Maize chloroplast protein synthesis elongation factors and methods of use for same

Abstract

The invention discloses a novel heat shock protein with high homology to chloroplast elongation factor EF-Tu. Also disclosed is a transgenic method for enhancing tolerance to heat and drought in female reproductive organs. It involves the temporal and spatial expression of novel heat shock EF-Tu in a plant organ or plant tissue. The invention also includes expression constructs, and methods for the production of crop plants with heritable phenotypes which are useful in breeding programs designed to increase heat and drought tolerance.

What is claimed is:  
     
         1 . A purified and isolated nucleotide sequence which encodes upon expression a regulatory protein characterized by the following: 
 (a) is approximately 45 kD;    (b) is expressed primarily under heat shock conditions;    (c) is localized in chloroplasts;    (d) has high homology to chloroplast elongation factor EF-Tu, from  E. coli  or tobacco;    said nucleotide sequence being capable of hybridizing under conditions of high stringency to SEQ ID NO:6 (putative coding sequence).    
     
     
         2 . The nucleotide sequence of  claim 1  wherein said sequence is SEQ ID NO:5.  
     
     
         3 . An expression construct comprising: 
 a nucleotide sequence according to  claim 1 , operatively linked to a regulatory region capable of directing expression of a protein in a plant cell.    
     
     
         4 . A vector capable of transforming or transfecting a host cell, said vector comprising an expression construct according to  claim 3 .  
     
     
         5 . The vector of  claim 4  wherein said vector is a plasmid based vector.  
     
     
         6 . The vector of  claim 4  wherein said vector is a viral based vector.  
     
     
         7 . A prokaryotic or eukaryotic host cell transformed or transfected with a vector according to  claim 4 .  
     
     
         8 . The host cell of  claim 7  wherein said cell is a plant cell.  
     
     
         9 . A regulatory protein which exhibits the following characteristics: 
 (a) is approximately 45 kD;    (b) is expressed primarily under heat shock conditions;    (c) is localized in plant chloroplasts;    (d) has high homology (80% or greater) to chloroplast elongation factor EF-Tu, from  E coli  or tobacco and comprises at least 80% amino acid sequence homology to SEQ ID NOS:1, 2, 3 or 4.    
     
     
         10 . The protein of  claim 9  wherein said protein comprises SEQ ID NO:1.  
     
     
         11 . The protein of  claim 9  wherein said protein comprises SEQ ID NO:2.  
     
     
         12 . The protein of  claim 9  wherein said protein comprises SEQ ID NO:3.  
     
     
         13 . The protein of  claim 9  wherein said protein comprises SEQ ID NO:4.  
     
     
         14 . A method for increasing plant tolerance to heat and drought comprising: 
 introducing to a plant cell a genetic construct comprising: 
 a nucleotide sequence which encodes a regulatory protein characterized by the following: 
 (a) is approximately 45 kD;  
 (b) is expressed primarily under heat shock conditions;  
 (c) is localized in chloroplasts;  
 (d) has high homology to chloroplast elongation factor EF-Tu, from  E. coli  or tobacco;  
 
 said nucleotide sequence being operably linked to promoter and regulatory regions capable of inducing expression in a plant.  
   
     
     
         15 . The method of  claim 14  wherein said expression construct elements cause expression during stress.  
     
     
         16 . The method of  claim 14  wherein said promoter is selected from the group consisting of: a constitutive, an inducible, and an organ specific promoter.  
     
     
         17 . The method of  claim 14  further comprising the step of: 
 selecting plants which are transformed with said construct.  
 
     
     
         18 . The method of  claim 14  wherein said expression construct further comprises a selectable marker gene.  
     
     
         19 . A method of identifying heat shock EF-Tu genes in plant species comprising: 
 screening the genome of said plant species for a sequence that is homologous to SEQ ID NO:5 or a region of at least 100 bases thereof.    
     
     
         20 . A transformed plant, which plant is substantially tolerant or resistant to one or more environmental conditions selected from the group consisting of excess heat and drought, wherein the cells of the plant comprise a recombinant DNA segment encoding EF-Tu, and wherein the EF-Tu is expressed so as to confer tolerance to the transformed plant to one or more of the environmental conditions that adversely affect cell metabolism, plant growth, or development of the corresponding untransformed plant.  
     
     
         21 . A transgenic plant containing a DNA construct encoding EF-Tu wherein said DNA construct is expressed so that the plant exhibits tolerance to one or more conditions selected from the group consisting of excess heat and drought, wherein said resistance is not present in a corresponding plant not containing the DNA construct.  
     
     
         22 . A transgenic plant according to  claim 21  wherein the DNA construct comprises a promoter.  
     
     
         23 . A seed produced by the transgenic plant of  claim 21  which comprises the DNA construct.  
     
     
         24 . A progeny transgenic plant derived from the transgenic plant of  claim 21  wherein said progeny plant expresses said DNA construct so that the progeny plant exhibits said tolerance.  
     
     
         25 . A transgenic plant according to  claim 21  wherein the plant is a maize plant.  
     
     
         26 . A seed derived from the progeny plant of  claim 24  wherein said seed comprises the DNA construct.  
     
     
         27 . A transgenic plant according to  claim 21  wherein the plant is obtainable by a process comprising the steps of: 
 bombarding intact regenerable plant cells with microprojectiles coated with the DNA construct;  
 identifying or selecting a population of transformed cells; and  
 regenerating a transgenic plant therefrom.  
 
     
     
         28 . A method of increasing the tolerance of a plant to heat or drought, comprising: 
 introducing into cells of a plant an expression cassette comprising a preselected DNA segment encoding EF-Tu, to yield transformed plant cells; and    regenerating a plant from said transformed cells, wherein the EF-Tu is expressed in the cells o f the plant so as to render the transformed plant substantially tolerant to drought or excess heat that inhibits the cell metabolism, growth, or development of an untransformed plant.    
     
     
         29 . A method according to  claim 28  further including the step of: 
 obtaining progeny from the transformed plant which comprise the DNA construct.  
 
     
     
         30 . A method according to  claim 29  wherein the progeny are obtained by crossing the transformed plant with an inbred line.  
     
     
         31 . A method according to  claim 29  further including the step of: 
 obtaining seed from the progeny and obtaining further progeny plants comprising the DNA construct from the seed.