Plants with improved nitrogen utilization and stress tolerance

Abstract

The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, or both and that have been transformed using a novel vector construct including nucleic acid sequences that modulate nitrogen use in plants. In various embodiments, the vector construct includes one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, or 38. The invention also relates to isolated vectors for transforming plants and to antibodies used for detecting transformed plants. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants or are modulated by nitrogen conditions.

1 . An expression vector comprising a nucleotide sequence selected from the group consisting of:
 a) a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38;   b) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, wherein said nucleotide sequence modulates nitrogen use in a plant;   c) a nucleotide sequence encoding an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 36; and,   (d) a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, wherein said nucleotide sequence modulates nitrogen use in a plant.   
     
     
         2 . An expression vector according to  claim 1 , further comprising a 5′ DNA promoter sequence and a 3′ terminator sequence, wherein the nucleotide sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence. 
     
     
         3 . An expression vector according to  claim 2 , wherein the promoter sequence is selected from the group consisting of constitutive plant promoters and tissue specific promoters. 
     
     
         4 . A polyclonal antibody, comprising a polyclonal antibody to an amino acid sequence encoded by a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36; and SEQ ID NO: 38. 
     
     
         5 . A plant, comprising a plant transformed with at least a first nucleotide sequence selected from the group consisting of:
 a) a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38;   b) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, wherein said nucleotide sequence modulates nitrogen use in a plant;   c) a nucleotide sequence encoding an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 36; and,   (d) a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, wherein said nucleotide sequence modulates nitrogen use in a plant.   
     
     
         6 . The plant of  claim 5 , wherein said plant further comprises at least a second nucleotide sequence selected from (a), (b), (c), or (d), wherein said first and said second nucleotide sequences are non-identical. 
     
     
         7 . A plant according to  claim 5 , wherein the plant is selected from the group consisting of corn (maize); sorghum; wheat; sunflower; tomato; crucifers; peppers; potato; cotton; rice; soybean; sugarbeet; sugarcane; tobacco; barley; and oilseed rape;  Brassica  sp.; alfalfa; rye; millet; safflower; peanuts; sweet potato; cassava; coffee; coconut; pineapple; citrus trees; cocoa; tea; banana; avocado; fig; guava; mango; olive; papaya; cashew;  macadamia ; almond; oats; vegetables; grasses; vegetables, including but not limited to, onions, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus  Curcumis  such as cucumber, cantaloupe, and muskmelon; ornamentals, including, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum; fruit trees; and conifers. 
     
     
         8 . A component part of a plant of  claim 7 . 
     
     
         9 . A plant seed produced from a plant of  claim 7 . 
     
     
         10 . A plant seed transformed with a vector of  claim 1 . 
     
     
         11 . A host cell, comprising a host cell transformed with at least a first nucleotide sequence selected from the group consisting of
 a) a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38;   b) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, wherein said nucleotide sequence modulates nitrogen use in a plant;   c) a nucleotide sequence encoding an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 36; and,   (d) a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, wherein said nucleotide sequence modulates nitrogen use in a plant.   
     
     
         12 . The host cell of  claim 11 , wherein said host cell further comprises at least a second nucleotide sequence selected from (a), (b), (c), or (d), wherein said first and said second nucleotide sequences are non-identical. 
     
     
         13 . A host cell according to  claim 11 , wherein the host cell is selected from the group consisting of bacterial cells and plant cells. 
     
     
         14 . A vector construct, comprising:
 a) at least a first nucleotide sequence encoding an amino acid sequence selected from the group consisting of:
 i) an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39; 
 ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, wherein said amino acid sequence modulates nitrogen use in a plant; 
 iii) an amino acid sequence encoded by a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38; and, 
 iv) an amino acid sequence encoded by a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, wherein said nucleotide sequence modulates nitrogen use in a plant; 
   b) a 5′ DNA promoter sequence; and,   c) a 3′ terminator sequence,   
       wherein the nucleotide sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence. 
     
     
         15 . A vector construct according to  claim 14 , further comprising at least a second nucleotide sequence encoding an amino acid sequence selected from (a)(i), (a)(ii), (a)(iii), or (a)(iv), wherein the amino acid sequences encoded by said first and said second nucleotide sequences are non-identical. 
     
     
         16 . A method of expressing a nucleic acid molecule modulated by nitrogen in a plant, said method comprising the steps of providing a transgenic plant or plant seed transformed with a vector construct according to  claim 1 , and growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed. 
     
     
         17 . A method according to  claim 16 , wherein expression of the nucleic acid molecule is effective in increasing nitrogen uptake of said transgenic plant or said plant grown from the transgenic plant seed. 
     
     
         18 . A method according to  claim 16 , wherein expression of the nucleic acid molecule is effective in increasing efficiency of nitrogen utilization of said transgenic plant or said plant grown from the transgenic plant seed. 
     
     
         19 . A method according to  claim 16 , wherein the plant is selected from the group consisting of rice, corn, soybean, canola, wheat, alfalfa, barley, rye, cotton, sunflower, peanut, sweet potato, bean, pea, potato, oilseed rape, sorghum, forage grass, pasture grass, turf grass, sugarcane. 
     
     
         20 . A method of according to  claim 16 , wherein expression of the nucleic acid molecule is effective in improving the stress tolerance of said transgenic plant or said plant grown from the transgenic plant seed. 
     
     
         21 . A method according to  claim 16 , wherein expression of the nucleic acid molecule is effective in altering the morphology of said transgenic plant or said plant grown from the transgenic plant seed.