Nitrate reductases from red algae, compositions and methods of use thereof

Abstract

The NR enzymes described herein were discovered in the red algae of Porphyra perforata (PpNR) and Porphyra yezoensis (PyNR). The present invention provides methods and compositions relating to altering NR 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 nitrogen fertility. The invention provides isolated nitrate reductase (NR) 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 NR enzyme show improved properties, for example, increased yield and growth.

What is claimed is: 
     
         1 . An isolated nitrate reductase (NR) polynucleotide comprising a member selected from the group consisting of:
 (a) a polynucleotide that encodes the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11 or 12;   (b) a polynucleotide comprising the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6; and   (c) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under stringent conditions to a polynucleotide of (a) or (b), 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   (d) a polynucleotide having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5 or 6, 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 nitrate reductase (NR) activity; and   (e) an isolated polynucleotide degenerate from any of (a) to (e) as a result of the genetic code;   (f) a polynucleotide complimentary to a polynucleotide of any one of (a) to (e).   
     
     
         2 . An isolated polynucleotide according to  claim 1  that encodes a NR polypeptide that confers increased yield or nitrogen utilization efficiency under lower fertility. 
     
     
         3 . A vector comprising at least one polynucleotide of  claim 1 . 
     
     
         4 . An expression cassette comprising at least one polynucleotide of  claim 1  operably linked to a promoter, wherein the polynucleotide is in sense orientation. 
     
     
         5 . A host cell into which is introduced at least one expression cassette of  claim 4 . 
     
     
         6 . The host cell of  claim 5  that is a plant cell. 
     
     
         7 . A transgenic plant comprising at least one expression cassette of  claim 4 . 
     
     
         8 . The transgenic plant of  claim 7 , wherein the plant is selected from the group consisting of: corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet. 
     
     
         9 . A seed from the transgenic plant of  claim 7 . 
     
     
         10 . The seed of  claim 9 , wherein the seed is corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley or millet. 
     
     
         11 . An isolated polypeptide selected from the group consisting of:
 a) an isolated polypeptide comprising any one of SEQ ID NOS: 7, 8, 9, 10, 11 or 12 said polypeptide having NR activity;   b) a polypeptide that is at least 70% identical to the amino acid sequence of any of SEQ ID NOS: 7, 8, 9, 10, 11 or 12 said polypeptide having NR activity;   c) a polypeptide that is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 70% identical to any one of SEQ ID NOS: 1, 2, 3, 4, 5 or 6 or a complement thereof, said polypeptide having NR activity;   d) a polypeptide that is encoded by a nucleic acid molecule that hybridizes with a nucleic acid probe consisting of the nucleotide sequence of any of SEQ ID NOS: 1, 2 or 3, or a complement thereof following at least one wash in 0.2×SSC at 55° C. for 20 minutes, said polypeptide having NR activity;   e) a fragment comprising at least 200 consecutive amino acids of any of SEQ ID NOS: 7, 8, 9, 10, 11, or 12, said polypeptide having NR activity.   
     
     
         12 . A recombinant expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide encodes the polypeptide of  claim 11 . 
     
     
         13 . A transformed host cell comprising the isolated polypeptide of  claim 11 . 
     
     
         14 . The host cell of  claim 13 , wherein the host cell is a transformed plant cell. 
     
     
         15 . The plant cell of  claim 14 , wherein the plant cell is selected from the group consisting of sorghum, maize, rice, wheat, soybean, sunflower, canola, alfalfa, barley and millet. 
     
     
         16 . A transformed plant regenerated from the plant cell of  claim 14 . 
     
     
         17 . The plant of  claim 16 , wherein the plant is sorghum, maize, rice, wheat, soybean, sunflower, canola, alfalfa, barley or millet. 
     
     
         18 . A transformed seed of the plant of  claim 16 . 
     
     
         19 . An isolated polypeptide encoded by the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5 or 6. 
     
     
         20 . The isolated polypeptide of  claim 11  wherein the expression of NR in a plant results in an increased yield in the plant as compared to a control plant, wherein the control plant that does not contain the polynucleotide encoding the NR. 
     
     
         21 . A method of modulating the level of nitrate reductase (NR) protein in a plant cell, comprising:
 (a) transforming a plant cell with a NR polynucleotide operably linked to a promoter, 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 . The method of  claim 21 , wherein the plant is corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley or millet. 
     
     
         23 . The method of  claim 21 , wherein NR protein is increased. 
     
     
         24 . The method of  claim 21 , wherein NR protein is decreased. 
     
     
         25 . A method of modulating the level of nitrate reductase (NR) protein in a plant, comprising:
 (a) stably transforming a plant cell with a NR polynucleotide operably linked to a promoter, 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.   
     
     
         26 . The method of  claim 25 , wherein the plant is corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley or millet. 
     
     
         27 . The method of  claim 25 , wherein NR protein is increased as compared to a control plant, wherein the control plant that does not contain the polynucleotide encoding the NR. 
     
     
         28 . The method of  claim 25 , wherein NR protein is decreased as compared to a control plant, wherein the control plant that does not contain the polynucleotide encoding the NR. 
     
     
         29 . A method for increasing yield in a plant, said method comprising the steps of:
 (a) introducing into plant cells a construct comprising a polynucleotide encoding a nitrate reductase (NR), wherein said NR polynucleotide is operably linked to a promoter functional in plant cells to yield transformed plant cells, and wherein the NR encoding the NR protein is selected from the group consisting of:
 (1) a polynucleotide that encodes the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11 or 12; 
 (2) a polynucleotide comprising the sequence set forth in the encoding region of SEQ ID NO: 7, 8, 9, 10, 11 or 12; and 
 (3) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under moderate stringency conditions to a polynucleotide of (a) or (b), 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 
 (4) a polynucleotide having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5 or 6, wherein the % sequence identity is based on the entire encoding region and is determined by BLAST 2.0 under default parameters; and 
 (5) an isolated polynucleotide degenerate from any of (1) to (4) as a result of the genetic code; 
 (6) a polynucleotide complimentary to a polynucleotide of any one of (1) to (5); 
   (b) regenerating a transgenic plant from said transformed plant cells, wherein said NR is expressed in the cells of said transgenic plant at levels sufficient to maintain or increase yield in said transgenic plant.   
     
     
         30 . The method of  claim 29 , 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. 
     
     
         31 . The method of  claim 29 , wherein the plant is grown under limited nitrogen fertility. 
     
     
         32 . The method of  claim 31 , wherein the nitrogen utilization efficiency of the plant is increased. 
     
     
         33 . The method of  claim 31 , wherein NR activity of the NR polynucleotide is increased compared to the activity of a NR endogenous to the plant. 
     
     
         34 . The method of  claim 29 , wherein the expression of the NR polynucleotide is driven by a phosphoenopyruvate decarboxylase (PEPC) promoter. 
     
     
         35 . The method of  claim 29 , wherein said polynucleotide encoding the NR is constitutively expressed. 
     
     
         36 . The method of  claim 29 , wherein said polynucleotide encoding the NR is inducibly expressed. 
     
     
         37 . The method of  claim 29 , wherein said polynucleotide encoding the NR is expressed in a cell-specific, tissue-specific, or organ-specific manner. 
     
     
         38 . The method of  claim 29 , wherein the plant is a dicotyledonous plant. 
     
     
         39 . The method of  claim 29 , wherein the plant is a monocotyledonous plant. 
     
     
         40 . The method of  claim 29 , wherein the yield of the plant is compared to a control plant, wherein the control plant does not contain the polynucleotide encoding the NR.