc. Natl. Acad. Sci. USA 87 (1990), 5139-5143; Galaktiniov, Cell 67 (1991), 1181-1194; Nagata, New Biol. 10 (1991), 959-968; Jinno, EMBO J. 13 (1994), 1549-1556).
  • fission yeast ⁇ S.pombe one CDC25 phosphatase has been isolated (Gould and Nurse, Nature 342 (1989), 39-45; Labib and Nurse (1993)).
  • nucleic acid molecule to be introduced into the plant cell or plant tissue or plant encodes an antisense RNA of said WEE kinase, MYT, MIK or functional analogue or equivalent thereof.
  • nucleic acid molecule is operativeiy linked to regulatory sequences allowing the expression of the nucleic acid molecule in the plant.
  • Said regulatory sequences comprise a promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions, protein and/or RNA stabilizing elements.
  • said regulatory sequence is a chimeric, tissue specific, constitutive or inducible promotor.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • the control sequence is a promoter
  • double-stranded nucleic acid is preferably used.
  • the nucleic acid molecule to be used in accordance with the present invention can be operably linked to poly-A signals ensuring termination of transcription and stabilization of the transcript, for example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV) and from the Nopaline Synthase promoter.
  • Additional regulatory elements may include transcriptional as well as translational enhancers.
  • a plant translational enhancer often used is the tobaccos mosaic virus (TMV) omega sequences, the inclusion of 15
  • an intron (lntron-1 from the Shrunken gene of maize, for example) has been shown to increase expression levels by up to 100-fold. (Mait, Transgenic Research 6 (1997), 143-156; Ni, Plant Journal 7 (1995), 661-676).
  • Any promoter that functions in the target cells can be used.
  • the use of the CaMV35S promoter per se known to the skilled person, resulted in plants with improved tolerance to salt stress (i.e., to a salt concentration in the growth medium of, e.g., 1 w/v % NaCI).
  • salt stress i.e., to a salt concentration in the growth medium of, e.g., 1 w/v % NaCI.
  • Such promoters can be taken for example from stress-related genes which are regulated directly or indirectly by an environmental, i.e., preferably abiotic, stress in a plant cell, including genes for which expression is increased, reduced or otherwise altered.
  • stress-related genes comprise genes the expression of which is either induced or repressed by anaerobic stress, flooding stress, cold stress, dehydration stress, drought stress, heat stress or salinity, amongst others.
  • the stress-related gene may encode an ANP selected from the group consisting of sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, fructose-1 ,6-diphosphate aldolase, glyceraldehyde-3- phosphate dehydrogenase, phosphoglycerate mutase, enolase, pyruvate decarboxylase, alcohol dehydrogenase and alanine amino transferase, amongst others.
  • Such promoters are known in the art (see also, e.g., Lang, and Palva, Plant Mol. Biol. 20 (1992), 951 and Table 1 below).
  • Trg-31 drought Chaudhary, Plant Mol. Biol. 30 (1996), 1247-57 osmotin osmotic Raghothama, Plant Mol. Biol. 23 (1993), 1117-28
  • said inducible promoter is inducible by abiotic stress, preferably, said abiotic stress is osmotic stress, preferably caused by salt.
  • nucleic acid molecules are comprised in an expression vector.
  • An "expression vector” is a construct that can be used to 17
  • Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA.
  • the above-described vectors of the invention comprise a selectable and/or scorable marker.
  • Selectable marker genes useful for the selection of transformed plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci.
  • npt which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485).
  • Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci.
  • mannose-6-phosphate isomerase which allows cells to utilize mannose
  • ODC omithine decarboxylase
  • DFMO omithine decarboxylase inhibitor
  • Blasticidin S Teamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
  • luciferase PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or ⁇ - glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907).
  • This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a vector of the invention.
