Methods for increasing plant cell proliferation by functionally inhibiting a plant cyclin inhibitor gene

Abstract

The present invention provides methods for modulating the growth and/or yield of plants. In particular the methods comprises the use of agents which functionally inhibit the expression of plant D-like cyclin inhibitors including isolated polynucleotide sequences which interact with DNA or RNA encoding proteins capable of binding plant D-like cyclins. Further, the present invention provides recombinant polynucleotide sequences, vectors and host cells which encode proteins capable of binding to and inactivating the activity of plant D-like cyclin/cyclin dependent kinase complexes preventing plant cells from exiting the cell cycle. Methods for determining and agents which are inhibitors of the BRO cyclin dependent kinase inhibitor proteins which are capable of modulating plant cell cycle progression are also provided. Methods for the production of transgenic plant cells and plants with increased growth rates and yields when compared to wild-type plants are also provided.

1 . A method for producing a hyperplastic variant plant, comprising functionally inactivating expression of one or more BRO genes, wherein said BRO genes comprises a polynucleotide that encodes a domain comprising 5 to 10 amino acid residues which is a cyclin binding domain having a consensus binding domain comprising the amino acid residues Glu Xaa 1  Xaa 2  Xaa 3  Xaa 4  Phe (SEQ ID NO: 9), wherein Xaa 1  can be Leu, Ile or another hydrophobic amino acid residue, Xaa 2  can be Glu or Asp, Xaa 3  can be Leu, Arg, Asp or any other amino acid residue, and Xaa 4  can be Phe, Leu, or another hydrophobic amino acid residue; and wherein said gene comprises a polynucleotide that encodes a cyclin dependent kinase binding domain comprising the consensus amino acid sequence Lys Tyr Asn Phe Asp Xaa 1  Xaa 2  Xaa 3  Xaa 4  Xaa 5  Pro Leu Xaa 6  Xaa 7  Gly Arg Tyr Xaa 8  Trp Xaa 9  Xaa 10  Leu Xaa 1  (SEQ ID NO: 10), wherein Xaa 1  can comprise Phe or Ile, Xaa 2  can comprise Glu or Val, Xaa 3  can comprise Lys or Asn, Xaa 4  can comprise Asp or Glu, Xaa 5  can comprise Glu or Lys, Xaa 6  can comprise Gly or Glu, Xaa 7  may be absent or can comprise Gly, Xaa 8  can comprise Glu or Lys, Xaa 9  can comprise Val or Asp, Xaa 10  can comprise Lys or Arg, and Xaa 11  can comprise Asn or Glu, wherein the hyperplasticity is relative to a wild-type plant. 
     
     
         2 . The method of  claim 1 , wherein the BRO gene is a BRO4 gene comprising a polynucleotide encoding the polypeptide having the amino acid sequence of SEQ ID NO:8. 
     
     
         3 . The method of  claim 1 , wherein the genome of said hyperplastic variant plant comprises a structurally disrupted plant D-like cyclin inhibitor gene. 
     
     
         4 . The method of  claim 1 , wherein an antisense or inverted repeat polynucleotide functionally inactivates expression of the plant D-like cyclin inhibitor gene in the plant. 
     
     
         5 . The method of  claim 1 , wherein the functionally inactivated plant D-like cyclin inhibitor gene is structurally disrupted by homologous recombination with a targeting construct. 
     
     
         6 . A polynucleotide targeting construct comprising a sequence that is homologous to a sequence present in a plant D-like cyclin inhibitor gene and which, when integrated at the corresponding plant D-like cyclin inhibitor gene locus, functionally inactivates plant D-like cyclin inhibitor protein expression. 
     
     
         7 . A polynucleotide targeting construct of  claim 6 , wherein said plant D-like cyclin inhibitor gene is a BOR gene. 
     
