WO2000056905A2 - Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture - Google Patents

Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture Download PDF

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WO2000056905A2
WO2000056905A2 PCT/EP2000/002441 EP0002441W WO0056905A2 WO 2000056905 A2 WO2000056905 A2 WO 2000056905A2 EP 0002441 W EP0002441 W EP 0002441W WO 0056905 A2 WO0056905 A2 WO 0056905A2
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plant
cyclin
cdk
cell cycle
nucleic acid
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PCT/EP2000/002441
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English (en)
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WO2000056905A3 (fr
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Lieven De Veylder
Veronique Katelijne Cecile Kristien Boudolf
Gerardus Theodorus Simon Beemster
Dirk INZÉ
Sylvia Burssens
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Cropdesign N.V.
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Priority to AU43960/00A priority Critical patent/AU4396000A/en
Priority to EP00925126A priority patent/EP1163341A2/fr
Priority to CA002367476A priority patent/CA2367476A1/fr
Publication of WO2000056905A2 publication Critical patent/WO2000056905A2/fr
Publication of WO2000056905A3 publication Critical patent/WO2000056905A3/fr

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • 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
    • 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 current invention relates to a method for modifying, preferably for enhancing or promoting plant growth and/or yield in plants and for modifying their architecture and to the transgenic plants obtainable by this method.
  • the invention concerns the simultaneous ectopic expression and/or overexpression of at least two cell cycle interacting proteins capable of forming a complex and specifically a CDK and an interacting cyclin; said co- overexpression results in an unexpected growth and architectural characteristics such as enhanced root and/or shoot growth in plants.
  • the eukaryotic cell division cycle can be divided in four main phases: the G1 phase or first gap phase, the S phase during which the DNA is duplicated, the G2 phase or second gap phase, and the M phase during which karyo- and cytokinesis take place.
  • the major checkpoints regulating the progression through the cell cycle are situated at the G1/S and G2/M transitions. If the conditions are inadequate for the cell to continue its cycle, a block can occur at one or both transition points.
  • the cell cycle can also be blocked at other transition points which had not until recently been considered important, namely M/G1.
  • CDKs cyclin dependent kinases
  • 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 et al., 1997).
  • Arabidopsis thaliana two CDKs, each belonging to a different family, have been characterised.
  • One such example is the CDC2ak ⁇ gene, which contains the conserved PSTAIRE amino acid motif, and is constitutively expressed during the cell cycle at transcriptional and protein level.
  • the associated kinase activity is maximal at the G1/S and G2/M transitions, suggesting a role at both checkpoints (Hemerly et al, 1993; Burssens et al., 1998; Segers et al; 1996).
  • CDC2bAt contains a PPTALRE motif and its mRNA levels are preferentially present during S and G2 phase (Segers et al, 1996 and references cited therein). The protein follows the transcriptional level but the CDC2b kinase activity becomes only maximal during mitosis, implying a role during the M phase. Cyciins
  • Cyciins share a highly homologous region of about 100 amino acids termed the "cyclin box" which is required for their interaction with the CDK catalytic subunit.
  • the plant A- and B- type cyciins are the so called 'mitotic cyciins' with an important function during mitosis, while the D-type cyciins (so called G1 cyciins) are thought to play a key role at the entry of S phase.
  • the transcriptional regulation of the mitotic cyciins CYCA2;1 and CYCB1;1 of A. thaliana has been analysed in detail in synchronised tobacco BY2-cells. Promoter activity of CYCA2; 1 is switched on upon entry of S phase and persists during G2 phase to be maximal at the end of G2 phase.
  • CYCB1;1 is expressed in a more narrow window of the cell cycle, namely upon exit of S phase and G2 phase with maximal mRNA levels at the entry of mitosis (Shaul et al 1996). Moreover, by developmental expression analysis, the presence of CYCB1;1 transcripts was exclusively linked with actively dividing cells (Ferreira et al, 1994), implying that CYCB1;1 is involved in the regulation of mitosis. Plant D cyciins, by analogy with their animal homologues, have been proposed to control the G1 progression in response to growth factors and nutrients (Dahl et al., 1995; Soni et al., 1995).
  • CDKs and cyciins interact to form functional protein complexes.
  • B ⁇ gre et al. (1997) have found that protein fractions from alfalfa extracts corresponding to monomeric CDKs are essentially devoid of kinase activity as measured by histone H1 phosphorylation and, on the other hand, alfalfa protein complexes immunoprecipitated with antibodies against the human cyclin A or alfalfa cyclin CYCB2;2 exhibit appropriate histone H1 kinase activity (Magyar et al., 1993; 1997).
  • a number of other cell cycle proteins have been shown to interact with one another to form active complexes. Some complexes relevant to the invention are involved in the initiation of DNA replication and ceil division including facilitating the entry into S phase of quielich cells (see Leatherwood 1998, Helin 1998 for reviews).
  • the complexes include ORC1/CDC6 or CDC7/DBF4 or E2F/DP. Importance of the cell cycle
  • CDK activity depends on the interaction with regulatory cyciins and is limiting for cell cycle progression. Different CDK/cyclin complexes act at different time-points of the cell cycle, although different CDK/cyclin complexes may also act at the same time-points of the cell cycle.
  • Complexes of A-type CDKs (such as CDC2a) with D-type cyciins are acting at the G1 phase and are involved in recruiting GO cells in the G1 phase of the cell cycle.
  • Complexes between A-type CDKs and A-type cyciins are operational in S- and G2 phase.
  • G2- and M-phase complexes between A-and B-type CDKs and B-type cyciins are controlling the progression of the cell cycle.
  • Two major checkpoints are operational during the cell cycle, one at the G1/S boundary and one at the G2/M boundary. At these checkpoints the activity of the appropriate CDK/cyclin complexes is controlled, either by interaction with inhibitory proteins (at the G1/S transition), or by inhibitory phosphorylations mediated by a Wee1 kinase (at the G2/M transition). Only when the conditions are favourable is the CDK/cyclin kinase activity restored either by inactivation of the inhibitor, or by dephosphorylation of the CDK/cyclin complex through the action of CDC25.
  • the current invention describes methods to overcome the inhibition of CDK/cyclin activity.
  • different effects are expected in plant growth, yield or architecture.
  • the technical problem underlying the present invention is to provide means and methods for enhancement of plant growth, and/or yield and/or modified architecture in particular in the entire plant, or specific parts of said plant, which are particularly useful in agriculture.
  • the present invention relates to a method for modifying plant growth and/or yield and/or architecture, in particular modifications to plant growth and development mediated by cell cycle protein complexes, thereby improving the agricultural and commercial value of these plants.
  • a method for modifying plant growth and/or yield and/or architecture in particular modifications to plant growth and development mediated by cell cycle protein complexes, thereby improving the agricultural and commercial value of these plants.
  • the overexpression of at least two proteins forming subunits of a protein complex in particular cells, tissues or organs of the plant would produce enhanced plant growth and/or yield and/or architecture compared to otherwise non-transformed plants.
  • the present invention relates to a method for modifying plant growth and/or yield and/or modifying architecture comprising introducing into a plant cell, plant tissue or plant (a) nucleic acid molecule(s) or regulatory sequence(s), wherein the introduction of the nucleic acid molecule(s) or regulatory sequence(s) result(s) in an increased or de novo expression of at least two cell cycle interacting proteins capable of forming a (heteromeric) complex in a plant cell.
  • the simultaneous ectopic expression and/or overexpression of more than one cell cycle interacting protein of which, preferably, at least one is a protein kinase and at least one is a protein forming a complex with such protein kinase and regulating the activity of said protein kinase, leads to enhanced plant growth and/or yield and/or modified architecture compared to wild type plants.
  • the -current invention demonstrates that it will be advantageous to use co-expression of both CYCB1 ;1 and CDC2a since this leads to growth stimulation under conditions in which CYCB1 ;1 alone does not stimulate growth.
  • the combined overexpression may therefore stimulate growth under a wider range of conditions, including environmental conditions, such as high or low availability of water and nutrients, high or low temperature, high or low light, etc.
  • the protein kinase is a cyclin dependent kinase (CDK).
  • CDK is a PSTAIRE type cyclin dependent kinase.
  • CDK is a CDC2a.
  • the CDK is a B- type cyclin dependent kinase namely CDC2b.
