EP1161541A1 - Procede de modification de la morphologie, biochimie ou physiologie de plantes, a l'aide de substrats comprenant cdc25 - Google Patents

Procede de modification de la morphologie, biochimie ou physiologie de plantes, a l'aide de substrats comprenant cdc25

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Publication number
EP1161541A1
EP1161541A1 EP00906072A EP00906072A EP1161541A1 EP 1161541 A1 EP1161541 A1 EP 1161541A1 EP 00906072 A EP00906072 A EP 00906072A EP 00906072 A EP00906072 A EP 00906072A EP 1161541 A1 EP1161541 A1 EP 1161541A1
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Prior art keywords
promoter sequence
plant
enhanced
gene
modified
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EP00906072A
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German (de)
English (en)
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EP1161541A4 (fr
Inventor
Peter Crook Lloyd John
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CropDesign NV
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CropDesign NV
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Priority claimed from PCT/AU2000/000135 external-priority patent/WO2000052172A1/fr
Publication of EP1161541A1 publication Critical patent/EP1161541A1/fr
Publication of EP1161541A4 publication Critical patent/EP1161541A4/fr
Withdrawn legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/8266—Abscission; Dehiscence; Senescence
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    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Definitions

  • the present invention relates generally to a method of modifying plant morphological, 5 biochemical and physiological properties or characteristics, such as one or more environmental adaptive responses and/or developmental processes, said method comprising introducing into a plant cell, tissue or organ a gene capable of expressing a cell cycle control protein, in particular a cyclin-dependent kinase (CDK), such as, for example a substrate of Cdc25 or a modified substrate of Cdc25, and, in particular
  • CDK cyclin-dependent kinase
  • the present invention extends to gene constructs which are useful for performing the inventive method and to transgenic plants produced therewith having altered morphological and/or biochemical and/or physiological properties compared to their otherwise isogenic counterparts.
  • cytokinins produces a variety of morphological, biochemical and physiological effects in plants, including the stimulation of organogenesis, shoot initiation from callus cultures, release of lateral buds from apical dominance, dwarf growth, alteration of source/sink relationships, stimulation of pigment synthesis, inhibition of root growth, and delay of senescence.
  • cytokinins have also been implicated in promoting the initiation of tuber formation in potato (International Patent Publication No. WO 93/07272) and in improving the resistance of potato plants to insects (United States Patent No. 5, 496, 732) and in inducing male sterility and partial female sterility in tobacco plants (European Patent No. EP-A-334, 383).
  • cytokinin on plant development and morphology may be attributed, at least in part, to modified biochemistry of the plant, such as a modification to the source/sink relationship in the plant or plant part.
  • Attempts to modify plant cytokinin-mediated and/or gibberellin-mediated growth and developmental responses employ the exogenous application of cytokinin and/or gibberellins respectively.
  • Such approaches are costly and produce undesirable pleiotropic side-effects on the plant tissue.
  • Modified plant growth or architecture has been shown to be effected by modifying expression of the G1 cyclin D3 (International Patent Publication No. WO98/42851).
  • G1 cyclins are required for the induction DNA replication (S phase), and the induction of G1 cyclin expression is dependent on mitogenic signals such as, for example, cytokinin and sucrose (Riou-Khamlichi et al. 1999, Soni et al. 1995).
  • mitogenic signals such as, for example, cytokinin and sucrose (Riou-Khamlichi et al. 1999, Soni et al. 1995).
  • prior art methods for modifying plant growth and architecture using these cyclin-encoding genes include pleiotropic effects.
  • the cell cycle is central to the process of growth and cell division in all multicellular organisms.
  • the four phases of the cell cycle in eukaryotic cells (S, G2, M and G1) are driven by a common class of heterodimeric serine/threonine protein kinases.
  • These kinases consist of one catalytic subunit (cyclin dependent kinases or CDKs) and one activating subunit (cyclins).
  • CDKs cyclin dependent kinases
  • cyclins activating subunit
  • a large number of putative cell cycle genes have been cloned in plants over the last 7 years.
  • a most recent count identified 17 types of cyclins and 5 types of CDKs in the model plant Arabidopsis thaliana. Identifying which of these genes are involved in the cell cycle is a major challenge to plant cell cycle work.
  • Substrates of Cdc25 including the A-type CDK, CDC2a, are homologues of the fission yeast p34 Cdc2/CDC28 protein kinases. These polypeptides are characterised by a PSTAIRE motif in the N-terminal protein sequence, which is considered to be essential for cyclin binding (Ducommun et al, 1991).
  • the switch that raises activity of Cdc2a at entry into mitosis is the Cdc25 catalysed removal of phosphate from threonine-14 and/or tyrosine-15 in Cdc2a.
  • Control of CDK activity can be achieved by cyclin association and phosphorylation.
  • the phosphorylation of CDK can either have an inhibitory effect of an activating effect on its activity depending on the position of the phosphorylation site.
  • the p34 Cdc2 protein is regulated by an activating phosphorylation during the transition from quiescence to active cycling at threonine-167 and by inhibitory phosphorylations at tyrosine-15 and/or threonine-14 during G2 (Jacobs, 1995).
  • Cdc2 CDK
  • Cdc25-catalysed removal of phosphate from threonine-14 and/or tyrosine-15 in Cdc2 occurs only once in the cell cycle, at the G2/M phase transition.
  • CDKs animal and plant cells
  • Cdc25 proteins animal cells
  • CDK2 the molecular switch of Cdc25-catalysed removal of phosphate from theonine-14 and/or tyrosine-15 in Cdc2 is also used at entry into S phase, however a separate CDK (CDK2) and a separate Cdc25 (Cdc25a) perform this function.
  • CDK2 the molecular switch of Cdc25-catalysed removal of phosphate from theonine-14 and/or tyrosine-15 in Cdc2 is also used at entry into S phase, however a separate CDK (CDK2) and a separate Cdc25 (Cdc25a) perform this function.
  • CDK2 the molecular switch of Cdc25-catalysed removal of phosphate from theonine-14 and/or tyrosine-15 in Cdc2 is also used at entry into S phase, however a separate CDK (CDK2) and a separate Cdc25 (Cdc25a) perform this function.
  • CDK2 the molecular switch
  • the present inventors sought to develop an improved method of increasing plant growth and vigour by modifying cytokinin- mediated and gibberellin-mediated plant growth and development.
  • a genetic sequence encoding a cyclin dependent kinase (CDK) protein or a modified form thereof into plant cells, tissues or organs in particular, a nucleotide sequence encoding a substrate of Cdc25 or a modified substrate of Cdc25, operably under the control of a plant expressible promoter sequence such as, for example, a cell-specific promoter, tissue-specific promoter, inducible promoter sequence, organ-specific promoter sequence, or a cell cycle specific gene promoter sequence, one or more cytokinin-mediated and/or gibberellin-mediated characteristics are modified in the transformed plants, without the undesirable pleiotrophy of the prior art.
  • a plant expressible promoter sequence such as, for example, a cell-specific promoter, tissue-specific promoter, inducible promoter sequence, organ-specific promoter sequence, or a cell cycle specific gene promoter sequence
  • the ectopic expression of a substrate of Cdc25 or a modified substrate of Cdc25, and, in particular, Cdc2a or a homologue, analogue or derivative thereof, can produce a range of desirable phenotypes in plants, such as, for example, by modifying one or more morphological, biochemical, or physiological characteristics as follows: (i) modification of the initiation, promotion, stimulation or enhancement of cell division; (ii) modification of the initiation, promotion, stimulation or enhancement of DNA replication;(iii) modification of the initiation, promotion, stimulation or enhancement of seed set and/or size and/or development; (iv) modification of the initiation, promotion, stimulation or enhancement of tuber formation; (v) modification of the initiation, promotion, stimulation or enhancement of shoot initiation and/or development; (vi) modification of the initiation, promotion, stimulation or enhancement of root initiation and/or development; (vii) modification of the initiation, promotion, stimulation or enhancement of lateral root initiation and/or development; (viii) modification of the initiation
  • the term "modification of the initiation, promotion, stimulation or enhancement" in relation to a specified integer shall be taken as a clear indication that the integer is capable of being enhanced, increased, stimulated, or promoted, or alternatively, decreased, delayed, repressed, or inhibited.
  • the targeted ectopic expression of a substrate of Cdc25, in particular Cdc2a or a homologue, analogue or derivative thereof, in one or more tissues of the plant produces the initiation, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment syntheses, and/or the modification of source/sink relationships, and/or the modification of root growth and/or the inhibition of apical dominance and/or the delay of senescence, amongst other characteristics.
  • the inventors found that, by ectopically-expressing a substrate of Cdc25 or a modified substrate of Cdc25, and, in particular, Cdc2a or a homologue, analogue or derivative thereof, operably under the control of a regulatable plant-expressible promoter sequence, such as, for example, a constitutive promoter sequence, the transformed plants exhibited increased growth and vigour; increased total biomass; increased cell number; reduced flowering time; increased branching; increased inflorescence formation; and increased seed set and/or increased seed yield and/or increased seed size.
  • a regulatable plant-expressible promoter sequence such as, for example, a constitutive promoter sequence
  • Cyclin-dependent kinases including Cdc25 and its substrates, such as, for example, Cdc2a, are intracellular proteins, which, unlike exogenously-applied cytokinin or cytokinin produced by ectopic expression of ipt or rolC genes, will only exert a localised effect at the site of protein synthesis.
  • the present invention may be applied to the modification of a wide range of biochemical, morphological and phenotypic characteristics in plants, depending upon the promoter sequence selected to regulate ectopic expression of the introduced Cdc2a-encoding genetic sequence or a homologue, analogue or derivative thereof.
  • one or more cytokinin-mediated and/or gibberellin-mediated effects are effectively modified using regulatable plant-operable promoter sequences selected from the group consisting of: cell-specific promoter sequences; tissue-specific promoter sequences; organ-specific promoter sequences; and cell cycle specific gene promoter sequences, such that the expression is targeted specifically to certain cells, tissues or organs of the transformed plants.
  • regulatable plant-operable promoter sequences selected from the group consisting of: cell-specific promoter sequences; tissue-specific promoter sequences; organ-specific promoter sequences; and cell cycle specific gene promoter sequences, such that the expression is targeted specifically to certain cells, tissues or organs of the transformed plants.
  • the modification of other characteristics in the plant can be performed using a constitutive promoter sequence.
  • the present invention is not limited by the nature of the promoter used and the selection of promoter is clearly a preferred embodiment of the present invention.
  • one aspect of the invention provides a method of modifying plant morphology and/or biochemistry and/or physiology comprising expressing in particular cells, tissues or organs of a plant, a genetic sequence encoding a cyclin-dependent kinase (CDK) protein, and, in particular a substrate of Cdc25 or a modified substrate of Cdc25, operably in connection with a plant-operable promoter sequence.
  • CDK cyclin-dependent kinase
  • Expression of the CDK is preferably carried out by introducing an isolated nucleic acid molecule comprising the CDK-encoding nucleotide sequence into a cell, tissue or organ of the plant, regenerating plant tissue or whole plants therefrom and then culturing those plant parts or whole plants under conditions suitable for activity of the promoter sequence to which said nucleotide sequence is operably connected.
  • the genetic sequence encoding the cyclin-dependent kinase protein is placed operably under the control of a plant-expressible promoter sequence selected from the list comprising strong constitutive promoter sequences, cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, cell-cycle-specific promoter sequences, and inducible promoter sequences (both pathogen-inducible and environmentally-inducible promoters are contemplated herein).
  • the present invention further encompasses the use of a promoter sequence in a gene construct wherein an excisable genetic element is inserted into said construct so as to inactivate expression of the CDK protein during transformation and regeneration steps.
  • excision of the excisable genetic element in the regenerated plant or progeny derived therefrom facilitates ectopic expression of the CDK protein.
  • Methods to induce excision of such genetic elements are known to those skilled in the art.
  • the excisable genetic element may be an autonomous or non- autonomous genetic element.
  • the substrate of Cdc25 or a modified substrate of Cdc25 is a Cdc2a polypeptide or modified Cdc2a polypeptide, and more preferably a Cdc2a polypeptide or modified Cdc2a polypeptide of plants, such as, but not limited to, the A. thaliana Cdc2a polypeptide or a biologically-active homologue, analogue or derivative thereof.