  • the present invention also relates to vectors, particularly plasmids, cosmids, 18
  • the present invention relates to a vector, at least comprising a stress-inducible promoter, preferably, a salt stress- inducible promoter, functional in plant cells, operably linked to a DNA sequence, coding for a mutated cdc2a gene of Arabidopsis thaliana or functionally equivalent gene of another species, preferably a plant species, the gene product thereof being a CDK mutein functional in the said plant cells and comprising, in the CDK mutein, a non-phosphorylatable amino acid residue at the position, corresponding to the tyrosine on position 15 of CDC2a.
  • a stress-inducible promoter preferably, a salt stress- inducible promoter
  • the mutein also comprises a non- phosphorylatable amino acid residue at the position of the mutein, corresponding to the threonine on position 14 of said CDC2a.
  • a plant CDK gene is used. Being transformed with a vector of this type, plant cells are capable of producing, at abiotic stress conditions, CDK muteins that are not susceptible to the above-discussed regulatory phosphorylation events, therefor leading to stress tolerant plants or plant cells.
  • the vector comprises a promoter as defined above, functional in plant cells, operably linked to a DNA sequence, coding for CDC25 or a functional analogue thereof, capable of dephosphorylating at least the tyrosine of at least one plant CDK, corresponding with the tyrosine on position 15 of CDC2a of Arabidopsis thaliana.
  • a vector can be used to transform plants in order to, as is discussed above, express CDC25 in plants, resulting in dephosphorylation of Y-15 and optionally the T-14 of the endogenous plant CDK, leading to improved stress tolerance.
  • Plasmids and vectors to be preferably employed in accordance with the present invention include those well known in the art.
  • the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for 19
  • the present invention furthermore relates to host cells comprising a vector as described above wherein the nucleic acid molecule is foreign to the host cell.
  • nucleic acid molecule is either heterologous with respect to the host cell, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host cell but located in a different genomic environment than the naturally occurring counterpart of said nucleic acid molecule.
  • the nucleic acid molecule may be either under the control of its own promoter or under the control of a heterologous promoter.
  • the vector or nucleic acid molecule according to the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • the said capacity is conferred to one or more cells of said plant by a) transforming one or more plant cells with a vector, at least comprising, under the control of a promoter functional in the said plant cells, a DNA sequence, coding for a mutated cdk gene of Arabidopsis thaliana or functional equivalent gene of another species, the gene product thereof being a CDK mutein functional in the said plant cells and comprising a non-phosphorylatable amino acid residue at the position of the CDK mutein, corresponding to the tyrosine on position 15 of CDC2a of Arabidopsis thaliana, preferably, the mutein also comprises a non-phosphorylatable amino acid residue at position 14 of the CDK mutein b) by regenerating a plant from one or more of the transformed plant cells, e.g., by the Agrobacterium tumefaciens transformation 20
  • mutein functional in plant cells
  • muteins are meant, which, when expressed in the said plant cells, lead to improved stress tolerance of the said cells.
  • Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacte ⁇ um tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment, pollen-mediated transformation, plant RNA virus-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods known in the art.
  • the vectors used in the method of the invention may contain further functional elements, for example "left border”- and “right border”-sequences of the T-DNA of Agrobacterium which allow for stably integration into the plant genome.
  • methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e., the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example co-transformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161 ; Peng, Plant Mol. Biol.
  • Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (PMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic Acid Res. 21
  • Agrobacterium tumefaciens Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example if a phenotype conferred by said strain is desired.
  • transformation refers to the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for the transfer.
  • the polynucleotide may be transiently or stably introduced into the host cell and may be maintained non-integrated, for example, as a plasmid, or alternatively, may be integrated into the host genome.
  • the resulting transformed plant cell or plant tissue can then be used to regenerate a transformed plant in a manner known by a skilled person.
  • ⁇ protein can be derived from any desired plant species. They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture interest, such as a crop plant, root plant, oil producing plant, wood producing plant, agricultured bioticultured plant, fruit-producing plant, fodder or forage legume, companion plant, or horticultured plant e.g., such a plant is wheat, barley, maize, rice, carrot, sugar beet, chicory, cotton, sunflower, tomato, cassava, grapes, soybean, sugar cane, flax, oilseed rape, tea, canola, onion, asparagus, carrot, celery, cabbage, lentil, broccoli, cauliflower, brussel sprout, artichoke, okra, squash, kale, collard greens, rye, sorghum, oats, tobacco, pepper, grape or potato. Additional species are not excluded. Crops grown on cultivated lands in arid or semi
  • the present invention relates also to transgenic plant cells which contain a nucleic acid molecule or regulatory sequence as defined above or vector according to the invention wherein the nucleic acid molecule or regulatory sequence is foreign to the transgenic plant cell.
