DWF12 and Mutants Thereof

Abstract

DWARF12 (DWF12) sequences, mutants and methods of using the same are disclosed. The dwf12 polynucleotides can be used in the production of transgenic plants which display at least one dwf12 mutant phenotype, so that the resulting plants have altered biochemistry, structure or morphology.

1 . An isolated dwf12 mutant polypeptide with at least about 70% sequence identity to the polypeptide depicted at positions 38-326 or 1-380 of  FIG. 3  or a polypeptide designated in Table 1, wherein said polypeptide comprises a mutation of at least one non-conservative substitution, addition or deletion of an amino acid in a region of the polypeptide corresponding to a region depicted in  FIG. 3 , wherein said region is one or more of (a) a casein kinase II phosphorylation domain spanning positions 104-106 of  FIG. 3 ; (b) a casein kinase II phosphorylation domain spanning positions 220-223 of  FIG. 3 ; (c) a casein kinase II phosphorylation domain spanning positions 261-264 of  FIG. 3 ; (d) a casein kinase II phosphorylation domain spanning positions 314-317 of  FIG. 3 ; (e) a casein kinase II phosphorylation domain spanning positions 353-356 (f) a protein kinase C phosphorylation domain spanning positions 104-106 of  FIG. 3 ; (g) a protein kinase C phosphorylation domain spanning positions 187-189 of  FIG. 3 ; (h) a protein kinase C phosphorylation domain spanning positions 310-312; (i) the active site lysine residue found at position 69 of  FIG. 3 ; (j) a tyrosine phosphorylation domain spanning positions 70-77 of  FIG. 3 ; (k) a protein kinase ATP-binding domain spanning positions 46-69 of  FIG. 3 ; (l) a eukaryotic protein kinase ATP-binding domain spanning positions 157-172 of  FIG. 3 ; (m) a eukaryotic protein kinase substrate-binding domain spanning positions 218-228 of  FIG. 3 ; (n) a eukaryotic protein kinase signature sequence spanning positions 316-327 of  FIG. 3 ; (o) a glycosylation domain spanning positions 197-200 of  FIG. 3 ; (p) a glycosylation domain spanning positions 376-379 of  FIG. 3 ; (q) the sequence CDFGSAK spanning positions 183-189 of  FIG. 3 ; or (r) the sequence SYICSR spanning positions 199-204 of  FIG. 3 . 
     
     
         2 . The mutant polypeptide of  claim 1 , wherein the mutation comprises a change in a casein kinase II phosphorylation domain. 
     
     
         3 . The mutant polypeptide of  claim 2 , wherein the mutation comprises a substitution of an acidic amino acid residue with a basic amino acid residue. 
     
     
         4 . The mutant polypeptide of  claim 3 , wherein the mutation comprises substitution of an Asp or Glu residue with a Lys, Arg or His residue. 
     
     
         5 . The mutant polypeptide of  claim 1 , wherein the mutation comprises a change in a protein kinase C phosphorylation domain. 
     
     
         6 . The mutant polypeptide of  claim 5 , wherein the mutation comprises a substitution of an acidic amino acid residue with a basic amino acid residue. 
     
     
         7 . The mutant polypeptide of  claim 3 , wherein the mutation comprises substitution of an Asp or Glu residue with a Lys, Arg or His residue. 
     
     
         8 . The mutant polypeptide of  claim 1 , wherein the mutation comprises a mutation to the sequence spanning positions 261-264 of  FIG. 3 . 
     
     
         9 . The mutant polypeptide of  claim 8 , wherein the mutation comprises substitution of Glu-263 or Glu-264 with a Lys residue. 
     
     
         10 . An isolated dwf12 mutant polynucleotide which encodes a polypeptide according to any of  claims 1 - 9 . 
     
     
         11 . The mutant polynucleotide of  claim 10 , wherein the polynucleotide is (a) a dwf12-1 polynucleotide comprising the dwf12-1 nucleotide sequence depicted in  FIGS. 1A-1F ; or (b) a dwf12-2 polynucleotide comprising the dwf12-2 nucleotide sequence depicted in  FIGS. 1A-1F . 
     
     
         12 . The mutant polynucleotide of either of  claims 10  or  11 , wherein the polynucleotide imparts at least one dwf12 mutant phenotype when expressed in a plant. 
     
     
         13 . The mutant polynucleotide of  claim 12 , wherein the mutant phenotype comprises a microscopic or macroscopic change in structure or morphology, or a biochemical difference, as compared to a progenitor, wild-type plant cultivated under the same conditions. 
     
     
         14 . The mutant polynucleotide of  claim 13 , wherein the change comprises reduced or increased SHAGGY kinase activity. 
     
     
         15 . The mutant polynucleotide of  claim 12 , wherein the change comprises accumulation of brassinolide. 
     
     
         16 . The mutant polynucleotide of  claim 12 , wherein the change comprises a short robust stature, short internodes, an increased number of inflorescences, small dark-green, round leaves, severe downward curling of leaves, infertility, and/or short siliques 
     
     
         17 . The mutant polynucleotide of  claim 16 , wherein the change comprises severe downward curling of leaves. 
     
     
         18 . A recombinant vector comprising:
 (i) the mutant polynucleotide of any of  claims 10 - 17 ; and   (ii) control elements operably linked to said polynucleotide whereby a coding sequence within said polynucleotide can be transcribed and translated in a host cell.   
     
     
         19 . The recombinant vector of  claim 18 , wherein said mutant polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter. 
     
     
         20 . A host cell comprising the recombinant vector of either of  claims 18  or  19 . 
     
