Heat stable mutants of starch biosynthesis enzymes

Abstract

The subject invention pertains to novel mutant polynucleotide molecules that encode enzymes that have increased heat stability. These polynucleotides, when expressed in plants, result in increased yield in plants grown under conditions of heat stress. The polynucleotide molecules of the subject invention encode maize endosperm ADP glucose pyrophosphorylase (AGP) and soluble starch synthase (SSS) enzyme activities. Plants and plant tissue bred to contain, or transformed with, the mutant polynucleotides, and expressing the polypeptides encoded by the polynucleotides, are also contemplated by the present invention. The subject invention also concerns methods for isolating polynucleotides and polypeptides contemplated within the scope of the invention. Methods for increasing yield in plants grown under conditions of heat stress are also provided.

We claim: 
     
         1 . A polynucleotide encoding a mutant starch biosynthesis protein, or a biologically-active fragment or variant of said mutant protein, wherein said mutant protein exhibits increased heat stability relative to the wild type protein. 
     
     
         2 . The polynucleotide according to  claim 1 , wherein said mutant protein encoded by said polynucleotide is a plant AGP protein. 
     
     
         3 . The polynucleotide according to  claim 2 , wherein said mutant protein encoded by said polynucleotide comprises an amino acid mutation in the large subunit of said protein. 
     
     
         4 . The polynucleotide according to  claim 2 , wherein said mutant protein encoded by said polynucleotide comprises an amino acid mutation in the small subunit of said protein. 
     
     
         5 . The polynucleotide according to  claim 3 , wherein said mutant protein encoded by said polynucleotide comprises an amino acid mutation wherein a histidine residue at position 333 in the amino acid sequence of said protein is replaced by an amino acid that confers heat stability to said protein. 
     
     
         6 . The polynucleotide according to  claim 5 , wherein said amino acid substituted for histidine at position number 333 is a glycine. 
     
     
         7 . The polynucleotide according to  claim 5 , wherein said amino acid substituted for histidine at position number 333 is a phenylalanine. 
     
     
         8 . The polynucleotide according to  claim 5 , wherein said amino acid substituted for histidine at position number 333 is a methionine. 
     
     
         9 . The polynucleotide according to  claim 1 , wherein said mutant protein encoded by said polynucleotide further comprises an amino acid mutation that confers increased seed weight to a plant expressing said polynucleotide. 
     
     
         10 . The polynucleotide according to  claim 9 , wherein said polynucleotide comprises the Rev6 mutation. 
     
     
         11 . The polynucleotide according to  claim 9 , wherein said polynucleotide encodes a maize large subunit AGP enzyme wherein at least one serine residue is inserted between amino acids 494 and 495 of the native AGP enzyme subunit. 
     
     
         12 . The polynucleotide according to  claim 9 , wherein said polynucleotide encodes a maize large subunit AGP enzyme wherein the amino acid pair tyrosine:serine is inserted between amino acids 494 and 495 of the native AGP enzyme subunit. 
     
     
         13 . The polynucleotide according to  claim 9 , wherein said polynucleotide encodes a maize large subunit AGP enzyme wherein the amino acid pair serine:tyrosine is inserted between amino acids 495 and 496 of the native AGP enzyme subunit. 
     
     
         14 . A method for increasing resistance of a plant to heat stress conditions, said method comprising incorporating the polynucleotide of  claim 1  into the genome of said plant and expressing the protein encoded by said polynucleotide molecule. 
     
     
         15 . The method according to  claim 11 , wherein said plant is a monocotyledonous plant. 
     
     
         16 . The method according to  claim 15 , wherein said monocotyledonous plant is selected from the group consisting of rice, wheat, barley, oats, sorghum, maize, lilies, and millet. 
     
     
         17 . The method according to  claim 14 , wherein said plant is  Zea mays.    
     
     
         18 . The method according to  claim 14 , wherein said plant is a dicotyledonous plant. 
     
     
         19 . The method according to  claim 18 , wherein said dicotyledonous plant is selected from the group consisting of peas, alfalfa, chickpea, chicory, clover, kale, lentil, prairie grass, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, and lettuce. 
     
     
         20 . A plant or plant tissue comprising the polynucleotide molecule of  claim 1 . 
     
     
         21 . The plant or plant tissue according to  claim 20 , wherein said plant or plant tissue is monocotyledonous. 
     
     
         22 . The plant or plant tissue according to  claim 21 , wherein said monocotyledonous plant or plant tissue is selected from the group consisting of rice, wheat, barley, oats, sorghum, maize, lilies, and millet. 
     
     
         23 . The plant or plant tissue according to  claim 20 , wherein said plant is  Zea mays  or said plant tissue is from  Zea mays.    
     
     
         24 . The plant or plant tissue according to  claim 20 , wherein said plant or plant tissue is dicotyledonous. 
     
     
         25 . The plant or plant tissue according to  claim 24 , wherein said dicotyledonous plant or plant tissue is selected from the group consisting of peas, alfalfa, chickpea, chicory, clover, kale, lentil, prairie grass, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, and lettuce. 
     
     
         26 . The plant tissue according to  claim 20 , wherein said plant tissue is a seed. 
     
     
         27 . A mutant starch biosynthesis protein encoded by the polynucleotide of  claim 1 . 
     
     
         28 . A method for identifying a polynucleotide encoding a mutant starch biosynthesis protein wherein said mutant starch biosynthesis protein exhibits increased heat stability relative to a wild type protein, said method comprising mutating a polynucleotide encoding a starch biosynthesis protein, expressing said mutated polynucleotide in a cell to produce a mutant starch biosynthesis protein, and determining whether said mutant starch biosynthesis protein exhibits increased heat stability relative to the wild type starch biosynthesis protein.