WO2002050292A2 - Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes - Google Patents

Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes Download PDF

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WO2002050292A2
WO2002050292A2 PCT/CA2001/001825 CA0101825W WO0250292A2 WO 2002050292 A2 WO2002050292 A2 WO 2002050292A2 CA 0101825 W CA0101825 W CA 0101825W WO 0250292 A2 WO0250292 A2 WO 0250292A2
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cyclin
plant
dependent kinase
kinase inhibitor
polypeptide
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PCT/CA2001/001825
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WO2002050292A3 (fr
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Hong Wang
Yongming Zhou
Larry C. Fowke
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Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agrifood Canada
University Of Saskatchewan Technologies Inc.
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Priority claimed from US09/733,507 external-priority patent/US7078591B2/en
Application filed by Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agrifood Canada, University Of Saskatchewan Technologies Inc. filed Critical Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agrifood Canada
Priority to CA002433048A priority Critical patent/CA2433048A1/fr
Priority to AU2002215793A priority patent/AU2002215793A1/en
Priority to US10/451,139 priority patent/US20040098763A1/en
Publication of WO2002050292A2 publication Critical patent/WO2002050292A2/fr
Publication of WO2002050292A3 publication Critical patent/WO2002050292A3/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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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 invention relates to the modification of growth and development of plants through transgenic modification.
  • CDKs are a group of related serine/threonine kinases having an activity that generally depends on their association with cyclins (Pines, 1995).
  • CDK1 p34 c a , or CDK1
  • Cdc2 p34 c a , or CDK1
  • CDC28 a Cdc2 homolog
  • yeast Saccharomyces cerevisiae L ⁇ rincz and Reed, 1984.
  • Cdc2 (or CDC28) kinase appears to be solely responsible for regulating the progression of the cell cycle.
  • Cdc2a also known as Cdc2aAt or Arath;CDKA;l
  • Cdc2aAt or Arath;CDKA;l resembles more closely Cdc2 homologues from other species because it has a conserved PSTAIRE motif and is able to genetically complement yeast cdc2 or CDC28 mutants (Ferreira et al., 1991; Hirahama et al., 1991), indicating some functional homology of A.
  • A. thaliana Cdc2a with the yeast Cdc2 kinase. Expression analyses showed that A. thaliana Cdc2a expression was correlated with the "competence" of a cell to divide and preceded the re-entry of differentiated cells into the cell division cycle (Martinez et al., 1992; Hemerly et al., 1993), and expression of a dominant negative Cdc2a mutant resulted in cell cycle arrest (Hemerly et al., 1995).
  • A. thaliana Cdc2b is atypical in that it has a PPTALRE motif in place of the PSTAIRE motif. Like Cdc2a, Cdc2b is also expressed in dividing plant cells.
  • Cdc2a is expressed constitutively throughout the cell cycle
  • Cdc2b is reportedly expressed preferably in S and G2 phases (Segers et al., 1996).
  • Cdc2a and Cdc2b other CDK-related genes have been isolated from Arabidopsis and other species (Joubes et al., 2000).
  • CDKs The activity of CDKs in controlling the cell cycle appears to be regulated by several other factors. Results from yeast and mammalian studies have demonstrated multiple pathways, both positive and negative, by which CDK activity can be modulated (Lees, 1995). In addition to binding by a cyclin, for example, activation of CDKs may also involve a CDK-activating kinase (CAK) which itself is a CDK, and CDC25 protein phosphatase.
  • CAK CDK-activating kinase
  • CDK inhibitors see reviews by Pines,
  • CDK inhibitors are understood to bind stoichiometrically to negatively regulate the activity of CDKs. It has been suggested that these inhibitors may be involved in animal development and cancer, in addition to their role in cell cycle regulation (Harper and Elledge, 1996). A plant CDK inhibitor activity was observed and was suggested to be involved in endosperm development in maize (Grafi and Larkins, 1995).
  • CDK inhibitors The activity of CDK inhibitors has been studied in animals.
  • Transgenic mice have been generated lacking p21, p27 and p57 CDK inhibitor genes.
  • the p21 knockout mice are reported to develop normally although they are deficient in Gl checkpoint control, such as cell cycle arrest in response to DNA damage (Deng et al., 1995).
  • Analysis of p27 knockout mice from three independent studies shows that transgenic mice lacking p27 display larger body size than control mice (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996).
  • the enhanced growth is reportedly due to an increase in cell number (Kiyokawa et al., 1996) and is gene dose-dependent (Fero et al., 1996).
  • mice lacking p57 show a range of developmental defects such as defective abdominal muscles, cleft palate and renal medullary dysplasia (Yan et al., 1997; Zhang et al., 1997). Developmental defects were observed in p27-/- mice, including impaired ovarian follicles (thus female sterility), impaired luteal cell differentiation and a disordered estrus cycle. These results may reflect a disturbance of the hypothalamic-pituitary-ovarian axis. In comparison, transgenic mice lacking p21 appear to develop normally at both gross anatomic and histologic levels (Deng et al., 1995).
  • mice lacking p57 an increase in apoptosis is observed in mice lacking p57.
  • the CDK inhibitor p27 was over-expressed in mouse hepatocytes (Wu et al., 1996), the result was a general decrease in overall number of adult hepatocytes leading to aberrant tissue organization, body growth and mortality.
  • Plants also have an inherent ability to incorporate additional growth into normal developmental patterns, as is illustrated by a study showing that ectopic expression of a mitotic cyclin driven by the Cdc2a promoter resulted in a larger but normal root system (Doerner et al., 1996). At this time, relatively little is known about the connections in plants between the regulatory genes controlling cell division patterns and cell cycle regulators such as CDKs and cyclins (Meyerowitz, 1997). A few studies of transgenic expression of cell cycle genes in plants are documented using various cell cycle genes other than CDK inhibitors.
  • a heterologous yeast cdc25 coding sequence a mitotic inducer gene, was introduced into tobacco plants under the control of a constitutive CaMV 35s promoter (Bell et al., 1993).
  • the transgenic tobacco plants showed abnormal leaves (lengthened and twisted lamina, pocketed interveinal regions), abnormal flowers, and also precocious flowering.
  • Analysis of cell size in the root meristem revealed that the transgenic plants expressing the yeast cdc25 had much smaller cells (Bell et al., 1993).
  • the wild type Cdc2a gene and variants of dominant negative mutations under the control of CaMN 35s promoter have been used to transform tobacco and Arabidopsis plants (Hemerly et al., 1995).
  • ICK1 cyclin-dependent kinase inhibitor gene ICK1 from a plant
  • ICK1 is different in sequence, structure and inhibitory properties from known mammalian CDK inhibitors. It has been shown that the recombinant protein produced from this gene in bacteria is able to inhibit plant Cdc2-like kinase activity in vitro (Wang et al., 1997).
  • Cytotoxin genes i.e. genes encoding a protein which will cause cell death, have been tested in transgenic plants for genetic ablation of specific cells or cell lines during development, including R ⁇ ase (Mariani et al., 1990), DTT (diphitheria toxin) chain A (Thorsness et al., 1991; Czako et al., 1992), Exotoxin A (Koning et al., 1992) and ribosomal inhibitor proteins (United States Patent No. 5,723,765 issued 3 March 1998 to Oliver et al).
  • cytotoxin genes i.e. genes encoding a protein which will cause cell death
  • the action of the cytotoxin may not be specific and may result in non-specific destruction of plant cells. This effect may be the result of diffusion of the cytotoxin, or of non-specific expression of the cytotoxin gene in non-target tissues.
  • Non-specific low-level expression of the cytotoxin may be a difficult problem to overcome, since most tissue-specific promoters have some levels of expression in other tissues in addition to a high level of expression in a particular tissue. Expression of a potent cytotoxin gene even at a low concentration may have a negative impact on growth and development in non-target tissues.
  • cytotoxic proteins of transgenic origin may also have a negative effect on the marketability of an edible plant, or plant product, even if the cytotoxin is demonstrably benign to consumers.
  • CDK inhibitors International Patent Publication No. WO 99/64599 of Wang et al, published 16 December 1999
  • D-rype cyclins International Patent Publication No. WO 98/42851 of Murray, published 1 October 1998)
  • CDKs and cyclins International Patent Publication No. WO 00/56905 of De Veylder et al, published 28 September 2000.
  • the invention provides methods of modifying plant or plant cell development using heterologous proteins that bind to or interact with CDK inhibitors.
  • the proteins that bind to CDK inhibitors may for example be used to counteract the effects of CDK inhibitors on plant growth and development.
  • the methods of the invention involve introducing into a plant cell a nucleic acid encoding a protein that binds to a cyclin-dependent kinase inhibitor, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or progeny of the plant cell, under conditions wherein the protein that binds to the cyclin-dependent kinase inhibitor is co-expressed with the cyclin-dependent kinase inhibitor in the plant cell or in the progeny of the transformed plant cell.
  • the step of introducing into the plant cell the nucleic acid encoding the protein that binds to the cyclin-dependent kinase inhibitor may for example be carried out by transforming the plant cell or by cross breeding.
  • the step of growing the plant cell or progeny of the plant cell may be carried out to produce a plant, and the protein that binds to the cyclin-dependent kinase inhibitor may be expressed in such a way that the development of the plant or progeny of the plant is altered.
  • the alteration may be to counteract the effect on the development of the plant that the cyclin-dependent kinase inhibitor would otherwise have had.
  • the word 'development' may encompass a wide variety of biological process, including growth, mo ⁇ hogenesis, multiplication, enlargement, differentiation or maturation of a cell.
  • the cyclin-dependent kinase inhibitor may for example be ICK1, ICK 2, ICN2 (which may also be known as ICK4), ICN6 (which may also be known as ICK5), ICN7 (which may also be known as ICK6), ICN8 (which may also be known as ICK7), ICDK (which may also be known as ICKCr) or homologues thereof.
  • the protein that binds to the CDK inhibitor may be a cyclin, such as cyclin D3.
  • the plant may for example be A. thaliana, or a member of the Brassica genus, or a canola variety.
  • tissue-specific promoters could be an NTM19 promoter, a promoter homologous to NTM19, an AP 3 promoter or a promoter homologous to AP3.
  • the invention provides methods of plant breeding that may include modulating phenotypic modification of plants over successive generations, including methods that may be used to at least partially reverse phenotypic modifications in alternative plant generations.
  • one generation of a plant may express a CDK inhibitor so that the development of the plant is modified by the expressed CDK inhibitor to provide a plant having an altered phenotype.
  • the plant having the altered phenotype may be crossed with a plant encoding a protein that binds to the CDK inhibitor.
  • the progeny of the cross may be selected to include plants wherein the altered phenotype, caused by the CDK inhibitor, is further altered by the expression of the protein that binds to the CDK inhibitor.
  • hybrid plants may be provided by the methods of the invention, wherein a first parent plant is homozygous for a sequence encoding a CDK inhibitor and a second parent plant is homozygous for a sequence encoding a protein that binds to the CDK inhibitor, so that the hybrid offspring of the first and second parent plants is heterozygous, having a copy of the sequence encoding the CDK inhibitor and a copy of the sequence encoding the protein that binds to the CDK inhibitor.
  • a male-sterile first parent plant may be provided which is homozygous for a sequence encoding a CDK inhibitor that confers the male-sterile phenotype.