  • the present invention clearly contemplates the use of functional homologues of nucleotide sequences encoding Cdc2a and, in particular, nucleotide sequences encoding a non-phosphorylateable, yet biologically-active, Cdc2a polypeptide, for example a Cdc2a polypeptide wherein threonine-14 and/or tyrosine-15 have been substituted with a non-phosphorylateable residue.
  • Preferred embodiments of the invention relate to the modification of one or more aspects of plant morphology and architecture by the ectopic expression of a Cdc25 substrate or modified Cdc25 substrate therein, including, but not limited to, increased growth and vigour and/or increased total biomass and/or increased cell number and/or modified flowering time and/or reduced branching and/or increased inflorescence formation and/or increased seed set and/or cytokinin-mediated effects and/or gibberellin-mediated effects.
  • the present invention clearly contemplates the broad application of the inventive method to the modification of a range of cellular processes, including but not limited to the modification of the initiation, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment syntheses, and/or the modification of source/sink relationships, and/or the modification of root growth and/or the inhibition of apical dominance and/or the delay of senescence.
  • the present invention clearly contemplated the broad application of the inventive method to the modification of a range of cellular processes, including but not limited to a modification of the inhibition, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment syntheses, and/or the modification of source/sink relationships, and/or the modification of root growth and/or the inhibition of apical dominance and/or the delay of senescence.
  • Cdc2a or a non-phosphorylateable Cdc2a protein is expressed in a whole plant under control of a regulatable promoter sequence, in particular a constitutive promoter sequence, such as, for example, the CaMV 35S promoter sequence, to increase growth and vigour and/or increase total biomass and/or increase cell number and/or modify flowering time and/or increase branching and/or increase inflorescence formation and/or increased seed set and/or seed size and/or seed yield.
  • a regulatable promoter sequence in particular a constitutive promoter sequence, such as, for example, the CaMV 35S promoter sequence
  • a substrate of Cdc25 or a modified substrate of Cdc25 is expressed operably under the control of a promoter derived from a stem-expressible gene, to increase the strength and thickness of a plant stem to confer improved stability and wind-resistance on the plant.
  • a substrate of Cdc25 or a modified substrate of Cdc25 is expressed in a tuber-forming plant operably under the control of a promoter derived from a stem-expressible gene or tuber-expressible gene, to increase improve tuber production in the plant.
  • a substrate of Cdc25 or a modified substrate of Cdc25 is expressed in a tree crop plant such as, but not limited to, Eucalyptus spp. or Populus spp., operably under the control of a promoter derived from a gene that is expressed in vascular tissue and/or cambium cells, to increase lignin content therein.
  • the ectopic expression of a CDK under control of a promoter that is operably in vascular tissue and preferably, in cambial cells produces thick-stemmed plants and a higher ratio of vascular tissue-to -pith cells within the stem thereby resulting in more lignin production and a modified ratio of spring wood to late wood.
  • cambial cells contain the highest levels of auxins and are therefore the preferential tissue for CDK overproduction.
  • a substrate of Cdc25 or a modified substrate of Cdc25 is expressed operably under the control of a promoter derived from a seed-expressible gene, to increase seed production in plants, in particular to increase seed set and seed yield. More preferably, the promoter is operable in the endosperm of the seed, in which case the combination of the cell cycle- control protein and endosperm-expressible promoter provides the additional advantage of increasing the grain size and grain yield of the plant.
  • a substrate of Cdc25 or a modified substrate of Cdc25 is expressed operably under the control of a promoter derived from a meristem-expressible gene or a shoot-expressible gene or a root-expressible gene, to reduce apical dominance and/or to promote bushiness of the plant and/or to increase or enhance the production of lateral roots and/or alter leaf shape.
  • a substrate of Cdc25 or a modified substrate of Cdc25 is expressed operably under the control of a promoter derived from a leaf-expressible gene, to alter leaf shape and/or to prevent or delay or otherwise reduce leaf chlorosis and/or leaf necrosis.
  • a substrate of Cdc25 or a modified substrate of Cdc25 such as, for example, Cdc2a or a non-phosphorylateable
  • Cdc2a protein is expressed under the control of a promoter that is operative in meristem tissue of grain crops, to stimulate cell division in the intercalary meristem of the youngest stem internode and produce greater elongation of the stem and/or to generate a more extensive photosynthetic canopy.
  • the present invention may be applied to the selection of any cell, tissue, organ or whole organism that exhibits cytokinin-mediated and/or gibberellin-mediated morphological characteristics and/or biochemical characteristics and/or physiological characteristics, from a background of cells, tissues, organs or whole organisms that do note exhibit such characteristics.
  • a second aspect of the invention provides a gene construct or vector comprising a nucleotide sequence that encodes a substrate of Cdc25 or a modified substrate of Cdc25, such as, for example, Cdc2a or a non-phosphorylateable Cdc2a protein, operably under the control of a plant-expressible promoter sequence selected from the group consisting of:
  • a plant-expressible cell-specific promoter sequence (i) a plant-expressible cell-specific promoter sequence; (ii) a plant-expressible tissue-specific promoter sequence; (iii) a plant-expressible organ-specific promoter sequence; (iv) a plant expressible inducible promoter sequence; (v) a plant-expressible constitutive promoter sequence, wherein the nucleotide sequence encoding said substrate or modified substrate, and the plant-expressible constitutive promoter sequence, are integrated into an excisable genetic element;
  • a plant-expressible constitutive promoter sequence wherein the nucleotide sequence encoding said substrate or modified substrate and said promoter sequence are such that expression of said substrate or modified substrate is capable of being modulated by an excisable genetic element; and (vii) a plant-expressible cell cycle specific gene promoter sequence.
  • the gene construct or vector according to this aspect of the invention is suitable for expression in a plant cell, tissue, organ or whole plant and more preferably, the subject gene construct or vector is suitable for introduction into and maintenance in a plant cell, tissue, organ or whole plant.
  • a third aspect of the invention extends to those plant cells, tissues, organs and whole plants that have been transformed or transfected with a gene construct of the invention and/or which exhibit one or more modified phenotypic characteristics as described herein by virtue of the ectopic expression of a substrate of Cdc25 or a modified substrate of Cdc25.
  • this aspect of the invention relates to those transformed plants that have been produced in accordance with the invention described herein and exhibit one or more plant morphological, biochemical, genetic, or physiological characteristics selected from the group consisting of: (i) cytokinin-mediated and/or gibberellin- mediated characteristics, such as, for example, modified stem length, tuber formation, lignin content, seed set and yield, bushiness, lateral root production, nitrogen fixation, leaf chlorosis, and leaf necrosis; (ii) increased growth and vigour; (iii) increased total biomass; (iv) increased cell number; (v) modified flowering time; (vi) increased branching; (vii) increased inflorescence formation; and (viii) increased seed set and/or seed size and/or increased seed yield; amongst others.
  • cytokinin-mediated and/or gibberellin- mediated characteristics such as, for example, modified stem length, tuber formation, lignin content, seed set and yield, bushiness, lateral root production, nitrogen fixation,
  • Transformed plants are preferably produced by the method of introducing into a plant cell, tissue or organ a nucleotide sequence capable of expressing a substrate of Cdc25 or a modified substrate of Cdc25, such as, for example, Cdc2a or a modified, non- phosphorylatable Cdc2a protein, and regenerating a whole plant therefrom, and more preferably, wherein the transformation procedure is performed without the application of exogenous cytokinin.
  • FIG. 1 is a photographic representation showing the stimulation of growth in A. thaliana plants that are homozygous at a single locus for the introduced plant- expressible A. thaliana Cdc2aAt gene, placed operably under control of the CaMV 35S promoter sequence.
  • Panel a shows genetically-untransformed plants.
  • Panel b shows clonal variants of a representative transformed homozygous plant line. Plants were grown photosynthetically in containers permeable to atmospheric gases on Murashige and Skoog (1962) inorganic medium.
  • Figure 2 is a photographic representation of a western blot showing increased Cdc2a protein levels in A. thaliana plants that are homozygous at a single locus for the introduced plant-expressible A. thaliana Cdc2aAt gene, placed operably under control of the CaMV 35S promoter sequence.
  • Figure 3 is a photographic representation showing the presence of the introduced Cdc2aA14F15 gene, operably in connection with the CaMV 35S promoter sequence, in the genomic DNA of transformed plants, as detected by PCR amplification.
  • Lane 1 DNA size standards; Lanes 2 and 5, control PCR reactions with no plant DNA added; Lanes 3 and 4, PCR reactions comprising untransformed plant DNA; Lanes 6 and 7, PCR reactions comprising DNA derived from transgenic plant lines. Amplification reactions were performed using the primers indicated at the bottom of the Figure, as follows:
  • Primer 1 (WT 5'): 5'-GTTGAGAAGATTGGTGAAGGAACTTA- 3'; Primer 2: (Cdc2aA14F15 5'): 5'-CCAAGATCCTTGAAGTATTCATGCTCC-3';
  • Primer 3 (common 3'): 5'-GTTGAGAAGATTGGTGAAGGAGCTTT-3'.
  • the arrow indicates the position of an expected 856 bp amplification product derived from the modified Cdc2a gene.
  • Figure 4 is a photographic representation showing the stimulation of growth in A. thaliana plants that are homozygous at a single locus for the introduced plant- expressible A. thaliana Cdc2aAtA14F15 gene (i.e. expressing a modified non- phosphorylateable Cdc2a protein), placed operably under control of the CaMV 35S promoter sequence.
  • Panel a shows genetically-untransformed plants.
  • Panels b, c and d show the representative transformed homozygous plant lines 1B1C1 , 1 B1 D4, and 1C2C5, respectively.
  • Transformed plants have roots and shoots that grow more rapidly than un-transformed plants, and have more highly-branched roots than untransformed plants. Plants were grown photosynthetically in containers permeable to atmospheric gases on Murashige and Skoog (1962) inorganic medium.
  • Figure 5 is a photographic representation of a western blot showing increased Cdc2a protein levels in A. thaliana plant lines that are homozygous at a single locus for the introduced plant-expressible A. thaliana Cdc2aAtA14F15 placed operably under control of the CaMV 35S promoter sequence.
  • Figure 6 is a photographic representation showing the unmodified cell size in the lower leaf epidermis of transgenic A. thaliana plants that are homozygous at a single locus for an introduced A. thaliana Cdc2aAtA14F15 gene.
  • Panel a wild type non- transformed plants.
  • Panels b, c and d show the transformed lines 1 BCA, 1 B1 D4 and 1C2C5, respectively.
  • the interlocking perimeters and size of wild type epidermal cells seen in panel a are not altered in the transgenic plants, although they have grown more rapidly, by more rapid cell formation. Growth of the transformed plants was due to increased cell division and not a greater cell size than that of the untransformed plants.
  • Figure 7 is a photographic representation showing the presence of the Cdc2aAtA14F15 transgene in A. thaliana plants, and enhanced growth of plants expressing the transgene.
  • Panels a and B show the inherited Cdc2aAtA14F15 transgene in F2 plants obtained by self-fertilisation of the primary transformant line YF- D, and the enhanced growth of those plants.
  • Panels C and D show the presence of the Cdc2aAtA14F15 transgene in the transgenic line YF-E, and the enhanced growth effects observed in plants expressing the transgene.
  • Panels a and C show Western blots of A. thaliana protein probed with an antibody that is specific for the 34 kDa A.
  • thaliana Cdc2a polypeptide Panels B and D show total protein transferred to PVDF membranes. Lanes shown contain protein from A. thaliana plants having defined phenotypes as follows: 1 , large (big) transgenic plants; 2, medium (mid)-sized transgenic plants; 3, small transgenic plants; and 4, non-transformed (WT) plants. Briefly, 50 ⁇ g total protein from plants was subjected to SDS/PAGE, transferred to PVDF membranes and stained (panels B, D), then de-stained and probed with anti-At Cdc2a-serum (panels a, C).
  • One aspect of the invention provides a method of modifying one or more plant morphological and/or biochemical and/or physiological characteristics comprising expressing in one or more particular cells, tissues or organs of a plant, a cell cycle control protein comprising a substrate of Cdc25 or a modified substrate of Cdc25 operably under the control of a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, and cell cycle-specific promoter sequences.
  • modifying one or more plant morphological and/or biochemical and/or physiological characteristics is meant that one or more morphological and/or biochemical and/or physiological characteristics of a plant is altered by the performance of one or more steps pertaining to the invention described herein.
  • Plant morphology or the term “plant morphological characteristic” or similar term will be understood by those skilled in the art to refer to the external appearance of a plant, including any one or more structural features or combination of structural features thereof.