  • the term "foreign" see supra.
  • the present invention relates to a transgenic plant cell comprising stably integrated into the genome a nucleic acid molecule, regulatory sequence, or a vector in accordance with the present invention or obtainable according to the method of the invention wherein the expression of the nucleic acid molecule or conferred by the regulatory sequence results in an increased or de novo expression of a non-phosphorylated form of CDK or of a dephosphorylating enzyme described above in transgenic plants compared to wild-type plants.
  • a plant cell having a nucleic acid molecule encoding a CDK mutein or corresponding dephosphorylating enzyme present in its genome can be used and modified such that said plant cell expresses the endogenous gene corresponding to this nucleic acid molecule under the control of regulatory sequences described above such as heterologous promoter and/or enhancer elements.
  • regulatory sequences described above such as heterologous promoter and/or enhancer elements.
  • a nucleic acid molecule encoding e.g., a CDK mutein using, e.g., gene targeting vectors
  • a nucleic acid molecule encoding e.g., a CDK mutein using, e.g., gene targeting vectors
  • a nucleic acid molecule encoding e.g., a CDK mutein using, e.g., gene targeting vectors
  • Suitable promoters and other regulatory elements such as enhancers include those mentioned herein before.
  • the present invention relates to a transgenic plant cell which contains stably integrated into the genome a nucleic acid molecule, regulatory sequence or vector described above or obtainable according to the method of the invention, wherein the presence, transcription and/or expression of the nucleic acid molecule, regulatory sequences or part thereof leads to reduction of the synthesis or the activity of proteins phosphorylating CDKs under abiotic stress conditions in transgenic plants compared to wild type plants.
  • said reduction is achieved by an antisense, sense, ribozyme, co-suppression, in vivo mutagenesis, antibody expression and/or dominant mutant effect.
  • nucleic acid molecules encoding an antisense RNA which is complementary to transcripts of an enzyme phosphorylating CDK in a plant is also the subject matter of the present invention.
  • complementarity does not signify that the encoded RNA has to be 100% complementary.
  • a low degree of complementarity is sufficient, as long as it is high enough in order to inhibit the expression of a protein phosphorylating CDK upon expression in plant cells.
  • the transcribed RNA is preferably at least 90% and most preferably at least 95% complementary to the transcript of the nucleic acid molecule encoding such a phosphorylating enzyme.
  • DNA molecules In order to cause an antisense- effect during the transcription in plant cells such DNA molecules have a length of at least 15 bp, preferably a length of more than 100 bp and most preferably a length or more than 500 bp, however, usually less than 5000 bp, preferably shorter than 2500 bp. Also DNA molecules can be employed which, during expression in plant cells, lead to the synthesis of an RNA which in the plant cells due to a co-suppression- effect reduces the expression of the nucleic acid molecules encoding the described 24
  • phosphorylating protein The principle of the co-suppression as well as the production of corresponding DNA sequences is precisely described, for example, in WO 90/12084.
  • DNA molecules preferably encode an RNA having a high degree of homology to transcripts of the genes encoding phosphorylating enzymes. It is, however, not absolutely necessary that the coding RNA is translatable into a protein.
  • the principle of co-suppression effect is known to the person skilled in the art and is, for example, described in Jorgensen, Trends Biotechnol. 8 (1990), 340- 344; Niebel, Curr. Top. Microbiol. Immunol. 197 (1995), 91-103; Flavell, Curr. Top. Microbiol. Immunol.
  • Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques it is possible to alter the specificity of ribozymes.
  • the second group consists of ribozymes which as a characteristic structural feature exhibit the so-called "hammerhead” motif.
  • the specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule.