  • the male-sterile parent may be crossed with pollen from a second parent plant that is homozygous for a sequence encoding a protein that binds to the CDK inhibitor, so that in the hybrid offspring the CDK inhibitor and the protein that binds to the CDK inhibitor are co-expressed and the hybrid FI plant is not male-sterile.
  • the invention also provides methods of modifying plant architecture and mo ⁇ hology for desired size and shape according to alternative embodiments of this invention.
  • Plant architecture, size and mo ⁇ hology may be modified by expressing a CDK inhibitor and a protein that binds to the CDK inhibitor.
  • Specific expression patterns conferred by a variety of promoters may be used to modify a specific tissue or organ or two or more tissues or organs. Therefore, plants of various shapes, sizes and appearances may be produced from plants of the same type. Specific modifications may be made for agriculture crops, horticultural plants or trees.
  • transgenic plants comprising (i.e. having or including, but not limited to) an expressible heterologous nucleic acid encoding a CDK inhibitor and a heterologous nucleic acid encoding a protein that binds to the CDK inhibitor, wherein the heterologous nucleic acids are introduced into the plant, or an ancestor of the plant, by transgenic or classical methods.
  • Plants of the invention may accordingly have a recombinant genome, with heterologous nucleic acids integrated into the recombinant genome.
  • the invention encompasses plant tissues, such as seeds, comprising heterologous nucleic acids encoding a CDK inhibitor and a protein that binds to the CDK inhibitor.
  • the invention may also provide methods of identifying proteins that bind to selected CDK inhibitors, and corresponding methods of identifying CDK inhibitors that bind to selected proteins, such as selected cyclins or cyclin fragments. Methods of the invention may also be used to identify interacting pairs of CDK inhibitors and proteins that bind to CDK inhibitors, so that such pairs of interacting proteins may be used in reversible methods of phenotypic modification in accordance with other aspects of the invention.
  • Figure 1 shows cDNA (Wang et al., 1997) and genomic sequences of ICKl, wherein: (A) shows the genomic organization ICKl (SEQ ID NO: 1). Open bars represent exons and filled bars represent introns; (B) shows features of the ICKl cDNA sequence (SEQ ID NO: 3) and ICKl deduced amino acid sequence (SEQ ID NO: 2). *
  • Figure 2 shows a CLUSTAL W alignment of an ICKl CDNA sequence (SEQ ID NO: 3) with alternative cDNAs: ICKlb (SEQ ID NO: 4) and ICKc (SEQ ID NO: 5).
  • Figure 3A shows the partial cDNA sequence of ICK2 (SEQ ID NO: 6).
  • Figure 3B shows the full-length cDNA sequence of ICK2 (SEQ ID NO: 18).
  • Figure 4 shows the cDNA sequence of ICN2 (SEQ ID NO: 7).
  • Figure 5 shows the cDNA sequence 0 ⁇ ICN6 (SEQ ID NO: 8).
  • Figure 6A shows the partial cDNA sequence of ICN7 (SEQ ID NO: 9).
  • Figure 6B shows the full-length cDNA sequence of ICN7 (SEQ ID NO: 20).
  • Figure 7A shows the amino acid sequence of ICK2 (SEQ ID NO.: 10) deduced from the partial cDNA.
  • Figure 7B shows the amino acid sequence of ICK2 (SEQ ID NO.: 19) deduced from the full-length cDNA.
  • Figure 8 A shows the deduced amino acid sequence of ICN2 (SEQ ID NO.: 11) as part of a functional fusion protein in the yeast two-hybrid system.
  • Figure 8B shows the amino acid sequence of ICN2 (SEQ ID NO.: 22), deduced from
  • ICN2 cDNA sequence taking into account the most probable translation start site yielding a sequence with 16 amino acid residues removed from the N-terminus compared to the sequence shown in Figure 8A.
  • Figure 9 A shows the deduced amino acid sequence of ICN6 (SEQ ID NO.: 12) as part of a functional fusion protein in the yeast two-hybrid system.
  • Figure 9B shows the amino acid sequence of ICN6 (SEQ ID NO.: 23), deduced from ICN6 cDNA sequence taking into account the most probable translation start site yielding a sequence with 12 amino acid residues removed from the N-terminus compared to the sequence shown in Figure 9A.
  • Figure 10A shows the amino acid sequence of ICN7 (SEQ ID NO.: 13) deduced from the partial cDNA.
  • Figure 10B shows the amino acid sequence of ICN7 (SEQ ID NO.: 21) deduced from the full-length cDNA.
  • Figure 11 shows the nucleic acid sequence of the ICDK cDNA (GenBank AJ002173; SEQ ID NO.: 14).
  • Figure 12 shows the deduced amino acid sequence of ICDK (SEQ ID NO.: 15).
  • Figure 13 shows the cDNA sequence of ICN8 (SEQ ID NO.: 16).
  • Figure 14 shows the deduced amino acid sequence of ICN8 (SEQ ID NO.: 17).
  • Figure 15A shows a ClustalW alignment of deduced amino acid sequences of: ICKl (SEQ ID NO: 2), ICK 2 (SEQ ID NO: 10), ICN2 (SEQ ID NO: 11), ICN6 (SEQ ID NO: 12), ICN7 (SEQ ID NO: 13), ICN8 (SEQ ID NO: 17), and ICDK (SEQ ID NO: 15).
  • Figure 15B shows an alternative alignment of deduced amino acid sequences of: ICKl (SEQ ID NO: 2), ICK 2 (SEQ ID NO: 19), ICN2 (SEQ ID NO: 22), ICN6 (SEQ ID NO: 23), ICN7 (SEQ ID NO: 21), ICN8 (SEQ ID NO: 17), and ICDK (SEQ ID NO: 15).
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter codes for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is hereby included by any reference to the displayed strand.
  • SEQ ID NO.: 1 shows the nucleic acid sequence of ICKl.
  • SEQ ID NO.: 2 shows the deduced amino acid sequence of ICKl.
  • SEQ ID NO.: 3 shows the nucleic acid sequence of the ICKl cDNA.
  • SEQ ID NO.: 4 shows the nucleic acid sequence of the ICKlb cDNA.
  • SEQ ID NO.: 5 shows the nucleic acid sequence of the ICKlc cDNA.
  • SEQ ID NO.: 6 shows the nucleic acid sequence of the ICK2 partial cDNA.
  • SEQ ID NO.: 7 shows the nucleic acid sequence of the ICN2 cDNA.
  • SEQ ID NO.: 8 shows the nucleic acid sequence of the 7CN6 ' cD ⁇ A.
  • SEQ ID NO.: 9 shows the nucleic acid sequence of the ICN7 partial cDNA.
  • SEQ ID NO.: 10 shows the amino acid sequence of ICK2 deduced from the partial cDNA.
  • SEQ ID NO.: 11 shows the deduced amino acid sequence of ICN2 as part of a functional fusion protein in the yeast two-hybrid system.
  • SEQ ID NO.: 12 shows the deduced amino acid sequence of ICN6 as part of a functional fusion protein in the yeast two-hybrid system.
  • SEQ ID NO.: 13 shows the amino acid sequence of ICN7 deduced from the partial cDNA.
  • SEQ ID NO. : 14 shows the nucleic acid sequence of the ICDK cDNA (GenBank
  • SEQ ID NO.: 15 shows the deduced amino acid sequence of ICDK.
  • SEQ ID NO.: 16 shows the cDNA sequence of ICN8.
  • SEQ ID NO.: 17 shows the deduced amino acid sequence of ICN8.
  • SEQ ID NO.: 18 shows the nucleic acid sequence of the ICK2 full-length cDNA.
  • SEQ ID NO.: 19 shows the amino acid sequence of ICK2 deduced from the full-length cDNA.
  • SEQ ID NO.: 20 shows the nucleic acid sequence of the ICN7 full-length cDNA.
  • SEQ ID NO.: 21 shows the amino acid sequence of ICN7 deduced from the partial cDNA.
  • SEQ ID NO.: 22 shows the amino acid sequence of ICN2 deduced from ICN2 cDNA sequence.
  • SEQ ID NO.: 23 shows the amino acid sequence of ICN6 deduced from 7CN6 ' cD ⁇ A sequence.
  • the invention provides methods of modifying plant or plant cell development.
  • the word 'development' encompasses a very wide variety of biological process, including growth, mo ⁇ hogenesis, multiplication, enlargement, differentiation or maturation of a cell or plant.
  • the invention provides methods of modifying plant development that include introducing into a plant cell a nucleic acid encoding a protein that binds to a cyclin-dependent kinase inhibitor polypeptide, where the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide.
  • the plant cell, or the progeny of the plant cell may then be grown under conditions so that the protein that binds to the CDK inhibitor and the CDK inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant. It has unexpectedly been discovered that this allows one to modulate the effect that the CDK inhibitor would otherwise have on development of the plant.
  • the effect of the CDK inhibitor may be at least partly reversed by the protein that binds to the CDK inhibitor.
  • the invention relates to CDK inhibitor polypeptides, such as ICKl.
  • a 'CDK inhibitor polypeptide' is any polypeptide capable of inhibiting a CDK, such as CDKs active during development of a plant or plant cell.
  • Proteins that bind to CDK inhibitors in the context of the invention are those proteins that show a sufficiently strong interaction with the CDK inhibitor of interest so that they are capable of modulating the desired effects of the invention.
  • the protein-protein interactions that mediate such binding will be sufficiently strong to be detected using a two-hybrid system, or a similar system for characterizing protein-protein interactions.
  • proteins that bind to a CDK inhibitor are identifiable as proteins that have at least one segment that, when used as 'prey' in a yeast two-hybrid assay, bind to at least a segment of a CDK inhibitor, to produce a positive signal in the two-hybrid assay, or an alternative assay of protein-protein interaction.
  • the yeast two-hybrid and interaction trap systems may, for example, be used to identify proteins or protein fragments that bind to CDK inhibitors, or to characterize protein-protein interactions in various aspects of the present invention.
  • Both the two-hybrid and interaction trap systems exploit the fact that the transcriptional activation and DNA binding domains of most eukaryotic transcriptional activators function when expressed as fusions with heterologous proteins (Brent and Ptashne, 1985, Cell 43 J29-36), and can transactivate when brought together by specific protein-protein interaction between separate fusion proteins (Chien, et al., 1991, Proc Natl Acad Sci U.S.A. 88:9578-82; Fields and Song, 1989, Nature 340:245-6).
  • a protein of interest is fused to the DNA-binding domain of GAL4 to create a "bait" fusion protein.
  • Proteins that interact with the bait protein termed the "prey” can be identified by their ability to cause transactivation of a GAL4-dependent reporter gene when expressed as a fusion to the C- terminal transactivation domain of GAL4 (Chien et al., 1991, Proc Natl Acad Sci U.S.A. 88:9578-82; Fields and Song, 1994, U.S. Pat. No. 5,283,173; Fields and Song, 1995, U.S. Pat. No. 5,468,614).
  • the "interaction trap” system employs the identical principle of using separate fusions with DNA-binding and transactivation domains, except that the bait is fused to LexA, which is a sequence-specific DNA binding protein from E. coli, and an artificial transactivation domain known as B42 (Ma and Ptashne, 1987, Cell 51 :113-9) is used for the "prey” fusions. Interaction between the bait and prey fusions is detected by expression of a LexA-responsive reporter gene (Brent et al., 1996, U.S. Pat. No. 5,580,736).