  • Such structural features include the shape, size, number, position, colour, texture, arrangement, and pattemation of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, stem, leaf, shoot, petiole, trichome, flower, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre, cambium, wood, heartwood, parenchyma, aerenchyma, sieve element, phloem or vascular tissue, amongst others.
  • plants may modify one or more plant morphological characteristics in response to the external stimuli, such as, for example, a plant pathogenic infection, or an external stress (drought, flooding, salt stress, dehydration, heavy metal contamination, mineral deficiency, etc).
  • a plant morphological characteristic that has been modified in response to one or more external stimuli is within the scope of the inventive method described herein, notwithstanding that the imposition of said external stimuli is not an essential feature of the present invention.
  • Plant biochemistry or the term “plant biochemical characteristic” or similar term will be understood by those skilled in the art to refer to the metabolic and catalytic processes of a plant, including primary and secondary metabolism and the products thereof, including any small molecules, macromolecules or chemical compounds, such as but not limited to starches, sugars, proteins, peptides, enzymes, hormones, growth factors, nucleic acid molecules, celluloses, hemicelluloses, calloses, lectins, fibres, pigments such as anthocyanins, vitamins, minerals, micronutrients, or macronutrients, that are produced by plants.
  • small molecules, macromolecules or chemical compounds such as but not limited to starches, sugars, proteins, peptides, enzymes, hormones, growth factors, nucleic acid molecules, celluloses, hemicelluloses, calloses, lectins, fibres, pigments such as anthocyanins, vitamins, minerals, micronutrients, or macronutrients, that are produced by plants.
  • Plant physiology or the term “plant physiological characteristic” or similar term will be understood to refer to the functional processes of a plant, including developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fibre production, secondary growth or wood production, amongst others; responses of a plant to externally-applied factors such as metals, chemicals, hormones, growth factors, environment and environmental stress factors (eg. anoxia, hypoxia, high temperature, low temperature, dehydration, light, daylength, flooding, salt, heavy metals, amongst others), including adaptive responses of plants to said externally- applied factors.
  • developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fibre production, secondary growth or wood production, amongst others
  • the ectopic expression of a Cdc25 substrate or modified Cdc25 substrate protein produces a wide range of desirable phenotypes in the plant, selected from the group consisting of:(i) enhanced growth and/or enhanced vigour of the plant; (ii) increased total biomass of the plant; (iii) increased cell number; (iv) reduced flowering time; (v) increased inflorescence formation; (vi) reduced time to seed set (vii) enhanced seed set; (viii) enhanced seed size; (ix) enhanced grain yield; (x) enhanced stem strength; (xi) enhanced stem thickness; (xii) enhanced stem stability; (xiii) enhanced wind-resistance of the stem; (xiv) enhanced tuber formation; (xv) enhanced tuber development; (xvi) increased lignin content; (xvii) enhanced ploidy of the seed; (xviii) enhanced endosperm size; (xix) reduced apical dominance; (xx) increased bushiness; (xxi) enhanced
  • the plant morphological, biochemical or physiological characteristic which is modified is a cytokinin-mediated or a gibberellin-mediated characteristic selected from the group consisting of: (i) enhanced stem thickness; (ii) enhanced stem stability; (iii) enhanced wind-resistance of the stem; (iv) enhanced tuber formation; (v) enhanced tuber development; (vi) increased lignin content; (vii) enhanced seed set;
  • expression of the cell cycle control protein preferably leads to the initiation, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment synthesis, and/or the modification of source/sink relationships, and/or the modification of root growth and/or the inhibition of apical dominance and/or the delay of senescence
  • the plant morphological, biochemical or physiological characteristic which is modified is selected from the group consisting of: (i) plant growth and vigour; (ii) total biomass; (iii) cell number; (iv) flowering time; (v) branching; (vi) inflorescence formation; and (vii) seed set and/or seed yield and/or seed size.
  • expression of the cell cycle control protein preferably leads to increased plant productivity such as, for example, increased growth and vigour; increased total biomass; increased cell number; reduced flowering time; increased branching; increased inflorescence formation; and increased seed set and seed size.
  • express or variations such as “expressing” and “expression” as used herein shall be taken in their broadest context to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA or the translation of a sense mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • expression comprising the production of a sense mRNA transcript
  • the word “express” or variations such as “expressing” and “expression” may also be construed to indicate the combination of transcription and translation processes, with or without subsequent post-translational events which modify the biological activity, cellular or sub-cellular localization, turnover or steady-state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • cell cycle may exert its effect upon the morphology, biochemistry or physiology of the cell at the transcriptional or post-transcriptional level.
  • the term "cell cycle” as used herein shall be taken to include the cyclic biochemical and structural events associated with growth and with division of cells, and in particular with the regulation of the replication of DNA and mitosis.
  • Cell cycle includes phases called: GO (gap 0), G1 (gap 1), DNA replication (S), G2 (gap 2), and mitosis including cytokinesis (M). Normally these four phases occur sequentially.
  • the cell cycle also includes modified cycles such as endomitosis, acytokinesis, polyploidy, polyteny, endopolyploidisation and endoreduplication or endoreplication.
  • cell cycle interacting protein means a protein which exerts control on or regulates or is required for the cell cycle or part thereof of a cell, tissue, organ or whole organism and/or DNA replication. It may also be capable of binding to, regulating or being regulated by cyclin dependent kinases or their subunits.
  • the term also includes peptides, polypeptides, fragments, variant, homologues, alleles or precursors (eg preproproteins or preproteins) thereof.
  • cell cycle control gene refers to any gene or mutant thereof which exerts positive or negative control on, or is required for, chromosomal DNA synthesis, mitosis (preprophase band, nuclear envelope, spindle formation, chromosome condensation, chromosome segregation, formation of new nuclei, formation of phragmoplast, etc) meiosis, cytokinesis, cell growth, or endoreduplication.
  • cell cycle control gene also includes any and all genes that exert control on a cell cycle protein as hereinbefore defined, including any homologues of CDKs, cyclins, E2Fs, Rb, CKI, Cks, cyclin D, cdc25, Wee1, Nim1, MAP kinases, etc.
  • a cell cycle control gene will exert such regulatory control at the post-translation level, via interactions involving the polypeptide product expressed therefrom.
  • cell cycle control genes are all genes involved in the control of entry and progression through S phase. They include, not exclusively, genes expressing "cell cycle control proteins” such as cyclin dependent kinases (CDK), cycline dependent kinase inhibitors (CKI), D, E and a cyclins, E2F and DP transcription factors, pocket proteins, CDC7/DBF4 kinase, CDC6, MCM2-7, Ore proteins, cdc45, components of SCF ubiquitin ligase, PCNA, and DNA-polymerase, amongst others.
  • CDK cyclin dependent kinases
  • CKI cycline dependent kinase inhibitors
  • D cycline dependent kinase inhibitors
  • E2F and DP transcription factors DP transcription factors
  • pocket proteins CDC7/DBF4 kinase
  • CDC6, MCM2-7 or MCM2-7
  • Ore proteins cdc45
  • cell cycle control protein includes cyclins a, B, C, D and E, including CYCA1 ;1 , CYCA2;1 , CYCA3;1 , CYCB1;1 , CYCB1 ;2, CYCB2;2, CYCD1 ;1 , CYCD2;1.
  • CYCD3;1 , and CYCD4;1 Evans et al. 1983; Francis et al. 1998; Labbe et al.
  • CKI cyclin dependent kinase inhibitor proteins
  • ICK1 Wang et al 1997), FL39, FL66, FL67 (PCT/EP98/05895), Sid , Far1 , Rum1 , p21 , p27, p57, p16, p15, p18, p19 (Elledge 1996; Pines 1995a,b), p14 and p14ARF; p13sud or CKSIAt (De Veylder et al 1997; Hayles and Nurse 1986) and nim-1 (Russell and Nurse 1987a; Russell and Nurse 1987b; Fantes 1989; Russell and Nurse 1986; Russell and Nurse 1987a; Russell and Nurse 1987b) homologues of Cdc2 such as Cdc2MsB (Hirt et al 1993) CdcM
  • cell cycle control proteins are involved in cyclin D-mediated entry of cells into G1 from GO include pRb (Xie et al, 1996; Huntley et al, 1998), E2F, RIP, MCM7, and the pRb-like proteins p107 and p130.
  • cell cycle control proteins are involved in the formation of a pre-replicative complex at one or more origins of replication, such as, but not limited to, ORC, CDC6, CDC14, RPA and MCM proteins or in the regulation of formation of this pre-replicative complex, such as, but not limited to, the CDC7, DBF4 and MBF proteins.
  • cell cycle control protein shall further be taken to include any one or more of those proteins that are involved in the turnover of any other cell cycle control protein, or in regulating the half-life of said other cell cycle control protein.
  • protein turnover is to include all biochemical modifications of a protein leading to the physical or functional removal of said protein. Although not limited to these, examples of such modifications are phosphorylation, ubiquitination and proteolysis.
  • Particularly preferred proteins which are involved in the proteolysis of one or more of any other of the above-mentioned cell cycle control proteins include the yeast-derived and animal-derived proteins, Skp1 , Skp2, Rub1 , Cdc20, cullins, CDC23, CDC27, CDC16, and plant-derived homologues thereof (Cohen-Fix and Koshland 1997; Hochstrasser 1998; Krek 1998; Lisztwan et al 1998) and Plesse et al in (Francis et al 1998)).
  • cell cycle control genes shall further be taken to include any one or more of those gene that are involved in the transcriptional regulation of cell cycle control gene expression such as transcription factors and upstream signal proteins. Additional cell cycle control genes are not excluded.
  • cell cycle control genes shall further be taken to include any cell cycle control gene or mutant thereof, which is affected by environmental signals such as for instance stress, nutrients, pathogens, or by intrinsic signals such as an animal mitogen or plant hormone (auxin, cytokinin, ethylene, gibberellic acid, abscisic acid and brassinosteroid).
  • environmental signals such as for instance stress, nutrients, pathogens, or by intrinsic signals such as an animal mitogen or plant hormone (auxin, cytokinin, ethylene, gibberellic acid, abscisic acid and brassinosteroid).
  • the present invention clearly extends to specific equivalents, in particular the use of any Cdc25 substrate or modified substrate that possesses the biological activity of a cell cycle control protein, notwithstanding the fact that it may not be regulatable directly by Cdc25, such as, for example, a non-phosphorylateable Cdc25 substrate.
  • the terms "substrate of Cdc25" and “Cdc25 substrate”, or similar term, shall be taken to refer to any protein that is regulated directly or indirectly by Cdc25, and more particularly, any protein that is dephosphorylated by Cdc25, including, but not limited to cyclin-dependent kinases (CDKs).
  • the CDK may be an A- type or a B-type CDK, and preferably, it is an A-type (or PSTAIRE-type) CDK, and more preferably, the CDK is a Cdc2 protein, in particular a Cdc2a protein.
  • a Cdc2 protein, such as Cdc2a is the key enzyme driving entry into S-phase and/or into mitosis (M-phase).
  • the inventors have also discovered that expression of a substrate of Cdc25, in particular a Cdc2 protein, in plants or plant cells can override the arrest of DNA replication (S phase) under growth-limiting conditions. Unexpectedly, the same process partially sustains endoreplication.
  • Preferred Cdc25 substrate proteins include Cdc2 and Cdc2a proteins.
  • modified Cdc25 substrates include non-phosphorylated and/or non- phosphorylateable Cdc25 substrate proteins, such as, for example non- phosphorylated or non-phosphorylateable Cdc2 proteins and/or non-phosphorylated or non-phosphorylateable Cdc2a proteins.
  • the modified Cdc25 substrate is the Cdc2aAt A14F15 polypeptide or a homologue, analogue or derivative thereof.
  • a Cdc2 protein shall be taken to include a reference to a Cdc2a protein, and, in particular, a reference to a Cdc2a polypeptide of plant origin, and including the A. thaliana Cdc2a polypeptide.
  • a modified Cdc2 protein shall be taken to include a reference to a modified Cdc2a protein, and, in particular, a reference to a modified Cdc2a polypeptide of plant origin, and including the A. thaliana Cdc2aAtA14Y15 polypeptide.
  • modified substrate of Cdc25 and “modified Cdc25 substrate” shall be taken to refer to any isoform of a substrate of Cdc25 that mimics the effect of Cdc25 activity, in particular a non-phosphorylated form of Cdc2 that is produced by the dephosphorylation and/or inhibition of phosphorylation of a Cdc2 polypeptide.
