Plant tolerance to stress through the control of chloroplast stability

Abstract

The chloroplast vesiculation (CV) gene encodes a protein associated with stress-induced chloroplast degradation. Inhibiting expression or activity of the protein inhibits or delays stress-induced chloroplast degradation and confers tolerance to a variety of stress conditions. Alternatively, enhancing CV expression or activity promotes chloroplast degradation and enhances nutrient assimilation in desired sink tissues, such as young leaves, fruit, or seeds.

1 . A method of preparing a transgenic plant having enhanced stress tolerance, the method comprising
 (a) introducing into a population of plants an expression cassette that inhibits expression of a chloroplast vesiculation (CV) gene; and   (b) selecting a plant having enhanced stress tolerance compared to a control plant that does not comprise the expression cassette.   
     
     
         2 . The method of  claim 1 , wherein the expression cassette comprises a nucleic acid sequence encoding a microRNA or an siRNA specific to the CV gene. 
     
     
         3 . The method of  claim 1 , wherein the CV gene encodes a CV protein comprising a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46. 
     
     
         4 . The method of  claim 1 , wherein the CV gene encodes a polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44. 
     
     
         5 . The method of  claim 1 , wherein the step of introducing is carried out using  Agrobacterium.    
     
     
         6 . The method of  claim 1 , wherein the expression cassette comprises a constitutive promoter. 
     
     
         7 . The method of  claim 1 , wherein the expression cassette comprises an inducible promoter. 
     
     
         8 . The method of  claim 7 , wherein the inducible promoter is induced in response to stress conditions. 
     
     
         9 . The method of  claim 1 , wherein the transgenic plant has enhanced tolerance to an abiotic stress. 
     
     
         10 . The method of  claim 9 , wherein the abiotic stress is high salt conditions and the step of selecting includes selecting plants having enhanced salt tolerance. 
     
     
         11 . The method of  claim 9 , wherein the abiotic stress is drought conditions and the step of selecting includes selecting plants having enhanced drought tolerance. 
     
     
         12 . A plant prepared by the method of  claim 1 . 
     
     
         13 . An isolated nucleic acid molecule comprising a plant promoter operably linked to a heterologous nucleic acid sequence encoding a microRNA or an siRNA specific to a target CV gene. 
     
     
         14 . The isolated nucleic acid molecule of  claim 13 , wherein the target CV gene encodes a CV protein comprising (i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44. 
     
     
         15 . The isolated nucleic acid molecule of  claim 13 , wherein the plant promoter is a constitutive promoter. 
     
     
         16 . The isolated nucleic acid molecule of  claim 13 , wherein the plant promoter is an inducible promoter. 
     
     
         17 . A method of preparing a transgenic plant having enhanced stress tolerance, the method comprising
 (a) introducing mutations in CV genes in a population of plants; and   (b) selecting a plant having enhanced stress tolerance compared to a control plant that does not comprise the mutation.   
     
     
         18 . The method of  claim 17 , wherein the mutations are introduced using chemical mutagenesis. 
     
     
         19 . The method of  claim 18 , wherein mutations in CV genes in the population of plants are identified using Targeting Induced Local Lesions in Genomes (TILLING). 
     
     
         20 . A plant prepared by the method of  claim 17 . 
     
     
         21 . A transgenic plant comprising an expression cassette comprising a plant promoter operably linked to a nucleic acid sequence encoding a microRNA or an siRNA specific to a target CV gene. 
     
     
         22 . The transgenic plant of  claim 21 , wherein the target CV gene encodes a CV protein comprising (i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44. 
     
     
         23 . A method of preparing a transgenic plant having enhanced nutrient assimilation, the method comprising
 (a) introducing into a population of plants an expression cassette comprising an inducible plant promoter operably linked to a heterologous CV polynucleotide sequence encoding a CV protein comprising (i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44; and   (b) selecting a plant having enhanced nutrient assimilation compared to a control plant that does not comprise the expression cassette.   
     
     
         24 . The method of  claim 23 , wherein the step of introducing is carried using  Agrobacterium.    
     
     
         25 . The method of  claim 23 , wherein the step of selecting is carried out by selecting plants with increased fruit yield compared to control plants. 
     
     
         26 . A transgenic plant prepared by the method of  claim 23 . 
     
     
         27 . A transgenic plant comprising an expression cassette comprising an inducible plant promoter operably linked to a heterologous CV polynucleotide sequence encoding a CV protein comprising (i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44. 
     
     
         28 . An isolated nucleic acid molecule comprising an expression cassette comprising an inducible plant promoter operably linked to a CV polynucleotide sequence encoding a CV protein comprising (i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44.