  • a cDNA library may be made using poly (A) mRNA isolated from whole plants at different stages of development and cloned in a suitable vector, such as Gal4 TA- (transcription-activation domain) pPC86 (Chevray and Nathans, 1992; available from GIBCO/BRL Life Technologies) or a similar vector (Koholmi et al., 1997).
  • the cDNA of the gene (such as Arabidopsis Cdc2a, cyclin D2 and cyclin D3) to be used for screening the library may be cloned in a suitable vector, such as the Gal4 DB- (DNA-binding domain) vector.
  • the yeast strain, such as MaV203, harboring the construct may be transformed using the library DNA.
  • a total of 1.8 X 10 transformants were subjected to two-hybrid selection on supplemented synthetic dextrose medium lacking leucine, tryptophan and histidine but containing 5 mM 3-amino-l,2,4-triazole.
  • the selected colonies were assayed for ⁇ -galactosidase activity using standard methods.
  • DNAs were isolated from positive clones and used to transform E. coli. Clones harboring the TA-fusion cDNAs were identified by PCR and plasmids were then isolated for DNA sequencing (Wang et al., 1997).
  • Interactions in the yeast two-hybrid system may, for example, be analyzed by either filter assay (Chevray and Nathans, 1992) using X-gal as the substrate or by a quantification assay using ONPG (ortho-nitrophenyl-beta-D-galactoside) as the substrate (Reynolds and Lundlad, 1994). Three or more independent transformants may be used for each interaction.
  • a plant cell or progeny of the plant cell may be grown to produce a plant, such as by regenerating a plant from a transformed cell or cell culture, or by propagating or growing whole plants from transformed plant parts.
  • the term 'progeny', with reference to a plant includes progeny produced sexually or asexually (for example by tissue culture-based propagation).
  • the term 'growing' with reference to the transformed cells or plants includes all methods for growing and propagating cells or plants, such as tissue culture or horticultural means of propagating plants or plant parts.
  • the growth of a plant cell, or progeny of a plant cell, in accordance with various aspects of the invention may be carried out so that an effect of a cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by a protein that binds to the cyclin-dependent kinase inhibitor polypeptide.
  • the 'effect' that is at least partially reversed may be any detectable physiological or genetic change caused by the expression of the CDK inhibitor.
  • 'reversal' it is meant that a measurable parameter of such a change is made to revert to a value that is closer to the value of the parameter in plants that do not manifest the change caused by the CDK inhibitor.
  • the size or weight of a plant may be reduced by a CDK inhibitor, compared to wild type plants, and a cyclin or a protein that binds to the CDK inhibitor may be used to increase the size or weight of the plant that expresses the CDK inhibitor.
  • the effect may relate to the modification of a plant tissue, such as the total or partial ablation of a developmental cell line by a CDK inhibitor, which may result in a phenotypic modification of the tissue in the plant, which may be at least partially restored by a cyclin or a protein that binds to the CDK inhibitor.
  • Nucleic acid coding sequences may be used in various aspects of the invention. Such sequences may be operatively linked to promoters, terminators, signal sequences, polyadenylation sequences, splice sites or alternative control sequences that may be used to facilitate expression of a coding sequence.
  • promoter means a sequence sufficient to direct transcription of a gene when the promoter is operably linked to the gene. The promoter is accordingly the portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not universally, located in the 5' non- coding regions of a gene.
  • a promoter and a gene are "operably linked” when such sequences are functionally connected so as to permit gene expression mediated by the promoter.
  • the term “operably linked” accordingly indicates that DNA segments are arranged so that they function in concert for their intended pu ⁇ oses, such as initiating transcription in the promoter to proceed through the coding segment of a gene to a terminator portion of the gene.
  • Gene expression may occur in some instances when appropriate molecules (such as transcriptional activator proteins) are bound to the promoter. Expression is the process of conversion of the information of a coding sequence of a gene into mRNA by transcription and subsequently into polypeptide (protein) by translation, as a result of which the protein is said to be expressed.
  • a gene or nucleic acid is "expressible” if it is capable of expression under appropriate conditions in a particular host cell.
  • promoters may for example be used that provide for preferential gene expression within a specific organ or tissue, or during a specific period of development.
  • promoters may be used that are specific for leaf (Dunsmuir, et al Nucleic Acids Res, (1983) 11:4177-4183), root tips (Pokalsky, et al Nucleic Acids Res, (1989) 17:4661-4673), fruit (Peat, et al Plant Mol. Biol, (1989) 13:639-651; United States Patent No. 4,943,674 issued 24 July, 1990; International Patent Publication WO-A 8 809 334; United States Patent No.
  • Promoters may be identified from genes which have a differential pattern of expression in a specific tissue by screening a tissue of interest, for example, using methods described in United States Patent No. 4,943,674 and European Patent Application EP-A 0255378.
  • Non-dividing plant cells may tolerate low level expression of CDK inhibitors without detectable effect.
  • the invention may be practiced in some embodiments using tissue specific promoters operably linked to CDK inhibitor encoding sequences, to give rise effects that may be reversed with a cyclin or protein that binds to the CDK inhibitor, even when the promoter mediates a tolerable basal level of CDK inhibitor expression in other tissues.
  • nucleic acid or amino acid sequences that are homologous to other sequences.
  • an amino acid or nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (for example, both sequences function as or encode a cyclin-dependent kinase inhibitor; as used herein, sequence conservation or identity does not infer evolutionary relatedness).
  • Nucleic acid sequences may also be homologous if they encode substantially identical amino acid sequences, even if the nucleic acid sequences are not themselves substantially identical, for example as a result of the degeneracy of the genetic code.
  • sequence identity may for example be at least 75%, at least 90% or at least 95%o.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al (1990), J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/).
  • the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • W word length
  • B BLOSUM62 scoring matrix
  • E expectation
  • P(N) the smallest sum probability
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1- preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • hybridize to each other under moderately stringent, or preferably stringent, conditions Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C, and washing in 0.2 x SSC/0.1% SDS at 42 °C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO , 7% SDS, 1 mM EDTA at 65 °C, and washing in 0.1 x SSC/0.1% SDS at 68 °C (see Ausubel, et al. (eds), 1989, supra).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • an alternative indication that two amino acid sequences are substantially identical is that one peptide is specifically immunologically reactive with antibodies that are also specifically immunoreactive against the other peptide.
  • Antibodies are specifically immunoreactive to a peptide if the antibodies bind preferentially to the peptide and do not bind in a significant amount to other proteins present in the sample, so that the preferential binding of the antibody to the peptide is detectable in an immunoassay and distinguishable from non-specific binding to other peptides.
  • the cyclin-dependent kinase inhibitors of the present invention may include non- naturally occurring sequences, such as functionally active fragments of naturally occurring sequences. For example, fragments of ICKl, or amino acid sequences homologous to those fragments, that have cyclin-dependent kinase inhibitory activity may be used in some embodiments of the invention.
  • the invention provides methods for identifying such fragments, for example by deletion mapping of active cyclin-dependent kinase inhibitors or cyclins.
  • cyclin-dependent kinase inhibitor or "cyclin” or "protein that binds to a CDK inhibitor” includes any polypeptide capable of having the relevant functioning, i.e. respectively inhibiting a cyclin-dependent kinase or at least partially reversing an effect of the CDK inhibitor.
  • the invention encompasses nucleic acid sequences encoding such alternative polypeptides.
  • heterologous refers to molecules or portions of molecules, such as DNA sequences, that are artificially introduced into a particular host cell or genome.
  • Heterologous DNA sequences may for example be introduced into a host cell by transformation.
  • Such heterologous molecules may include sequences derived from the host cell, and thereafter reintroduced into the host cell or a cell from the same cell line or species.
  • Heterologous DNA sequences may become integrated into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination events, but they remain heterologous because of their different 'artificial' origin.
  • reversal of an effect of a CDK inhibitor may be used to enhance growth during plant development.
  • a CDK inhibitor such as ICKl
  • Such growth enhancement may be tissue-specific.
  • the expression of cyclins or proteins that bind to CDK inhibitors may be made to be tissue-specific by operably linking the relevant coding sequences to tissue-specific promoters.
  • a variety of plants can be used for identifying proteins that are useful in various embodiments of the present invention, such as CDK inhibitors, cyclins and proteins that bind to CDK inhibitors.
  • Arabidopsis thaliana "Columbia” may be used as a convenient model system for identifying proteins that are useful in various embodiments of the present invention.
  • Arabidopsis plants are generally grown in pots placed in growth chambers. Other plants may also of course be used in various embodiments of the invention in accordance with known growth and transformation techniques.
  • Standard methods are available for cloning genomic and cDNA nucleotide sequences for use in identifying and characterizing proteins and nucleic acids useful in various embodiments of the invention.
  • General molecular techniques may for example be performed by procedures generally described by Ausubel et al. (1995). Sequence analyses, including determination of sequence homology, may be performed using a variety of software, such as LASERGENE (DNASTAR). Database searches may also use a variety of software tools, such as the BLAST program (NCBI). Alternative equivalent methods or variations thereof may be used in accordance with the general knowledge of those skilled in this art.
  • genomic DNA was isolated from leaf tissue of Nicotiana tabacum CV "Xanthi" based on a described procedure (Dellaporta et al., 1983).
  • the promoter sequence was amplified by 30 cycles of PCR using sequence-specific primers with inco ⁇ orated restriction sites.
  • Pfu DNA polymerase (Stratagene), which has a higher replication fidelity than the Taq DNA polymerase, may be used.
  • the amplified DNA fragment may be cloned into a suitable vector, such as pGEM3Zf(+) (Promega). Plasmids may then be purified and the cloned DNA fragment sequenced.
  • a cDNA clone ICDK (Fountain et al., 1999) (GenBank Accession AJ002173, SEQ ID No. 15 and SEQ ID No.16) was identified from Chenopodium rubrum, which encodes a protein sharing sequence similarity with ICKl and thus also with ICK2, ICN2, ICN6 and ICN7 (Table 1).
  • RNA was isolated from seedlings and leaves of Chenopodium rubrum.
  • the full-length coding region of ICDK cDNA was cloned by RNA RT-PCR (e.g. using ThermoScript RT-PCR System, GIBCO/BRL Life Technologies) with sequence-specific primers.
  • the amplified fragment was cloned and sequenced.
  • the sequence data showed that the cloned cDNA was identical to ICDK of C. rubrum.
  • a putative protein is identified encoded by a sequence BAC clone F24L7 (GenBank AC003974), which share some homology with ICKl (Wang et al., 1997).
  • Specific primers were designed and used to clone the cDNA corresponding to the genomic sequence by RNA RT-PCR. This cDNA was found to encodes a protein able to interact with Arabidopis CycD3, it is designated as ICN8.
  • ICKl cDNA (SEQ ID NO: 1 ; Wang et al., 1997) was amplified by PCR and transcriptionally linked with the 35S promoter in a binary vector pBI121 (Clontech). The chimeric gene ends with a nopaline synthase terminator.
  • the Arabidopsis CycD2 (Arath; CycD2;l, GenBank accession number X83370), CycD3 (Arath;CycD3;7, GenBank accession number X83371) and Cdc2a (Cdc2aAt or Arath;CDKA; 1 , GenBank accession number M59198 or X57839) were cloned by polymerase chain reaction (PCR) using Arabidopsis cDNA based on published sequence information (Hirayama et al, 1991 for Cdc2a, GenBank X57839; Soni et al., 1995 for CycD2 and CycD3, X83370 and X83371).