  • the second protein is a cyclin.
  • the cyclin is a G1 cyclin such as a D- type cyclin (e.g. CYCD4;1) or an E-type cyclin.
  • the cyclin is a mitotic cyclin such as a B-type cyclin like CYCB1 ;1.
  • cell cycle interacting proteins to be coexpressed and forming the relevant cell cycle complex are ORC1 with CDC6 or CDC7 with DBF4 or E2F with DP.
  • both proteins of the cell cycle complex are ectopically expressed under the control of a constitutive promoter such as the 35S promoter.
  • a constitutive promoter such as the 35S promoter.
  • both proteins may also be expressed under the control of other promoters such as tissue specific promoters, which may be the same for both proteins or which may be different as long as those promoters are driving simultaneous expression of both proteins in at least one tissue. Growth stimulation occurs in particular in those tissues in which both proteins are simultaneously (over)expressed.
  • tissue specific promoters which may be the same for both proteins or which may be different as long as those promoters are driving simultaneous expression of both proteins in at least one tissue. Growth stimulation occurs in particular in those tissues in which both proteins are simultaneously (over)expressed.
  • the man skilled in the art will see various ways of implementing said method in plants.
  • plants are transformed separately with one protein of the cell cycle complex, e.g. or protein kinase such as CDC2a, and with the other interacting cell cycle protein of the complex such as a regulatory protein of such protein or protein kinase, e.g. CYCD4;1.
  • one protein of the cell cycle complex e.g. or protein kinase such as CDC2a
  • the other interacting cell cycle protein of the complex such as a regulatory protein of such protein or protein kinase, e.g. CYCD4;1.
  • Such plants are subsequently crossed and the offspring plants that contain both transgenes are selected and demonstrated to exhibit modified growth, yield and/or architectural characteristics in comparison with a wild type plant.
  • plants exhibiting simultaneous ectopic expression and/or overexpression of the two genes according to the present invention will be obtained via so called cotransformation.
  • Each gene will be present in a different vector or gene construct (e.g. an Agrobacterium vector) and during the transformation step both vectors will be used in combination.
  • the success rate of cotransformation will be highest when both vectors contain a different marker gene (e.g. bar, nptll, hyg,..) and when the selection will be performed with both selective agents; it is also possible to use only one selectable marker gene and its corresponding selective agent and then to identify cotransformants by means of genetic analysis (e.g. PCR based methods).
  • each gene will be present on the same vector or gene construct and the plant transformed with such a vector.
  • vectors will be constructed in accordance with the invention.
  • Such vectors will contain a nucleic acid molecule, e.g. a gene, encoding one protein of the cell cycle complex, e.g. encoding a protein kinase, under the control of a given promoter sequence as well as a nucleic acid molecule, e.g. a gene, encoding the other interacting cell cycle protein of the said complex, e.g. regulating the protein kinase activity, under the control of a given promoter sequence.
  • a nucleic acid molecule e.g. a gene
  • encoding the other interacting cell cycle protein of the said complex e.g. regulating the protein kinase activity
  • control sequences may be present.
  • the promoters of both genes may be identical or may be different as long as there is simultaneous expression in at least one tissue. Bidirectional promoters such as the TR promoter may also be used to drive expression of both genes.
  • transgenic plants, plant tissues, plant organs or plant cells obtained by the method according to the invention are obtainable from a monocotyiedonous plant or dicotyledonous plant.
  • the invention also relates to a transgenic plant cell comprising an overexpressed protein complex obtainable according to any of the methods of the present invention.
  • a transgenic plant or plant tissue comprising said plant cells and harvestable parts or propagation material of those plants are part of the invention too.
  • the invention also relates to the vectors necessary to obtain transformed plants in accordance with the previous embodiments of the invention, those vectors are characterized by the fact that they contain both a protein kinase gene and a gene encoding a regulatory protein regulating the activity of said protein kinase.
  • the invention is also related to utilisation in hybrid seeds in the following way.
  • Two transgenes of interest each present in a homozygous condition in one of the parents of a hybrid, will be present in combination and in a heterozygous condition in the hybrid seed, thus providing the hybrid seed with the benefit of accelerated growth based on the simultaneous ectopic expression and/or overexpression of the two transgenes.
  • Seed harvested from the F1 hybrid plants will segregate for both transgenes and only 9 out of 16 plants of the F2 generation will possess the two transgenes, thus resulting in additional protection of the value of the hybrid seed.
  • the present invention relates to composition comprising the above-described nucleic acid molecules, regulatory sequences or vectors, containing the same identified by the method of the present invention.
  • the invention relates to the use of the transformed cells or the above described nucleic acid molecules, regulatory sequences or vectors for the production of more biomass, secondary metabolites or additives for plant culturing in plant cell culture.
  • the present invention relates to a method for modifying plant growth and/or yield and/or modifying architecture comprising introducing into a plant cell, plant tissue or plant (a) nucleic acid molecule(s) or regulatory sequence(s), wherein the introduction of the nucleic acid molecule(s) or regulatory sequence(s) result(s) in an increased or de novo expression of at least two cell cycle interacting proteins capable of forming a (heteromeric) complex in a plant cell.
  • growth is a concept well known to the person skilled in the art and includes increased crop growth and/or enhanced biomass.
  • modifying plant growth and/or yield refers to a general alteration in the growth of the plant, its tissues or organs or the yield as examplified below.
  • modifying plant growth relates to an acceleration, enhancement or promotion of plant growth.
  • “Architecture” refers to the general morphology of a plant including any one of more structural features including the shape, size, number, colour, texture, arrangement and pattemation of any cell, tissue or organ or groups of cells, tissues, or organs of plants including the root, leaf, shoot, fruit, petiole, trichome, flower, sepals, petal, hypocotyl, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre nodule, cambium, wood, heartwood, parenchyma, erenchyma, selve element, phloem, or vascular tissue amongst others.
  • Modifying yield refers to an altered, preferably increased or enhanced biomass of either the total plant or specific tissues or organs of plants such as root, leaf, shoot, fruit, petiole, trichome, flower, sepals, petal, hypocotyl, stigma, style, stamen, pollen, ovule, seed, bulb, embryo, endosperm, seed coat, aleurone, fibre, nodule, cambium, wood, heartwood, parenchyma, erenchyma, seive element, phloem, or vascular tissue.
  • Yield also refers to accumulation of metabolites and/or the sink/source relationships in the total plant or specific cells, tissues or organs of the plant such as root, leaf, shoot, fruit, petiole, trichome, flower, sepals, petal, hypocotyl, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre, nodule, cambium, wood, heartwood, parenchyma, erenchyma, seive element, phloem, or vascular tissue. This means, for instance that increased growth and/or yield results from increased growth rate or increased root size or shoot growth or, alternatively, in an increased yield because of enhanced fruit growth.
  • plants may modify one or more plant growth and/or architectural and/or yield characteristics in response to external stimuli, such as, for example, a plant pathogenic infection, or an external stress or environmental stress (e.g. anoxia, hypoxia, high temperature, low temperatures, light, daylength, drought, flooding, salt stress, dehydration, heavy metal contamination, nutrient/mineral deficiency, amongst others).
  • external stimuli such as, for example, a plant pathogenic infection, or an external stress or environmental stress (e.g. anoxia, hypoxia, high temperature, low temperatures, light, daylength, drought, flooding, salt stress, dehydration, heavy metal contamination, nutrient/mineral deficiency, amongst others).
  • anoxia e.g. anoxia, hypoxia, high temperature, low temperatures, light, daylength, drought, flooding, salt stress, dehydration, heavy metal contamination, nutrient/mineral deficiency, amongst others.
  • cell cycle shall be taken to include the cyclic biochemical and structural events associated with growth and with division of cells, and in particular with the regulation of the replication of DNA and mitosis.
  • Cell cycle includes phases called: GO (gap 0), G1 (gap 1), DNA replication (S), G2 (gap 2), and mitosis including cytokinesis (M). Normally these four phases occur sequentially.
  • the cell cycle also includes modified cycles such as endomitosis, acytokinesis, polyploidy, polyteny, endopolyploidisation and endoreduplication or endoreplication.
  • cell cycle interacting protein means a protein which exerts control on or regulates or is required for the cell cycle or part thereof, of a cell, tissue, organ or whole organism and/or DNA replication. It may also be capable of binding to, regulating or being regulated by cyclin dependent kinases or their subunits.
