Modulation of plant cyclin-dependent kinase inhibitor activity

Abstract

In various aspects, the invention provides methods of modifying plant development using cyclin-dependent kinase (CDK) inhibitors, cyclins and proteins that bind to CDK inhibitors, including methods of genetic modification utilizing nucleic acids encoding such proteins. In alternative aspects, the invention may provide methods of plant breeding that facilitate the modulation of plant phenotype in alternative generations. The invention may also provide plants produced by the methods of the invention, including transgenic plants having recombinant genomes that include heterologous sequences encoding CDK inhibitors, cyclins and proteins that bind to CDK inhibitors.

What is claimed is:  
     
         1 . A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a protein that binds to or interacts with a cyclin-dependent kinase inhibitor polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the protein that binds to the cyclin-dependent kinase inhibitor polypeptide and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the protein that binds to the cyclin-dependent kinase inhibitor polypeptide.  
     
     
         2 . The method of  claim 1 , wherein the effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is to inhibit development of a differentiated tissue in the plant, and the inhibition of development of the differentiated tissue is at least partly reversed by the protein that binds to the cyclin-dependent kinase inhibitor polypeptide.  
     
     
         3 . The method of  claim 2 , wherein the cyclin-dependent kinase inhibitor polypeptide is expressed in the proliferative tissue of the plant.  
     
     
         4 . The method of  claim 2  or  3 , wherein the protein that binds to the cyclin-dependent kinase inhibitor polypeptide is expressed in the proliferative tissue of the plant  
     
     
         5 . The method of any one of claims  1  through  4  wherein the cyclin-dependent kinase inhibitor polypeptide is a plant cyclin-dependent kinase inhibitor polypeptide.  
     
     
         6 . The method of any one of claims  1  through  5 , wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor is at least 80% identical, when optimally aligned, to a sequence encoding a cyclin-dependent kinase inhibitor selected from the group consisting of ICK1, ICK2, ICN2 (ICK4), ICN6 (ICK5), ICN7 (ICK6) and ICN8 (ICK7).  
     
     
         7 . The method of any one of claims  1  through  5 , wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor is at least 80% identical, when optimally aligned, to a coding sequence selected from a group consisting of the coding sequences of ICK1, ICK2, ICN2, ICN6, ICN7 and ICN8.  
     
     
         8 . The method of any one of claims  1  through  5 , wherein the cyclin-dependent kinase inhibitor polypeptide comprises a peptide sequence that is at least 80% identical, when optimally aligned, to a peptide sequence selected from the group consisting of the peptide sequences of ICK1, ICK2, ICN2, ICN6, ICN7 and ICN8.  
     
     
         9 . The method of any one of claims  1  through  5 , wherein the cyclin-dependent kinase inhibitor polypeptide is selected from a group consisting of ICK1, ICK2, ICN2, ICN6, ICN7 and ICN8.  
     
     
         10 . The method of any one of claims  1  through  9 , wherein the protein that binds to the CDK inhibitor is a cyclin or a polypeptide fragment of the cyclin.  
     
     
         11 . The method of  claim 10 , wherein the cyclin is a D-type cyclin, cyclin D2 or cyclin D3.  
     
     
         12 . The method of  claim 10  or  11 , wherein the polypeptide fragment of the cyclin comprises at least 10 amino acids.  
     
     
         13 . The method of any one of claims  1  through  9 , wherein the protein that binds to the CDK inhibitor is a cyclin-dependent kinase (CDK) or a polypeptide fragment of the CDK.  
     
     
         14 . The method of  claim 13 , wherein the CDK is Arabidopsis Cdc2a.  
     
     
         15 . The method of  claim 13  or  14 , wherein the polypeptide fragment of the CDK comprises at least 10 amino acids  
     
     
         16 . The method of any one of claims  1  through  15 , wherein the plant is a dicotyledon or monocotyledon plant  
     
     
         17 . The method of any one of claims  1  through  15 , wherein the plant is a member of the Cruciferae family.  
     
     
         18 . The method of any one of claims  1  through  15 , wherein the plant is a member of the Brassica genus.  
     
     
         19 . The method of any one of claims  1  through  15 , wherein the plant is a member of the tobacco family.  
     
     
         20 . The method of any one of claims  1  through  19 , wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide is operably linked to a constitutive promoter.  
     
     
         21 . The method of any one of claims  1  through  19 , wherein the nucleic acid encoding the protein that binds to the cyclin-dependent kinase inhibitor polypeptide is operably linked to a constitutive promoter.  
     
     
         22 . The method of any one of claims  1  through  19 , wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide is operably linked to a tissue-specific promoter.  
     
     
         23 . The method of any one of claims  1  through  19 , wherein the nucleic acid encoding the protein that binds to the cyclin-dependent kinase inhibitor polypeptide is operably linked to a tissue-specific promoter.  
     