  • the Arabidopsis AP3 promoter was cloned by PCR from Arabidopsis thaliana "Columbia" genomic DNA, on the basis of the published sequence (Irish and Yamamoto, 1995; GenBank U30729). The promoter was linked transcriptionally with ICKl cDNA.
  • the tobacco NTM19 promoter was linked transcriptionally with ICKl cDNA. The NTM19 promoter from tobacco has been shown to activate gene expression at early stages of microspore development (Custers et al., 1997; Oldenhof et al., 1996). The resulting plasmids were introduced into Agrobacterium tumefaciens strain GV3101 (bearing helper plasmid pMP90; Koncz and Schell 1986).
  • plant cells may be transformed with heterologous nucleic acids.
  • heterologous denotes any nucleic acid that is introduced by transformation, or a sequence that is descended from a sequence introduced by transformation into a progenitor cell. Transformation techniques that may be employed include plant cell membrane disruption by electroporation, microinjection and polyethylene glycol based transformation (such as are disclosed in Paszkowski et al. EMBO J. 3:2717 (1984); Fromm et al, Proc. Natl Acad. Sci. USA 82:5824 (1985); Rogers et al, Methods Enzymol 118:627 (1986); and in U.S. Patent Nos.
  • transformation may be caried out by infiltration.
  • seeds (Tl generation) collected from infiltrated Arabidopsis plants may be surface-sterilized and placed onto MS medium containing 50 ⁇ g/ml kanamycin.
  • the antibiotic timentin may also be included in the medium to prevent any bacterial growth, which could occur due to carrier-over from the infiltration.
  • the vast majority of germinating seedlings will typically not be transformed, and will became pale and eventually stop growing, transformed seedlings will be green and display normal growth due to the presence of the selectable marker gene. After 4-5 weeks in the selection medium, transformants may be transferred to soil in pots.
  • the presence of the DNA insertion encoding a CDK inhibitor (ICKl) was confirmed by extracting the genomic DNA and then using it for PCR amplification.
  • ICKl CDK inhibitor
  • Transformed plant cells may be cultured to regenerate whole plants having the transformed genotype and displaying a desired phenotype, as for example modified by the expression of a heterologous CDK inhibitor during growth or development.
  • a variety of plant culture techniques may be used to regenerate whole plants, such as are described in Gamborg and Phillips, "Plant Cell, Tissue and Organ Culture, Fundamental Methods", Springer Berlin, 1995); Evans et al. "Protoplasts Isolation and Culture", Handbook of Plant Cell Culture, Macmillian Publishing Company, New York, 1983; or Binding, "Regeneration of Plants, Plant Protoplasts", CRC Press, Boca Raton, 1985; or in Klee et al, Ann. Rev. of Plant Phys. 38:467 (1987).
  • Standard techniques may be used for plant transformation, such as transformation of Arabidopsis.
  • the 35S-ICK1, 35S-CycD2, 35S-CycD3 and 35S-GUS constructs were tested in A. thaliana by in planta transformation techniques.
  • Wild type (WT) A. thaliana seeds of ecotype "Columbia” were planted in 4" pots containing soil and plants grown in a controlled growth chamber or greenhouse.
  • the vacuum infiltration method of in planta transformation (Bechtold et al., 1993) was used to transform .4.
  • pMP90 is a disarmed Ti plasmid with intact vir region acting in trans, gentamycin and kanamycin selection markers as described in Koncz and Schell (1986). Following infiltration, plants were grown to maturity and seeds (Tl) were collected from each pod individually. Seeds were surface-sterilized and screened on selective medium containing 50 mg/L kanamycin with or without 200-300 mg/L timentin. After about four weeks on selection medium, the non-transformed seedlings died. The transformed seedlings were transferred to soil in pots. Leaf DNA was isolated (Edwards et al., 1991) and analyzed by PCR for the presence of the DNA insertion. Genomic DNA was also isolated and used in Southern hybridization (Southern, 1975) to determine the copy number of the inserted sequence in a given transformant. To determine the segregation, T2 seeds were collected from Tl plants.
  • Tl plant was male sterile
  • crosses were made using the WT A. thaliana pollen to obtain seeds.
  • T2 seeds were surface-sterilized and screened on selective medium.
  • Alternative embodiments of the invention may make use of techniques for transformation of Brassica. Such as transformation of B. napus cv. Westar and B. carinata cv. Dodolla by co-cultivation of cotyledonary petioles or hypocotyl explants with A. tumefaciens bearing the plasmids described herein. Transformation of 5. napus plants may, for example, be performed according to the method by Moloney et al. (1989). Modifications of that method may include the introduction of a 7-day explant-recovery period following co-cultivation, on MS medium with the hormone benzyladenine (BA), and the antibiotic timentin for the elimination of Agrobacterium. Transformation of B.
  • BA hormone benzyladenine
  • carinata plants may be performed according to the method by Babic et al. (1998).
  • Cotyledonary petiole explants may be dipped in suspension of Agrobacterium bearing the desired constructs and placed on 7-cm filter paper (Whatman no. 1) on top of the regeneration medium for 2 days. After co- cultivation, explants may be transferred onto the selection medium containing 50 mg/L kanamycin. Regenerated green shoots may first be transferred to a medium to allow elongation and then to a rooting medium all containing 50 mg/L kanamycin. Putative transformants with roots (TO) may be transferred to soil. Genomic DNA may be isolated from developing leaves for PCR and Southern analyses. Seeds (Tl) from transgenic plants may then be harvested. Other techniques known in the art may be used to transform plants of other species such as tobacco.
  • Transgenic plants may be analysed for changes in growth and development. For example, seeds of T2 transgenic 35S-ICK1 lines were planted in soil. Wild type plants and transgenic plants carrying the 35S-GUS construct were used as controls. At the three-week stage, the above ground portion (including cotyledons, leaves and shoot) was removed and the fresh weight determined. The number of days to flower and leaf number (rosette plus inflorescence leaves on the primary axis) were obtained. Changes in the development of a particular organ can also be analysed. For instance, the leaf mo ⁇ hology and width/length ratio were determined. Similarly, the length of roots and shoots could be determined. Transgenic plants may be observed and characterized for alteration of traits such as petals, male sterility and ability to set seeds. For example, to determine the development of floral organs, flowers at different stages of development may be dissected and examined under a stereomicroscope.
  • RNA isolation and northern blotting analysis may be useful in various embodiments of the invention.
  • various tissues may be taken from Arabidopsis plants (Wang et al., 1995).
  • Total RNA was isolated using TRIzol reagent (GIBCO BRL).
  • TRIzol reagent GIBCO BRL
  • the indicated amount of RNA was fractionated in a 1.2% agarose gel and transferred onto Hybond-N+ nylon membrane (Amersham).
  • the RNA was crosslinked to the membrane by UV-light (Stratalinker, Stratagene) and hybridized with P-labeled probes.
  • the membranes may be wrapped and used to expose Hyperfilm MP (Amersham) film.
  • membranes may be stripped by treating with a boiling solution of 0.1X SSC and 0.1% SDS for 5 min. Quantification of hybridized signal was performed using a phosphoimager and the accompanying software.
  • kinases may be purified from A. thaliana tissues or cultured B. napus cells. Plant materials may be homogenized in 2 mis per gram tissue of ice cold extraction buffer consisting of 25 mM Tris pH 8.0, 100 mM NaCl, 10 mM DTT, 5 mM NaF, 1 mM Na 3 VO , 1 mM ⁇ -glycerophosphate, 2.5 mM EDTA, 400 ⁇ g/ml AEBSF [4-(2-aminoethyl)-enzensulfonyl fluoride], 1 ⁇ g/ml leupeptin and 1 ⁇ g/ml pepstatin.
  • AEBSF 4-(2-aminoethyl)-enzensulfonyl fluoride
  • the homogenate was centrifuged at 12,000g at 4°C for 30 min.
  • the supernatants may be used to purify Cdc2-like protein kinases using pl3 suc -conjugated agarose beads (Oncogene Sciences).
  • the required amount of supernatant 150 ⁇ g protein for each reaction was added to the beads and tumbled at 4°C for 2 h.
  • the beads may be washed twice in a washing buffer consisting of 50 mM Tris pH 7.4, 250 mM NaCl, 0.1% NP-40, 2.5 mM EDTA, 1 mM DTT and inhibitor cocktail of (in final concentrations) 10 ⁇ g/ml apotinin, 10 ⁇ g/ml antipain, 10 ⁇ g/ml soybean trypsin inhibitor, 10 mM ⁇ - glycerophosphate, 1 mM NaF and 0.2 mM Na 3 VO 4 .
  • a washing buffer consisting of 50 mM Tris pH 7.4, 250 mM NaCl, 0.1% NP-40, 2.5 mM EDTA, 1 mM DTT and inhibitor cocktail of (in final concentrations) 10 ⁇ g/ml apotinin, 10 ⁇ g/ml antipain, 10 ⁇ g/ml soybean trypsin inhibitor, 10 mM ⁇ - glycerophosphate, 1 mM NaF and 0.2
  • Beads may then be washed twice in the kinase assay buffer (50 mM Tris pH 7.4, 10 mM MgCl, 2 mM EGTA, 2 mM DTT and the inhibitor cocktail).
  • the recombinant protein was added to the reactions and incubated (tumbling slowly) for 1.5 h at 4°C.
  • the kinase reaction was initiated by adding 1 ⁇ g/ ⁇ l histone HI (Sigma), 25 ⁇ M ATP and 0.05 ⁇ Ci/ul 32 P- ⁇ -ATP (final concentrations), and stopped after 20 min incubation by adding the sample buffer. Denatured supernatant was resolved by SDS-PAGE.
  • the structure of the transgenic plants may be characterized by scanning electron microscopy (SEM) and light microscopy.
  • SEM scanning electron microscopy
  • plant tissue samples were taken from 35S-ICK1 and control plants grown under the same conditions.
  • SEM of tissues such as leaves and hypocotyls
  • the epoxy replica method Green and Linstead, 1990; Fowke et al., 1994
  • the impression moulds were prepared using dental impression material (GC Exaflex vinyl silicone). The moulds were then used to make replicas of original samples with epoxy cement.
  • the epoxy replicas and critical point-dried specimens were mounted on SEM stubs, coated with gold in an Edwards Sputter Coater (Model S150B) and then examined in a Philips 505 Scanning Electron Microscope.
  • samples leaf tissues
  • 1.5-2.5 ⁇ m sections were stained with toluidine blue before examination. Determination of organ size and cell size may be useful in various embodiments of the invention. In one example, the leaf size and cell size of Arabidopsis 35S-ICK1 transgenic and controls plants were analysed.
  • Leaves (number 5-8) of 30-day plants grown in a growth chamber were used, as leaves of younger plants (e.g. 15-day) may continue to expand. At this stage, both the 35S-ICK1 and control plants were flowering. Leaves were excised and scanned first on an Epson flat bed scanner (model 1200S) to determine the leaf size. Epoxy replicas of the adaxial surface were prepared and SEM photographs taken. Two non-marginal sectors from the widest part of each leaf were photographed and about twenty pavement cells of each photographed sector were chosen at random. Guard cells were excluded from measurements. The surface area of each cell was determined by Image J (http:/rsb.Mo.rim.gov/ij/docs/intro.html). Results from the same transformed lines were pooled for each leaf number. To determine the leaf size, the margins of scanned individual leaf images were traced and areas were determined using ImageJ.