  • a modified substrate of Cdc25 is a mutated A-type CDK, namely a Cdc2a protein, wherein threonine-14 and/or tyrosine-15 have been substituted with non- phosphorylatable residues, such as phenylalanine and/or alanine.
  • a modified substrate of Cdc25 is a mutated B-type CDK, in particular Cdc2b, wherein threonine-14 and/or tyrosine-15 have been substituted with non- phosphorylatable residues, such as phenylalanine and/or alanine.
  • modified substrate of Cdc25 also refers to a homologue, analogue or derivative of a substrate of Cdc25 that mimics the effect of Cdc25, such as, for example, a modified Cdc2a protein with increased activity compared to the wild-type Cdc2a protein, which preferably results in earlier switching from quiescence to active cycling and/or early entry of cells into S-phase and/or into mitosis (M-phase).
  • a modified substrate of Cdc25 encompasses any Cdc2 protein, including Cdc2a, or a homologue, analogue or derivative thereof which is non- phosphorylateable, or dephosphorylated, such that the modification removes the inhibitory effect of the phosphates on Cdc2 (or Cdc2a) and/or removes inhibition of Cdc2 (or Cdc2a) resulting from phosphorylation by wee-1 and/or mik-1 , or otherwise increases Cdc2 (or Cdc2a) activity.
  • a particularly preferred modified substrate of Cdc25 is a modified Cdc2a protein wherein both threonine-14 and tyrosine-15 have been substituted with alanine and phenylalanine, respectively, to produce Cdc2aA14F15.
  • a non-phosphorylatable form of Cdc2a is free of the phosphate at the tyrosine at position 15 (i.e. tyrosine-15 or Y15) and optionally (though not necessarily) free of the phosphate at threonine at position 14 (i.e. threonine-14 or T14).
  • the present inventors have shown that the ectopic expression of a modified substrate of Cdc25 which consists of the Cdc2aA14F15 polypeptide, produces growth effects and/or one or more cytokinin-like effects in the plants, especially increased meristem activity and outgrowth of lateral buds, leading to increased branching of plants that is increased relative to that observed following constitutive Cdc25 expression in plants. Without being bound by any theory or mode of action, these effects are presumably the result of the ectopically-expressed protein being both active and expressed at a high level.
  • the present invention encompasses de novo and/or increased expression of a substrate of Cdc25, or a modified substrate of Cdc25, such as a non-phosphorylatable Cdc2 or a modified Cdc2 protein with increased/earlier activity, or alternatively by raising Cdc2 activity by introducing additional copies of the Cdc2 gene to increase the amount of Cdc2 enzyme formed.
  • the substitution or deletion of the phosphorylation sites of a Cdc2 protein mimics the effect of a constitutive phosphatase activity, such as the effect of the Cdc25 protein phosphatase (p80 Cdc25 ) activity.
  • the substitution or deletion of the phosphorylation sites of a Cdc2a protein further mimics the effect of down-regulated kinase activity, such as a down- regulation of the wee-1 kinase and/or mik-1.
  • down-regulated kinase activity such as a down- regulation of the wee-1 kinase and/or mik-1.
  • modified Cdc2a activity effects described herein can also be obtained, albeit only in part and without the benefits derived from increasing the amount of active Cdc2a protein, by the regulated expression of Cdc25.
  • the modified Cdc2a activity effects described herein can also be obtained by down- regulating, or inhibiting, wee-1 and/or mik-1 kinase activity, such as, for example, by using antisense molecules, ribozymes, cosuppression molecules, gene targeting molecules, or gene silencing molecules, etc., which target the wee-1 and/or mik-1 genes or gene products, respectively.
  • the present invention involves providing de novo and/or increased expression of a substrate of Cdc25 or a modified substrate of Cdc25, such as, for example, a non- phosphorylatable Cdc2a or a modified Cdc2a with increased activity, or alternatively by raising Cdc2a activity by introducing additional copies of the Cdc2a gene to increase the amount of Cdc2a enzyme formed.
  • a substrate of Cdc25 or a modified substrate of Cdc25 such as, for example, a non- phosphorylatable Cdc2a or a modified Cdc2a with increased activity, or alternatively by raising Cdc2a activity by introducing additional copies of the Cdc2a gene to increase the amount of Cdc2a enzyme formed.
  • Cdc2a biological activity of Cdc2a depends upon the presence of one or more cyclins in the cell, such as, for example, cyclin A and/or cyclin B. Accordingly, increased cyclin levels in plants to achieve the altered morphological, biochemical or physiological properties described herein are clearly within the scope of the present invention. In the use of cyclins to achieve these improvements, it is preferred that the ectopic expression of one or more cyclins in the plant accompanies the ectopic expression of Cdc2a or a modified substrate of Cdc25 described herein, in particular a modified Cdc2a protein such as Cdc2aA14F15.
  • the present invention provides a method of modifying one or more plant morphological and/or biochemical and/or physiological characteristics comprising expressing in one or more particular cells, tissues or organs of a plant, a Cdc2 protein or a homologue, analogue or derivative thereof, operably under the control of a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, and cell cycle-specific promoter sequences.
  • "Homologues" of Cdc2 are those peptides. oligopeptides, polypeptides, proteins and enzymes which contain amino acid substitutions, deletions and/or additions relative to a non-mutant or wild-type Cdc2 polypeptide, without altering one or more of its cell cycle control properties.
  • amino acids present in Cdc2 can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break ⁇ -helical structures or ⁇ -sheet structures, and so on.
  • Substitutional variants are those in which at least one residue in the Cdc2 amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the Cdc2 protein. Insertions can comprise amino- terminal and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.
  • Deletional variants are characterised by the removal of one or more amino acids from the Cdc2 sequence.
  • Amino acid variants of the Cdc2 polypeptide may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations.
  • the manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.
  • techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis or other site-directed mutagenesis protocol.
  • the Cdc2 protein is the A. thaliana Cdc2a protein or a homologue, analogue or derivative thereof, in particular a non-phosphorylateable homologue, analogue or derivative.
  • the Cdc2 homologue comprises a non-phosphorylateable Cdc2 protein, such as a Cdc2 protein wherein threonine-14 and/or tyrosine-15 is substituted with a non-phosphorylateable residue.
  • the Cdc2 homologue is the Cdc2aA14F15 protein kinase (Hemerly et al, 1995).
  • the Cdc25 protein can dephosphorylate T14 and/or Y15 residues of a Cdc2 protein, to activate a Cdc2 protein kinase activity.
  • the wee-1 and/or mik-1 proteins phosphorylate T14 and/or Y15 residues of a Cdc2 protein to inactivate a Cdc2 protein kinase activity.
  • the Cdc2aA14F15 mutant protein is a non- phosphorylateable mutant that escapes regulation by phosphorylation and/or dephosphorylation.
  • the positions of the said tyrosine-15 and threonine-14 are conserved, for example, as in the A. thaliana Cdc2a protein.
  • Those skilled in the art will be aware of the possibility that, for example, during the course of evolution, the respective positions of these consensus amino acids have become altered. Such a shift may occur, for example, as a result of one or more deletions or additions at the N-terminus of the protein.
  • tyrosine at position 15 or equivalent terms such as “tyrosine-15” or “Y15”; and “threonine at position 14" or equivalent terms such as “threonine-14" or “T14”, refer herein to encompass the residues that are generally present at positions 15 and 14 of the respective CDK.
  • tyrosine at position 15 or equivalent terms such as “tyrosine-15” or “Y15”; and “threonine at position 14" or equivalent terms such as “threonine-14” or “T14”, refer further to tyrosine and threonine residues present in a CDK polypeptide at a different position to that which normally occurs, that possesses the inhibitory phosphorylatable characteristic of the Y15 and/or T14 residues with respect to CDK activity.
  • Determination of phosphorylation sites in CDKs corresponding to tyrosine at position 15 and threonine at position 14 of Cdc2a can be done for example by computer assisted identification of such sites in the amino acid sequence of a given CDK using eg BLAST2 (Basic Local Alignment Search Tool) which can be used to search for local sequence alignments. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologues. Phosphorylation sites can also be determined using anti-phospho-tyrosine and anti- phospho-threonine antibodies as described for instance by Zhang et al. (1996).
  • Analogues of a Cdc2 protein are defined as those peptides, oligopeptides, polypeptides, proteins and enzymes which are functionally equivalent to a Cdc2 protein.
  • Analogues of a Cdc2 protein include those Cdc25 substrates and modified Cdc25 substrates that comprise peptides, polypeptides, proteins, and enzymes that are capable of functioning in a plant cell and/or plant tissue and/or plant organ and/or whole plant to produce the same modified plant morphological and/or biochemical and/or physiological characteristics as the ectopic expression of a Cdc2 protein, including any functional homologues, mutants, derivatives, parts or fragments of Cdc2.
  • any Cdc2 analogue may be substrate of Cdc25 or modified substrate of Cdc25, including the fission yeast Cdc2 protein, or a Cdc2 protein of plants, such as, for example, a monocotyledonous plant or dicotyledonous plant species, and, in particular, tobacco, A. thaliana, maize, wheat, rice, barley, or other plant referred to herein.
  • the Cdc2 analogue is a substrate of Cdc25 or a modified substrate of Cdc25 other than a cyclin B protein or a homologue or derivative thereof.
  • “Derivatives" of a Cdc2 protein are those peptides, oligopeptides, polypeptides, proteins and enzymes which comprise at least about five contiguous amino acid residues of a naturally-occurring Cdc2 polypeptide, in particular the A. thaliana Cdc2a polypeptide, but which retain activity in the induction of one or more of the morphological and/or biochemical and/or phenotypic characteristics that are capable of being modulated by expression of the full-length Cdc2 polypeptide as described herein.
  • a "derivative" of a Cdc2 protein may further comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring Cdc2 polypeptide.
  • a derivative may comprise one or more non-amino acid substituents compared to the amino acid sequence of a naturally-occurring Cdc2 polypeptide, for example a reporter molecule or other ligand, covalently or non- covalently bound to the amino acid sequence such as, for example, a reporter molecule which is bound thereto to facilitate its detection.
  • Cdc2 polypeptides include those incorporating single or multiple substitutions, deletions and/or additions therein, such as carbohydrates, lipids and/or proteins or polypeptides.
  • Naturally-occurring or altered glycosylated or acylated forms of the Cdc2 polypeptide are also contemplated by the present invention.
  • homopolymers or heteropolymers comprising one or more copies of the Cdc2 polypeptide are within the scope of the invention, the only requirement being that such molecules retain activity in the induction of one or more of the morphological and/or biochemical and/or phenotypic characteristics that are capable of being modulated by expression of the full-length Cdc2 polypeptide as described herein.
  • either the protein may be introduced directly to said cell, such as by microinjection means or alternatively, an isolated nucleic acid molecule encoding said protein may be introduced into the cell, tissue or organ in an expressible format.
  • “expressible format” is meant that the isolated nucleic acid molecule is in a form suitable for being transcribed into mRNA and/or translated to produce a protein, either constitutively or following induction by an intracellular or extracellular signal, such as an environmental stimulus or stress (anoxia, hypoxia, temperature, salt, light, dehydration, etc) or a chemical compound such as an antibiotic (tetracycline, ampicillin, rifampicin, kanamycin) hormone (eg.
  • gibberellin gibberellin, auxin, cytokinin, glucocorticoid, etc), hormone analogue (iodoacetic acid (IAA), 2,4-D, etc) , metal (zinc, copper, iron, etc), or dexamethasone, amongst others.
  • expression of a functional protein may also require one or more post- translational modifications, such as glycosylation, phosphorylation, dephosphorylation, or one or more protein-protein interactions, amongst others. All such processes are included within the scope of the term "expressible format".
  • expression of a Cdc25 substrate or modified Cdc25 substrate protein in a specific plant cell, tissue, or organ is effected by introducing and expressing an isolated nucleic acid molecule encoding said protein, such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame, to said cell, tissue or organ, wherein said nucleic acid molecule is placed operably in connection with a plant-expressible promoter sequence.
  • an isolated nucleic acid molecule encoding said protein such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame
  • promoter includes the transcriptional regulatory sequences derived from a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • promoter also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or a -10 box transcriptional regulatory sequences.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected.