  • the term also includes peptides, polypeptides, fragments, variants, homologues, alleles or precursors (e.g. preproproteins or preproteins) thereof.
  • the cell cycle interacting protein is preferably of plant origin, although it may also be the yeast homologues thereof.
  • the cell cycle interacting protein is preferably a protein kinase, in particular a cyclin dependent kinase (CDK) for example an A-type (CDC2a) or a B-type (CDC2b) CDK.
  • CDK cyclin dependent kinase
  • the cell cycle interacting protein is also preferably a cyclin, including cyciins A, B, C, D and E, and in particular CYCA1 ; 1 , CYCA2;1 , CYCA3;1 , CYCB 1 ;1 , CYCB1 ;2, CYCB2;2, CYCD1 ;1 , CYCD2;1.
  • CYCD3; 1 , and CYCD4;1 Evans et al. 1983; Francis et al. 1998; Labbe et al. 1989; Murray and Kirschner 1989; Renaudin et al 1996; Soni et al 1995; Sorrell et al 1999; Swenson et al 1986). Cyciins are also referred to in Table 1.
  • the cell cycle interacting protein preferably includes proteins involved in the control of entry and progression through S phase in particular ORC1 , CDC6, (Nevins, 1992; Liang, 1995) CDC7, DBF4 kinase, E2F (WO 99/58681 ; WO 99/53075) and DP (WO 99/53075).
  • the complex formation in the cell leads to the induction of potential processes of cell division, preferably cell proliferation.
  • cell cycle interacting proteins include, but are not limited to protein kinases e.g., cyclin-dependent kinases (CDKs), and their activating associated subunits, namely cyciins (CYCs).
  • CDKs protein kinases
  • CYCs cyciins
  • Other cell cycle interacting proteins capable of forming a (heteromeric) complex include ORC1 and CDC6, CDC7 and DBF4, and E2F and DP.
  • cell cycle complex The resulting complex of at least two cell cycle interacting proteins capable of forming a (heteromeric) complex is termed a "cell cycle complex".
  • a preferred cell cycle complex is a CDK/cyclin complex, namely the complex formed when a preferably functional cyclin associates with its appropriate CDK, preferably a functional form thereof.
  • Such complexes may be active in phosphorylating proteins and may or may not contain additional protein species.
  • preferred ceil cycle complexes are ORC1/CDC6 or CDC7/DBF4 or E2F/DP.
  • the invention includes modified forms of a cell cycle interacting protein, including homologues or analogues (as defined below) thereof.
  • a preferred modified cell cycle interacting protein is a modified CDK protein wherein the modification removes the inhibitory effect of phosphates on CDC2, in particular the threonine-14 and/or tyrosine-15 have been substituted with non- phosphorylatable residues such as phenylalanine and/or alanine.
  • modified CDKs include Cdc2aA14F15 and Cdc2bA14F15.
  • Another form of modification includes the mutation of the amino acid residue responsible for ATP binding, namely the D residue is replaced with an N residue to form for example Cdc2b.N161 , Cdc2f.N164, or Cdc2aN147.
  • modified cell cycle interacting protein is a modified cyclin protein wherein the modification results in the stablisation of the cyclin.
  • modification may be the result of the mutation or complete or partial removal of the destruction box (D box) motif (RxxLxx[L/l]xN) in the cyclin N-terminal domain (where R and L residues are highly conserved and x stands for any amino acid) (Plesse et al 1998).
  • modified cyciins for use in the present invention include CYCA2;2 ⁇ 64, CYCA2;3 ⁇ 63, CYCB2;1 ⁇ 44 (where ⁇ - is the truncated form lacking the N-terminal part containing the destruction box).
  • “Homologues” of cell cycie interacting proteins are those peptides, oiigope ' ptides, polypeptides, proteins and enzymes which contain amino acid substitutions, deletions and/or additions relative to a non-mutant or wild-type cell cycle interacting protein polypeptide, without altering one or more of its cell cycle control properties.
  • amino acids present in the protein can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break helical structures or sheet structures, and so on.
  • Substitutional variants are those in which at least one residue in the cell cycle interacting protein amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the cell cycle interacting protein. Insertions can comprise amino- terminal and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.
  • Deletional variants are characterised by the removal of one or more amino acids from the cell cycle interacting protein sequence.
  • Amino acid variants of the cell cycle interacting polypeptide may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations.
  • the manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.
  • techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis or other site-directed mutagenesis protocol.
  • Analogues of a cell cycle interacting protein are defined as those peptides, oligopeptides, polypeptides, proteins and enzymes which are functionally equivalent to a cell cycle interacting protein.
  • Analogues of a cell cycle interacting protein include those CDKs, cyciins etc and modified versions thereof that comprise peptides, polypeptides, proteins, and enzymes that are capable of functioning in a plant cell and/or plant tissue and/or plant organ and/or whole plant to produce the same modified plant growth and/or yield and/or architectural characteristics as the ectopic expression and/or (over)expression of such cell cycle interacting proteins forming a cell cycle complex.
  • 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.
  • the present invention denotes nucleic acid molecules which enhances expression of said cell cycle interacting proteins.
  • said nucleic acid molecules comprise a coding sequence of a mentioned protein or of a regulatory protein, e.g., a transcription factor, capable of inducing the expression of said cell cycle interacting protein.
  • regulatory sequence denotes a nucleic acid molecule increasing the expression of the said protein(s), e.g. of cell cycle interacting protein(s), due to its integration into the genome of a plant cell in close proximity to the gene(s), e.g. encoding said cell cycle interacting protein(s).
  • Such 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.
  • nucleic acid molecule(s) leads to de novo expression, or if the mentioned regulatory sequence(s) is used to increase in expression of said proteins, resulting in an increased amount of active protein in the cell.
  • the present invention is aiming at providing de novo and/or increased activity of e.g., cell cycle interacting proteins.
  • transgenic lines overexpressing both CDC2a and CYCB1 ;1 were obtained by crossing a line homozygous for a 35ScaMV-CDC2a construct with a line homozygous for a modified 35S- CYCB1 ;1 construct, it is clear for a person skilled in the art that the same effect could also be obtained by introducing in one plant e.g., a DNA construct in which both the CDC2a and CYCB1 ;1 are placed under a constitutive or tissue specific promoter.
  • nucleic acid molecule(s) encode(s) said cell cycle interacting protein(s) and the regulatory sequence(s) is (are) capable of increasing the expression of a gene encoding said cell cycle interacting protein(s).
  • a nucleic acid molecule comprises a coding sequence for a cell cycle interacting protein as defined herein.
  • 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.
  • one of said cell cyclin interacting proteins is a protein kinase. Therefore, in a preferred embodiment of the method of the present invention one of said cell cycle interacting proteins is a protein kinase.
  • said protein kinase is a cyclin-dependent kinase (CDK) and the other of said cell cycle interacting proteins is a cyclin.
  • CDKs and cyciins that can be employed according to the invention are described in Table 1 , in Segers et al., 1997 or Renaudin et al., 1996, (the disclosure contents of which are hereby incorporated by reference). Preferred coexpression of combinations of CDKs and cyciins in a cell cycle complex and the resulting phenotype are described below:
  • A-type CDK with a cyclin D2 or cyclin D4 Without being bound by any theory or mode of action overexpression of A-type CDKs and cyclin D2 and/or cyclin D4 shortens the G1 phase and overrides the checkpoints, which monitors the availability of sugars. As such plants complete their lifecycle (from seed to seed) faster. It also is expected that plants can complete extra rounds of cell division resulting in an enhanced production of biomass.
  • meristem specific promoters e.g. promoters active in root- or shoot meristems, or subdomains thereof, promoters active during early seed development, cambium specific promoters etc. growth of particular organs can be enhanced.
  • A-type CDK with a cyclin D4 Without being bound by any theory or mode of action overexpression of A-type CDKs and cyclin D4 elevates the threshold that cells needs to re-enter the cell cycle. As such plants cells are more easily regenerated and form more structures (such as lateral roots) of which the formation is dependent on the re-activation of the cell cycle (GO' to G1 transition). 3.