     
         24 . The method of  claim 22  or  23 , wherein the tissue-specific promoter is an AP3 promoter.  
     
     
         25 . The method of  claim 22  or  23 , wherein the tissue-specific promoter is an NTM19 promoter.  
     
     
         26 . The method of any one of claims  2  through  25  wherein the differentiated tissue in the plant is a male reproductive tissue.  
     
     
         27 . The plant produced by the method of any one of claims  1  through  26 .  
     
     
         28 . A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a cyclin polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the cyclin polypeptide and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the cyclin polypeptide.  
     
     
         29 . The method of  claim 28 , wherein the cyclin polypeptide is a full length cyclin or a polypeptide fragment of the full length cyclin.  
     
     
         30 . The method of  claim 29 , wherein the full length cyclin is selected from the group consisting of cyclin D2 and cyclin D3.  
     
     
         31 . The method of  claim 29  or  30 , wherein the polypeptide fragment of the cyclin comprises at least 10 amino acids.  
     
     
         32 . A plant having a recombinant genome comprising: 
 a) a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor; and,    b) a heterologous nucleic acid encoding a protein that binds to the cyclin-dependent kinase inhibitor.    
     
     
         33 . The plant of  claim 32 , wherein the protein that binds to the cyclin-dependent kinase inhibitor is a cyclin or a polypeptide fragment of the cyclin.  
     
     
         34 . A plant having a recombinant genome comprising: 
 a) a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor; and,    b) a heterologous nucleic acid encoding a cyclin or a polypeptide fragment of the cyclin.    
     
     
         35 . The plant of  claim 33  or  34 , wherein the cyclin is selected from the group consisting of cyclin D2 and cyclin D3.  
     
     
         36 . A plant tissue derived from the plant of any one of claims  27  or  32  through  35 .  
     
     
         37 . The plant tissue of  claim 36  wherein the tissue is a seed.  
     
     
         38 . A method of growing a plant, wherein the plant comprises a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide and a heterologous nucleic acid encoding a protein that binds to the cyclin-dependent kinase inhibitor polypeptide, the method comprising growing the plant under conditions so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the protein that binds to the cyclin-dependent kinase inhibitor polypeptide.  
     
     
         39 . A method of growing a plant, wherein the plant comprises a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide and a heterologous nucleic acid encoding a cyclin polypeptide, the method comprising growing the plant under conditions so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the cyclin polypeptide.  
     
     
         40 . A method of plant breeding comprising: 
 a) providing a first parent plant having a heterologous nucleic acid sequence encoding a cyclin-dependent kinase inhibitor polypeptide;    b) providing a second parent plant having a heterologous nucleic acid sequence encoding a protein that binds to the cyclin-dependent kinase inhibitor polypeptide;    c) crossing the first and second parent plants to obtain a hybrid offspring plant having a copy of the heterologous sequence encoding the cyclin-dependent kinase inhibitor and a copy of the heterologous sequence encoding the protein that binds to the cyclin-dependent kinase inhibitor.    
     
     
         41 . A method of plant breeding comprising: 
 a) providing a first parent plant having a heterologous nucleic acid sequence encoding a cyclin-dependent kinase inhibitor polypeptide;    b) providing a second parent plant having a heterologous nucleic acid sequence encoding a cyclin polypeptide;    c) crossing the first and second parent plants to obtain a hybrid offspring plant having a copy of the heterologous sequence encoding the cyclin-dependent kinase inhibitor and a copy of the heterologous sequence encoding the cyclin polypeptide.    
     
     
         42 . A method of plant breeding comprising: 
 a) providing a first parent plant having a heterologous nucleic acid sequence encoding a cyclin-dependent kinase inhibitor polypeptide;    b) providing a second parent plant having a heterologous nucleic acid sequence encoding a cyclin-dependent kinase polypeptide;    c) crossing the first and second parent plants to obtain a hybrid offspring plant having a copy of the heterologous sequence encoding the cyclin-dependent kinase inhibitor and a copy of the heterologous sequence encoding the cyclin-dependent kinase polypeptide.    
     
     
         43 . A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a cyclin-dependent kinase, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the cyclin-dependent kinase and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the cyclin-dependent kinase.  
     
     
         44 . A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a recombinant polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the recombinant polypeptide and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the recombinant polypeptide.  
     
     
         45 . The method of  claim 44  wherein the polypeptide is an antibody such as single-chain antibody.  
     
     
         46 . The method of  claim 44  wherein the polypeptide is a peptide selected from a peptide library.  
     
     
         47 . A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a polypeptide that modulates the degradation of a cyclin-dependent kinase inhibitor polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the polypeptide that modulates the degradation of the cyclin-dependent kinase inhibitor and the cyclin-dependent kinase inhibitor polypeptide are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the polypeptide that modulates the degradation of the cyclin-dependent kinase inhibitor.