  • copper-responsive, glucocorticoid-responsive or dexamethasone-responsive regulatory elements may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible, glucocorticoid- inducible, or dexamethasone-inducible expression respectively, on said nucleic acid molecule.
  • the promoter is a plant-expressible promoter sequence.
  • plant-expressible is meant that the promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, preferably a monocotyledonous or dicotyledonous plant cell and in particular a dicotyledonous plant cell, tissue, or organ. Accordingly, it is within the scope of the invention to include any promoter sequences that also function in non-plant cells, such as yeast cells, animal cells and the like.
  • plant-operable promoter sequence and "promoter sequence operable in a plant” or similar term shall be taken to be equivalent to the term “plant-expressible promoter sequence”.
  • the promoter is a regulatable promoter sequence.
  • a "regulatable promoter sequence” is a promoter that is capable of conferring expression on a structural gene sequence in a particular cell, tissue, or organ or group of cells, tissues or organs of a plant, optionally under specific conditions.
  • a regulatable promoter sequence may be a promoter sequence that confers expression on a gene to which it is operably connected in a particular location within the plant or alternatively, throughout the plant.
  • promoters include strong constitutive promoter sequences, cell-specific promoter sequences, inducible promoter sequences, tissue- specific promoter sequences, organ-specific promoter sequences, cell cycle-specific gene promoter sequences, and constitutive promoter sequences that have been modified to confer expression in a particular part of the plant at any one time, such as by integration of said constitutive promoter within an excisable genetic element.
  • constitutive will be known by those skilled in the art to indicate that expression is observed predominantly throughout the plant, albeit not necessarily in every cell, tissue or organ under all conditions.
  • a strong constitutive promoter is one which confers a high level of ectopic expression on a structural gene to which it is operably connected, predominantly throughout the plant, albeit not necessarily in every cell, tissue or organ under all conditions.
  • cell-specific shall be taken to indicate that expression is predominantly in a particular plant cell or plant cell-type, albeit not necessarily exclusively in that plant cell or plant cell-type.
  • tissue-specific shall be taken to indicate that expression is predominantly in a particular plant tissue or plant tissue-type, albeit not necessarily exclusively in that plant tissue or plant tissue-type.
  • organ-specific shall be taken to indicate that expression is predominantly in a particular plant organ albeit not necessarily exclusively in that plant organ.
  • cell cycle specific or similar shall be taken to indicate that expression is predominantly under control of the cell cycle, or capable of being cyclic such that it occurs in one or more phases of the cell cycle, albeit not necessarily in consecutive phases of the cell cycle, or in cycling cells.
  • an "inducible promoter” is a promoter the transcriptional activity of which is increased or induced in response to a developmental, chemical, environmental, or physical stimulus.
  • the present invention does not require the exclusive expression of the cell cycle control protein in a cell, tissue or organ of a plant, in order to induce ectopic effects therein, however the expression of the cell cycle control protein should be regulatable such that it can be conferred in specific cells, tissues, or organs of the plant, or alternatively, induced under predetermined conditions, or during particular developmental stages of the plant.
  • the activity of the promoter sequence is particularly preferred for the activity of the promoter sequence to be regulated by integrating the promoter sequence and cell cycle control gene in one or more excisable genetic elements.
  • an excisable genetic element shall be taken to refer to any nucleic acid which comprises a nucleotide sequence which is capable of integrating into the nuclear, mitochondrial, or plastid genome of a plant, and subsequently being autonomously mobilised, or induced to mobilise, such that it is excised from the original integration site in said genome.
  • autonomously mobilised is meant that the genetic element is excised from the host genome randomly, or without the application of an external stimulus to excise.
  • the genetic element is preferably induced to mobilise, such as, for example, by the expression of a recombinase protein in the cell which contacts the integration site of the genetic element and facilitates a recombination event therein, excising the genetic element completely, or alternatively, leaving a "footprint", generally of about 20 nucleotides in length or greater , at the original integration site.
  • the excisable genetic element comprises a transposable genetic element, such as, for example, Ac, Ds, Spm, or En, or alternatively, on or more loci for interaction with a site-specific recombinase protein, such as, for example, one or more lox or frt nucleotide sequences.
  • a transposable genetic element such as, for example, Ac, Ds, Spm, or En, or alternatively, on or more loci for interaction with a site-specific recombinase protein, such as, for example, one or more lox or frt nucleotide sequences.
  • Du Pont de Nemours and Company published 29 April, 1987 discloses a method for producing site-specific recombination of DNA in yeast utilising the cre/lox system, wherein yeast is transformed with a first DNA sequence comprising a regulatory nucleotide sequence and a ere gene and a second DNA sequence comprising a pre-selected DNA segment flanked by two lox sites such that, upon activation of the regulatory nucleotide sequence, expression of the ere gene is effected thereby producing site-specific recombination of DNA and deletion of the pre-selected DNA segment.
  • United States Patent No. 4,959,317 (E.I. Du Pont de Nemours and Company) filed 29 April 1987 and International Patent Application No. PCT/US90/07295 (E.I. Du Pont de Nemours and Company) filed 19 December, 1990 also disclose the use of the cre/lox system in eukaryotic cells.
  • a requirement for the operation of site-specific recombination systems is that the loci for DNA recombination and the recombinase enzyme contact each other in vivo, which means that they must both be present in the same cell.
  • the prior art means for excising unwanted transgenes from genetically-transformed cells all involve either multiple transformation events or sexual crossing to produce a single cell comprising both the loci for DNA recombination and the site-specific recombinase.
  • a “site-specific recombinase” is understood by those skilled in the relevant art to mean an enzyme or polypeptide molecule which is capable of binding to a specific nucleotide sequence, in a nucleic acid molecule preferably a DNA sequence, hereinafter referred to as a "recombination locus” and induce a cross-over event in the nucleic acid molecule in the vicinity of said recombination locus.
  • a site-specific recombinase will induce excision of intervening DNA located between two such recombination loci.
  • recombination locus and “recombination loci” shall be taken to refer to any sequence of nucleotides which is recognized and/or bound by a site-specific recombinase as hereinbefore defined.
  • a number of different site specific recombinase systems can be used, including but not limited to the Cre/lox system of bacteriophage P1 , the FLP/FRT system of yeast, the Gin recombinase of phase Mu, the Pin recombinase of E.coli, the PinB, PinD and PinF from Shigella, and the R/RS system of the psR1 plasmid. Some of these systems have already been used with high efficiency in plants, such as tobacco, and A. thaliana.
  • Preferred site-specific recombinase systems contemplated for use in the gene constructs of the invention, and in conjunction with the inventive method are the bacteriophage P1 Cre/lox system, and the yeast FLP/FRT system.
  • the site specific recombination loci for each of these two systems are relatively short, only 34 bp for the lox loci, and 47 bp for the frt loci.
  • the recombination loci are lox sites, such as lox P, lox B, Lox L or lox R or functionally-equivalent homologues, analogues or derivatives thereof.
  • Lox sites may be isolated from bacteriophage or bacteria by methods known in the art (Hoess et al, 1982). It will also be known to those skilled in the relevant art that lox sites may be produced by synthetic means, optionally comprising one or more nucleotide substitutions, deletions or additions thereto.
  • the relative orientation of two recombination loci in a nucleic acid molecule or gene construct may influence whether the intervening genetic sequences are deleted or excised or, alternatively, inverted when a site-specific recombinase acts thereupon.
  • the recombination loci are oriented in a configuration relative to each other such as to promote the deletion or excision of intervening genetic sequences by the action of a site-specific recombinase upon, or in the vicinity of said recombination loci.
  • the present invention clearly encompasses the use of gene constructs which facilitate the expression of a site-specific recombinase protein which is capable of specifically contacting the excisable genetic element, in conjunction with the gene constructs containing the cell cycle control protein-encoding gene.
  • a single gene construct may be used to express both the site-specific recombinase protein and the cell cycle control protein, or alternatively, these may be introduced to plant cells on separate gene constructs.
  • the recombinase gene could already be present in the plant genome prior to transformation with the gene construct of the invention, or alternatively, it may be introduced to the cell subsequent to transformation with the gene construct of the invention, such as, for example, by a separate transformation event, or by standard plant breeding involving hybridisation or cross-pollination.
  • the recombinase gene is supplied to the transgenic plants containing a vector backbone sequence flanked by recombination sites by sexual crossing with a plant containing the recombinase gene in it's genome.
  • Said recombinase can be operably linked to either a constitutive or an inducible promoter.
  • the recombinase gene can alternatively be under the control of single subunit bacteriophage RNA polymerase specific promoters, such as a T7 or a T3 specific promoter, provided that the host cells also comprise the corresponding RNA polymerase in an active form.
  • Yet another alternative method for expression of the recombinase consists of operably linking the recombinase open reading frame with an upstream activating sequence fired by a transactivating transcription factor such as GAL4 or derivatives (US5801027, WO97/30164, WO98/59062) or the Lac repressor (EP0823480), provided that the host cell is supplied in an appropriate way with the transcription factor.
  • a substantially purified recombinase protein could be introduced directly into the eukaryotic cell, eg., by micro-injection or particle bombardment.
  • the site-specific recombinase coding region will be operably linked to regulatory sequences enabling expression of the site-specific recombinase in the eukaryotic cell.
  • the site-specific recombinase sequences is operably linked to an inducible promoter.
  • Dual-specific recombinase systems can also be employed, which may employ a recombinase enzyme in conjunction with direct or indirect repeats of two different site- specific recombination loci corresponding to the dual-specific recombinase, such as that described in International Patent Publication No. WO99/25840.
  • transposase catalyzes a recombination reaction only between two transposon border sequences.
  • transposon/transposase systems can be used including but not limited to the Ac/Ds system, the Spm system and the Mu system. All of these systems are operable in Zea mays, and at least the Ac/Ds and the Spm system function in other plants.
  • Preferred transposon sequences for use in the gene constructs of the invention are the Ds-type and the Spm-type transposons, which are delineated by border sequences of only 11 bp and 13 bp in length, respectively.
  • the present invention clearly encompasses the use of gene constructs which facilitate the expression of a transposase enzyme which is capable of specifically contacting the transposon border sequence, in conjunction with the gene constructs containing thecell cycle control protein-encoding gene.
  • a single gene construct may be used to express both the transposase and the cell cycle control protein, or alternatively, these may be introduced to plant cells on separate gene constructs.
  • the transposase-encoding gene could already be present in the plant genome prior to transformation with the gene construct of the invention, or alternatively, it may be introduced to the cell subsequent to transformation with the gene construct of the invention, such as, for example, by a separate transformation event, or by standard plant breeding involving hybridisation or cross-pollination.
  • a substantially purified transposase protein could be introduced directly into the eukaryotic cell, eg., by micro-injection or particle bombardment.
  • the transposase coding region will be operably linked to regulatory sequences enabling expression of the transposase in the eukaryotic cell.
  • the transposase-encoding sequence is operably linked to an inducible promoter.
  • transposon border sequences are organized as inverted repeats flanking the excisable genetic element.
  • transposons often re-integrate at another locus of the host's genome, segregation of the progeny of the hosts in which the transposase was allowed to act might be necessary to separate transformed hosts containing only the gene(s) of interest and transformed hosts containing only the cell cycle control protein-encoding gene.
  • the site-specific recombinase gene or transposase gene present in the host's genome can be removed by segregation of the progeny of the hosts to separate transformed hosts containing only the gene(s) of interest and transformed hosts containing only the site-specific recombinase gene or transposase gene.
  • said site-specific recombinase gene or transposase gene are included in the same or in a different excisable genetic element as thecell cycle control protein-encoding gene.
  • the promoter is a cell-specific, tissue-specific, organ-specific, or cell cycle-specific promoter sequence.
  • the plant morphological, biochemical or physiological characteristic which is modified is selected from the group consisting of: (i) plant growth and vigour; (ii) total biomass; (iii) cell number; (iv) flowering time; (v) branching; (vi) inflorescence formation; and (vii) seed set and/or seed size and/or seed yield
  • the promoter is a strong constitutive promoter sequence.
  • the present invention is not to be limited by the choice of promoter sequence.
  • the promoter selected for regulating expression of the cell cycle control protein in a plant cell, tissue or organ will confer expression in a range of cell-types or tissue-types or organs, sufficient to produce the desired phenotype, whilst avoiding undesirable phenotypes produced in other cell-types or tissue-types or organs.