  • DNA sequence 25 In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a gene encoding a kinase for CDK, for example a DNA sequence 25
  • Sequences encoding the catalytic domain may for example be the catalytic domain of the satellite DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224 and Steinecke, EMBO J. 11 (1992), 1525-1530) or that of the satellite DNA of the TobR virus (Haseloff and Gerlach, Nature 334 (1988), 585-591).
  • the DNA sequences flanking the catalytic domain are preferably derived from the above-described DNA molecules of the invention.
  • ribozymes in order to decrease the activity in certain proteins in cells is also known to the person skilled in the art and is, for example, described in EP-B1 0 321 201.
  • the expression of ribozymes in plant cells was, for example, described, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).
  • the kinase activity of enzymes capable of phosphorylating CDK in the plant cells of the invention can also be decreased by the so-called "in vivo mutagenesis", for which a hybrid RNA-DNA oligonucleotide ("chimeroplast”) is introduced into cells by transformation of cells TIBTECH 15 (1997), 441-447; WO95/15972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273 (1996), 1386-1389).
  • Part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous enzyme capable of phosphorylating CDK, in comparison to the said nucleic acid sequence protease it displays, however, a mutation or contains a heterologous region which is surrounded by the homologous regions.
  • the mutation contained in the DNA component of the RNA-DNA oligonucleotide or the heterologous region can be transferred to the genome of a plant cell. This results in a decrease of the activity.
  • nucleic acid molecules encoding antibodies specifically recognizing an enzyme capable of phosphorylating a CDK in a plant or parts, i.e., specific fragments or epitopes, of such a protein can be used for inhibiting the activity of the protein in plants.
  • These antibodies can be monoclonal antibodies, polyclonal antibodies or 26
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
  • antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • nucleic acid molecules encoding mutant forms of a protein capable of phosphorylating a CDK in a plant protease can be used to interfere with the activity of the wild type protein.
  • Such mutant forms preferably have lost their biological activity, e.g., kinase activity and may be derived from the corresponding wild-type protein by way of amino acid deletion(s), substitution(s), and/or additions in the amino acid sequence of the protein.
  • Mutant forms such proteins also encompass hyper-active mutant forms of such proteins which display, e.g., an increased substrate affinity and/or higher substrate turnover of the same.
  • hyper-active forms may be more stable in the cell due to the incorporation of amino acids that stabilize proteins in the cellular environment.
  • These mutant forms may be naturally occurring or genetically engineered mutants, see also supra.
  • nucleic acid and amino acid sequences for proteins capable of phosphorylating CDK in a plant can be arrived, for example, from the above- 27
  • any combination of the above-identified strategies can be used for the generation of transgenic plants, which due to the presence of non-phosphorylated form of CDK display a novel or enhanced abiotic stress tolerance.
  • Such combinations can be made, e.g., by (co-)transformation of corresponding nucleic acid molecules into the plant cell, plant tissue or plant, or may be achieved by crossing transgenic plants that have been generated by different embodiments of the method of the present invention.
  • the plants obtainable by the method of the present invention can be crossed with other transgenic plants so as to achieve a combination of abiotic stress tolerance and another genetically engineered trait, see also infra.
  • the present invention also relates to transgenic plants and plant tissue comprising transgenic plant cells according to the invention.
  • Said transgenic plant cell comprises at least one nucleic acid molecule or regulatory sequence as defined above or obtainable by the method of the present invention.
  • the present invention relates to transgenic plants and plant tissue obtainable by 28
  • transgenic plants may display various idiotypic modifications due to their abiotic stress tolerance, preferably display accelerated and/or enhanced plant growth, root growth and/or yield compared to the corresponding wild type plant.
  • the plant cells, plant tissue, in particular, transgenic plants of the invention display a certain degree of (higher) abiotic stress resistance compared to the corresponding wild-type plants.
  • abiotic stress for the meaning of "abiotic stress"; see supra.
  • the transgenic plant displays increased tolerance to osmotic stress, preferably to salt stress.