  • A-type CDK with a D-type cyclin The G1/S checkpoint as well as the G2/M checkpoint is of importance for the arrest of cell division under environmental stress conditions. It is anticipated that overexpression of A-type CDKs and D- type cyciins would result in plants with growth characteristics that are more tolerant to stress conditions which cause a cell cycle arrest at the G1/S boundary.
  • A-type CDK with a B-type cyclin or a B-type CDK with a B-type cyclin overexpression of A-type and/or B-type CDKs with B-type cyciins overrides the G2/M checkpoints.
  • Such plants are expected to have growth characteristics less sensitive to environmental stress conditions, such as osmotic stress and complete faster the G2 phase. Furthermore, cells will become less dependent upon the continuous availability of cytokinins.
  • A-type CDK with a D-type cyclin Without being bound by any theory or mode of action the G1 phase is thought to be of importance for growth control of plant cells and an arrest of cells at the G1 phase (e.g. through the use of a dominant negative A-type CDK) cause cells to become larger.
  • A-type CDKs and D-type cyciins enhances the progression through the G1 phase and reduces the average cell size.
  • More cells per unit surface results in a modification of the tissue texture (relative more cell wall material, more membranes etc.) and has an important impact on quality traits. For example the difference between spring and summer wood in trees is largely a consequence of differences in cell size.
  • fibrelength of cells e.g. cotton fibres
  • B-type CDK with an A-type' cyclin plant A-type cyciins are expressed from mid S till early M phase, therefore it can be expected that the co-expression of CDC2bAt with CYCA results in an enhanced progression though these cell cycle phases. Also cells may become less sensitive to the G2/M checkpoint control, making the plants putatively less sensitive to stress conditions and plant growth regulators which operate at this transition point. The result would be that these plants grow better in suboptimal conditions, compared to normal plants, noting that suboptimal conditions may occur frequently even under good agronomical conditions. A higher yield is anticipated under most, but particularly under suboptimal/marginal, agricultural conditions.
  • Table 2 also illustrates preferred CDK/cyclin cell cycle complexes for use in the performance of the application:
  • nucleic acid molecule(s) encode(s) at least a catalytic and/or regulatory subunit of said cell cycle interacting protein(s).
  • Components of CDK/cyclin complexes that can be employed in accordance with the method of the present invention and how to obtain them are known to the person in skilled and are described, e.g., in WO 98/41642, WO 92/09685 the disclosure of which is hereby incorporated by reference.
  • One aspect of the invention provides a method of modifying plant growth and/or yield and/or architecture by expressing in particular cells, tissues or organs of a plant, at least two subunits of a cell cycle complex operably under the control of a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissues-specific promoter sequences and organ-specific promoter sequences.
  • a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissues-specific promoter sequences and organ-specific promoter sequences. Examples of such promoters include promoters which are:
  • stem-expressible and more specifically in the stem cambium to increase strength and thickness of a plant stem to confer improved stability and wind-resistance on the plant
  • tuber expressible to increase or improve tuber production in the plant
  • seed expressible to increase seed production in plants in particular to increase seed set and/or seed production and/or seed yield.
  • endosperm expressible those skilled in the art will be aware that grain yield in crop plants is largely a function of the amount of starch produced in the endosperm of the seed. The amount of protein produced in the endosperm is also a contributing factor to grain yield. In contrast that embryo and aleurone layers contribute little in terms of the total weight of the mature grain. Therefore endosperm-expressible promoters provide the advantage of increasing grain size and grain yield of the plant.
  • root expressible to increase or enhance the production of roots or storage organs derived from roots
  • nodule expressible to increase the nitrogen-fixing capability of a plant.
  • embryo expressible embryo size being important for growth after germination
  • promoters suitable for use in gene constructs of the present invention include those listed in Table 3, amongst others.
  • the promoters listed in Table 3 are provided for the purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention.
  • Table 4 describes constitutive promoters for use in the present invention.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected.
  • copper-responsive, glucocorticoid- responsive or dexamethasone-responsive regulatory elements may be placed adjacent to a heteroiogous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible, giucocorticoid-inducible, or dexamethasone-inducible expression respectively, on said nucleic acid molecule.
  • nucleic acid molecule(s) is (are) operatively linked to control sequences allowing the expression of the nucleic acid molecule(s) in the plant.
  • control sequences comprise a promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions, protein and/or RNA stabilizing elements.
  • said control sequence is a chimeric, tissue specific, constitutive or inducible promoter.
  • both proteins are expressed under the control of a promoter which is active in non differentiated plant cells or in plant protoplasts growing in an artificial medium.
  • the increased growth rate of the cells results in increasing growth of the plant cells in plant cell culture, thus allowing the production of more biomass in plant cell culture.
  • Plant cell production in plant culture can be useful for the production of certain secondary metabolites of plants which may be useful in the pharmaceutical, cosmetics,* food industry etc.
  • the present invention further relates to a nucleic acid molecule encoding at least two cell cycle interacting proteins as mentioned above.
  • the present invention also relates to vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that comprise the nucleic acid molecule or at least two nucleic acid molecules and/or regulatory sequences according to the invention.
  • vectors particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that comprise the nucleic acid molecule or at least two nucleic acid molecules and/or regulatory sequences according to the invention.
  • Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994).
  • Plasmids and vectors to be preferably employed in accordance with the present invention include those well known in the art.
  • nucleic acid molecules and vectors of the invention can be reconstituted into " liposomes for delivery to target cells.
  • the vector of the present invention comprises separate nucleic acid molecules encoding at least one of said cell cycle interacting proteins.
  • nucleic acid molecule present in the vector is linked to (a) control sequence(s) which allow the expression of the nucleic acid molecule or of cell cycle interacting proteins in a host cell, e.g. prokaryotic and/or eukaryotic cells.
  • control sequence refers to regulatory DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term “control sequence” is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components. Preferably, said control sequence comprises a constitutive, chimeric, tissue specific or inducible promoter.
  • 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, it is obvious for a skilled person that double-stranded nucleic acid is used.
  • the vector of the invention is preferably an expression vector.
  • An "expression vector” is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host.
  • 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.
  • Regulatory elements ensuring expression in prokaryotic and/or eukaryotic cells are well known to those skilled in the art.
  • eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript, for example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV).
  • CaMV Cauliflower Mosaic Virus
  • TMV tobaccos mosaic virus
  • Additional regulatory elements may include transcriptional as well as translational enhancers.
  • the above- described vectors of the invention comprises 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. Adv.) 13 (1994), 143-149); 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).
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • mannose-6- phosphate isomerase which allows cells to utilize mannose
  • ODC ornithine decarboxylase
  • DFMO ornithine decarboxylase
  • deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
  • Useful scorable marker are also known to those skilled in the art and are commercially available.
  • said marker is a gene encoding luciferase (Giacomin, 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).
  • luciferase PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121
  • green fluorescent protein Gerdes, FEBS Lett. 389 (1996), 44- 47
  • ⁇ -glucuronidase Jefferson, EMBO J. 6 (1987), 3901-3907.
  • the vectors used according to a method of the present invention can carry nucleic acid molecules encoding the above-mentioned enzymes or enzymatical fragments thereof and fusions of targeting signals to these molecules.
  • said nucleic acid molecules may be under the control of the same regulatory elements or may be separately controlled for expression.
  • the nucleic acid molecules encoding e.g. the domains of cell cycle interacting protein(s) can be expressed in the form of a single mRNA as transcriptional and optionally translational fusions.
  • domains are produced as separate polypeptides or in the latter option as a fusion polypeptide that is further processed into the individual proteins, for example via a cleavage site for proteinases that has been incorporated between the amino acid sequences of both proteins.
  • the resultant protein domains can then self- assemble in vivo.
  • the domains may also be expressed as a bi- or multifunctional polypeptide, preferably disposed by a peptide linker which advantageously allows for sufficient flexibility of both proteins.
  • said peptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of one of said proteins and the N-terminal end of the other of said proteins when said polypeptide assumes a conformation suitable for biological activity of both proteins when disposed in aqueous solution in the plant cell.
  • dicistronic mRNA Reinitiation
  • Brinck-Peterson 1996
  • Hotze (1995) bifunctional proteins are discussed in Lamp (1998) and Dumas (1997) and for linker peptide and protease refer to Doskeland (1996).
  • the present invention furthermore relates to host cells comprising a vector as described above or the mentioned complex overexpressed in a plant cell according to the invention wherein the nucleic acid molecule is foreign to the host cell.