  • a Cdc25 substrate or modified Cdc25 substrate including a Cdc2 protein or a modified Cdc2 protein, such as, for example, the Cdc2a or modified Cdc2a polypeptides and homologues, analogues and derivatives thereof, using publicly-available or readily-available sources, such as those listed in Table 1.
  • Placing a nucleic acid molecule under the regulatory control of a promoter sequence, or in operable connection with a promoter sequence, means positioning said nucleic acid molecule such that expression is controlled by the promoter sequence.
  • a promoter is usually, but not necessarily, positioned upstream, or at the 5'-end, and within 2 kb of the start site of transcription, of the nucleic acid molecule which it regulates.
  • heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting (i.e., the gene from which the promoter is derived). As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting (i.e., the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in gene constructs of the present invention include those listed in Table 1 , amongst others.
  • the promoters listed in Table 1 are provided for the purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention.
  • the promoter is a tissue-specific inducible promoter sequence, such as but not limited to a light-inducible rbcs-1A or rbcs-3A promoter, anoxia-inducible maize Adh1 gene promoter (Howard etal, 1987; Walker et al, 1987), hypoxia-inducible maize Adh 1 gene promoter (Howard et al, 1987; Walker et al, 1987), and the temperature-inducible heat shock promoter.
  • tissue-specific inducible promoter sequence such as but not limited to a light-inducible rbcs-1A or rbcs-3A promoter, anoxia-inducible maize Adh1 gene promoter (Howard etal, 1987; Walker et al, 1987), hypoxia-inducible maize Adh 1 gene promoter (Howard et al, 1987; Walker et al, 1987), and the temperature-inducible heat shock promoter.
  • Such environmentally- inducible promoters are reviewed
  • the promoter is a chemically-inducible promoter, such as the 3- ⁇ - indoylacrylic acid-inducible Tip promoter; IPTG-inducible lac promoter; phosphate-inducible promoter; L-arabinose-inducible araB promoter; heavy metal- inducible metallothionine gene promoter; dexamethasone-inducible promoter; glucocorticoid-inducible promoter; ethanol-inducible promoter (Zeneca); the N.N-diallyl- 2,2-dichloroacetamide-inducible glutathione-S-transferase gene promoter (Wiegand et al, 1986); or any one or more of the chemically-inducible promoters described by Gatz et al. (1996; 1998), amongst others.
  • a chemically-inducible promoter such as the 3- ⁇ - indoylacrylic acid-inducible Tip promoter; IP
  • the promoter is a wound-inducible or pathogen-inducible promoter, such as the phenylalanine ammonia lyase (PAL) gene promoter (Ebel etal, 1984), chalcone synthase gene promoter (Ebel et al, 1984) or the potato wound- inducible promoter (Cleveland et al, 1987), amongst others.
  • PAL phenylalanine ammonia lyase
  • the promoter is a hormone-inducible promoter, such as the abscisic acid-inducible wheat 7S globulin gene promoter and the wheat Em gene promoter (Marcotte et a/., 1988); an auxin-responsive gene promoter, such as, for example, the SAUR gene promoter, the parAs and parAt gene promoters(van der Zaal et al, 1991 ; Gil et al, 1994; Niwa et al, 1994); or a gibberellin-inducible promoter such as the Amy32b gene promoter (Lanahan et al. 1992), amongst others.
  • a hormone-inducible promoter such as the abscisic acid-inducible wheat 7S globulin gene promoter and the wheat Em gene promoter (Marcotte et a/., 1988
  • an auxin-responsive gene promoter such as, for example, the SAUR gene promoter, the parAs and parAt gene promoters(van der Za
  • the promoter is a cell cycle-specific gene promoter, such as the Cdc2a gene promoter sequence described by Chung and Parish (1995), or the PCNA gene promoter sequence described by Kosugi et al. (1991), and Kosugi and Ohashi (1997), amongst others.
  • the promoter is a strong constitutive plant- expressible promoter sequence such as the CaMV 35S promoter sequence, CaMV 19S promoter sequence, the octopine synthase (OCS) promoter sequence, or nopaline synthase (NOS) promoter sequence (Ebert et al. 1987), amongst others.
  • a strong constitutive plant- expressible promoter sequence such as the CaMV 35S promoter sequence, CaMV 19S promoter sequence, the octopine synthase (OCS) promoter sequence, or nopaline synthase (NOS) promoter sequence (Ebert et al. 1987), amongst others.
  • such sequences may be modified by the addition of nucleotide sequences derived from one or more of the tissue-specific promoters listed in Table 1 , or alternatively, nucleotide sequences derived from one or more of the above- mentioned tissue-specific inducible promoters, to confer tissue-specificity thereon.
  • the CaMV 35S promoter may be modified by the addition of maize Adh1 promoter sequence, to confer anaerobically-regulated root-specific expression thereon, as described previously (Ellis et al, 1987). Such modifications can be achieved by routine experimentation by those skilled in the art.
  • a method of modifying a morphological or physiological characteristic of a plant selected from the group consisting of: (i) increased growth and vigour; (ii) increased total biomass; (iii) increased cell number; (iv) modified flowering time; (v) increased branching; (vi) increased inflorescence formation; and (vii) increased seed set comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of the CaMV 35S promoter sequence.
  • a method of increasing the strength and/or thickness and/or stability and/or wind-resistance of a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein, therein operably under the control of a stem-expressible promoter sequence.
  • the stem-expressible promoter sequence is derived from the rbcs-1A gene, the rbcs-3A gene, the AtPRP4 gene, the T. bacilliform virus gene, or the sucrose- binding protein gene set forth in Table 1 , or a stem-specific or stem-expressible homologue, analogue or derivative thereof.
  • a method of increasing tuber formation and/or development in a tuberous crop plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a tuber-specific promoter sequence.
  • the tuberous crop plant is potato and the tuber-specific promoter is the potato patatin gene promoter. Additional species and promoters are not excluded.
  • a method of modifying the lignin content of a woody crop plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein, therein operably under the control of a cambium-specific or vascular-tissue-specific promoter sequence.
  • the promoter is a cinnamoyl alcohol dehydrogenase (CAD) gene promoter, laccase gene promoter, cellulose synthase gene promoter and xyloglucan endotransglucosylase (XET) gene promoter sequences, amongst others.
  • CAD cinnamoyl alcohol dehydrogenase
  • laccase gene promoter laccase gene promoter
  • cellulose synthase gene promoter xyloglucan endotransglucosylase (XET) gene promoter sequences, amongst others.
  • XET xyloglucan endotransglucosylase
  • Preferred target plant species according to this embodiment are woody plants of economic/ agronomic value, in particular hardwood crop plants such as, but not limited to Eucalyptus spp., Populus spp., Quercus spp., Acer spp., Juglans spp., Fagus spp., Acacia spp., or teak, amongst others. More preferably, this embodiment of the invention is applicable to modifying the lignin content of Eucalyptus spp., in particular E. globulus and E. robusta; or Quercus spp., in particular Q. dentata, Q. ilex, Q. incana, and Q.
  • Cdc2a or a variant thereof under control of a promoter that is operable in vascular tissue and preferably, in cambial cells, will produce thick-stemmed plants and a higher ratio of vascular tissue-to-pith cells within the stem, thereby resulting in more lignin production.
  • cambial cells contain the highest levels of auxins and are therefore the preferential tissue for the overexpression of a Cdc2 protein or a modified Cdc2 protein.
  • a method of increasing seed set and/or seed production and/or seed size and/or grain yield in a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a seed-specific promoter sequence.
  • the seed-specific promoter is operable in the seeds of monocotyledonous plants, for example the barley blz2 gene promoter, the barley Amy32b gene promoter, Cathepsin ⁇ -like gene promoter, wheat ADP-glucose pyrophosphorylase gene promoter, maize zein gene promoter, or rice glutelin gene promoter.
  • the seed-specific promoter is operable in the seeds of dicotyledonous plant species, for example the legumin gene promoter, napA gene promoter, Brazil Nut albumin gene promoter, pea vicilin gene promoter and sunflower oleosin gene promoter, amongst others.
  • grain yield in crop plants is largely a function of the amount of starch produced in the endosperm of the seed.
  • the amount of protein produced in the endosperm is also a contributing factor to grain yield.
  • the embryo and aleurone layers contribute little in terms of the total weight of the mature grain.
  • endoreplication and endoreduplication are generally considered as an important factor for increasing yield (Traas et al 1998).
  • enhancing, promoting or stimulating this process is likely to result in increased grain yield.
  • Enhancing, promoting or stimulating cell division during seed development is an alternative way to increase grain yield.
  • the present invention clearly encompasses the use of a Cdc25 substrate or modified Cdc25 substrate, such as a Cdc2 protein or modified Cdc2 protein, to modify endoreplucation and/or stimulate cell division.
  • a Cdc2-encoding gene preferably the Cdc2a or Cdc2aA14F15 gene, is placed operably in connection with a promoter that is operable in the endosperm of the seed, in which case the combination of the cell cycle- control protein and endosperm-expressible promoter provides the additional advantage of increasing the grain size and grain yield of the plant.
  • Endosperm-specific promoters that can be used to drive expression of a Cdc25 substrate or modified Cdc25 substrate protein have been identified.
  • the components of the promoters responsible for specific expression have been identified (Grosset et al (1997) and are interchangeable between agriculturally important cereals (Olsen et al 1992; Russell and Fromm, 1997).
  • promoters can be used, including the barley blz2 gene promoter, the rice prolamin NRP33 promoter, the rice REB promoter, the zein (ZmZ27) gene promoter, the rice glutelin 1 gene (osGT1) promoter, the rice small subunit ADP-glucose pyrophosphorylase (osAGP) promoter, the maize granule- bound starch synthase (Waxy) gene (zmGBS) promoter surveyed by Russell and Fromm (1997), the Brazil Nut albumin gene promoter, and the pea vicilin gene promoter, amongst others.
  • Promoters derived from those genes that are expressed in the endosperm during nuclear proliferation are also useful for driving expression of a Cdc25 substrate or modified Cdc25 substrate protein, such as, for example a Cdc2a protein or a homologue, analogue or derivative thereof, preferably Cdc2aA14F15. Promoters derived from those genes that are expressed in the endosperm at the stage when nuclear proliferation is ending could be ideal for extending this period. ln a preferred embodiment, the promoter sequence is derived from the wheat END1 gene described by Doan et al. (1996).
  • cytokinin level in the endosperm the number of endosperm cells formed during seed development and grain size, in which cytokinin activates Cdc2a kinase or modified Cdc2a kinase to drive nuclear division.
  • ectopic expression of the gene encoding a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein, in the endosperm enhances nuclear proliferation, resulting in increased grain size, without incurring the non-specific side effects that application of cytokinin or expression of the ipt gene would produce in the plant.
  • a further advantage of the present inventive approach is that the activity of cytokinin metabolising enzymes is circumvented by the ectopic expression of a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein, in the endosperm, thereby raising the activity of Cdc25 substrates or modified Cdc25 substrates therein.
  • a Cdc2 protein preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein, in the endosperm, thereby raising the activity of Cdc25 substrates or modified Cdc25 substrates therein.
  • cytokinin degrading enzymes including cytokinin oxidase (Chatfield and Armstrong 1987; reviewed by Morris et al 1993).
  • a method of inhibiting or reducing apical dominance or increasing the bushiness of a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a meristem-specific promoter sequence or a stem-specific promoter sequence.
  • a method of increasing lateral root production in a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a root-specific promoter sequence.
  • Preferred promoter sequences according to this embodiment of the present invention include any one of the root-expressible or root-specific promoters listed in Table 1 and in particular, the tobacco auxin-inducible gene promoter described by Van der Zaal et al (1991) that confers expression in the root tip of plants, in particular dicotyledonous plants.
  • a method of increasing the nitrogen-fixing capability of a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a nodule- specific promoter sequence.
  • Preferred nodule-specific promoter sequences according to this embodiment of the present invention are listed in Table 1. Additional promoters that are suited for this purpose include the hemoglobin gene promoters derived from Frankia spp., A. thaliana or other plants.
  • a method of preventing or delaying or otherwise reducing leaf chlorosis and/or leaf necrosis in a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a leaf-specific promoter sequence.
  • Preferred promoters for use according to this embodiment of the present invention include the SAM22 promoter, rbcs-1A and rbcs-3A gene promoters listed in Table 1.
  • the SAM22 gene promoter is particularly preferred in light of the developmental regulation of the SAM22 gene and its induction in senescent leaves.
