Functional expression of yeast nitrate transporter (ynt1) in maize to improve nitrate uptake

Abstract

The present invention provides methods and compositions relating to altering NT activity, nitrogen utilization and/or uptake in plants. The invention relates to a method for the production of plants with maintained or increased yield under low or normal nitrogen fertility. The invention provides isolated nitrate transporter (NT) nucleic acids and their encoded proteins. The invention further provides recombinant expression cassettes, host cells, and transgenic plants. Plants transformed with nucleotide sequences encoding the NT enzyme show improved properties, for example, increased yield.

1 . An isolated or recombinant nucleic acid comprising a nitrate transporter (NT) polynucleotide or nitrate reductase (NR) polynucleotide;
 wherein said NT polynucleotide comprises a member selected from the group consisting of,   (a) a polynucleotide comprising a nucleotide that has been substituted, wherein the nucleotide substitution is one or more of the substitutions shown in  FIG. 3 , and wherein the polynucleotide encodes a polypeptide having NT activity,   (b) a polynucleotide comprising the sequence set forth in SEQ ID NO: 3,   (c) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under stringent conditions to a polynucleotide of (a), wherein the conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C., and wherein the polynucleotide encodes a polypeptide having NT activity,   (d) a polynucleotide having at least 70% sequence identity to SEQ ID NO: 3, wherein the % sequence identity is based on the entire encoding region and is determined by BLAST 2.0 under default parameters, and wherein the polynucleotide encodes a polypeptide having NT activity,   (e) an isolated polynucleotide degenerate from any of (a) to (d) as a result of the genetic code, and   (f) a polynucleotide complimentary to a polynucleotide of any one of (a) to (e); and   
       wherein said NR polynucleotide comprises a member selected from the group consisting of,
 (g) a polynucleotide that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 or 9; 
 (h) a polynucleotide comprising the sequence set forth in SEQ ID NO: 4 or 9; and 
 (i) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under stringent conditions to a polynucleotide selected from the group consisting of (g) and (h), wherein the conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C., and wherein the polynucleotide encodes a polypeptide having NR activity and comprising the A561G substitution, the S561D substitution, or the A561 and S561 D substitutions; and 
 (j) a polynucleotide having at least 70% sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 4 and 9, wherein the % sequence identity is based on the entire encoding region and is determined by BLAST 2.0 under default parameters, and wherein the polynucleotide encodes a polypeptide having NR activity and comprising the A561G substitution, the S561D substitution, or the A561 and S561 D substitutions; and 
 (k) an isolated polynucleotide degenerate from any of (g) to (j) as a result of the genetic code; 
 (l) a polynucleotide complimentary to a polynucleotide of any one of (g) to (k). 
 
     
     
         2 . The polynucleotide of  claim 1 , wherein the polynucleotide encodes a NT polypeptide that confers to a plant increased yield or nitrogen utilization efficiency under lower fertility. 
     
     
         3 . A vector comprising at least one polynucleotide, or an expression cassette comprising at least one polynucleotide operably linked to a promoter; wherein the polynucleotide is the polynucleotide of  claim 1 . 
     
     
         4 . A host cell comprising at least one expression cassette of  claim 3 . 
     
     
         5 . The host cell of  claim 4 , wherein the host cell is a dicotyledonous or monocotyledonous plant cell. 
     
     
         6 . A transgenic plant comprising at least one expression cassette of  claim 3 . 
     
     
         7 . A seed produced by the transgenic plant of  claim 6 , wherein the seed comprises the expression cassette. 
     