  • nucleic acid molecule is either heteroiogous 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. This means that, if the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural location in the genome of said host cell, in particular it is surrounded by different genes. In this case the nucleic acid molecule may be either under the control of its own promoter or under the control of a heteroiogous 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 animai cells.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • a composition comprising vectors wherein each vector contains at least one nucleic acid molecule encoding at least one cell cycle interacting protein is disclosed. The expression of said vectors results in the production of at least two cell cycle interacting proteins and assembly of the same in a complex in vitro or in vivo.
  • Another object of the invention is a method for the preparation of a cell cycle complex which comprises the cultivation of host cells according to the invention which, due to the presence of a vector or a nucleic acid molecule according to the invention, are able to express such a cell cycle complex, under conditions which allow expression of the cell cycle complex and recovering of the so-produced complex from the culture.
  • the present invention relates to a cell cycle complex obtainable by said method of the present invention or encodable by the nucleic acid molecule of the present invention.
  • polypeptide refers to a polymer of amino acids (amino acid sequence) and does not refer to a specific length of the molecule. Thus peptides and oligopeptides are included within the definition of polypeptide.
  • polypeptides for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • 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 Agrobacterium 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. 12(1984), 8711 ; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467- 8471 ; Koncz, Plant Mol. Biol.
  • Agrobacterium tumefaciens is preferred in the method of the invention
  • other Agrobacterium strains such as Agrobacterium rhizogenes
  • Methods for the transformation using biolistic methods are well known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1 , 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY (1995).
  • Plants may also be transformed by an in planta method using Agrobacterium tumefaciens such as that described by Bechtoid et al, (1993) or Clough et al (1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed without the exogenous application of cytokinin and/or gibberellin.
  • Agrobacterium tumefaciens such as that described by Bechtoid et al, (1993) or Clough et al (1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed without the exogenous application of cytokinin and/or gibberellin.
  • 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.
  • the plants which can be modified according to the invention and which either show overexpression of a cell cycle complex according to the invention 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, fodder or forage legume, companion plant, or horticultured plant e.g., such a plant is wheat, barley, maize, rice, carrot, sugar beet, cichorei, 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 or potato.
  • a plant is wheat, barley, maize, rice, carrot, sugar be
  • the present invention also relates to transgenic plants and plant tissue comprising transgenic plant cells according to the invention. Due to the (over)expression of the protein complex of the invention, e.g., at developmental stages and/or in plant tissue in which they do not naturally occur or are present at low levels, these transgenic plants may show various growth, yield or architectural modifications in comparison to wild-type plants.
  • the present invention relates to a transgenic plant cell displaying de novo expressed cell cycle interacting protein complex or an increased amount of said complex compared to a corresponding wild type plant cell.
  • 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 the method of the present invention.
  • said transgenic plants may display various idiotypic modifications, preferably display modified and/or accelerated and/or enhanced plant growth, root growth and/or yield compared to the corresponding wild type plant.
  • Preferred characteristics of the transgenic plants in the present invention is for example that the displays and increased cell division rate.
  • the present invention contemplates the broad application of the inventive method to the modification of a range of cellular processes, including but not limited to the initiation, promotion, stimulation or enhancement of cell division, seed development, tuber formation, shoot initiation, leaf initiation, root growth, the inhibition of apical dominance etc.
  • 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 transformed plants with the same characteristics and/or can be used to introduce the same characteristic in other varieties of the same or related species. 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 for instance corn, wheat, barley, rice, oilseed crops, cotton, tree species, sugar beet, cassava, tomato, potato, numerous other vegetables or fruits.
  • the invention also relates to harvestable parts and to propagation material of the transgenic plants according to the invention which either contain transgenic plant cells over-expressing the protein complex 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 relates to the use of the above described nucleic acid molecules, regulatory sequences, and vectors for increasing cell division rates in plants, plant cells or plant tissue.
  • said increased cell division rates result in increased biomass, plant growth, root and/or shoot growth, increased seed setting.
  • said increased cell division rates result in increased plant growth, modified architecture and/or yield, e.g. of harvestable material, which is displayed for instance by (but not limited to) increased or enhanced biomass, root growth, shoot growth, seed set, seed production, grain yield, fruit size, nitrogen fixing capacity, nodule size, tuber formation, stem thickness, endosperm size, number of fruit per plant etc.
  • the method of the present invention provides plant cells, plant tissue and plants with novel phenotypes due to the increased or de novo formation of complexes of cell cycle interacting proteins.
  • the plants, plant tissue and plant cells of the present invention will allow the understanding of function of cell cycle protein complexes during this cell division may also open up the way for finding compounds that interfere with formation of such complexes.
  • the present invention provide a basis for the development of mimetic compounds that may be inhibitors or regulators of cell cycle interacting proteins or their encoding genes. It will be appreciated that the present invention also provides cell based screening methods that allow a high-throughput-screening (HTS) of compounds that may be candidates for such inhibitors and regulators.
  • HTS high-throughput-screening
  • the present invention relates to a composition
  • a composition comprising the nucleic acid molecule, the plant cell or the vector of the present invention or the mentioned vector comprised in the composition of the present invention or the mentioned nucleic acid molecules or regulatory sequences.
  • the present invention relates to the use of the mentioned nucleic acid molecule or the mentioned regulatory sequence or the nucleic acid molecule, the vector or the plant cell of the present invention or the mentioned vectors of the composition of the present invention for the production of more biomass, of secondary metabolites or additives for plant culturing in plant cell culture.
  • FIG. 1 Southern blots of the wild type (C24), and transgenic CycB1;1 overexpressing A. thaliana lines (Cyc 28.10 and Cyc 5.9). Genomic DNA extracted from C24, Cyc 28.10 and Cyc 5.9 was digested with the indicated enzymes, separated on a 3% agarose gel and blotted. The membranes are hybridised with a probe derived from the CycB1 ;1 cDNA at high stringency.
  • RNA gel blot analysis RNA was extracted from control plants (C24), homozygous Cyc 5.9 plants, homozygous Cyc 28.10 plants, and heterozygous Cyc 5.9 x CDC2aAt plants. 20 ⁇ g of RNA was separated on a 0.8% agarose gel and blotted on a nitro-cellulose membrane. Equal loading was confirmed by methylene blue staining. The biot was hybridised using an antisense CYCB1 ;1 probe.
  • the filters were probed using a CYCB1 ;1 (diluted 1/500) or a CDC2aAt (diluted 1/5000) specific antibody.
  • a CYCB1 ;1 diluted 1/500
  • a CDC2aAt diluted 1/5000 specific antibody.
  • second antibody an anti-rabbit antibody coupled to peroxidase was used (diluted 1/10000). The detection was performed using the chemoluminescent procedure (Pierce, Rockford, IL).
  • Root elongation rates as a function of time after sowing. Genotypes are indicated as in Fig 4. Data are averages ⁇ SE (n 8 -13) from the same roots as in Fig 4.
  • Seeds of wild type A. thaliana (C24) and all transgenic lines are harvested from plants growing in the same conditions (22 °C and continuous light: 110 ⁇ E nr ⁇ 2 s "1 PAR) and stored at 4°C after harvesting from completely dried plants. For each line to be tested the following screens were performed comparing wild-type and transgenic.
  • the position of the tip of the main root was marked daily at the bottom of the plate with a razorblade.
  • the plates were digitised using a flatbed scanner with overhead illumination attached to a PC. Files with a resolution of 9.775 pixels per mm were saved in Tiff format.
  • Root elongation rate for each root as a function of time was determined by dividing daily growth by the time interval between successive marks.
  • cortical cell length was determined over the whole growth zone. Using the same setup as for the mature cell length, a series of partially overlapping images was recorded covering the whole of the growth zone and part of the mature region. Each series was transformed into a single composite image, which was then used to measure the length of all cells in each cortical file, starting from the quiescent centre. These data were transformed to express the length of each cell as a function of its midpoint. Interpolation and smoothing of these data was performed with a specially created algorithm, which repeatedly fits polynomials to small sections of the data to estimate the midpoint of such section (Beemster and Baskin, 1998). Data obtained with this algorithm are equidistally spaced and were averaged between replicate roots. All data processing was done using the spreadsheet program Excel (Microsoft Corporation). This procedure gives, in addition to the mature cell length, also an idea about size of meristematic cells, and size of the meristem.
