Identification And Gene Mapping Of Starch Accumulation And Early Senescence Leaf Mutant Esl9 In Rice

Leaf,as the main place of photosynthesis,is an important organ of plant growth and development.Early senescence of leaf will directly affect plant photosynthesis,growth and metabolic activities,thus affecting the yield and quality traits.A new leaf senescence mutant esl9?early senescence leaf 9?which may be caused by excessive accumulation of starch in leaves was discovered from the progeny of indica maintainer line 1B mutated by ethyl methane sulfonate?EMS?.In this study,the phenotype of the mutant was identified and the agronomic traits were investigated.Meanwhile we determinated the photosynthetic pigment contents,the senescence-related physiological indexes and the expression of SAGs,also observed the cell structure.In addition,the starch content of leaf was tested and the leaf was dyed by iodine-iodide kalium to check whether there was excessive starch in leaves of the mutant esl9,also we analyzed the expression of starch metabolism-related genes.In order to seek the target gene of esl9,the mutant gene was preliminarily mapped and refined.These results will lay a foundation for cloning and functional analysing ESL9.The main studies are as follow: 1 Phenotype and main agronomic traitsThe leaf of esl9 was pale green at seedling stage;chlorosis occurred at leaf tip and gradually extended to the middle-upper parts of leaf at tillering stage.However,the leaf base remained green until maturity.Compared with the wild-type,many agronomic traits of esl9 decreased extremely significantly,such as the effective panicle,the grain number per panicle,the filled grain number per panicle,the seed setting rate and the primary branch number per panicle,except the plant height,the panicle length and the 1000-grain weight deduced significantly.The number of grains per panicle was only 17.35% of the wild-type,whose reduction was the largest among these agronomic traits.2 Photosynthetic pigments and net photosynthetic rateCompared with the wild-type,the photosynthetic pigment contents of the mutant esl9 decreased in varying degrees at seedling stage or tillering stage.In the second leaf of esl9,the contents of four kinds of photosynthetic pigments were all less than half of those in the wild-type respectively.At the same time,the net photosynthetic rate of esl9 was extremely significantly lower than that of the wild-type.3 Cytological observationMany excessive starch granules could be seen in the chloroplast of mutant esl9 through the transmission electron microscope.Moreover,the chloroplast structure of esl9 was destroyed and its auto-fluorescence weakened.4 Starch content and expression of genes participated in starch metabolismThe results of iodine-iodide kalium dyeing showed that more starch granules accumulated in the esl9 leaves,especially in the second leaf.Meanwhile the starch content of the second leaves was determinated,and results showed that the starch content of esl9 was extremely significantly higher than that of the wild-type in the morning or evening.However,the starch content in the morning was lower than that in the evening,indicating that in the esl9 leaf,the starch had a process of accumulation and it also could be degraded in the evening,but not completely,so excess starch accumulated gradually and eventually caused excessive accumulation.Quantitative RT-PCR results indicated that genes responsible for starch synthesis were up-regulated and genes participated in the triose phosphate distribution path were down-regulated.We made an inference that gene mutation changed the distribution of triose phosphate,causing the starch synthesis enhanced and starch degradation also been promoted,but the decomposition rate was not as good as the synthesis rate,therefore the starch accumulated excessively in the esl9 leaves.5 Physiological indexes related to senecenceCompared with the wild-type,activities of protective enzymes,SOD and CAT,both reduced in esl9,and the activity of POD elevated slightly.The contents of reactive oxygen species?ROS?,such as,O₂^-,·OH and H₂O₂,heightened compared with those in the wild-type.6 Expression of SAGs in riceQuantitative RT-PCR results of SAGs indicated that the expression of the positive regulatory factors of senescence up-regulated and the negative regulatory factors down-regulated,further accelerating the chlorophyll degradation,the destruction of chloroplast and free radical accumulation,promoting aging finally.7 Genetic analysisHybrid combinations were formulated with 56 S and esl9.The leaf of the F1 group plants was yellow.Plants showed obvious character segregation in F2 group,both yellow leaf and green leaf,corresponding 1941 and 596 single plants respectively.The separation ratio was 3:1.As a consequence,the phenotype of esl9 was controlled by a dominant nuclear gene.8 Gene mapping and candidate genesThe target gene ESL9 was mapped between SSR markers S11-110 and S11-87 with a physical distance of 304.9 kb on chromosome 11 through the preliminary and fine mapping.There were 41 annotated genes in this interval,among these,nine genes coding expressed protein,four coding leucine rich repeat family protein,two coding tropinone reductase 2,one coding hypothetical protein and the others coding transposon protein and retrotransposon protein.No reported senescence-related genes were found in this interval.The four genes that code leucine rich repeat family protein and participate in the programmed cell death,containing NB-ARC domain,were made a sequencing comparison.However,there was no differential locis between the wild-type and the mutant esl9.These results will lay a foundation for cloning and functionally analysing ESL9.