  • In vitro binding assays may be useful in various aspects of the invention, for example to assay the interaction of a CDK inhibitor, or fragments of a CDK inhibitor, and a particular kinase, cyclin or protein that binds to the CDK inhibitor.
  • S-Met labeled ICKl protein may be expressed from a T7 promoter construct using an in vitro coupled rabbit reticulocyte transcription/translation system ('TNT', Promega).
  • Ni + -NTA beads Qiagen
  • NNN NETN buffer lacking EDTA
  • Equilibrated beads may be incubated with His 6 -CycD3 protein NTN buffer for 2 h followed by washing with 2 X 1 ml NTN buffer. Binding experiments may be carried out in NTN containing 10 ⁇ l beads, plus 5 ⁇ l S-Met labeled protein. The binding reaction was incubated at 10°C for 2 h, followed by washing with 3 X 0.5 ml NTN buffer. Washed beads may be eluted with 10 ⁇ l SDS-containing denaturing buffer at 100°C for 5 min. The bound 5 S-Met labeled proteins may be analyzed by SDS-PAGE and autoradiography. In the present invention, 'proteins that bind to a CDK inhibitor' include proteins identifiable by such assays.
  • Deletion constructs may be useful for domain mapping to determine the functional domains of a CDK inhibitor, cyclin or protein that binds to a CDK inhibitor.
  • N-terminal deletion constructs of ICKl were made using cDNAs with deletions of various lengths from the N-terminal end.
  • the C-terminal deletion constructs were prepared by PCR using Pfu DNA polymerase with sequence-specific primers and the resulting DNA fragments were cloned into the yeast two-hybrid activation-domain (AD) vector (Fields and Song, 1989).
  • the deletion clones may be verified by DNA sequencing.
  • the constructs may be used to transform a suitable yeast strain.
  • the yeast strain harbouring a deletion construct was further transformed with either Cdc2a or CycD3 cloned in a BD- (binding domain) vector.
  • Interactions in the yeast two-hybrid system may then, for example, be analyzed by X-gal filter assay (Chevray and Nathans, 1992) and by liquid culture assays for relative ⁇ - galactosidase activity (Reynolds and Lundlad, 1994). Three or more independent transformants may be used for each interaction.
  • Tissue-specific or inducible expression may for example be determined by activity of a reporter gene/protein, by northern hybridization or by in situ hybridization.
  • activity of the GUS reporter gene was analysed by histochemical staining following the method as described (Jefferson, 1987) using 5-bromo-4-chloro-3-indolyl glucuronidase (X-gluc) as the substrate. Fresh tissues were removed from plants.
  • Tissues were placed in a staining solution consisting of 1 mg ml "1 X-gluc, 100 mM PO (pH 7.0), 0.5 mM K 3 [Fe(CN) 6 ], 0.5 mM K4[Fe(CN) 6 ]- 10 mM Na 2 EDTA and 0.02% Triton X-100, and were infiltrated under partial vacuum (50-64 cm Hg). They were then incubated in the staining solution at 37°C. After the staining, tissues may be cleared with 70% ethanol.
  • the cyclin-dependent kinase inhibitors may be used to modify plant development.
  • Arabidopsis thaliana plants were used as hosts to express the plant cyclin- dependent kinase inhibitor ICKl.
  • Fifty independent transgenic 35S-ICK1 plants were analysed. Majority of transgenic lines expressing ICKl displayed modifications in plant growth and development. These plants were smaller in size. Most organs were smaller. The mo ⁇ hology of leaves and floral organs were also altered. These plants also showed reduced number of leaves and early flowering in comparison to control plants. These modifications of size and mo ⁇ hology in transgenic 35S-ICK1 plants made it easy to distinguish them from the wild type plants.
  • transgenic plants were obtained expressing CycD3 or CycD2 of Arabidopsis.
  • the homozygous 35S-ICK1 lines were crossed with 35S-CycD lines.
  • the FI plants from such crosses displayed characteristics similar to wild type plants rather than to the parental 35S-ICK1 plants. For instance, the FI plants were much bigger than 35S-ICK1 parental plants.
  • 35S-ICK1 parental plants there was strong serration in leaves while FI plants displayed leaf mo ⁇ hology as the wild type plants.
  • the FI plants also showed normal flower mo ⁇ hology rather than the modified flower mo ⁇ hology observed in the 35S-ICK1 plants. These results suggest that the expression of CycD3 or CycD2 had at least partially restore the modifications resulted from the expression of ICKl in these plants. In another example at least partial restoration may be achieved by the expression of a CDK.
  • Transgenic Arabidopsis plants expressing Cdc2a were obtained. The homozygous 35S-ICK1 lines were crossed with 35S-Cdc2a lines. The FI plants from such crosses were analysed similarly.
  • restoration of the phenotypic effects as a result of expressing a plant CDK inhibitor may be accomplished by expression of a protein that mediates specific degradation of the CDK inhibitor.
  • a protein that mediates specific degradation of the CDK inhibitor It is generally known that many cell cycle regulators are controlled by protein degradation and more specifically through ubiquitin-mediated proteolysis (King et al., 1996; Pagano et al., 1997). The ubiquitin- mediated proteolysis pathway is conserved in plants (Callis, 1997). Published results suggest that plant cell cycle regulators may also be degradated by the ubiqutin-proteasome pathway (Genschik et al., 1998).
  • ubiquitination involves sequential transfer of an activated ubiquitin to the protein through a cascade of three different classes of enzymes El, E2 and E3.
  • E3s also collectively known as ubiqutin ligase transfer ubiquitin to specific substrates.
  • at least partial restoration of the phenotypic effects due to expression of a CDK inhibitor may be restored by expressing in the plant an E3-like protein that interacts with and transfers the activated ubiquitin to the CDK inhibitor.
  • restoration of the phenotypic effects as a result of expressing a plant CDK inhibitor may be accomplished by expression of a recombinant antibody that specifically recognizes the CDK inhibitor or a recombinant peptide that specifically interacts with the CDK inhibitor.
  • Techniques are available for selecting such a recombinant antibody or peptide.
  • bacterial phage can be used to display recombinant antibodies (e.g. McCafferty et al., 1990; Winter et al., 1994; Gavilondo and Larrick, 2000) or recombinant peptides (e.g. Cwirla et al., 1990; Burritt et al., 1996).
  • the immunoglobulin variable (V) domain genes of both heavy (H) and light (L) chains which determine the functional structure of the antigen-binding site, can be amplified by PCR and reconstructed into large single-chain Fv antibody (scFv) libraries.
  • Single chain antibodies that specifically recognize a plant CDK inhibitor can be selected by affinity binding of filamentous phage that display the recombinant antibodies.
  • recombinant antibodies may be displayed on bacteria (Daugherty et al., 1998), yeast (Kieke et al., 1997) and ribosomes (Hanes et al., 1997).
  • Specific peptides that interact with a plant CDK inhibitor may be selected from recombinant peptide libraries (Burritt et al., 1996).
  • the DNA sequences encoding the recombinant antibodies or peptides may be expressed in plants (e.g. Hiatt et al, 1989; Tavladoraki et al., 1993; Artsaenko et al., 1995) to modulate the function of the target CDK inhibitor.
  • transgenic Nicotiana tobaccum plants were obtained with 35S-ICK1 construct. Twenty one independent transformants were analysed. The insert number was analysed by Southern hybridization. ICKl expression was anlaysed by northern hybridization. Results show that at least twelve plants had significant levels of ICKl expression. Some transgenic plants showed modified development including smaller plant size and smaller organs such as leaves.
  • the progeny plants may be further analysed for modifications. For instance, five tobacco 35S-ICK1 Tl lines were used to determine and verify modifications. The non-transformed tobacco plants and two Tl lines carrying 35S- GUS construct were used as controls. For each line, five progeny plants were grown in pots.
  • the length of three longest leaves on each plant was measured.
  • the average length of these three leaves for the Wt and two 35S-GUS Tl lines were 23.2, 23.8 and 23 J cm respectively.
  • the average leaf length for four 35S-ICK1 Tl lines was 17.5, 19.5, 14.6 and 12.1 cm respectively.
  • T2 plants of the Tl transgenic 35S-ICK1 lines were also shorter on average than the control plants when measured at the same time.
  • Plant mo ⁇ hology of the 35S-ICK1 tobacco plants was also modified. For instance, leaf may be mottled in color.
  • the expression of a CDK inhibitor and a protein that can bind to the CDK inhibitor can be achieved in a variety of plant species.
  • tobacco plants were transformed with 35S-CycD2 and 35S-CycD3 construsts. The insert number was analysed by Southern hybridization. Northern analysis using RNA isolated from leaf tissues showed that many of these plants had a significant level of cyclin expression while controls (non-transformed plants and plants carrying 35S-GUS construct) had no detected level of transcript.
  • Transgenic lines carrying 35S-CycD2 or 35S-CycD3 construct were crossed with homozygous 35S-ICK1 lines. FI seeds from these crosses were planted. At least partial restoration of plant development modifications as described above due to ICKl expression was observed.
  • transgenic Brassica napus plaints expressing ICKl were obtained.
  • the presence of heterologous transgene was verified by PCR and southern hybridization. Modifications of plant development were observed in the original (Tl) and subsequent generations of plants.
  • a variety of plant species may be transformed by techniques known in the art to express a CDK inhibitor, a cyclin or a protein that binds to a CDK inhibitor.
  • Plants e.g. T2 derived from the original transformants (Tl) may be studied to determine the segregation of the inserted gene and also to verify whether the particular phenotype is co-inherited with the inserted gene.
  • T2 seeds of four transgenic Arabidopsis 35S-ICK1 lines and seeds of control Arabidopsis plants were sterilized and placed onto the selective medium containing 50mg/L kanamycin. Four duplicate plates were used for each type of plant.
  • the number of resistant susceptible seedlings for four independent Arabidopsis 35S-ICK1 lines was 103/27, 89/29, 116/36 and 108/33, indicating that there was one insert in each of these transgenic lines.
  • Seedlings of non-transformed control were all susceptible while seedlings of a homozygous control transgenic line were all resistant.
  • seven transgenic tobacco lines were determined for segregation and five showed a segregation ratio close to 3 : 1.
  • Genomic DNA may be isolated and the copy number of the transgene may be determined by Southern hybridization. For instance, of the four transgenic Arabidopsis 35-ICK1 lines that showed 3:1 segregation, two had a single copy of the transgene, one had two copies and one had three copies.
  • the expression of a CDK inhibitor, cyclin or protein that binds to a CDK inhibitor in particular plant tissues may be assayed to determine, for example, whether that CDK inhibitor will have utility as a division or growth modulator when expressed in such tissues.
  • the expression of ICKl was analyzed in independent transgenic Arabidopsis 35S-ICK1 plants.
  • the ICKl expression level increased significantly in transgenic 35S-ICK1 Arabidopsis plants as shown by several independent analyses.
  • Increased ICKl expression was observed in original Tl transformants and was similarly observed in the progeny T2 plants, indicating that the increased level was due to transgene integration.
