Methods of modulating glucosinolate production in plants

Abstract

The present invention relates to methods for modulating glucosinolate production in plants, specifically by modulating CYP83A1 expression. The present invention also relates to transgenic plants that overexpress and underexpress CYP83A1.

1 . A transgenic plant with altered CYP83A1 expression relative to the corresponding wild-type plant.  
     
     
         2 . The transgenic plant of  claim 1 , wherein CYP83A1 is overexpressed.  
     
     
         3 . The transgenic plant of  claim 1 , wherein CYP83A1 is underexpressed.  
     
     
         4 . A method of producing a transgenic plant with altered CYP83A1 expression relative to the wild-type plant, said method comprising: 
 (a) introducing an expression construct that comprises a polynucleotide encoding a CYP83A1 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide into a plant cell to produce a transformed plant cell; and    (b) producing a transgenic plant from the transformed plant cell with altered CYP83A1 expression.    
     
     
         5 . The method of  claim 4 , wherein CYP83A1 is overexpressed.  
     
     
         6 . The method of  claim 4 , wherein CYP83A1 is underexpressed.  
     
     
         7 . The method of  claim 4 , wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.  
     
     
         8 . A method of producing a transgenic plant with altered CYP83A1 expression relative to the wild-type plant, said method comprising: 
 (a) introducing a polynucleotide that inhibits expression of a CYP83 μl polynucleotide into a plant cell to produce a transformed plant cell; and    (b) producing a transgenic plant from the transformed plant cell with altered CYP83A1 expression.    
     
     
         9 . A method for altering the biochemical activity of a cell, said method comprising: 
 (a) introducing an expression construct that comprises a polynucleotide encoding a CYP83A1 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide into a plant cell to produce a transformed plant cell; and    (b) growing the cell under conditions such that the biochemical activity of the cell is altered.    
     
     
         10 . The method of  claim 9 , wherein the expression construct is introduced into the cell ex vivo.  
     
     
         11 . The method of  claim 9 , wherein the expression construct is introduced into the cell in vivo.  
     
     
         12 . A method for altering the biochemical activity of a cell, said method comprising: 
 (a) introducing a polynucleotide that inhibits expression of a CYP83A1 polynucleotide into a plant cell to produce a transformed plant cell; and    (b) growing the cell under conditions such that the biochemical activity of the cell is altered.    
     
     
         13 . The method of  claim 12 , wherein the polynucleotide is introduced into the cell ex vivo.  
     
     
         14 . The method of  claim 12 , wherein the polynucleotide is introduced into the cell in vivo.  
     
     
         15 . A method of producing a transgenic plant with altered expression of a cytochrome P450 that catalyzes the conversion of an aldoxime to a glucosinolate, the method comprising: 
 (a) introducing an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide, into a plant cell to produce a transformed plant cell; and    (b) producing a transgenic plant from the transformed plant cell with altered cytochrome P450 expression.    
     
     
         16 . The method of  claim 15 , wherein the cytochrome P450 is CYP83A1.  
     
     
         17 . The method of  claim 16 , wherein CYP83A1 is overexpressed.  
     
     
         18 . The method of  claim 16 , wherein CYP83A1 is underexpressed.  
     
     
         19 . The method of  claim 15 , wherein the 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 . The method of  claim 15 , wherein the glucosinolate is aliphatic, aromatic or indolic.  
     
     
         21 . The method of  claim 20 , wherein the glucosinolate is aliphatic.  
     
     
         22 . The method of  claim 21 , wherein the aliphatic glucosinolate is obtained from a corresponding aldoxime.  
     
     
         23 . The method of  claim 22 , wherein the aldoxime is obtained from the conversion of an aliphatic amino acid or a chain-elongated form thereof to the corresponding N-hydroxy amino acid, and the conversion of the N-hydroxyamino acid to the aldoxime.  
     
     
         24 . The method of  claim 23 , wherein the aliphatic amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine and chain-elongated forms thereof.  
     
     
         25 . The method of  claim 20 , wherein the glucosinolate is aromatic.  
     
     
         26 . The method of  claim 25 , wherein the aromatic glucosinolate is obtained from the corresponding aldoxime.  
     
     
         27 . The method of  claim 26 , wherein the aldoxime is obtained from the conversion of an aromatic amino acid to the corresponding N-hydroxy amino acid, and the conversion of the N-hydroxyamino acid to the aldoxime.  
     
     
         28 . The method of  claim 27 , wherein the aromatic amino acid is phenylalanine or tyrosine.  
     
     
         29 . A method of producing a cytochrome P450 that catalyzes the conversion of aldoxime to a corresponding aci-nitro and the conversion of the aci-nitro to a corresponding S-alkyl-thiohydroximate and the conversion of the S-alkyl-thiohydroximate to glucosinolate, the method comprising: 
 (a) introducing an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide, into a host cell to produce a transformed host cell;    (b) expressing the cytochrome P450 in the host cell; and    (c) isolating the expressed the cytochrome P450.    
     
     
         30 . The method of  claim 29 , wherein the cytochrome P450 is CYP83A1.  
     
     
         31 . The method of  claim 30 , wherein CYP83 μl is overexpressed.  
     
     
         32 . The method of  claim 29 , wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.  
     
     
         33 . The method of  claim 29 , wherein the aldoxime is obtained from the conversion of an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, tyrosine, and phenylalanine to a corresponding N-hydroxy amino acid, and the conversion of N-hydroxyamino acid to the aldoxime.  
     
     
         34 . A method for producing a glucosinolate, the method comprising contacting an acetaldoxime with a cytochrome P450, and isolating the glucosinolate.  
     
     
         35 . The method of  claim 34 , wherein the cytochrome P450 is CYP83A1.  
     
     
         36 . The method of  claim 34 , wherein the cytochrome P450 is overexpressed in a transformed host cell.  
     
     
         37 . The method of  claim 36 , wherein the transformed host cell comprises an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing the polypeptide.  
     
     
         38 . The method of  claim 37 , wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.