  • An increase in tolerance to salt stress is understood to refer to the capability of the transgenic plant to grow on a medium such as soil, comprising a higher content of salt in the order of at least about 10% compared to a medium the corresponding non-transformed wild-type plants is capable to grow on, which already provides for beneficial effects on the vitality of the plant such as, e.g., improved growth.
  • the transgenic plant of the invention is capable of growing on a medium or soil comprising at least about 50%, preferably more than about 75%, particularly preferred at least about more than 100% and still more preferable more than about 200% salt than medium or soil the corresponding wild-type plant is capable of growing on.
  • the above- described transgenic plants are capable of growing on medium or soil containing 40, more preferably 100, still more preferably 200, and even more advantageously 300 mM salt.
  • Said salt can be for example, water soluble inorganic salts such as sodium sulfate, magnesium sulfate, calcium sulfate, sodium chloride, magnesium chloride, calcium chloride, potassium chloride etc., salts of agricultural fertilizers and salts associated with alkaline or acid soil conditions.
  • said salt is NaCI.
  • Any transformed plant obtained according to the invention can be used in a conventional breeding scheme or in in vitro plant propagation to produce more 29
  • transgenic plants of the present invention can be used to introduce the same characteristic in other varieties of the same or related species.
  • characteristic of the transgenic plants of the present invention to maintain rapid/high growth rates under stress conditions can be combined with various approaches to confer biotic or abiotic stress tolerance with the use of other stress tolerance genes.
  • Some examples of such stress tolerant genes are provided in Table 2 and see generally Holmberg and Bulow, Trends Plant Sci. 3, 61-66 (1998).
  • Most prior art approaches which include the introduction of various stress tolerance genes have the drawback that they result in reduced growth (compared to non-transgenic controls) under normal, non-stressed conditions, namely stress tolerance comes at the expense of growth and productivity.
  • transgenic plants which have the ability to grow under abiotic stress conditions and display further new phenotype characteristics compared to naturally occurring wild-type plants, for example, due to the presence of another transgene.
  • the above-described nucleic acid molecules and regulatory sequences can be used in combination with other transgenes that confer another phenotype to the plant.
  • the result of the present invention displays at least two new properties compared to a naturally occurring wild-type plant, that is increased tolerance to abiotic stress, in particular osmotic stress preferably to high salinity and; a phenotype that is due to the presence of a further nucleic acid molecule in said plants.
  • said phenotype is conferred by the (over)expression of homologous or heterologous genes or suppression of endogenous genes of the plant or their gene products.
  • Ribozymes of different kinds which are capable of specifically cleaving the (pre)-mRNA of a target gene are described in, e.g., EP-B1 0 291 533, EP-A10 321 201 and EP-A2 0 360 257. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460.
  • ribozyme mediated virus resistance is described in Feyter (Mol. Gen. Genet. 250 (1996), 329-228).
  • the method of the present invention can be employed to produce transgenic stress tolerant plant with any further desired trait (see for review TIPTEC Plant Product & Crop Biotechnology 13 (1995), 312-397) comprising (i) herbicide tolerance (DE-A-3701623; Stalker, Science 242 (1988), 419), (ii) insect resistance (Vaek, Plant Cell 5 (1987), 159- 169), (iii) virus resistance (Powell, Science 232 (1986), 738-743; Pappu, World Journal of Microbiology & Biotechnology 11 (1995), 426-437; Lawson, Phytopathology 86 (1996) 56 suppl.), (vi) ozone resistance (Van Camp, Biotech.
  • the present invention relates to any plant cell, plant tissue, or plant which due to genetic engineering displays abiotic stress tolerance obtainable in accordance with the method of the present invention and comprising a further nucleic acid molecule conferring a novel phenotype to the plant such as one of those described above.
  • the combination of the approaches can be done by crossing plants displaying the individual phenotypes referred to above. Such plants are also part of the invention. Seeds obtained from the transformed plants genetically also contain the same characteristic and are part of the invention.
  • the present invention is in principle applicable to any plant and crop that can be transformed with any of the transformation method known to those skilled in the art and includes those mentioned hereinbefore for instance corn, wheat, barley, rice, oilseed crops, cotton, tree species, sugar beet, cassava, tomato, potato, numerous other vegetables, fruits.