  • the increased expression was detected in different tissues analysed, as expected since the 35S promoter activates gene expression in most tissues. Expression of the CDK gene Cdc2a did not decrease and perhaps showed a slight increase.
  • a ubiquitin gene UBQ11 (Callis et al., 1993) remained more consistent.
  • the CDK activity may also be assayed.
  • the pi 3 uc -associated Cdc2-like histone HI kinase activity was analysed with the same source tissues that were used in gene expression analyses as described above. Results show that, coinciding with increased ICKl expression, the Cdc2-like kinase activity decreased significantly in comparison to control plants. This decrease was observed in independent transgenic 35S-ICK1 plants and also in different tissues. As there was no decrease in the level of expression for positive cell cycle regulators such as Cdc2a, it is concluded that the decreased Cdc2 kinase activity is directly due to inhibition by increased ICKl expression in these 35S-ICK1 plants.
  • CDK inhibitors may not inhibit CDK from mammalian and yeast cells (Wang et al., 1997).
  • recombinant ICKl is effective in vitro in inhibiting the histone HI kinase activity of pl3 suc -associated kinases from A. thaliana and heterologous Brassica napus (Wang et al., 1997; 1998)
  • CDK inhibitor-binding proteins for use in various aspects of the invention may be identified using a yeast two hybrid screening protocol with a variety of bait fusion protein sequences.
  • ICKl may be used as a bait to identify cyclin homologs or proteins that interact with ICKl .
  • Interactions in the yeast two-hybrid system may then, for example, be analyzed by X-gal filter assay (Chevray and Nathans, 1992) and by liquid culture assays for relative activity (Reynolds and Lundlad, 1994).
  • X-gal filter assay Chevray and Nathans, 1992
  • Li culture assays for relative activity Reynolds and Lundlad, 1994.
  • further in vitro binding assays may be conducted as described (Wang et al., 1998). Similar assays may be used to identify CDK inhibitors capable of interacting with other cellular targets.
  • the regions of a proteins that are functionally involved in interactions with other proteins in various aspects of the invention may be mapped by deletion mapping using a variety of techniques, such as the yeast two-hybrid system and variations thereof.
  • Such in vitro assay results may be verified by in vivo tests, since the persistence of interactions in the two-hybrid system may be affected by possible alterations in functionality of plant proteins expressed in yeast.
  • an in vitro assay to determine the functional significance of the C-terminal domain and other regions of ICKl, three N-terminal and three C-terminal deletion mutants were assessed for their interactions with Cdc2a and CycD3 in the two-hybrid system.
  • ⁇ -galactosidase activity for the interaction of ICKl with CycD3 resulted from the deletion of amino acids 109-153, whereas the deletion of amino acids 176-191 had a more detrimental effect on the interaction with Cdc2a.
  • the functional importance of a portion of a CDK inhibitor may also be assayed by analyzing transgenic plants expressing a modified version of the inhibitor. Deletion constructs of ICKl may be used to transform Arabidopsis plants. The changes in these transgenic plants expressing variants of ICKl may be compared with plants expressing an ICKl with unmodified functionality.
  • a series of deletions may also be made to map the regions in CycD3 that is responsible for interacting with ICKl and Cdc2a.
  • One aspect of the invention utilizes functionally important regions of a protein.
  • the functionally important regions of a CDK inhibitor, cyclin or protein that binds to a CDK inhibitor may be determined through routine assays. Randomly selected portions of a protein, such as a CDK inhibitor, may be selected for use in assays to determine whether the selected region is capable of functioning as required in the context of the present invention.
  • regions of ICKl may be used, such as the 109-153 region and/or the 163-191 region, with or without additional regions from ICKl or other CDK inhibitors, provided the recombinant protein meets the functional requirements of the present invention (which may be determined through routine screening of functionality) .
  • the modifications of plant development by expression of a CDK inhibitor, a cyclin or a protein that binds to a CDK inhibitor may involve modification of cell number and/or cell size.
  • analyses of the structure and cell size in 35S-ICK1 and control plants by scanning electron microscopy and light microscopy showed that the cells of 35S-ICK1 plants were on average larger than the corresponding cells in control plants in different tissues examined (leaves, hypocotyl, root and flower organs).
  • the leaf and cell size of35S-ICKl and control plants were quantified using fully expanded leaves (the 5 to 8 leaves) of 30-day plants.
  • the average leaf size of the 35S-ICK1 plants (lines) used was between 3.4% to 57.1% of the leaf size of the wild type plants. Pavement cells on the adaxial surface in similar areas of leaves were measured. Cells in leaves of different 35S-ICK1 lines were 1J-2J times larger than the cells of control plants.
  • Various aspects of the invention may be used to obtain a wide variety of phenotypic variations in plant mo ⁇ hology or other characteristics, which are at least partially reversible effects.
  • various promoters with tissue-specific expression patterns or with inducible expression may be used.
  • Arabidopsis plants were transformed with NTM19-ICK1, NTM19-CycD3 and NTM19-GUS constructs respectively.
  • the pattern of gene expression directed by the NTM19 promoter (Oldenhof et a., 1996) was confirmed by histochemical staining of the GUS reporter gene in transgenic NTM19-GUS plants.
  • These plants including NTM19-ICK1 plants developed normally before fertilization and silique (pod) development.
  • the transgenic plants with male sterility may set seeds after pollination, using pollen from non-transformed plants, indicating that the female reproduction system is unaffected in these male sterile plants. Apart from these specific modifications, these transgenic plants otherwise grew and developed normally.
  • transgenic B. napus and Arabidopsis plants were obtained with a chimeric gene construct consisting of the Bgpl promoter and ICKl.
  • the Bgpl promoter has previously been shown to direct strong exogenous GUS reporter gene expression in pollen of Arabidopsis and Nicotiana tobacum plants (Xu et al., 1993).
  • Some showed reduced seed setting with four transformants showing a greater reduction.
  • the degree of reduction varied from an amount of about half the seed-setting in normal plants (in term of number of seeds per pods) to nearly complete sterility. All of over twenty transgenic Arabidopsis plants were normal and showed no significant reduction in seed-setting.
  • transgenic Brassica napus plants were obtained with AP3-ICK1 construct. Some of the plants showed much reduced petal size and significant reduction in seed-setting, with one plant showing almost complete sterility.
  • the transgenic Brassica phenotypes were consistent with the pattern of AP3 promoter-directed gene expression, i.e. stronger expression in petal and stamen primordia and possibly low levels of expression in the inner integument or ovule (Day et al., 1995). Of fifty-two primary transformants, some transformants showed changes in petal mo ⁇ hology and development with four transformants displaying significant alterations. Plants showing a strong phenotype in modification of petal mo ⁇ hology also had reduced seed-setting.
  • RNA samples were isolated from the leaf and mature anthers of a transgenic plant showing sterility phenotype and the control B. napus (Westar) plant. For each sample, 15 ⁇ g of RNA was loaded and separated by electrophoresis. RNA transfer and hybridization were performed as described.
  • ICKl expression was analysed in plants transformed by AP3-ICK1 construct. RNA samples were isolated from the leaf, sepal, petal, anther and whole young flower of the transgenic plant and the control plant. The highest level of ICKl expression was shown to be in the petals of the transgenic plants. There was no detectable signal under the conditions used for the tissues from the control plant.
  • a variety of plant CDK inhibitors may be used in the invention. They include ICKl , ICK2, ICN2, ICN4, ICN6, ICN7, ICN8 and ICDK.
  • ICKl ICK2, ICN2, ICN4, ICN6, ICN7, ICN8 and ICDK.
  • ICDK a putative CDK inhibitor gene ICDK (AJ002173, SEQ ID No. 15 and SEQ ID No.16) was identified from Chenopodium rubrum (Fountain et al., 1999), as it shares some similar properties as ICKl and thus with ICK2, ICN2, ICN6 and ICN7 as well (Table 1). C. rubrum seeds were collected in Saskatchewan, Canada. RNA was isolated from seedlings and leaves. The full-length coding region of ICDK cDNA was cloned using RNA RT-PCR.
  • the sequence data showed that the cloned cDNA was identical to ICDK of C. rubrum in the database.
  • a construct consisting of the 35S promoter and ICDK was prepared.
  • the Agrobacterium strain harboring the 35S-ICDK was used to transform Arabidopsis. Selection for transformants was performed as described elsewhere herein.
  • thirty eight (38) independent transgenic 35S-ICDK plants twelve (12) showed serrated leaves and twenty two (22) showed modified flowers, which were observed in 35S-ICK1 plants. Expression of ICDK in these plants was confirmed by northern analysis.
  • a construct consisting of 35S promoter and ICN2 was prepared and used to transform Arabidopsis plants.
  • CDK inhibitors and CDK inhibitor genes sharing functional and sequence similarity with ICKl may be identified using an approach similar to the approach used to isolate ICKl, based for example on their interactions with either Arabidospis Cdc2a or a D-class cyclin (e.g. cyclin D3 or cyclin D2).
  • a D-class cyclin e.g. cyclin D3 or cyclin D2.
  • the sequences of ICK2 (SEQ ID NO: 6), ICN2 (SEQ ID NO: 7), ICN6 (SEQ ID NO: 8), and ICN7 (SEQ ID NO: 9) are shown in Figs 2 through 6. Additional CDK inhibitors may identified by other techniques known in the art.
  • BAC clone F24L7 (GenBank Accession AC003974) encodes a putative protein that shares some similarity with the ICK/ICN proteins.
  • a corresponding cDNA clone to this segment of genomic DNA was identified and sequenced.
  • the protein encoded by this clone interacted with CycD3 as the ICN proteins do by yeast two-hybrid assay and thus it is designated as ICN8.
  • ICN8 cDNA sequence is given in Figure 7.
  • a BAC clone from Oryza sativa contains a segment that encodes a protein (GenBank AAG16867) sharing similarity with ICK/ICN proteins.
  • partial cDNA sequences may be identified first. Full-length cDNA sequences can then be identified using routine techniques known to the art. For instance, partial cDNAs can be used as probes to screen cDNA libraries to obtain full-length cDNA sequences. If the 5'-part or 3'-part of the full cDNA is missing, particular techniques of polymerase chain reaction such as 5' RACE and 3' RACE methods can be used to identify the sequences. Alternatively, other techniques can also be used to identify full-length cDNA sequences for instance by searching sequences databases using the partial cDNA sequences. Originally, for example, the cDNA sequences for ICK2 and ICN6 were partial sequences. Subsequently, full-length cDNA sequences were identified.
  • genes share at least two functional properties with ICKl: First, all of these genes encode proteins able to interact with either Cdc2a or a D-class cyclin or both. Such interactions may enable them to regulate the activity of plant CDKs in alternative embodiments of the invention. Second, these ICK/ICN proteins all share some sequence similarity in the region of ICKl that is functionally important in some embodiments for its interaction with Cdc2a and cyclin D3 (discussed above). These homologous genes or proteins may be used in some embodiments, in a manner similar to ICKl, to modulate plant growth and development. One or more such genes or proteins may be used in some embodiments alone or in combination to provide temporal and spatial regulation of cell cycle initiation and progressing during plant development in accordance with this invention.
  • an assay is provided to determine if a CDK inhibitor interacts with a known protein, which are thereby identified as proteins that bind to the CDK inhibitor.
  • a CDK inhibitor interacts with a known protein, which are thereby identified as proteins that bind to the CDK inhibitor.