     
         21 . A method of producing a recombinant polypeptide comprising:
 (a) providing a population of host cells according to  claim 20 ; and   (b) culturing said host cells under conditions whereby a recombinant polypeptide encoded by the coding sequence present in said recombinant vector is expressed.   
     
     
         22 . A transgenic plant comprising a dwf12 mutant polynucleotide, wherein said mutant polynucleotide encodes for a dwf12 mutant polypeptide according to any of  claims 1 - 9 . 
     
     
         23 . The transgenic plant of  claim 22 , wherein the dwf12 mutant polynucleotide is (a) a dwf12-1 polynucleotide comprising the dwf12-1 nucleotide sequence depicted in  FIGS. 1A-1F ; or (b) a dwf12-2 polynucleotide comprising the dwf12-2 nucleotide sequence depicted in  FIGS. 1A-1F . 
     
     
         24 . A method of producing a transgenic plant comprising:
 (a) introducing a dwf12 mutant polynucleotide according to any of  claims 10 - 17  into a plant cell to produce a transformed plant cell; and   (b) producing a transgenic plant from the transformed plant cell.   
     
     
         25 . A method of producing a transgenic plant comprising:
 (a) introducing the recombinant vector of either of  claims 18  or  19  into a plant cell to produce a transformed plant cell; and   (b) producing a transgenic plant from the transformed plant cell.   
     
     
         26 . A method for producing a transgenic plant having an altered phenotype relative to the corresponding wild-type plant comprising:
 (a) introducing a dwf12 mutant polynucleotide according to any of  claims 10 - 17  into a plant cell; and   (b) producing a transgenic plant from the plant cell, said transgenic plant having an altered phenotype relative to the corresponding wild-type plant.   
     
     
         27 . A method for producing a transgenic plant having an altered phenotype relative to the corresponding wild-type plant comprising:
 (a) introducing the recombinant vector of either of  claims 18  or  19  into a plant cell to produce a transformed plant cell; and   (b) producing a transgenic plant from the plant cell, said transgenic plant having an altered phenotype relative to the corresponding wild-type plant.   
     
     
         28 . The method of either of  claims 26  or  27 , wherein the mutant polynucleotide is overexpressed. 
     
     
         29 . The method of either of  claims 26  or  27 , wherein expression of DWF12 is inhibited. 
     
     
         30 . The method of either of  claims 26  or  27 , wherein at least first and second polynucleotides are introduced into the plant cell, said first and second polynucleotides operably linked to at least first and second tissue-specific promoters, wherein said first polynucleotide is overexpressed and said second polynucleotide inhibits expression of DWF12. 
     
     
         31 . A method of modulating an endogenous DWF12 polypeptide in a transgenic plant, said method comprising:
 (a) introducing a dwf12 mutant polynucleotide according to any of  claims 10 - 17  into a plant cell; and   (b) producing a transgenic plant from the plant cell, said transgenic plant having altered endogenous DWF12 activity relative to the corresponding wild-type plant.   
     
     
         32 . A method of modulating an endogenous DWF12 polypeptide in a transgenic plant, said method comprising:
 (a) introducing the recombinant vector of either of  claims 18  or  19  into a plant cell; and   (b) producing a transgenic plant from the plant cell, said transgenic plant having altered endogenous DWF12 activity relative to the corresponding wild-type plant.   
     
     
         33 . The method of  claim 32 , wherein the mutant polynucleotide is overexpressed. 
     
     
         34 . The method of  claim 32 , wherein expression of the mutant polynucleotide is inhibited. 
     
     
         35 . A method for altering the biochemical activity of a cell comprising:
 (a) introducing at least one mutant polynucleotide according to any of  claims 1 - 17  into the cell to produce a transformed cell; and   (b) causing expression of said mutant polynucleotide such that the biochemical activity of the cell is altered.   
     
     
         36 . A method for altering the biochemical activity of a cell comprising:
 (a) introducing the recombinant vector of either of  claims 18  or  19  into the cell to produce a transformed cell; and   (b) causing expression of said mutant polynucleotide such that the biochemical activity of the cell is altered.   
     
     
         37 . The method of either of  claims 35  or  36 , wherein introducing into the cell is done ex vivo. 
     
     
         38 . The method of either of  claims 35  or  36 , wherein introducing into the cell is done in vivo. 
     
     
         39 . The method of  claims 35  or  36 , wherein more than one dwf12 mutant polynucleotide is introduced into the cell. 
     
     
         40 . The method of any of  claims 24 - 39 , wherein expression of SHAGGY kinase is altered. 
     
     
         41 . A method for modulating a trait in a plant, wherein said trait is selected from the group consisting of (a) biomass; (b) height; (c) tissue or organ size; (d) fertility; (e) fecundity; (f) leaf curling; (g) cell elongation; and (h) stress tolerance. 
     
     
         42 . The method of  claim 41 , wherein the size of the leaves, seeds, roots, flowers, stems, petioles, internodes, and/or hypocotyls are altered. 
     
     
         43 . The method of  claim 41 , wherein leaf curling in the rosette and/or cauline is produced. 
     
     
         44 . The method of  claim 41 , wherein the trait is modulated by modulating expression of an endogenous SHAGGY kinase by inserting a mutant polynucleotide into a cell, wherein said polynucleotide encodes for a SHAGGY kinase exhibiting reduced activity as compared to the corresponding endogenous SHAGGY kinase. 
     
     
         45 . The method of  claim 44 , wherein the mutant polynucleotide encodes a mutant dwf12 polypeptide with reduced activity. 
     
     
         46 . The method of  claim 44 , wherein the mutant polynucleotide encodes a mutant dwf12 polypeptide with enhanced activity. 
     
     
         47 . The method of  claim 44 , wherein the SHAGGY kinase modulated is involved in a BR signaling pathway.