  • A. thaliana Cdc2a or a homologue analogue or derivative thereof, in particular Cdc2aA14F15 is expressed in one of the specialised minority of plant tissues in which the activation of cell cycle progression that is generally contributed by cytokinin is in part performed by other hormones.
  • An example of such a tissue is the youngest stem internode of cereal plants in which gibberellic acid stimulates cell division.
  • the present invention preferably provides a method of stimulating cell division in the intercalary meristem of the youngest stem internode to produce greater elongation of the stem and/or to generate a more extensive photosynthetic canopy of a plant comprising ectopically expressing a Cdc2 protein, preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a meristem specific promoter sequence.
  • a Cdc2 protein preferably a modified Cdc2 protein, more preferably a modified Cdc2a protein or a homologue, analogue or derivative thereof, and, in particular, a Cdc2aA14F15 protein therein operably under the control of a meristem specific promoter sequence.
  • Preferred promoters for use according to this embodiment of the invention include meristem promoters listed in Table 1 and in particular the Proliferating Cell Nuclear Antigen (PCNA) promoter of rice described by Kosugi et al (1991).
  • PCNA Proliferating Cell Nuclear Antigen
  • a Cdc25 substrate or modified Cdc25 substrate protein in particular the Cdc2a or Cdc2aA14F15 protein, is expressed under the operable control of a plant-expressible promoter sequence.
  • this is generally achieved by introducing a gene construct or vector into plant cells by transformation or transfection means.
  • the nucleic acid molecule or a gene construct comprising same may be introduced into a cell using any known method for the transfection or transformation of said cell.
  • a cell is transformed by the gene construct of the invention, a whole organism may be regenerated from a single transformed cell, using methods known to those skilled in the art.
  • transfect is meant that the gene construct or vector or an active fragment thereof comprising the gene encoding the Cdc25 substrate or modified Cdc25 substrate, in particular, the gene encoding a Cdc2 protein or a homologue, analogue or derivative thereof, and preferably a gene encoding Cdc2aA14F15, operably under the control of the plant-expressible promoter sequence, is introduced into said cell without integration into the cell's genome.
  • transform is meant that the gene construct or vector or an active fragment thereof comprising the gene encoding the Cdc25 substrate or modified Cdc25 substrate, in particular, the gene encoding a Cdc2 protein, or a homologue, analogue or derivative thereof, and preferably a gene encoding Cdc2aA14F15, operably under the control of the plant-expressible promoter sequence, is stably integrated into the genome of the cell.
  • the present invention provides a method of modifying one or more plant morphological and/or biochemical and/or physiological characteristics comprising
  • a plant cell, tissue or organ a gene construct or vector comprising a nucleotide sequence that encodes a Cdc25 substrate or modified Cdc25 substrate, such as a Cdc2 protein or modified Cdc2 protein or a homologue, analogue, or derivative thereof, in particular Cdc2a or Cdc2aA14F15 protein, operably in connection with a plant-expressible promoter sequence selected from the list comprising strong constitutive promoter sequences; cell-specific promoter sequences, tissue-specific promoter sequences, cell cycle-specific gene promoter sequences, inducible promoter sequences and organ-specific promoter sequences; and
  • the inventive method comprises regenerating a whole plant from the transformed cell.
  • Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI 2 and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment, electroporation (Fromm et al, 1985), microinjection of DNA (Crossway et al, 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et a/.(1985), Herrera-Estrella et al. (1983a, 1983b, 1985), amongst others.
  • a microparticle is propelled into a cell to produce a transformed cell.
  • Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp etal. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No.4,945,050).
  • the gene construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • microparticles suitable for use in such systems include 1 to 5 m gold spheres.
  • the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
  • a whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • existing meristematic tissue e.g., apical meristem, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and hypocotyl meristem.
  • organogenesis means a process by which shoots and roots are developed sequentially from meristematic centres.
  • embryogenesis means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1) transformed plant may be selfed to give homozygous second generation (orT2) transformant, and the T2 plants further propagated through classical breeding techniques.
  • the generated transformed organisms contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed root stock grafted to an untransformed scion ).
  • the transformed plants are produced by a method that does not require the application of exogenous cytokinin and/or gibberellin during the tissue culture phase, such as, for example, an in planta transformation method.
  • plants are transformed by an in planta method using Agrobacterium tumefaciens such as that described by Bechtold et al, (1993) or Clough et al (1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed without the exogenous application of cytokinin and/or gibberellin.
  • Agrobacterium tumefaciens such as that described by Bechtold et al, (1993) or Clough et al (1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and
  • cytokinin modifies the phosphorylation of a Cdc25 substrate, in particular a Cdc2 protein and, as a consequence, the transformation and/or regeneration of plants in the absence of cytokinin facilitates the recovery of plants which express said Cdc25 substrate and functional homologues, analogues and derivatives thereof at a sufficiently high level to show significant differences from wild-type non-transformed plants.
  • the transformation of plants using gene constructs comprising nucleotide sequences encoding substrates or modified substrates of Cdc25, in particular a Cdc2 protein, such as, but not limited to, Cdc2a or modified Cdc2a, and, in particular Cdc2aA14F15, placed operably under the control of strong, constitutive promoter sequences, are particularly preferred for the purposes of modifying plant morphological, biochemical and physiological properties or characteristics in accordance with the present invention.
  • a further aspect of the present invention clearly provides the gene constructs and vectors designed to facilitate the introduction and/or expression and/or maintenance of the cell cycle control protein-encoding sequence and plant-expressible promoter into a plant cell, tissue or organ.
  • the gene construct of the present invention may further comprise one or more terminator sequences.
  • Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
  • terminators particularly suitable for use in the gene constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence (t3'Bt2), the Zea mays zein gene terminator sequence, the rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences, amongst others.
  • NOS nopaline synthase
  • OCS Agrobacterium tumefaciens octopine synthase
  • CaMV Cauliflower mosaic virus
  • t3'Bt2 Oryza sativa ADP-glucose pyrophosphorylase terminator sequence
  • Zea may
  • the gene constructs of the invention may further include an origin of replication sequence which is required for maintenance and/or replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.
  • an origin of replication sequence which is required for maintenance and/or replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.
  • Preferred origins of replication include, but are not limited to, the r -ori and co/E1 origins of replication.
  • the gene construct may further comprise a selectable marker gene or genes that are functional in a cell into which said gene construct is introduced.
  • selectable marker gene includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct of the invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp r ), tetracycline resistance gene (Tc r ), bacterial kanamycin resistance gene (Kar ), phosphinothricin resistance gene, neomycin phosphotransferase gene (t?pfll), hygromycin resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene, amongst others.
  • Amicillin resistance Amicillin resistance
  • Tc r tetracycline resistance gene
  • Kar bacterial kanamycin resistance gene
  • phosphinothricin resistance gene phosphinothricin resistance gene
  • neomycin phosphotransferase gene t?pfll
  • hygromycin resistance gene ⁇ -glucuronidase
  • a further aspect of the invention clearly extends to a plant cell, tissue, organ or whole plant that has been transformed or transfected with an isolated nucleic acid molecule that comprises a nucleotide sequence which encodes a cell cycle control protein, wherein the expression of said nucleotide sequence is placed operably under the control of a plant-expressible cell-specific promoter sequence, plant-expressible tissue- specific promoter sequence, plant-expressible cell cycle specific gene promoter sequence, or plant-expressible organ-specific promoter sequence or inducible promoter.
  • the invention extends to a plant cell, tissue, organ or whole plant that has been transformed or transfected with an isolated nucleic acid molecule that comprises a nucleotide sequence which encodes the cell cycle control protein, placed operably under the control of a plant-expressible constitutive promoter sequence, such that said plant-expressible constitutive promoter sequence and said nucleotide sequence encoding a cell cycle control protein are integrated into a transposable genetic element.
  • the present invention is applicable to any plant, in particular a monocotyledonous plants and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp.,
  • the present invention clearly extends to any plant produced by the inventive method described herein, and any and all plant parts and propagules thereof.
  • the present invention extends further to encompass the progeny derived from a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by the inventive method, the only requirement being that said progeny exhibits the same genotypic and/or phenotypic characteristic(s) as that (those) characteristic(s) that has (have) been produced in the parent by the performance of the inventive method.
  • genotypic characteristic is meant the composition of the genome and, more particularly, the introduced gene encoding the cell cycle control protein.
  • phenotypic characteristic is meant one or more plant morphological characteristics and/or plant biochemical characteristics and/or plant physiological characteristics that are produced by ectopic expression of a cell cycle control protein in a plant.
  • the plant is produced according to the inventive method is transfected or transformed with a genetic sequence, or amenable to the introduction of a protein, by any art-recognised means, such as microprojectile bombardment, microinjection, /-gro- ⁇ acter/um-mediated transformation (including in planta transformation), protoplast fusion, or electroporation, amongst others.
  • any art-recognised means such as microprojectile bombardment, microinjection, /-gro- ⁇ acter/um-mediated transformation (including in planta transformation), protoplast fusion, or electroporation, amongst others.
  • Transformation procedures A binary plasmid vector containing the A. thaliana Cdc2a gene (hereinafter "Cdc2aAf) under control of the CaMV 35S promoter, and a kanamycin resistance gene selectable marker was introduced into the bacterium Agrobacterium tumefaciens by triparental mating. The transformed A. tumefaciens was then introduced into wounded roots from which genetically-transformed kanamycin resistant callus was derived, and shoot and root formation was stimulated by adjustment of auxin to cytokinin ratio during culture to regenerate transgenic plants, essentially as described by Hemerly et al (1995).
  • Transgenic plants were derived from separate plant populations and are therefore the products of independent transformation events. Individuals having the introduced gene construct at a single point of insertion were selected according to their segregation ratio for the kanamycin-resistance phenotype when selfed. In particular, those plants giving a 3:1 (resistance: sensitivity) segregation ratio were selected. The progeny of these homozygous plants were analysed further for their growth and vigour characteristics compared to otherwise isogenic non-transformed plants.
  • Plants were grown in containers that are permeable to atmospheric gases, on Murashige and Skoog (1962) mineral salts medium containing 0.75 %(w/v) agar, and without added sugar, at 25°C, and a moderate light intensity of about 80 ⁇ mol m ⁇ 2 sec "1 .
  • Cdc2a protein was determined by western blotting using two antibodies: (i) anti-(PSTAIR) antibodies, which are specific for all CDKs having the PSTAIR amino acid sequence motif, including Cdc2a, modified Cdc2a and any other endogenous CDK present in the cell; and
  • anti-(Cdc2a carboxyl terminal peptide) antibodies which are specific for wild- type Cdc2a and is able to discriminate between endogenous (wild-type) Cdc2a and modified Cdc2a having an altered C-terminus.
  • Flowering time for both transformed and non-transformed plants was determined as the number of days from germination to the opening of the first flower on a plant and reflexing of the petals of said flower, following growth at 21 °C in a photoperiod comprising 18 hour light (80 ⁇ mol m "2 sec "1 ) and 6 hour dark.
  • Time to seed set for both transformed and non-transformed plants was determined as the number of days from germination to the attainment of a first silique of length greater than 7 mm.
  • silique elongation in A. thaliana is an indicator of seed development and formation in that plant.
  • C24 WT and Cdc2a transgenics Shoot biomass, branching, fruit (silique) number and seed size
  • EXAMPLE 2 Modification of growth, vigour, flowering time and seed set by ectopic expression of modified Cdc2a (Cdc2aA14F15) under control of the CaMV 35S promoter in plants
  • a binary plasmid vector containing a modified A. thaliana Cdc2a gene which expresses a non-phosphorylateable Cdc2a protein (hereinafter "Cdc2aA14F15"; Hemerly et al, 1995) under control of the CaMV 35S promoter, and a kanamycin resistance gene selectable marker was introduced into Agrobacterium tumefaciens by triparental mating. The transformed A. tumefaciens was then introduced into the developing flower bud of A.
  • thaliana plants and the seeds developing therefrom were germinated on mineral salts medium supplemented with thiamine and myoinositol (Murashige and Skoog 1962), and containing kanamycin (50 ⁇ g/ml) and 30 mM sucrose. Selection was made at an early stage of growth purely on the basis of kanamycin resistance.
  • PCR was performed using amplification primers that specifically detect the presence of either the wild-type Cdc2a gene or alternatively, the presence of the mutant Cdc2aA14F15 gene.
