Use of nap gene to manipulate leaf senescence in plants

Abstract

The present invention discloses transgenic plants having an altered level of NAP protein compared to that of a non-transgenic plant, where the transgenic plants display an altered leaf senescence phenotype relative to a non-transgenic plant, as well as mutant plants comprising an inactivated NAP gene, where mutant plants display a delayed leaf senescence phenotype compared to that of a non-mutant plant. The present invention also discloses methods for delaying leaf senescence in a plant, as well as methods of making a mutant plant having a decreased level of NAP protein compared to that of a non-mutant plant, where the mutant plant displays a delayed leaf senescence phenotype relative to a non-mutant plant. Methods for causing precocious leaf senescence or promoting leaf senescence in a plant are also disclosed. Also disclosed are methods of identifying a candidate plant suitable for breeding that displays a delayed leaf senescence and/or enhanced yield phenotype.

1 .- 46 . (canceled) 
     
     
         47 . A method of making a mutant plant having a decreased level of NAP protein compared to that of a non-mutant plant, wherein the mutant plant displays a delayed leaf senescence phenotype relative to a non-mutant plant, said method comprising:
 providing at least one cell of a non-mutant plant containing a gene encoding a functional NAP protein;   treating said at least one cell of a non-mutant plant under conditions effective to inactivate said gene, thereby yielding at least one mutant plant cell containing an inactivated NAP gene; and   propagating said at least one mutant plant cell into a mutant plant, wherein said mutant plant has a decreased level of NAP protein compared to that of the non-mutant plant and displays a delayed leaf senescence phenotype relative to a non-mutant plant.   
     
     
         48 . The method according to  claim 47 , wherein the functional NAP protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     
     
         49 . The method according to  claim 47 , wherein said treating comprises subjecting the at least one cell of the non-mutant plant to a chemical mutagenizing agent under conditions effective to yield at least one mutant plant cell containing an inactive NAP gene. 
     
     
         50 . The method according to  claim 49 , wherein the chemical mutagenizing agent is ethylmethanesulfonate. 
     
     
         51 . The method according to  claim 47 , wherein said treating comprises subjecting the at least one cell of the non-mutant plant to a radiation source under conditions effective to yield at least one mutant plant cell containing an inactive NAP gene. 
     
     
         52 . The method according to  claim 51 , wherein the radiation source is effective in producing ultraviolet rays, gamma rays, or fast neutrons. 
     
     
         53 . The method according to  claim 47 , wherein said treating comprises inserting an inactivating nucleic acid molecule into the gene encoding the functional NAP protein or its promoter under conditions effective to inactivate the gene. 
     
     
         54 . The method according to  claim 53 , wherein said inactivating nucleic acid molecule is a transposable element. 
     
     
         55 . The method according to  claim 54 , wherein the transposable element is selected from the group consisting of an Activator (Ac) transposon, a Dissociator (Ds) transposon, and a Mutator (Mu) transposon. 
     
     
         56 . The method according to  claim 47 , wherein said treating comprises subjecting the at least one cell of the non-mutant plant to  Agrobacterium  transformation under conditions effective to insert an  Agrobacterium  T-DNA sequence into the gene, thereby inactivating the gene. 
     
     
         57 . The method according to  claim 56 , wherein said treating comprises subjecting the at least one cell of the non-mutant plant to site-directed mutagenesis of the NAP gene or its promoter under conditions effective to yield at least one mutant plant cell containing an inactive NAP gene. 
     
     
         58 . The method according to  claim 56 , wherein said treating comprises mutagenesis by homologous recombination of the NAP gene or its promoter. 
     
     
         59 . The method according to  claim 56 , wherein said treating comprises targeted deletion of a portion of the NAP gene sequence or its promoter. 
     
     
         60 . The method according to  claim 56 , wherein said treating comprises targeted insertion of a nucleic acid sequence into the NAP gene or its promoter. 
     
     
         61 . The method according to  claim 47 , wherein the non-mutant plant is a crop plant. 
     
