Positional Cloning And Functional Analysis Of Early Senescence Gene ES5 In Rice (Oryza Sativa L.)

Leaf senescence,which affects the plant growth and yield in rice,is an ideal target for rice improvement and remarkable advances have been made to identify the mechanisms behind this process.It is very important for the growth and development of the plant.In the present study we have reported a novel early senescence mutant es5?early leaf senescence 5?which was isolated from a mutant library of ethyl methanesulfonate?EMS?treated japonica rice cultivar Jiahe212.We carried out phenotypic characterization,agronomic traits evaluation,physiological and biochemical characterization,detection of cell death occurrence and ROS accumulation,genetic mapping of the ES5 gene,functional complementation and lipid metabolites profiling.We have successfully cloned the ES5 gene encoding a phosphatidylserine synthase enzyme.Our study provides evidence that the mutation of ES5 causes the accumulation of phosphatidylserine and impacts on early senescence in rice?Oryza sativa L.?.The results obtained in this experiment are as follows:1.The es5 mutant showed a leaf yellowing phenotype after the 4-leaf stage.This leaf yellowing phenotype became severe at the maturity stage.This phenotype was confirmed by the higher accumulation of reactive oxygen species?ROS?and malondialdehyde?MDA?,the disintegration of chloroplasts,reduction in chlorophyll content and photosynthetic rate.Early senility also affected the agronomic performances negatively such as reduced plant height,number of effective tillers plant^-1,seed setting rate,and thousand-grain weight.Moreover,senescence-associated genes and chlorophyll degradation related genes were up-regulated and photosynthesis-related genes were down-regulated.2.The candidate region for ES5 was fine mapped on the long arm of chromosome 5 with a physical distance of 37.57 kb.The candidate region harbors 6 open reading frames?ORFs?.The DNA sequencing result showed that the es5 mutant has a‘G-to-A'substitution in the ORF2,which was confirmed by the cDNA sequencing.Functional complementation of the ES5 gene in the es5 mutant background completely rescued the wild-type phenotype,which proves that‘G-to-A'substitution in the ES5 caused the early senescence phenotype.3.ES5 encodes a membrane-bound enzyme phosphatidylserine synthase?PSS?which is involved in the biosynthesis of a minor membrane phospholipid phosphatidylserine?PS?.We have measured the PSS and PS content in the wild-type,es5 and complemented plants and found that PSS and PS content are significantly higher in the es5 compared to wild-type and complemented plants.This result implies that the altered level of PS may play an important role in cell death signaling and impacts on early senescence in rice?Oryza sativa L.?.4.We also performed lipid metabolites profiling and found that a total of 1514 metabolites expressed differentially in wild-type vs es5 leaves,of which 1220 were upregulated and 294 were down-regulated.KEGG analysis with differentially expressed metabolites revealed that glycerophospholipid had significant enrichment in KEGG pathways which implies that altered glycerophospholipid metabolism may initiate the early leaf senescence in es5 mutants.