  • the invention also relates to harvestable parts and to propagation material of the transgenic plants according to the invention which contain transgenic plant cells according to the invention.
  • Harvestable parts can be in principle any useful parts of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots etc.
  • Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
  • the present invention generally relates to the use of the above described nucleic acid molecules, regulatory sequences, and vectors for conferring abiotic stress tolerance to a plant and/or as a selectable marker for plants.
  • the above described nucleic acid molecules, regulatory sequences and vectors in accordance with the method of the invention for conferring abiotic stress tolerance can be used as selectable markers in plants according to other systems which for example employ (over)expression of enzymes or muteins thereof capable of conferring tolerance (i.e., resistance) to plant cell killing effects of, e.g., herbicides.
  • EPP 5-enolpyruvlyshikimate-3-phosphate
  • the nucleic acid molecules, regulatory sequences and vectors described above can be used for conferring tolerance against abiotic stress, in particular salt stress as shown in the appended examples.
  • transgenic plants obtained in accordance with the method of the present invention can be easily selected for in the green house on soil, which contains, for example 40 to 300 mM salt, e.g., NaCI.
  • the present invention relates to the use of a nucleic acid molecule or regulatory sequence capable of counteracting stress- induced down-regulation of cell division for the production for osmotic stress tolerant plants.
  • the present invention relates to the use of a plant obtainable by the method of the invention or a plant as described hereinbefore for culturing on soil with 40 mM to 300 mM salt content.
  • Fig. 1 Recovery from salt stress of wild type (WT), CDC2aWT, YF2 and YF5 35
  • Fig. 2 Differential growth upon salt stress of the WT, CDC2aWT, YF2 and YF5 lines.
  • Fig. 3 Histone H1 CDK activity of stressed and non-stressed WT, CDC2aWT and YF2 lines.
  • Fig. 4 Percentage of G2 Arabidopsis cells with a 4C content with and without addition of NaCI to the medium (— . 0% NaCI; — 0.5% NaCI).
  • Example 1 Morphological alterations in response to salt stress and correlation to the expression of cell cycle regulatory genes
  • Example 2 Improved tolerance to salt stress of Arabidopsis thaliana containing a CDC2a-Y15F/T14A mutant gene under the control of a CaMV35S promoter
  • Arabidopsis plants (ecotype C24) were engineered containing a mutated form of the CDC2aAt gene in which the phosphorylation sites T1 (Threonine at amino acid position number 14) and the Y15 (Tyrosine at amino acid position number 15) were changed in A14 (Alanine at amino acid position number 14) and F15 (Phenylalanine at amino acid position number 15) under control of a CaMV35S promoter.
  • the overexpression of mutant CDC2a-Y15F/T14A in Arabidopsis lines did not show drastic changes in development. Only a tendency for loss for apical dominance could be noticed.
  • the salt stressed mutant plants had bigger and more elongated leaves than control non transformed plants and plants overexpressing the wild type CDC2aAt gene. Furthermore, compared to wild type plants (WT) and transgenic Arabidopsis plants ectopically expressing the wild type form of CDC2aAt (CDC2aWT), the YF lines displayed an enhanced shoot growth after cultivation in the presence of NaCI. Additionally the YF lines recovered faster upon release from salinity than the CDC2aWT and WT plants (Fig. 1 ). As mentioned before, CDC2aWT and YF lines contain respectively the wild type and mutated CDC2aAt form in which Thr14 and Tyr15 were substituted for Ala14 and 38
  • Leaf epidermal cell numbers and hypocotyl lengths were measured. Ten days old Arabidopsis seedlings, grown in vitro on solid K1 medium, were transferred to the same medium with or without the addition of 1% NaCI respectively for seven days. Measurements of epidermal cell numbers and individual cell surfaces were obtained from digitalized camera-lucida drawings made from the adaxial leaf surface of the third leaf, using DIC optics on a Leitz Diaplan microscope (Leitz, Wetzlar, Germany). The third leaf was chosen based on the expression of the CycB1;1:gus marker, representative for actively dividing cells (Ferreira, Plant Cell 6 (1994), 1763-1774), at the moment of transfer to the saline environment.