     
         62 . The method according to  claim 61 , wherein the crop plant is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, kidney bean, pea, chicory, lettuce, endive, cabbage, bok choy, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, peach, strawberry, grape, raspberry, pineapple, soybean,  Medicago , tobacco, tomato, sorghum, and sugarcane. 
     
     
         63 . The method according to  claim 47 , wherein the non-mutant plant is an ornamental plant. 
     
     
         64 . The method according to  claim 63 , wherein the ornamental plant is selected from the group consisting of  Arabidopsis thaliana, Saintpaulia, Populus , petunia, pelargonium, poinsettia, chrysanthemum, carnation, zinnia, turfgrass, lily, and nightshade. 
     
     
         65 . A mutant plant produced according to the method of  claim 47 . 
     
     
         66 . A mutant plant seed produced by growing the mutant plant according to  claim 65  under conditions effective to cause the mutant plant to produce seed. 
     
     
         67 . A method for causing precocious leaf senescence or promoting leaf senescence in a plant, said method comprising:
 transforming a plant cell with a nucleic acid molecule encoding a NAP protein capable of causing leaf senescence in a plant operably associated with a promoter to obtain a transformed plant cell;   regenerating a plant from the transformed plant cell; and   inducing the promoter under conditions effective to cause premature or precocious leaf senescence in the plant.   
     
     
         68 . The method according to  claim 67 , wherein the nucleic acid molecule encodes a NAP protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     
     
         69 . The method according to  claim 67 , wherein the plant cell is from a crop plant. 
     
     
         70 . The method according to  claim 69 , wherein the crop plant is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, kidney bean, pea, chicory, lettuce, endive, cabbage, bok choy, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, peach, strawberry, grape, raspberry, pineapple, soybean,  Medicago , tobacco, tomato, sorghum, and sugarcane. 
     
     
         71 . The method according to  claim 70 , wherein the crop plant is cotton. 
     
     
         72 . The method according to  claim 67 , wherein the plant cell is from an ornamental plant. 
     
     
         73 . The method according to  claim 72 , wherein the ornamental plant is selected from the group consisting of  Arabidopsis thaliana, Saintpaulia, Populus , petunia, pelargonium, poinsettia, chrysanthemum, carnation, zinnia, turfgrass, lily, and nightshade. 
     
     
         74 . The method according to  claim 67 , wherein said transforming is carried out by a method selected from the group consisting of  Agrobacterium  mediated transformation, vacuum infiltration, biolistic gene transformation, electroporation, microinjection, chemical-mediated transformation, and laser-beam transformation. 
     
     
         75 . A plant produced by the method of  claim 67 . 
     
     
         76 . A method of identifying a candidate plant suitable for breeding that displays a delayed leaf senescence and/or enhanced yield phenotype, said method comprising:
 analyzing the candidate plant for the presence, in its genome, of an inactivated NAP gene.   
     
     
         77 . The method according to  claim 76 , wherein the method identifies a candidate plant suitable for breeding that displays a delayed leaf senescence phenotype. 
     
     
         78 . The method according to  claim 76 , wherein the method identifies a candidate plant suitable for breeding that displays an enhanced yield phenotype. 
     
     
         79 . The method according to  claim 76 , wherein the method identifies a candidate plant suitable for breeding that displays a delayed leaf senescence and enhanced yield phenotype. 
     
     
         80 . The method according to  claim 76 , wherein the plant is a crop plant. 
     
     
         81 . The method according to  claim 80 , wherein the crop plant is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, kidney bean, pea, chicory, lettuce, endive, cabbage, bok choy, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, peach, strawberry, grape, raspberry, pineapple, soybean,  Medicago , tobacco, tomato, sorghum, and sugarcane. 
     
     
         82 . The method according to  claim 76 , wherein the plant is an ornamental plant. 
     
     
         83 . The method according to  claim 82 , wherein the ornamental plant is selected from the group consisting of  Arabidopsis thaliana, Saintpaulia, Populus , petunia, pelargonium, poinsettia, chrysanthemum, carnation, zinnia, turfgrass, lily, and nightshade.