  • Total leaf (shoot) area was determined from the scanned images of the root systems described above. This was done by thresholding the image so as to select the entire shoot of each plantlet and then using the "analysis particles" routine. Obtained area values contain both leaf blade, petiole and hypocotyl. They are an underestimation for the true value of the area of these parts as the blades are aligned randomly instead of parallel to the field of view and there is overlap between various plant parts. It is obvious that the degree of underestimation increases for larger plants (more and larger organs) and therefore observed differences between genotypes are a conservative estimation of the true magnitude of differences in leaf area.
  • Plants are germinated on agar as in Screen 1 , transferred to soil at two weeks and all seeds are harvested when the plants have completely dried. For each plant the total seed weight is then determined. Finally seed size is determined by placing between 100 and 300 seeds per parental plant on the flatbedscanner. Images are scanned at 2400 dpi and analysed using the program Photoshop with a set of additional image analysis plug-ins (The image processing toolkit version 3.0, Reindeer Games, Inc). The procedure is as follows: First the image is thresholded to select the seeds. Then touching seeds are separated using the watershed routine. After that all size/shape parameters are determined using the features/measure all command. From the resulting file the columns containing area, length, breadth, formfactor and roundness are selected. Outliers (dust and contamination particles) are removed based on their deviating formfactor and roundness factor. Of the remaining seeds the distribution is plotted and mean, median, average, standard deviation and standard error of the mean are determined.
  • Vector pcyc1T735 gift of Dr. Paulo Ferreira, Departemento de Bioquimica Medica, UFRJ, Rio de Janeiro, Brazil
  • a PUC 19 vector containing 1.2Kb CYCB1;1 cDNA with a T7 leader peptide and a NOS terminator, was digested with the restriction enzymes Ncol and Xbal.
  • the PGSC TCyd vector was mobilised by the helper plasmid pRK 2013 into Agrobacterium tumefasciens C58C1 RifR, harbouring the plasmid PGV 2260 (Deblaere, 1985).
  • A. thaliana plants (ecotype C24) were then transformed by root transformation (Valvekens, 1988).
  • Transgenic plants were selected on hygromycine containing media and later transferred to soil for optimal seed production.
  • a segregation analysis of ten independent lines was performed in the F1 generation based on hygromycine resistance, and out of two parental lines with single locus insertion (1/4 segregation; line 5 and 28) two homozygous daughter lines (5.9 and 28.10) of the F2 generation were selected.
  • Root growth and cell length distribution was determined using Screen 1 described above.
  • Leaf (shoot) area was determined using Screen 2 described above.
  • Variations in root elongation can be due to differences in cell expansion or cell division characteristics.
  • the length distribution of cortical cells was analysed along the root. Typically small meristematic cells are found at the tip of the root. In both wild type and roots from F1 seedlings from the CYCB1;1 and CDC2a overexpressing lines this region is approximately 500 Dm long (Fig 6).
  • a region of rapidly growing cells is located between approximately 500 and 1750 ⁇ m from the quiescent centre of the root for both genotypes. Basal to 1750 ⁇ m, cells have reached their mature cell size. Given the small sample size of 2 roots per genotype and the large standard errors for the F1 line, it is uncertain if the observed differences in cell size are real.
  • the area of the shoot in lateral projection mirrors the differences found in root growth (cf. Figs 3 and 6). Although only a rough measure for shoot area, these data indicate that the growth increase in the CYCB1;1 and CDC2a overexpressing lines is not restricted to the roots, but also occurs in the aerial parts of the plant.
  • Example 3 In order to establish that the observed growth enhancement is not dependent on the specific growth conditions utilised for Example 3, an experiment similar to Example 3 is performed, but with a day/night cycle of 8/16 hrs. In addition to this, leaf growth under natural light conditions on soil (natural light intensity is too high for root growth) is investigated. For this, seeds of the same crosses as used in Example 3 are sown directly into potting soil and placed in glasshouse. Leaf area are measured at 1 weekly intervals from 5 representative plants of each cross. For this the leaf blades are dissected and placed on a flatbed scanner, which make a digital image of the leaves. Total leaf area is determined for each plant by measuring the combined blade area's using the thresholding option of the image analysis program Scion Image.
  • transgenic plants e.g. Cdc2b and CycA
  • media with 0.5X NaCl - thus providing an environmental stress or more particularly a salt stress.
  • the response of the transgenic plants to this stress condition are assessed using the various Screens described previously.
  • the template is total RNA isolated from a rice IR 52 cell suspension culture (Lee et al. 1989) that has been previously reversed transcribed as a bulk.
  • the PCR conditions chosen to amplify this sequence are: 40 cycles of denaturation at 92° for 10 sec, annealing at 60° for 10 sec, extension at 72° for 60 sec. Concerning the oligos for cycOs2, they match ATGGAGAACATGAGATCTGA (SEQ ID NO:3) for the 5'end and TTACAGTGCCACGCTCTTGAG (SEQ ID NO:4) for the 3'side of the sequence.
  • the expected size of the amplification product is 1259 bp.
  • the following PCR conditions are used: 45 cycles of denaturation at 92° for 10 sec, annealing at 53° for 10 sec, and extension at 72° for 90 sec.
  • the Pfu polymerase is used in both cases to generate blunt end fragments.
  • the maize ubiquitin promoter from plasmid pAHC17 (Christensen, 1996) is excised as Pstl fragment (made blunt ended with Pfu polymerase) and subcloned into the Xbal site (filled-in) of the binary vector pBIBHYG to give the vector pBHU.
  • the cDNA of cdc2Os-1 and cyclinOs2 are cloned into the Sacl site (trimmed off) of pPHU to produce pBHU-cdc2 and pBHU-cyc2.
  • the vectors are then introduced into an Agrobacterium tumefaciens octopine strain, via electroporation (McCormac, 1998).
  • A. tumefaciens bearing either pBHU-cdc2 or pBHU-cyc2 are used to produce transgenic rice expressing either cdc2Os-1 or cyclinOs2 under control of the ubiquitin promoter, following Hiei Y., (1994). Lines expressing highest levels of the transgenes are crossed to produce transgenic lines co-expressing both transgenes.
  • transgenic rice plants overproducing both cdc20s-1 and cyclinOs2 will display increased growth rates and robustness. Since the growth stimulating effects observed in Arabidopsis have rather general character and not confined to a particular organ or tissue, we expect also the transgenic rice to show an increase in the grain size.
  • Mutant alleles and wild type genes of CDC2bAt and CDC2fAt were cloned under the control of the CaMV 35S promoter and transferred to a binary vector.
  • the CDC2bAt and CDC2fAt genes are cloned in a kanamycin- containing vector.
  • the mutant alleles include dominant negative forms of CDKs (CDC2b.N161 and CDC2fAt.N164 - in both constructs the D residue was replaced with an N residue; this mutation has been shown to inactivate the kinase causing an arrest of the cell cycle) and positive forms of the CDKs.
  • the following cyclin genes are cloned in a hygromycin vector allowing the coexpression of the cyciins and CDK combinations by crossing.
  • Ncol and BamHl restriction sites were introduced in the cDNAs of CDC2bAt, CDC2bAt.A14F15 and CDC2bAt.N161 by performing PCRs with the following primers: 5'-GGCCATGGAGAAGTACGAGAAGC-3' (SEQ ID NO:5) (containing a Ncol restriction site) and 5'-
  • Clal and Sail restriction sites were introduced in the cDNAs of CDC2fAt, CDC2fAt.A26F27 and CDC2fAt.N164 by performing PCRs with the following primers: 5'-GGATCGATATGGACGAGGGAGTTATAGC-3' (SEQ ID NO.7) (containing a Clal restriction site) and 5'-
  • CYCA2;2 constructs in order to introduce the Clal and Sail restriction sites and a HA tag into the cDNAs of CYCA2;2 and CYCA2;2 ⁇ 64, PCRs were performed using the following primers:
  • CYCA2;3 constructs in order to introduce the Sail restriction sites and a HA tag into the cDNAs of
  • PCRs were performed using the following primers:
  • the 3'NOS was removed from pH35S by BamHl and Xbal digestion and cloned into the BamHl and Xbal restriction sites of the vector pLBR19, resulting into the pLBR19/NOS vector.