     
         8 . A hyperplastic plant having one or more functionally inactivated BRO genes, comprising a polynucleotide that encodes an amino acid sequence domain comprising 5 to 10 amino acid residues which is a cyclin binding domain having a consensus binding domain comprising the amino acid residues Glu Xaa 1  Xaa 2  Xaa 3  Xaa 4  Phe (SEQ ID NO: 9), wherein Xaa 1  can be Leu, Ile or another hydrophobic amino acid residue, Xaa 2  can be Glu or Asp, Xaa 3  can be Leu, Arg, Asp or any other amino acid residue, and Xaa 4  can be Phe, Leu, or another hydrophobic amino acid residue; and wherein said gene comprises a cyclin dependent kinase binding domain comprising the consensus amino acid sequence Lys Tyr Asp Phe Asp Xaa 1  Xaa 2  Xaa 3  Xaa 4  Xaa 5  Pro Leu Xaa 6  Xaa 7  Gly Arg Tyr Xaa 8  Trp Xaa 9  Xaa 10  Leu Xaa 11  (SEQ ID NO: 10), wherein Xaa 1  can comprise Phe or Ile, Xaa 2  can comprise Glu or Val, Xaa 3  can comprise Lys or Asn, Xaa 4  can comprise Asp or Glu, Xaa 5  can comprise Glu or Lys, Xaa 6  can comprise Gly or Glu, Xaa 7  may be absent or can comprise Gly, Xaa 8  can comprise Glu or Lys, Xaa 9  can comprise Valor Asp, Xaa 10  can comprise Lys or Arg, and Xaa 11  can comprise Asn or Glu, the hyperplasticity being relative to a wild-type plant. 
     
     
         9 . The hyperplastic plant of  claim 8 , wherein the BRO gene is a BRO4 gene comprising a polynucleotide encoding the polypeptide having the amino acid sequence of SEQ ID NO:8. 
     
     
         10 . A method for increasing the growth rate of a plant, comprising functionally inactivating expression of a plant D-like cyclin inhibitor gene in a plant wherein the growth rate of the plant is increased relative to a wild-type plant of the same species having the functional plant D-like cyclin inhibitor gene. 
     
     
         11 . The method of  claim 10 , wherein the expression of the plant D-like cyclin inhibitor is functionally inactivated by an antisense or an inverted repeat polynucleotide. 
     
     
         12 . A method for increasing the proportion of dividing cells in a plant cell population comprising:
 exposing said population of cells to an inhibitor of a plant D-like cyclin inhibitor in an amount sufficient to increase the proportion of dividing cells to non-dividing cells relative to said proportion in a population of untreated cells.   
     
     
         13 . The method according to  claim 12 , wherein the cell population comprises protoplasts, seeds, root cells, meristem cells or leaf cells. 
     
     
         14 . An inhibitor of a plant D-like cyclin inhibitor which comprises an oligonucleotide that specifically binds to DNA encoding BRO4 or an RNA transcribed therefrom and inhibits expression of BRO4 protein. 
     
     
         15 . The method of  claim 1 , wherein the plant D-like cyclin inhibitor gene is an ICK1 gene. 
     
     
         16 . The hyperplastic plant of  claim 8 , wherein the plant D-like cyclin inhibitor gene is an ICK1 gene. 
     
     
         17 . A method for increasing cell proliferation in a plant, comprising functionally inactivating expression of one or more BRO genes, wherein said BRO genes comprise a polynucleotide that encodes a domain comprising 5 to 10 amino acid residues which is a cyclin binding domain having a consensus binding domain comprising the amino acid residues Glu Xaa 1  Xaa 2  Xaa 3  Xaa 4  Phe (SEQ ID NO: 9), wherein Xaa 1  can be Leu, Ile or another hydrophobic amino acid residue, Xaa 2  can be Glu or Asp, Xaa 3  can be Leu, Arg, Asp or any other amino acid residue, and Xaa 4  can be Phe, Leu, or another hydrophobic amino acid residue; and wherein said gene comprises a polynucleotide that encodes a cyclin dependent kinase binding domain comprising the consensus amino acid sequence Lys Tyr Asn Phe Asp Xaa 1  Xaa 2  Xaa 3  Xaa 4  Xaa 5  Pro Leu Xaa 6  Xaa 7  Gly Arg Tyr Xaa 8  Trp Xaa 9  Xaa 10  Leu Xaa 11  (SEQ ID NO: 10), wherein Xaa r  can comprise Phe or Ile, Xaa 2  can comprise Glu or Val, Xaa 3  can comprise Lys or Asn, Xaa 4  can comprise Asp or Glu, Xaa 5  can comprise Glu or Lys, Xaa 6  can comprise Gly or Glu, Xaa 7  may be absent or can comprise Gly, Xaa 8  can comprise Glu or Lys, Xaa 9  can comprise Val or Asp, Xaa 10  can comprise Lys or Arg, and Xaa 11  can comprise Asn or Glu, wherein the increase in cell proliferation is relative to a wild-type plant. 
     
     
         18 . The method of  claim 17 , wherein an antisense or inverted repeat polynucleotide functionally inactivates expression of the plant BRO gene. 
     
     
         19 . The method of  claim 17 , wherein the plant BRO gene is a BRO4 gene comprising a polynucleotide encoding the polypeptide having the amino acid sequence of SEQ ID NO: 8.