  • H1 kinase activities of the CDK complexes were determined (Fig. 3). Arabidopsis seedlings were grown for ten days in sterile conditions on filters on solid K1 medium and subsequently transferred to liquid K1 medium for two hours. The plants were then transferred to fresh liquid K1 medium with or without the addition of 1 % NaCI. Samples were taken after 3 and 24hrs and H1 kinase activities were measured (Azzi, Eur. J. Biochem. 203 (1992), 353-360). Protein concentrations were determined using the Protein Assay kit (Bio-Rad, Kunststoff, Germany), using Bovine Albumin Serum as a standard.
  • Histone H1 kinase assays were performed with the CDK complexes purified from crude extracts by p13SUC1 affinity (Azzi, Eur. J. Biochem. 203 (1992), 353-360), using 50 ⁇ g of total proteins and 20 ⁇ l of a
  • the enhanced growth of the YF plants demonstrates the importance of a regulatory control mechanism upon abiotic stress such as salt stress that inhibits CDC2aAt activity by alteration of phosphorylation status of the CDC2aAt complex.
  • abiotic stress such as salt stress that inhibits CDC2aAt activity by alteration of phosphorylation status of the CDC2aAt complex.
  • the activity of CDC2aAt is maximal at the G1/S and G2/M transitions, suggesting a functional involvement at both checkpoints.
  • the nuclear content of Arabidopsis cell suspensions was analyzed after addition of NaCI.
  • the Arabidopsis cell line (Axelos, Mol. Gen. Genet. 219 (1989), 106-112) was subcultured every seven days in Gamborg B5 medium (Sigma) supplemented with 0.2 mg/l ⁇ -naphtalenic acid. NaCI (0.5 %) was added 48hrs after subculturing in fresh medium. To determine the DNA nuclear content of the cells, 1ml of the cell suspensions was centrifuged and the nuclei were released (Glab, FEBS Lett.
  • MIK1 acts cooperatively with the WEE1 protein kinase in the inhibitory Tyr15 phosphorylation of CDC2 (Lundgren, Cell 64 (1991), 1111-1122).
  • WEE1 a MYT1 protein kinase that phosphorylates CDC2 on both Thr14 and Tyr15, has been discovered in Xenopus (Mueller, 41
  • - Freezing stress -6 to -4°C - Growth under stress conditions: Transfer of ten-days-old Arabidopsis seedlings (WT, CDC2aAtWT, YF2 and YF5) grown in sterile conditions on solid K1 medium to a growth chamber where the temperature is lowered to, for example, 4°C (cold shock) or increased to 28°C (heat shock). Observations can be made daily after 4 to 10 days transfer to observe growth differences.
  • - Recovery of stress conditions Release of stressed plants (cold or heat shock) described above after 4 to ten days to normal growth conditions. Observations can made daily up to 15 days after the moment of release. 42
  • the period of exposure to a stress and the empirical values of the stress (e.g., 40 ⁇ C vs. 6°C) will depend upon the species of plant being tested, however, a person skilled in the art is able to easily determined these periods or values.

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Abstract

L'invention concerne un procédé permettant d'obtenir des végétaux tolérants aux agressions abiotiques, en particulier aux agressions osmotiques, ce procédé consistant à conférer à ces végétaux la capacité de contrarier la phosphorylation, due aux agressions susmentionnées, des protéines de la kinase dépendante de la cycline (CDK). L'invention concerne également des vecteurs destinés à conférer la capacité susmentionnée.
PCT/EP1999/002696 1998-04-21 1999-04-21 Vegetaux tolerants aux agressions WO1999054489A1 (fr)

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JP2000544818A JP2002512040A (ja) 1998-04-21 1999-04-21 ストレス耐性植物
AU39283/99A AU758559B2 (en) 1998-04-21 1999-04-21 Stress tolerant plants

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