  • the obtained PCR fragments were cut with Sail and cloned into the Sail restriction site of pLBR19/NOS.
  • the cassettes 35S-CYCA2;3-3'NOS and 35S- CYCA2;3 ⁇ 63-3'NOS were removed from pLBR19/NOS, blunt ended and cloned into the SNABI restriction site of the pGSC1704 binary vector.
  • CYCB2;1 constructs in order to introduce the Sail and BamHl restriction sites and a HA tag into the cDNAs of CYCB2;1 and CYCB2;1 ⁇ 44, PCRs were be performed using the following primers:
  • the obtained PCR fragments were cut with Sail and BamHl and cloned into the Sail and BamHl restriction sites of pLBR19/NOS.
  • the cassettes 35S-CYCB2;1-3'NOS and 35S-CYCB2;1 ⁇ 44-3'NOS were removed from pLBR19/NOS, blunt ended and cloned into the SNABI restriction site of the pGSC1704 binary vector.
  • CYCB-type ⁇ construct in order to introduce the Ncol and BamHl restriction sites and a HA tag into the cDNA of CYCB-type ⁇ , a PCR was performed using the following primers:
  • GCGCCATGGGCTACCCTTACGATGTTCCAGATTACGCTCCACATATCCG TGATGAGG-3' (SEQ ID NO:18) (containing a Ncol site and a HA tag in fusion with the truncated CYCS-t pe-d), and 5'- GCGGATCCATTCTTCTCCCATTTTGG-3' (SEQ ID NO:19) (containing a BamHl restriction site).
  • the PCR fragment was cut with Ncol and BamHl and cloned into the Ncol and BamHl sites of pH35S.
  • the cassette 35S-CYCB-type ⁇ -3'NOS were removed from pH35S by EcoRl and BamHl digestion, blunt ended and cloned into the SNABI restriction site of the pGSC1704 binary vector.
  • Transgenic plants are analysis for various growth and cell division characteristics or phenotypes according to the Screens described in the General Methodology above.
  • the CDK containing fractions are first concentrated on DEAE 650S (TSK) therefore the pH is raised to 9.3 and the sample is applied onto the column (HR5/5 Pharmacia). Bound proteins are eluted in one step through injection of 0.5M NaCL in DEAE buffer (pH 7.8).
  • the concentrated CDK fraction is further separated by size on a gel filtration column: Superdex 200pg Pharmacia (1.7x100cm column Omnifit) or a Sephacryl S200 Pharmacia (1.5x100cm column Pharmacia).
  • the columns are equilibrated in S200 buffer when fractions (5ml) are collected or in DEAE buffer when the eluting proteins are immediately eluted onto DEAE 650S (TSK) (HR5/5 columns), in the latter case the pH of the size exclusion buffer was raised to 9.3 (cf. concentration step).
  • CDK containing fractions (5ml) which are later bound on DEAE or those CDK complexes which are directly bound to DEAE when eluting from the size exclusion column are eluted in a similar way: a 10 column-volume gradient of 0-500mM NaCl is applied and the eluting complexes are collected.
  • a final purification step consists of hydrophobic interaction chromatography: The conductivity of the purified fractions is raised tolOOmS by adding saturated ammonium sulphate and the samples are individually applied onto Ether PW-5 or Phenyl PW-5 (TSK). The bound complexes are eluted with a decreasing (NH 4 ) 2 S0 4 gradient and can be tested on their kinase activity.
  • Cyclin A2:1 is present in a 100kDa complex with an as yet unidentified protein(s). However it is not yet clear whether the latter one is a CDC homologue or an (un)related protein.
  • Plant cyciins A unified nomenclature for plant A-, B- and D-type cyciins based on sequence organisation. Plant Mol. Biol. 32, 1003-1018. Riou-Khamlichi C; Huntley R; Jacqmard A; Murray JA (1999) Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science

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Abstract

L'invention concerne un procédé pour favoriser ou modifier la croissance et/ou le rendement de végétaux et/ou leur architecture, qui consiste en une expression plus importante d'au moins deux protéines interagissant dans le cycle cellulaire, notamment d'une protéine kinase telle que CDK et d'une cycline. On obtient par ce procédé des plantes transgéniques qui manifestent des taux de croissance et de division cellulaire plus élevés. En outre, l'invention concerne les parties récoltables et le matériel de propagation de la plante susmentionnée ainsi que l'utilisation des cellules, des tissus et des plantes de l'invention dans la production de biomasse, de métabolites secondaires ou d'additifs destinés à la culture de végétaux.
PCT/EP2000/002441 1999-03-19 2000-03-20 Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture WO2000056905A2 (fr)

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AU43960/00A AU4396000A (en) 1999-03-19 2000-03-20 Method for enhancing and/or improving plant growth and/or yield or modifying plant architecture
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CA002367476A CA2367476A1 (fr) 1999-03-19 2000-03-20 Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture

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Cited By (19)

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Publication number Priority date Publication date Assignee Title
WO2001023594A2 (fr) * 1999-09-27 2001-04-05 Pioneer Hi-Bred International, Inc. Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire
WO2002050292A2 (fr) * 2000-12-08 2002-06-27 Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agrifood Canada Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes
WO2003012035A2 (fr) * 2001-07-27 2003-02-13 Icon Genetics, Inc. Usage commercial d'arabidopsis pour la production de proteines diagnostiques et therapeutiques humaines et animales
WO2004087929A2 (fr) * 2003-03-31 2004-10-14 Cropdesign N.V. Plantes a caracteristiques de croissance ameliorees et leur procede de production
WO2005024029A3 (fr) * 2003-09-05 2005-09-01 Cropdesign Nv Plantes a caracteristiques de croissance modifiees, et leur procede de production
EP1580275A1 (fr) * 2004-03-22 2005-09-28 CropDesign N.V. Plantes présentant des caractéristiques de croissance améliorée et procédé de fabrication associé
WO2005093077A1 (fr) * 2004-03-22 2005-10-06 Cropdesign N.V. Plantes presentant des caracteristiques de croissance ameliorees et leur procede de production
WO2006013010A2 (fr) 2004-07-31 2006-02-09 Metanomics Gmbh Preparation d'organismes a croissance plus rapide et/ou a meilleur rendement
EP1711592A2 (fr) * 2003-12-30 2006-10-18 ArboGen, LLC Genes de cycles cellulaires et leurs procedes d'utilisation associes
WO2007141189A3 (fr) * 2006-06-08 2008-02-14 Basf Plant Science Gmbh végétaux aux caractéristiques de croissance améliorées et procédé d'obtention
WO2008138975A1 (fr) * 2007-05-15 2008-11-20 Cropdesign N.V. ACCROISSEMENT DU RENDEMENT DE PLANTES PAR MODULATION DU ZmPKT
US7589256B2 (en) 2003-02-17 2009-09-15 Metanomics Gmbh Preparation of organisms with faster growth and/or higher yield
US20090271895A1 (en) * 2004-07-16 2009-10-29 Cropdesign N.V. Plants having improved growth characteristics and method for making the same
WO2010023320A2 (fr) * 2008-08-29 2010-03-04 Basf Plant Science Company Gmbh Végétaux présentant des caractéristiques associées au rendement améliorées, et leur procédé de production
US7732663B2 (en) 1998-06-08 2010-06-08 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Cyclin-dependent kinase inhibitors as plant growth regulators
EP2272345A1 (fr) 2009-07-07 2011-01-12 Bayer CropScience AG Processus d'amélioration de la croissance de semis et/ou levée précoce de cultures
US20110145949A1 (en) * 2008-08-20 2011-06-16 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
US8991098B2 (en) 2008-09-16 2015-03-31 Basf Plant Science Gmbh Method for improved plant breeding
US9538124B2 (en) 2004-09-16 2017-01-03 Cropdesign N.V. Root evaluation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1934259B (zh) * 2004-03-22 2012-07-25 克罗普迪塞恩股份有限公司 具有改良的生长特性的植物以及制备所述植物的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992009685A1 (fr) * 1990-11-29 1992-06-11 The Australian National University Procede de regulation de la proliferation et de la croissance des cellules vegetales