     
         8 . A method for increasing yield in a plant, said method comprising the steps of:
 (a) introducing into a plant cell a construct comprising an NT polynucleotide, wherein said NT polynucleotide is operably linked to a promoter functional in plant cells to yield a transformed plant cell, and wherein the NT polynucleotide is selected from the group consisting of:
 (i) a polynucleotide comprising a nucleotide that has been substituted and wherein the nucleotide substitution is one or more of the substitutions shown in  FIG. 3 , and wherein the polynucleotide encodes a polypeptide having NT activity; 
 (ii) a polynucleotide that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; 
 (iii) a polynucleotide comprising the sequence set forth in the coding region of SEQ ID NO: 1 or 3; 
 (iv) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under moderate stringency conditions to a polynucleotide selected from the group consisting of (i) and (ii), wherein the conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C., and wherein the polynucleotide encodes a polypeptide having NT activity; and 
 (v) a polynucleotide having at least 70% sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 and 3, wherein the % sequence identity is based on the entire encoding region and is determined by BLAST 2.0 under default parameters, wherein the polynucleotide encodes a polypeptide having NT activity; and 
 (vi) a polynucleotide degenerate from any of (i) to (v) as a result of the genetic code; and 
   (b) regenerating a transgenic plant from said transformed plant cell, wherein said NT is expressed in said transgenic plant at levels sufficient to maintain or increase yield in said transgenic plant;   
       wherein increased yield comprises enhanced root growth, increased seed size, increased shoot biomass, increased seed weight, increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size or chlorophyll content, wherein the yield of the plant is compared to a control plant, and wherein the control plant does not contain the NT polynucleotide. 
     
     
         9 . The method of  claim 8 , wherein the plant is grown under limited nitrogen fertility. 
     
     
         10 . The method of  claim 8 , wherein the nitrogen utilization efficiency of the plant is increased. 
     
     
         11 . The method of  claim 8 , wherein NT activity of the NT polypeptide encoded by the NT polynucleotide is increased compared to the activity of a NT polypeptide endogenous to the plant. 
     
     
         12 . The method of  claim 8 , wherein the expression of the NT polynucleotide is driven by a cell-specific, tissue-specific, organ-specific, constitutive, or inducible promoter, or wherein the expression of the NT polynucleotide is driven by a root-preferred promoter selected from the group consisting of tubulin promoter (pTUB); maize ubiquitin promoter (ZM UBI), maize root metallothionein promoter (ZM-RM2); lipid transfer protein 2 promoter (LTP2); banana streak virus promoter truncated version promoter (BSV(TR)), maize NAS2 promoter (ZM-NAS2), and banana streak virus promoter full version (BAV (FL). 
     
     
         13 . The method of  claim 8 , wherein the construct further comprises a second polynucleotide, wherein the second polynucleotide encodes a nitrate transporter, a nitrate reductase or a root protein, and wherein said second polynucleotide is operably linked to a second promoter functional in plant cells. 
     
     
         14 . The method of  claim 13 , wherein the second polynucleotide is selected from the group consisting of YNT1, optimized or partially optimized YNT1, YNR1, PPNR A551G and ZM-CKXg, and wherein the second promoter is selected from the group consisting of maize root metallothionein promoter (ZM-RM2); maize NAS2 promoter, banana streak virus promoter truncated version promoter (BSV (TR)), maize phosphoenolpyruvate carboxylase promoter (ZM-PEPC); and maize ubiquitin promoter (ZM-UBI). 
     
     
         15 . A method of modulating the level of NT protein in a plant cell, comprising:
 (a) transforming a plant cell with a NT polynucleotide of  claim 8  operably linked to a promoter that drives expression in a plant, wherein the polynucleotide is in sense orientation; and   (b) expressing the polynucleotide for a time sufficient to modulate the NT protein in the plant cell.   
     
     
         16 . A method of modulating the level of NT protein in a plant, comprising:
 (a) stably transforming a plant cell with an NT polynucleotide of  claim 8  operably linked to a promoter that drives expression in a plant, wherein the polynucleotide is in sense orientation; and   (b) regenerating the transformed plant cell into a transformed plant that expresses the NT polynucleotide in an amount sufficient to modulate the level of NT protein in the plant.   
     
