Novel polypeptides and polynucleotides relating to the a- and b-subunits of glutamate dehydrogenases and methods of use

Abstract

Amino acid and nucleotide sequences relating to the glutamate dehydrogenase (GDH) enzyme are described. The GDH enzymes described herein were discovered in the alga Chlorella sorokiniana in the form of seven different inducible isoenzymes. These isoenzymes are found in the algae as chloroplast-localized hexamers composed of alpha- and beta-subunits. Plants transformed with nucleotide sequences encoding the alpha- or beta-subunits of the enzyme show improved properties, for example, increased growth and improved stress tolerance. A heterohexamer having both α- and β-subunits can have higher aminating:deaminating activity ratio than α-homohexamers or β-homohexamers.

1 . A method for modulating nitrogen metabolism in plant cells, said method comprising the steps of transforming a plant cell with a polynucleotide encoding a polypeptide having glutamate dehydrogenase activity, and culturing said cell under conditions whereby descendant cells are produced which comprise said polynucleotide and wherein said polynucleotide is expressed, whereby nitrogen metabolism is modulated.  
     
     
         2 . The method of  claim 1 , further comprising the step of regenerating said descendant cells to form a plant.  
     
     
         3 . The method of  claim 1 , wherein said nitrogen metabolism comprises increasing the assimilation of inorganic nitrogen into organic nitrogen.  
     
     
         4 . The method of  claim 1 , wherein said polypeptide is selected from the group consisting of an alpha subunit of glutamate dehydrogenase, a beta subunit of glutamate dehydrogenase, and fragments or mutants thereof which exhibit glutamate dehydrogenase activity.  
     
     
         5 . The method of  claim 1 , wherein said polypeptide is operably linked to a chloroplast transit peptide.  
     
     
         6 . The method of  claim 5 , wherein the chloroplast transit peptide comprises the amino terminus of SEQ ID NO. 2 or SEQ ID NO. 4, or a fragment or mutant thereof sufficient to exhibit chloroplast transit activity.  
     
     
         7 . The method of  claim 1  wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 24, SEQ ID NO. 26, and fragments or mutants thereof.  
     
     
         8 . The method of  claim 1 , wherein said polynucleotide is operably linked to a plant polyadenylation sequence.  
     
     
         9 . The method of  claim 1 , further comprising engineering said polynucleotide to maximize expression in said plant cell, said engineering comprising determining favored codon usage in said plant cell and altering said polynucleotide by increasing the frequency of favored codons.  
     
     
         10 . A method of modulating the properties of a plant comprising culturing a plant comprising cells that comprise a polynucleotide encoding a polypeptide having glutamate dehydrogenase activity under conditions where said polynucleotide is expressed in said cells, whereby properties of said plant are modulated.  
     
     
         11 . The method of  claim 10 , wherein at least one property modulated by said method is selected from the group consisting of increased crop yield, altered ammonium assimilation, altered osmotic stress tolerance, and altered composition of said plant.  
     
     
         12 . The method of  claim 12 , wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 24, SEQ ID NO. 26, and fragments or mutants thereof sufficient to encode a polypeptide having glutamate dehydrogenase activity.  
     
     
         13 . A plant produced by the method of  claim 2 , comprising cells which express a polynucleotide encoding a polypeptide exhibiting glutamate dehydrogenase activity, whereby nitrogen metabolism in said cells is modulated.  
     
     
         14 . A descendant of the plant of  claim 13 , said descendant comprising cells expressing said polypeptide and wherein nitrogen metabolism of said cells of said descendant is thereby modulated.