Genetic Analysis And Gene Mapping Of Premature Senescence Leaf Mutants Es5 And G398 In Rice

The plant senescence,an important period of plant development,is a programmed cell death process commonly subjected to genetic and external factors?high temperature,drought,pests,diseases,hormones,light and so on?.The leaf,an important source of plant organs,provides the energy and a large amount of organic material for plant.Rice is a principal cereal crop and premature leaf senescence in rice affects plant yield and quality.Study on rice premature senescence related genes is helpful in clarifying the molecular mechanisms of rice premature senescence and it is also important in rice genetic improvement.1.es5 was isolated from the progeny of Oryza sativa subsp.Geng cv.Jiahe212 treated by ethyl methane sulfonate?EMS?.Leaf senescence in es5 occurred at 4-leaf-stage.The etiolated area of the rice leaf increased gradually with the growth and development.The top second leaf,third leaf and forth leaf showed serious aging in heading stage.Compared with the wide-type,Chl a,Chl b,carotenoid content and photosynthetic capacity of mutant significantly decreased and the plant height,the number of productive panicles per plant,the number of spikelet per panicle,1000-grain weight and seed setting rate also significantly reduced.The results of Evans blue and DAB staining assay indicated that there were more dead cells and H₂O₂ in leaves of es5.The SOD activities and MDA content increased significantly in es5 while the soluble protein content significantly decreased in heading stage.Dissolved cytoplasm,abnormal chloroplast structure,dissolved chloroplast membrane,blurred base,loose lamellar lamellae,abnormal thylakoid development and more starches grains and osmiophilic granules were observed in the senescent leaf region of es5 by TEM.The genetic analysis revealed that the phenotype of es5 was controlled by single recessive nuclear gene,and ES5 was fine mapped in a 52.7 Kb interval including 8open reading frames between BF-10 and RM3664 on chromosome 5 by map-based cloning strategy.qRT-PCR analysis showed that senescence-associated genes Osh36,Osh69,OsI85,RCCR1 were up regulated significantly in es5.2.The early senescence mutant g398 was isolated from EMS treatment rice mutant library?Oryza sativa subsp.Xian cv.ZH8015?.There was no difference in the phenotype between mutant g398 and wide-type ZH8015 before the tillering stage and the leaves in the lower part of mutant g398 were yellowish at late tillering stage.Most lower leaves of the mutant g398 were from yellow to red-brown after heading,and the flag leaves also turned yellow.The lower leaves of mutant g398 became withered at the mature stage.Compared with the Wild-type,the plant height,the grain length,the grain width,the number of productive panicles per plant and 1000-grain weight significantly reduced.The length and width of flag leave and top second leaf in mutant g398 were significantly reduced and there was no significant difference in panicle length between mutant g398 and ZH8015.Histochemical analysis showed that there were more dead cells and reactive oxygen in leaves of mutant g398.The results of chlorophyll and photosynthetic indexes showed that the net photosynthetic capacity and chlorophyll content in the leaves of the mutant g398 decreased significantly at 7 days after heading.Enzyme activity and senescence associated index analysis results showed that the content of H₂O₂ and MDA in mutant g398 were significantly higher than that in wild-type leaves,while the protein content and CAT enzyme activity were significantly reduced,and the T-AOC and T-SOD activities were significantly increased.The results of transmission electron microscopy?TEM?showed that abnormal chloroplast structure resulted in the poor photosynthesis in mutant g398 leaves.The genetic analysis revealed that the phenotype of g398 was controlled by single recessive nuclear gene,the target gene was located within the437Kb physical interval between the MA2-43 and MA2-74 markers on the long arm of the chromosome5.qRT-PCR analysis showed that the expression of senescence-associated genes SGR,RCCR1,and OsPAO were significantly upregulated in the mutant g398,and the senescence marker genes OsH36,OsI57,OsH69,and OsI85 were significantly up-regulated.The chlorophyll degradation-related gene NYC1,NOL,and NYC4 were significantly down-regulated except NYC3.Chlorophyll synthesis-related genes were significantly down-regulated and the expression of some photosynthetic genes were significantly reduced.