  • the presence of the wild-type Cdc2a gene was confirmed using the primer pair:
  • Primer 1 (WT 5'): 5'-GTTGAGAAGATTGGTGAAGGAACTTA-3'; and Primer 3:(common 3'): 5'-GTTGAGAAGATTGGTGAAGGAGCTTT-3'.
  • Reactions were commenced by the addition of DNA, or alternatively, by the addition of water for negative controls samples.
  • Amplification reactions were performed in a total volume of 100 ⁇ l comprising 1 ⁇ l plant DNA (0.15 ⁇ g/ ⁇ l); 74 ⁇ l sterile H 2 O; 10 ⁇ l Taq polymerase buffer; 10 ⁇ l 50% (v/v) glycerol; 2.5 ⁇ l deoxynucleoside triphosphate mix (2.5 mM each of dATP, dTTP, dCTP, dGTP); 1 ⁇ l forward primer (25 ⁇ M stock); 1 ⁇ l reverse primer (25 ⁇ M stock); and 0.5 ⁇ l Taq polymerase (2.5 U). Reaction conditions were as follows:
  • the product size expected to be amplified is 856 bp in length.
  • Data presented in Figure 3 indicate that a band of the expected size was present in one putatively transformed sample using primers that specifically detect the mutant Cdc2aA14F15 gene (see lane 7), but not using primers that detect the wild-type Cdc2a gene (Lane 6).
  • a band of the same size was only detected in non-transformed plants using primers that detect the wild-type Cdc2a gene (Lane 3). No amplification products were detected in control samples having no added DNA.
  • Plants were grown in containers that are permeable to atmospheric gases, on
  • Chlorophyll a (Chi a) and chlorophyll b (Chi b) levels were estimated by absorbance at 663.8 nm and 646.8 nm, respectively, as described by Porra et al (1989).
  • Chlorophyll content ( ⁇ g per gram fresh weight) of shoot tissue in A. thaliana that ectopically express the Cdc2aA14F15 gene
  • DAPI 4,6-diamidino-2-phenylindole
  • the level of Cdc2a protein in the transformed plants was determined using western blotting as described in Example 1.
  • the transgenic line 5A2 8C does not show any elevation of Cdc2a protein level and may have integrated only the Kanamycin resistance gene with the modified Cdc2a gene.
  • the Cdc2a protein is increased in plants for several reasons.
  • Third, the CaMV 35S promoter which regulates expression of both the introduced Cdc2a gene and the modified Cdc2aA14F15 gene is stronger than the endogenous Cdc2a gene promoter.
  • Cell size for transformed and non-transformed plants was determined by examining the lower leaf epidermis of unfixed turgid leaves using a Zeiss Axiovert 35M at X40 magnification, with DIC optics and Metamorph 4.0 software.
  • Results are presented in Figure 6.
  • 1 cm corresponds to 25 ⁇ m of leaf.
  • Data presented in Figure 6 indicate that there is no difference in cell size between transformed and non-transformed lines. Accordingly, we conclude that the increased size and biomass of plants transformed with either the mutant Cdc2aA14F15 gene or the wild-type Cdc2a gene is due to an increase in cell number. Such an increase in cell number may be the result of more rapid cell cycling and/or an increase in the number of cells dividing (i.e. larger meristematic centres).
  • Flowering times and seed set We also determined the times to flowering and seed set for the transformed and non- transformed plants.
  • Flowering time for both transformed and non-transformed plants was determined as the number of days from germination to the opening of the first flower on a plant and reflexing of the petals of said flower, following growth at 21 °C in a photoperiod comprising 18 hour light (80 ⁇ mol m 2 sec "1 ) and 6 hour dark.
  • Time to fruit (silique) formation for both transformed and non-transformed plants was determined as the number of days from germination to the attainment of a first silique of length greater than 7 mm.
  • silique elongation in A. thaliana is an indicator of seed development and formation in that plant.
  • flowering time is reduced by up to about 10% in the homozygous, transformed lines of plants, compared to otherwise isogenic non- transformed A. thaliana Columbia plants. This corresponds to a reduction in at least up to 3-4 days in the time to flowering for these transformed plants. Similar results were obtained for the time to seed set in the transformed plant lines. However, these data are qualified by the fact the, at the time these measurements were taken, not all of the control wild-type non-transgenic plants had flowered. As a consequence, the real reduction in both flowering time and seed set for the transformed lines is greater than that indicated in Table 10.
  • the transformed lines that grew the fastest in the first 11 days from germination are generally not earlier-flowering than those lines that grew more slowly.
  • the earliest-to-flower transformed lines were about twice the size of the control plants at 11 days, whilst the larger plants flowered more slowly. Possibly, in the larger plants, increased growth potential had been more directed to producing lateral branches than to early flower production.
  • C24 WT and Cdc2aA14F15 transgenics Shoot biomass, branching, fruit
  • the A. thaliana Cdc2a coding sequence is cloned between the promoter of a class I patatin gene ( Liu et al, 1991) and the transcription termination signals of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens.
  • the B repeat region and the distal region of the A repeat of the patatin promoter is used, without the proximal region of the A repeat.
  • the proximal region of the A repeat of the patatin promoter confers sucrose-responsiveness in various tissues, which is not a desirable characteristic for our purposes (Grierson et al, 1994).
  • This construct is placed in a binary vector, mobilized to Agrobacterium tumefaciens, and the introduced into potato plants.
  • the Cdc2a protein is expressed under the control of the Class I patatin promoter when the first stolon starts to tuberize, consistent with the expression pattern for the patatin gene (Liu et al, 1991). At this stage, expression is associated with both internal and external phloem. After tuber induction has occurred, promoter activity is found both in tuberized stolons and in non-tuberized stolons. Expression then expands to the entire storage parenchyma, cortex and pith, but remains absent from the periderm.
  • Class II patatin promoters are expressed in the periderm and as such are complementary to the Class I promoters (K ⁇ ster-T ⁇ pfer et al.,; Liu et al, 1991 ; Nap et al, 1992), it is beneficial to have Cdc2a expression driven by both Class I and Class II promoters within the same plant. Because the Class I patatin promoter is not expressed before the first stolon initiates tuberization, no effects of Class I patatin- Cdc2a transgenes is seen on tuber initiation.
  • the Class I patatin promoter drives Cdc2a expression very early after tuber initiation onwards, allowing a maximal impact of Cdc2a activity on organ formation and, as a consequence, on tuber size.
  • the A. thaliana Cdc2a coding sequence is cloned between the promoter of the soybean SAUR gene (Li et al, 1992 ) and the transcription termination signals of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens.
  • the SAUR promoter is inducible by auxins.
  • This chimeric gene construct is introduced between the T-DNA borders of the binary vector pBI121 or similar vector and mobilised into Agrobacterium tumefaciens. Poplar is transformed by Agrobacterium-med ⁇ ated transformation using standard procedures.
  • Transgenic poplar trees containing this construct show increased lignin content, correlated with an increased stem diameter and the higher ratio of vascular tissue to pith and cortex cells.
  • a similar phenotype in poplar is produced when the Cdc2a expression is driven by the rolB promoter of Agrobacterium rhizogenes (Nilsson et al, 1997), that is expressed in cambial cells (i.e. the dividing cells of the vascular tissue).
  • the A. thaliana Cdc2a coding sequence is placed operably in connection with the endosperm-specific Itr1 promoter from barley, or a synthetic promoter containing the endosperm box (GCN motif) of the barley Hor2 gene (Vicente-Carbajosa et al, 1998).
  • the Cdc2a structural gene is placed upstream of the transcription termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene.
  • Cereals, in particular rice, maize, wheat and bariey are transformed using standard procedures, in particular microprojectile bombardment or Agrobacter/i/m-mediated transformation systems, with the gene constructs.
  • the A. thaliana Cdc2a coding sequence is placed operably in connection with the shoot meristem-specific LEAFY promoter (Weigel et al, 1992), or the KNOTTED-like A. thaliana knatl promoter (Accession number AJ131822), or the KNOTTED-like Malus domestica kn1 promoter ( Accession No. Z71981), or the A. thaliana CLAVATA1 promoter (Accession number AF049870).
  • the Cdc2a structural gene is placed upstream of the transcription termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene.
  • NOS nopaline synthase
  • Transformed plants exhibit cytokinin-like effects at the level of the shoot (and flower) meristem, resulting in reduced apical dominance.
  • Cdc2a under the control of the cab-6 or ubi7 promoters reduces leaf necrosis and chlorosis in lettuce plants
  • the A. thaliana Cdc2a coding sequence is placed operably in connection with the leaf- specific cab-6 gene promoter derived from Pinus (Yamamoto et al, 1994) or senescence-specific ubi7 gene promoter (Garbarino et al, 1995).
  • the Cdc2a structural gene is placed upstream of the transcription termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene. Lettuce is transformed as described by Bechtold et al, 1993.
  • Leaf deterioration (chlorosis and necrosis) in lettuce for example as a consequence of post-harvest storage, is delayed in transformed lettuce plants compared to non- transformed control plants.
  • transgenic lines YF-D and YF-E showed a 75% incidence of kanamycin resistance among progeny when the initial hemizygous individual was self-fertilised, indicative of a single point of insertion of the transgenic DNA.
  • Plants of the transgenic lines YF-D and YF-E were categorised after 11 days of growth, on the basis of whole-seedling fresh weight, and seedling size class (big, mid or small). In each category, 14 individuals were pooled; this being the largest number of individuals that in 99% of cases would be present in a total population of 100 individuals in which a single transgene insertion was segregating.
  • Plants were also subjected to western blotting as described for Figure 5, to determine Cdc2a protein expression level, and to confirm the presence of the functional transgene.
  • proteins were separated using SDS/PAGE transferred to PVDF membranes and stained to visualise the transferred proteins, using 0.1 % (w/v) polyvinylpyrrolidone-40 followed by immersion in 0.75 mM ferrozine, 20 mM FeCI 2 , 2% (v/v) glacial acetic, followed by immersion in 100 mM ferrocyanide, 60 mM FeCI 2 , 100 mM Na-Acetate, buffered at pH4 with glacial acetic acid.
  • FIG. 7 shows the inheritance of the Cdc2aAt.A14F15 transgene and enhanced growth effects seen in A. thaliana seedlings from self-fertilisation of initial transgenic isolate line YF-D (panels A, B) and a different transgenic isolate line YF-E (Panels C, D).
  • Panels (A) and (C) show a Western blot probed with antibody specific for the cdc2a.At gene product (the 34 kDa Cdc2a protein is indicated by the arrows), to detect the expression of the transgene (lanes 1-3), relative to the amount of protein detected in a non- transgenic isogenic plant (lane 4).
  • the fresh weight of the plants measured as the weight of a whole seedling including root and shoot, and the size of the plant, is positively correlated with the amount of Cdc2a protein expressed, as detected in Western blots.
  • the capacity of the Cdc2aAt.A14F15 transgene to be segregated out of the slower- growing plants further indicates a single point of insertion of the introduced DNA and confirms that the presence and expression of the transgene induces faster growth of plants.
  • Fresh weight and size of transgenic plants expressing Cdc2aAtA14F15 is correlated with the amount of Cdc2aAtA14F15 protein expressed in plants

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Abstract

L'invention concerne un procédé d'accélération et d'accroissement de la production de la biomasse, des branches, fleurs et fruits, ainsi que de modification d'une ou plusieurs propriétés ou caractéristiques morphologiques, biochimiques et physiologiques, telles qu'au moins une réponse adaptative à l'environnement et/ou des processus de développement de plantes, ce procédé consistant à faire s'exprimer de manière ectopique, dans des plantes, un substrat de Cdc25 ou un substrat modifié de Cdc25, notamment une protéine Cdc2 ou un homologue, analogue ou dérivé de celle-ci.
EP00906072A 1999-02-26 2000-02-25 Procede de modification de la morphologie, biochimie ou physiologie de plantes, a l'aide de substrats comprenant cdc25 Withdrawn EP1161541A4 (fr)

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AU780301B2 (en) * 1999-09-27 2005-03-17 Pioneer Hi-Bred International, Inc. Enhanced stress tolerance in maize via manipulation of cell cycle regulatory genes
AU2005274622A1 (en) 2004-08-18 2006-02-23 Alellyx S.A. Polynucleotides, DNA constructs and methods for the alteration of plant lignin content and/or composition
KR101581039B1 (ko) 2005-02-28 2016-01-05 22엔디 센츄리 리미티드, 엘엘씨 식물의 니코틴 알칼로이드 수준의 감소
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