WO1997002354A1 (fr) * 1995-06-30 1997-01-23 Medical Research Council INTERACTION DE LA PROTEINE p53 AVEC UN FACTEUR DE TRANSCRIPTION DP-1
WO1998003631A1 (fr) * 1996-07-18 1998-01-29 The Salk Institute For Biological Studies Procede d'accroissement de la croissance et du rendement de plantes
WO1998041642A1 (fr) * 1997-03-14 1998-09-24 Cropdesign N.V. Procede et moyens de modulation des proteines du cycle cellulaire des plantes et utilisation de ces moyens pour controler la croissance cellulaire des plantes
WO1999013083A2 (fr) * 1997-09-05 1999-03-18 Cropdesign N.V. Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
WO1999058681A2 (fr) * 1998-05-08 1999-11-18 Consejo Superior De Investigaciones Cientificas Cellules de plantes transgeniques exprimant un peptide e2f vegetal recombinant

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992009685A1 (fr) * 1990-11-29 1992-06-11 The Australian National University Procede de regulation de la proliferation et de la croissance des cellules vegetales
WO1997002354A1 (fr) * 1995-06-30 1997-01-23 Medical Research Council INTERACTION DE LA PROTEINE p53 AVEC UN FACTEUR DE TRANSCRIPTION DP-1
WO1998003631A1 (fr) * 1996-07-18 1998-01-29 The Salk Institute For Biological Studies Procede d'accroissement de la croissance et du rendement de plantes
WO1998041642A1 (fr) * 1997-03-14 1998-09-24 Cropdesign N.V. Procede et moyens de modulation des proteines du cycle cellulaire des plantes et utilisation de ces moyens pour controler la croissance cellulaire des plantes
WO1999013083A2 (fr) * 1997-09-05 1999-03-18 Cropdesign N.V. Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
WO1999058681A2 (fr) * 1998-05-08 1999-11-18 Consejo Superior De Investigaciones Cientificas Cellules de plantes transgeniques exprimant un peptide e2f vegetal recombinant

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DOONAN J: "PLANT GROWTH: ROOTS IN THE CELL CYCLE" CURRENT BIOLOGY,GB,CURRENT SCIENCE,, vol. 6, no. 7, 1 July 1996 (1996-07-01), pages 788-789, XP002045511 ISSN: 0960-9822 *
FOWLER M R ET AL: "The plant cell cycle in context." MOLECULAR BIOTECHNOLOGY, vol. 10, no. 2, October 1998 (1998-10), pages 123-153, XP000939061 ISSN: 1073-6085 *
FRANCIS DENNIS: "Regulation of the cell cycle plant development" BIOSIS, ACTA PHARMACEUTICA, vol. 45, no. 2, 1995, XP002140190 *
GENSCHIK PASCAL ET AL: "Cell cycle-dependent proteolysis in plants: Identification of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132." PLANT CELL, vol. 10, no. 12, December 1998 (1998-12), pages 2063-2075, XP002146545 ISSN: 1040-4651 *
RIOU-KHAMLICHI C ET AL: "Cytokinin activation of Arabidopsis cell division through a D-type cyclin" SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 283, no. 5407, 5 March 1999 (1999-03-05), pages 1541-1544, XP002134537 ISSN: 0036-8075 cited in the application *
SAUTER MARGRET: "Differential expression of a CAK (cdc2-activating kinase)-like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin." PLANT JOURNAL, vol. 11, no. 2, 1997, pages 181-190, XP002146544 ISSN: 0960-7412 *
SEGERS GERDA ET AL: "The Arabidopsis cyclin-dependent kinase gene cdc2bAt is preferentially expressed during S and G-2 phases of the cell cycle" PLANT JOURNAL,GB,BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, vol. 10, no. 4, 1996, pages 601-612, XP002138663 ISSN: 0960-7412 cited in the application *

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US7732663B2 (en) 1998-06-08 2010-06-08 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Cyclin-dependent kinase inhibitors as plant growth regulators
WO2001023594A2 (fr) * 1999-09-27 2001-04-05 Pioneer Hi-Bred International, Inc. Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire
WO2001023594A3 (fr) * 1999-09-27 2001-12-06 Pioneer Hi Bred Int Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire
WO2002050292A2 (fr) * 2000-12-08 2002-06-27 Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agrifood Canada Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes
WO2002050292A3 (fr) * 2000-12-08 2002-09-19 Ca Minister Agriculture & Food Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes
WO2003012035A2 (fr) * 2001-07-27 2003-02-13 Icon Genetics, Inc. Usage commercial d'arabidopsis pour la production de proteines diagnostiques et therapeutiques humaines et animales
WO2003012035A3 (fr) * 2001-07-27 2005-05-19 Icon Genetics Inc Usage commercial d'arabidopsis pour la production de proteines diagnostiques et therapeutiques humaines et animales
EP2322633A2 (fr) 2003-02-17 2011-05-18 Metanomics GmbH Préparation d'organismes dotés d'une croissance plus rapide et/ou d'un meilleur rendement
US7589256B2 (en) 2003-02-17 2009-09-15 Metanomics Gmbh Preparation of organisms with faster growth and/or higher yield
WO2004087929A2 (fr) * 2003-03-31 2004-10-14 Cropdesign N.V. Plantes a caracteristiques de croissance ameliorees et leur procede de production
US8299319B2 (en) 2003-03-31 2012-10-30 Cropdesign N.V. Plants having improved growth characteristics and a method for making the same
WO2004087929A3 (fr) * 2003-03-31 2004-12-23 Cropdesign Nv Plantes a caracteristiques de croissance ameliorees et leur procede de production
CN102174545A (zh) * 2003-09-05 2011-09-07 作物培植股份有限公司 具有改变的生长特性的植物及其制备方法
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US20070199085A1 (en) * 2003-09-05 2007-08-23 Willem Broekaert Plants Having Modified Growth Characteristics And Method For Making Same
WO2005024029A3 (fr) * 2003-09-05 2005-09-01 Cropdesign Nv Plantes a caracteristiques de croissance modifiees, et leur procede de production
CN1845997B (zh) * 2003-09-05 2012-07-25 作物培植股份有限公司 具有改变的生长特性的植物及其制备方法
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EP1711592A4 (fr) * 2003-12-30 2007-08-22 Arborgen Llc Genes de cycles cellulaires et leurs procedes d'utilisation associes
JP2011097941A (ja) * 2003-12-30 2011-05-19 Arborgen Inc 細胞周期遺伝子および関連した使用方法
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US7598084B2 (en) 2003-12-30 2009-10-06 Arborgen Llc Modifications of plant traits using cyclin A
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WO2005093077A1 (fr) * 2004-03-22 2005-10-06 Cropdesign N.V. Plantes presentant des caracteristiques de croissance ameliorees et leur procede de production
US7579518B2 (en) 2004-03-22 2009-08-25 Cropdesign N.V. Plants having improved seed yield and expressing a nucleic acid encoding a small subunit ribosomal (S3A) protein and method for making the same
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WO2006013010A2 (fr) 2004-07-31 2006-02-09 Metanomics Gmbh Preparation d'organismes a croissance plus rapide et/ou a meilleur rendement
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WO2008138975A1 (fr) * 2007-05-15 2008-11-20 Cropdesign N.V. ACCROISSEMENT DU RENDEMENT DE PLANTES PAR MODULATION DU ZmPKT
US20110145949A1 (en) * 2008-08-20 2011-06-16 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
WO2010023320A3 (fr) * 2008-08-29 2010-05-20 Basf Plant Science Company Gmbh Végétaux présentant des caractéristiques associées au rendement améliorées, et leur procédé de production
US20110214207A1 (en) * 2008-08-29 2011-09-01 Basf Plant Science Company Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
US8946512B2 (en) 2008-08-29 2015-02-03 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and a method for making the same
WO2010023320A2 (fr) * 2008-08-29 2010-03-04 Basf Plant Science Company Gmbh Végétaux présentant des caractéristiques associées au rendement améliorées, et leur procédé de production
US8991098B2 (en) 2008-09-16 2015-03-31 Basf Plant Science Gmbh Method for improved plant breeding
US10244692B2 (en) 2008-09-16 2019-04-02 BASF Plan Science GmbH Method of improved plant breeding
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EP2272345A1 (fr) 2009-07-07 2011-01-12 Bayer CropScience AG Processus d'amélioration de la croissance de semis et/ou levée précoce de cultures

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