     
         17 . A method of modulating nitrate uptake in plants, comprising:
 (a) introducing into a plant cell a recombinant expression cassette comprising an NT polynucleotide of  claim 8  operably linked to a promoter that drives expression in a plant; and   (b) culturing the plant under plant cell growing conditions; wherein the nitrogen uptake in the plant cell is modulated.   
     
     
         18 . The method of  claim 17 , wherein the plant displays at least one phenotypic change when compared to a control plant, said phenotypic change selected from the group consisting of:
 (i) enhanced root growth;   (ii) increased stay green;   (iii) increased fresh weight when the plant is grown under limited nitrogen fertility; and   (iv) increased root architecture.   
     
     
         19 . A method of uncoupling in a plant nitrate signalling from nitrate uptake comprising expressing in a plant a microbial nitrate transporter that is encoded by an NT polynucleotide of  claim 8 . 
     
     
         20 . An isolated polypeptide selected from the group consisting of:
 (a) an polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 or 9;   (b) a polypeptide that is at least 70% identical to at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 5 and 9, wherein said polypeptide has NR activity and comprises the A561G substitution, the S561D substitution, or the A561 and S561 D substitutions;   (c) a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or 8;   (d) a polypeptide that is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 70% identical to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 4 and 8, wherein said polypeptide has NR activity and comprises the A561G substitution, the S561D substitution, or the A561 and S561 D substitutions;   (e) a polypeptide that is encoded by a nucleic acid molecule that hybridizes with a nucleic acid probe consisting of at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 4 or 8, following at least one wash in 0.2×SSC at 55° C. for 20 minutes, wherein said polypeptide has NR activity and comprises the A561G substitution, the S561D substitution, or the A561 and S561 D substitutions;   (f) a fragment comprising at least 200 consecutive amino acids of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 5 and 9, wherein said polypeptide has NR activity and comprises the A561G substitution, the S561D substitution, or the A561 and S561 D substitutions.   
     
     
         21 . A method of modulating the level of NR protein in a plant cell, comprising:
 (a) transforming a plant cell with a NR polynucleotide of  claim 8  operably linked to a promoter that drives expression in a plant, wherein the polynucleotide is in sense orientation; and   (b) expressing the polynucleotide for a time sufficient to modulate the NR protein in the plant cell.   
     
     
         22 . A method of modulating the level of NR protein in a plant, comprising:
 (a) stably transforming a plant cell with a NR polynucleotide of  claim 8  operably linked to a promoter that drives expression in a plant, wherein the polynucleotide is in sense orientation; and   (b) regenerating the transformed plant cell into a transformed plant that expresses the NR polynucleotide in an amount sufficient to modulate the level of NR protein in the plant.   
     
     
         23 . A method for increasing yield in a plant, said method comprising the steps of:
 (a) introducing into a plant cell a construct comprising an NR polynucleotide of  claim 8  operably linked to a promoter functional in plant cells, so as to yield a transformed plant cell;   (b) regenerating a transgenic plant from said transformed plant cell, wherein NR protein is expressed in the transgenic plant at levels sufficient to maintain or increase yield in said transgenic plant;   wherein increased yield comprises enhanced root growth, increased seed size, increased seed weight, the plant has seed with increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size or chlorophyll content, wherein the yield of the plant is compared to a control plant, and wherein the control plant does not contain the polynucleotide encoding the NR   
     
     
         24 . The method of  claim 23 , wherein the plant is grown under limited nitrogen fertility. 
     
     
         25 . The method of  claim 23 , wherein the nitrogen utilization efficiency of the plant is increased. 
     
     
         26 . The method of  claim 23 , wherein NR activity of the NR polynucleotide is increased compared to the activity of a NR endogenous to the plant. 
     
     
         27 . The method of  claim 23 , wherein the expression of the NR polynucleotide is driven by a phosphoenolpyruvate decarboxylase (PEPC) promoter. 
     
     
         28 . The method of  claim 23 , wherein said polynucleotide encoding the NR is expressed in a cell-specific, tissue-specific, organ-specific, constitutive, or inducible manner.