  • the full-length cDNA of the gene to be analyzed may be cloned in a GAL4-binding domain vector using PCR and gene specific primers with flanking restriction sites.
  • Such constructs may be used to transform the yeast carrying the CDK inhibitor of interest, such as ICKl in a GAL4-activation domain vector.
  • the interactions of ICKl with a number of cell cycle-related genes from A. thaliana were examined in accordance with the invention (Table 2).
  • yeast two-hybrid assay results indicate that in particular embodiments of the invention, ICKl protein may interact with Cdc2a but not with Cdc2b. Similarly, ICKl may interact with D-type cyclins, CycDl, CycD2 and CycD3, while not interacting with A/B- class mitotic cyclins, CycA2, CycBl and CycB2 (Table 2).
  • the yeast two-hybrid assay results also indicate that ICKl may not interact in some embodiments with PCNA, also a cell cycle protein, and ATMAP2, a kinase sharing some similarity with Cdc2 kinase. Table 2. Analyses of ICKl interactions with other proteins in the yeast two-hybrid system
  • Cdc2a and Cdc2b The interactions of additional plant CDK inhibitors can also be determined using similar methods. For instance, the interactions of ICKl, ICK2, ICN2, ICN6, ICN7, ICN8 and ICDK with Arabidopsis CDKs (Cdc2a and Cdc2b) and the D-type cyclins were analysed using the yeast two-hybrid system. As summarized in Table 3, CKS1 At (De Veylder et al. 1997), one of the controls used, was able to interact with Arabidopsis Cdc2a and Cdc2b.
  • yeast strain MaV203
  • AD constructs listed in the first row were transformed first with AD constructs listed in the first row, and then with BD constructs listed in the first column of the table following standard protocol.
  • Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1 and for two alternative cyclin D expression constructs designated 35S-CycD2 and 35S-CycD3 (for expression respectively of cyclin D2 and cyclin D3).
  • the ICKl cDNA was linked to the CaMV 35S promoter in a vector (pBI121) for plant expression. Transformation of Arabidopsis was performed based on the infiltration method (Bechtold et al., 1993) except the surfactant Silwet-40 (0.01-0.05%) (Clough et al, 1998) was added to the final suspension for infiltration. Seeds (Tl) were harvested and selected on Vi MS basal medium (Sigma) containing 50 mg/1 kanamycin and 300 mg/1 Timentin. Transformants were transferred to soil and grown in growth chambers.
  • Table 4 shows growth and development of transgenic 35S-ICK1 Arabidopsis plants.
  • 35S-ICK1 and control plants were grown in growth chamber.
  • shoot above-ground tissues
  • fresh weight 21 -day plants was determined.
  • flowering time and leaf number (rosette plus inflorescence leaves on the primary axis) were obtained.
  • SD standard deviation. ** indicates significance (t-test) from the Wt control plants at PO.001 level and * indicates significance at PO.05 level.
  • the 35S-ICK1 plants showed profound changes in mo ⁇ hology of organs such as leaves. Depending on transgenic lines, there was a range of changes in leaf shape, in addition to a reduction in size. In some lines, leaves were significantly serrated. In wild type plants, only slight serration occurred in adult leaves. The expression of ICKl resulted in much more prominent serration of the leaves and this characteristic was observed in almost all leaves in 35S-ICK1 plants with strong phenotype of growth inhibition. The smaller leaves and shorter leaf petioles gave 35S-ICK1 plants a more compact appearance. Root growth of 35S-ICK1 plants was similarly affected.
  • the ICKl expression level increased significantly in transgenic 35S-ICK1 Arabidopsis plants as shown from several independent northern analyses. Increased ICKl expression was observed in original Tl transformants and was similarly observed in the progeny T2 plants, indicating that the increased level was due to transgene integration. The increased expression was detected in tissues analysed including shoots, roots, leaves, stems and flowers, as expected since the 35S promoter activates gene expression in most tissues.
  • the pl3 -associated Cdc2-like histone HI kinase activity was analysed with the same source tissues that were used in gene expression analyses. Results show that, coinciding with increased ICKl expression, the Cdc2-like kinase activity decreased significantly in comparison to control plants. This decrease was observed in independent 35S-ICK1 tranformants and different tissues. Results also show that there was no decrease in the expression level of positive cell cycle regulators such as Cdc2a. It is concluded that the decreased Cdc2 kinase activity is directly due to inhibition by increased ICKl expression in these 35S-ICK1 plants. Cell number and cell size were affected
  • 35S-ICK1 and control plants were examined by scanning electron microscopy and light microscopy. It was consistently observed that the cells of 35S-ICK1 plants in all tissues examined (leaves, hypocotyl, root and flower organs) were on average slightly larger than the corresponding cells in control plants.
  • Chenopodium rubrum CDK inhibitor gene ICDK (ICKCr) were introduced into Arabidopsis plants and the transformed plants were characterized, using the 35S-ICK1 transformants as comparisons for determining the effects of other plant CDK inhibitor genes.
  • RNA samples were prepared from transgenic and control plants. Northern analyses showed that specific transgenes were overexpressed respectively in the plants transformed with 35S-ICK1, 35S-ICN2 and 35S-ICDK constructs compared to the controls. There was a low background level of ICKl and ICN2 in wild type Arabidopsis plants but there was no signal for ICDK in Arabidopsis plants except those transformed with 35S-ICDK.
  • the 35-ICDK or 35S-ICN2 plants with phenotypic changes were smaller in size than the wild type plants and 35S-GUS plants.
  • 35S-ICK1 transformants described above the major changes in mo ⁇ hology for plants carrying 35S-ICN2 and 35S-ICDK included leaf serration and modified flowers. Transformed plants showing these phenotypes flowered earlier than control plants.
  • Mature rosette leaves from four-week wild type and transgenic T3 plants were analysed for DNA content (ploidy level) of isolated nuclei by flow cytometry.
  • Wild type Arabidopsis leaf tissue showed a similar profile of nuclear DNA content to that described by Galbraith et al (1991) with four major peaks at 2C, 4C, 8C and 16C levels and a minor peak at the 32C level occasionally.
  • decreased ploidy level was observed in transgenic lines expressing one of the plant CDK inhibitors. The extent of decrease varied with different transgenic lines. Quantitative analyses were performed using transgenic 35S-ICK1, 35S-ICN2 and 35S-ICDK lines with a strong phenotype for the respective construct as well as controls.
  • DNA content was determined using nuclei isolated from comparable leaves of wild type and transgenic plants expressing plant CDK inhibitors as described. Each datum for a particular DNA content peak represents the average of 5-8 individual plants measured and is expressed relatively in percentage with a total value for all peaks to be 100%.
  • Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1, for two alternative cyclin D expression constructs designated 35S-CycD2 and 35S-CycD3 (for expressing respectively of Arabidopsis cyclin D2;l and cyclin D3;l), and for a CDK expression construct designated 35S-Cdc2a (for expressing Arabidopsis Cdc2a).
  • Crosses were made between 35S-ICK1 lines, expressing the CDK inhibitor ICKl, and the cyclin expressing lines.
  • FI plants were analyzed, demonstrating that cyclin D2 (CycD2) and cyclin D3 (CycD3) can reverse the inhibition of cell division and plant growth that is otherwise mediated by ICKl over-expression.
  • Transgenic Arabidopsis plants were obtained as described. Crosses were made between homozygous 35S-ICK1 (200-13 female parent) and the rest of plant lines (male parent).
  • Plant growth was anayzed in Petri plates.
  • FI and transformed parent seeds were sterilized and plated on medium (1/2 MS, 1% sucrose, pH5.6, 0.8% agar) containing 300 mg/L timentin and 50mg/L kanamycin, and Wt seeds were plated on the same medium but without kanamycin. After 14 days, the fresh weight of seedlings was determined. Results
  • Table 7 Fresh weight of FI Arabidopsis plants from crosses between 35S-ICK1 female arent line and C cD male arent lines 3
  • Type 1 refers to the plants with the phenotype of 200-13-1 which have changed leaf shape compared with wild type, such as narrowed and serrated leaves at the investigation time (see example 1).
  • Type 2 refers to the plants with wild type mo ⁇ hology although their size may vary. In general type 2 plants are larger than type 1 plants.
  • Wt seedlings were grown on medium without kanamycin. As shown in Table 7, there were two distinct phenotypes in crosses involving CycD3 expressing male parent lines: i.e. 200-13-1 X 294-2 and 200-13-1 X 297-18. The two phenotypes were distinguishable by both mo ⁇ hology and seedling size even before weighing. The two types had a segregation ratio close to 1:1.
  • transgenic Arabidopsis plants were obtained expressing 35S-Cdc2a (Cdc2a from Arabidopsis). Homozygous lines were selected. Crosses were made between the homozygous 35S-ICK1 and 35S-Cdc2a lines. The FI plants were analysed as above. The data indicate that the modified plant phenotype due to ICKl expression is at least partially restored by the expression of Cdc2a.
  • the 35S-ICK1 line was also crossed with a transgenic line transformed with 35S-GUS and a transgenic line transformed with 35S-antisense-ICKl .
  • the FI plants from the cross [35S-ICK1 X 35S-GUS] displayed the typical phenotype of ICKl overexpression i.e. smaller plants, serrated leaves and modified flowers.
  • the FI plants from the cross [35S-ICK1 X 35S-antisense-ICKl] were indistinguishable from the Wt plants.
  • the invention facilitateaties modulation of the effects of the CDK inhibitor to different extents. Differing extents of modulation may for example be accomplished by various levels of expression of the gene encoding the modulatory protein.
  • the plant CDK inhibitor and the modulatory protein may be regulated differently (for instance by using different promoters), and the their transgenic expression may be optimized (for instance by selecting transgenic lines) so that desired effects on growth may be achieved for whole plants or for certain plant organs.
  • Transgenic Arabidopsis plants overexpressing ICKl that displayed a much smaller plant size had larger cells than Wt plants (Example #1). The cell size was thus examined in
  • the FI plants from the crosses [35S-ICK1 X Wt] and [35S-ICK1 X 35S-GUS] had larger cells than control plants, as observed for ICKl -overexpressing plants.
  • the FI plants from the cross [35S-ICK1 X 35S-antisense-ICKl] had leaf cells similar in size to control plants.
  • the FI plants from the cross [35S-ICK1 X 35S-CycD3] had smaller cells than the control plants, despite the fact that these FI plants were smaller than control plants.
  • the SEM results of leaf epidermal cells and light microscopy results of leaf transverse sections are consistent with each other. The present results show that expression of CycD3 in these FI plants had modified the cell size in comparison to Wt plants and plants expressing ICKl alone.
  • a plant CDK inhibitor such as ICKl inhibited endoreduplication and thus decreased the ploidy level in Arabidopsis leaves, which normally have cells of mixed polyploidy. Therefore the ploidy level in the FI plants was determined using comparable leaf tissues (leaves no. 7 and 8; see Example #1) from plants grown under identical conditions. As shown in Table 8, the FI plants from the cross [35S-ICK1 X 35S- antisense-ICKl] has similar ploidy profile as the Wt plants with four peaks of nuclear contents at 2C, 4C and 8C peaks and 16C.
  • the FI plants from the crosses [35S-ICK1 X Wt] and [35S-ICK1 X 35S-GUS showed a significant reduction in ploidy level with essentially two peaks and mostly at 2C level.
  • the FI plants from the crosses [35S-ICK1 X 35S-CycD2] and [35S-ICK1 X 35S-CycD3] had a ploidy profile between the [35S-ICK1 X Wt] type and the [35S-ICK1 X 35S-antisense-ICKl] type. They had major 2C and 4C peaks with a minor 8C peak.
  • the data on ploidy level were consistent with the phenotype of these plants, indicating that the effect on endoreduplication from ICKl overexpression was partially reversed by the expression of Arabidopsis CycD3 or CycD2.
  • DNA content was determined using nuclei isolated from the control and FI plants containing corresponding genes as indicated. An average for each peak was obtained from 5-6 individual plants measured and expressed relatively in percentage with a total value for all peaks to be 100%). The data presented are means plus standard deviation.
  • gene expression was determined in FI plants. High levels of ICKl expression were observed in FI plants from crosses of
  • Transgenic Nicotiana tobacum plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1 and for two alternative cyclin D expression constructs designated 35S-D2 and 35S-D3 (for expressing respectively of Arabidopsis cyclin D2;l and cyclin D3;l). Phenotypic effects were determined in these transgenic plants expressing one of these genes. These analyses were performed for the pu ⁇ ose of determining the effects of interaction in the plant of a CDK inhibitor and a protein that binds to the inhibitor.
  • a number of independent tobacco transformants were obtained harboring either of 35S-ICK1, 35S-CycD2 and 35S-CycD3 constructs.
  • Gene expression in these transgenic plants was analysed by northern hybridization.
  • the transgenic 35S-ICK1 tobacco plants shown several major changes. The plants were smaller in comparison to same-aged control plants. They had smaller leaves and shorter internodes. Thus, they appeared stockier that control plants.
  • the Cy ⁇ D-expressing transgenic plants displayed a ranged of other modifications. These modifications were similar in types between CycD2 and CycD3- expressing plant, but were different from the changes observed in 35S-ICK1 plants.
  • the main changes include faster growth, taller plants and bigger leaves. The leaves curled downward, and stems were curled or twisted. These modifications were observed in the first and subsequent generations of transgenic plants.
  • the leaf length for three longest leaves of individual T2 plants (10 weeks) was measured. Five to six plants were per line of plant were used for the measurement. The mean value and standard deviation for each line of plant are given as below. Table 9: Average leaf length (cm) of three longest leaves of T2 plants (10 weeks). For each line, 5-6 plants were used for measurement.
  • T3 plants of homozygous T2 35S-ICK1 tobacco lines were grown in soil in greenhouse.
  • Transgenic 35S-ICK1 plants were much smaller. For instance, after 45 days, individual plants were removed from pots and the fresh weight of the plant (above ground) was determined. Typically, 6-8 plants were used for each type and the data are summarized below in Table 10.
  • Plant type Tl lines Average ⁇ SD
  • Cellular structure and cell size can also be analysed as described in Example #1. For instance, tobacco plants of Wt, 35S-GUS and 35S-ICK1 lines were grown. Leaf samples were taken from comparable mature leaves of 14- week plants. The samples were fixed, dehydrated and embedded as described. Examination by light microscopy and electron microscopy revealed that cells in 35S-ICK1 plants were larger, as observed in Arabidopsis plants overexpressing ICKl. The results show that cells were larger in 35S-ICK1 tobacco plants than control plants. This effect on cell size in tobacco plants from expression of a plant CDK inhibitor was similar to what observed in Arabidopsis plants. EXAMPLE 4
  • Transgenic Nicotiana tobacum plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1 and for two alternative cyclin D expression constructs designated 35S-D2 and 35S-D3 (for expression respectively of cyclin D2 and cyclin D3).
  • Crosses were made between 35S-ICK1 lines, expressing the CDK inhibitor ICKl, and the cyclin expressing lines.
  • FI plants were analyzed, demonstrating that cyclin D2 (CycD2) and cyclin D3 (CycD3) can reverse the inhibition of cell division and plant growth that is otherwise mediated by ICKl over-expression.
  • FI plants from crosses between 35S-ICK1 and Wt lines showed consistently the phenotype similar to the 35S-ICK1 parent. For example, they had smaller leaves, and shorter in height. Overall, they were smaller than Wt plants.
  • FI plants from crosses between 35S-ICK1 and 35S-CycD lines showed significant restoration of the modified phenotype that is observed the 35S-ICK1 plants. For instance they were taller and had larger leaves than 35S-ICK1 plants.
  • some FI plants were not entirely similar to CycD parents but displayed intermediate characteristics between ICKl and CycD parents. Crosses were made between a homozygous 35S-ICK1 line and a heterozygous 35S-CycD3 line.
  • EXAMPLE 5 Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression during early stages of microspore development using construct designated NTM19-ICK1.
  • the promoter NTM19 from tobacco has been shown to activate gene expression at early stages of microspore development (Custers et al., 1997; Oldenhof et al., 1996).
  • the NTM19 promoter was cloned from tabacco genomic DNA using PCR with primers sequence-specific to the NTM19 promoter (Oldenhof et al., 1996).
  • the ICKl cDNA was linked to the NTM19 promoter in a vector for plant expression.
  • NTM19-GUS, and NTM19-CycD3 (from Arabidopsis) constructs were also prepared. Transformation of Arabidopsis plants was as described.
  • the specificity of NTM19 promoter-directed gene expression was verified using NTM19-GUS plants. GUS activity was detected histochemically using 5-bromo-4-chloro-3-indolyl glucuronidase (X-gluc) as substrate following the method as described (Jefferson et al., 1987).
  • NTM19-GUS expressing plants confirmed that NTM19 activated gene expression during early stage of microspore development (Custers et al., 1997; Oldenhof et al., 1996).
  • SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box.
  • Floral dip a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743
  • Plant Cyclins a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization. Plant Mol Biol 32: 1003-1018
  • a Brassica S- locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana. Dev Biol 143: 173-184

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Abstract

Selon divers aspects, l'invention concerne des procédés qui permettent de modifier la croissance végétale par le biais d'inhibiteurs de CDK, de cyclines et de protéines assurant la liaison avec les inhibiteurs de ce type de kinase, y compris des procédés relatifs à la modification génétique faisant appel à des acides nucléiques qui codent les protéines considérées. Selon d'autres aspects, l'invention concerne différents procédés de sélection des plantes qui facilitent la modulation du phénotype végétal dans les générations alternatives. L'invention concerne également des plantes produites selon les procédés décrits, y compris les plantes transgéniques à génomes de recombinaison qui englobent des séquences hétérologues codant les inhibiteurs de CDK, des cyclines, et des protéines assurant la liaison avec les inhibiteurs de CDK.
PCT/CA2001/001825 1998-06-08 2001-12-18 Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes WO2002050292A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002433048A CA2433048A1 (fr) 2000-12-18 2001-12-18 Modulation d'activite d'inhibiteur de kinase dependante des cyclines (cdk) dans les plantes
AU2002215793A AU2002215793A1 (en) 2000-12-18 2001-12-18 Modulation of plant cyclin-dependent kinase inhibitor activity
US10/451,139 US20040098763A1 (en) 1998-06-08 2001-12-18 Modulation of plant cyclin-dependent kinase inhibitor activity

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/733,507 US7078591B2 (en) 1998-06-08 2000-12-08 Cyclin-dependent kinase inhibitors as plant growth regulators
US25590800P 2000-12-18 2000-12-18
US60/255,908 2000-12-18

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WO2002050292A2 true WO2002050292A2 (fr) 2002-06-27
WO2002050292A3 WO2002050292A3 (fr) 2002-09-19

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005085452A1 (fr) * 2004-03-10 2005-09-15 Cropdesign N.V. Plantes presentant un rendement ameliore et procede d'elaboration correspondant
JP2009502175A (ja) * 2005-07-29 2009-01-29 ターゲット・グロース・インコーポレーテッド 野生型ｋｒｐによる活性サイクリン−ｃｄｋ複合体阻害の、優性ネガティブ変異ｋｒｐタンパク質の防御
CN112011547A (zh) * 2020-07-23 2020-12-01 华中农业大学 一种控制油菜叶形的主效基因及其应用

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WO1999014331A2 (fr) * 1997-09-16 1999-03-25 Cropdesign Nv Inhibiteurs de la kinase cycline-dependante et utilisations de ceux-ci
WO1999064599A1 (fr) * 1998-06-08 1999-12-16 Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Inhibiteurs de la kinase dependant de la cycline, utilises comme regulateur de croissance des plantes
WO2000056905A2 (fr) * 1999-03-19 2000-09-28 Cropdesign N.V. Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture

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WO1999014331A2 (fr) * 1997-09-16 1999-03-25 Cropdesign Nv Inhibiteurs de la kinase cycline-dependante et utilisations de ceux-ci
WO1999064599A1 (fr) * 1998-06-08 1999-12-16 Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Inhibiteurs de la kinase dependant de la cycline, utilises comme regulateur de croissance des plantes
WO2000056905A2 (fr) * 1999-03-19 2000-09-28 Cropdesign N.V. Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture

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COCKCROFT C E ET AL: "CYCLIN D CONTROL OF GROWTH RATE IN PLANTS" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 405, no. 6786, 2000, pages 575-579, XP000938947 ISSN: 0028-0836 *
DEN BOER BART G W ET AL: "Control of plant growth and development through manipulation of cell-cycle genes." CURRENT OPINION IN BIOTECHNOLOGY, vol. 11, no. 2, April 2000 (2000-04), pages 138-145, XP002199592 ISSN: 0958-1669 *
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, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 10, no. 12, December 1998 (1998-12), pages 2063-2075, XP002146545 ISSN: 1040-4651 *
MIRONOV VLADIMIR ET AL: "Cyclin-dependent kinases and cell division in plants: The Nexus." PLANT CELL, vol. 11, no. 4, April 1999 (1999-04), pages 509-521, XP002126400 ISSN: 1040-4651 *
RIOU-KHAMLICHI C ET AL: "Cytokinin activation of Arabidopsis cell division through a D-type cyclin" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 283, no. 5407, 5 March 1999 (1999-03-05), pages 1541-1544, XP002134537 ISSN: 0036-8075 *
WANG ET AL: "ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid" PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 15, no. 4, 1 August 1998 (1998-08-01), pages 501-510, XP002095094 ISSN: 0960-7412 *
WANG HONG ET AL: "Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology." PLANT JOURNAL, vol. 24, no. 5, December 2000 (2000-12), pages 613-623, XP002199593 ISSN: 0960-7412 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005085452A1 (fr) * 2004-03-10 2005-09-15 Cropdesign N.V. Plantes presentant un rendement ameliore et procede d'elaboration correspondant
US7872172B2 (en) 2004-03-10 2011-01-18 Cropdesign N.V. Plants having improved yield and method for making the same
JP2009502175A (ja) * 2005-07-29 2009-01-29 ターゲット・グロース・インコーポレーテッド 野生型ｋｒｐによる活性サイクリン−ｃｄｋ複合体阻害の、優性ネガティブ変異ｋｒｐタンパク質の防御
CN112011547A (zh) * 2020-07-23 2020-12-01 华中农业大学 一种控制油菜叶形的主效基因及其应用
CN112011547B (zh) * 2020-07-23 2022-04-15 华中农业大学 一种控制油菜叶形的主效基因及其应用

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