Identification And Gene Fine Mapping Of Early Senescent Leaf Mutant Esl10 In Rice

Premature senescence in leaves leads to lower photosynthetic capacity and influences the production of photosynthetic products.Therefore,research on the senescence mechanism may contribute to genetic improvement in rice?Oryza sativa L.?.The novel early senescent leaf mutant Esl10 was identified among progeny of the rice indica maintainer line Xinong 1B raised from seeds treated with ethyl methane sulfonate?EMS?.Compared with the wild type,the leaves of Esl10 began to yellow at the four-leaf stage,and a large amount of starch and sucrose accumulated in the leaves.In this study,we analyzed the phenotypes and agronomic characters,investigated the photosynthetic characteristics and the photosynthetic pigment contents,observed the cell structure,made iodide staining and measured the starch and sucrose contents of leaves.Meanwhile we analyzed the relative expression levels of genes associated with starch metabolism and glucose metabolism,the senescence-related physiological indexes and the expression of pigment synthesis and photosynthesis,and undertook gene fine mapping of the Esl10 mutant.The main results are as follows:1 Phenotype analysis and investigation of agronomic traitsUnder paddy field conditions,the Esl10 mutant plants remained green until the four-leaf stage,when the premature leaf senescence phenotype was first evident.Yellowing gradually extended from the tip to the middle portion of the leaf,whereas the leaf base was slightly yellowed.The trait lasted until maturity.Compared with the wild type,among the measured traits,the plant height,panicle length,1000-grain weight,and grain number per panicle of the Esl10 mutant decreased significantly and were respectively 92.63,88.27,88.91,and 86.78%of the corresponding wild type values.In addition,the effective number of panicles,filled grain number per panicle,primary branch number per panicle,tiller number,and seed setting rate of Esl10declined more severely to 64.29,65.61,76.23,71.59,and 77.27%of the corresponding wild type values,respectively.2 Analysis of photosynthetic pigments and photosynthetic parametersCompared with the wild type,the decreases in the contents of chlorophyll a,chlorophyll b,total chlorophyll and carotenoids in the leaves of Esl10 at the seedling stage were highly significantly.The contents of photosynthetic pigments in different leaves of Esl10 at the tillering stage differed to varying degrees.In the second leaf,there were highly significant reduction in chlorophyll a,chlorophyll b,total chlorophyll,and carotenoids contents.Analysis of photosynthetic parameters at the heading stage showed that,compared with the wild type,there was highly significant reduction in the net photosynthetic rate in the mutant,and the stomatal conductance decreased significantly.Moreover,there was highly significant increase in the intercellular CO₂ concentration.3 Cytological observationCompared with wild type,the profile of the mesophyll cells of the Esl10 leaves was relatively obscure,appearance was dry.Fluorescence microscopy revealed that the chlorophyll autofluorescence of Esl10 mesophyll cells was extremely weak,suggesting that chlorophyll a serious degradation of mesophyll cells in Esl10 leaves.4 Iodine-potassium iodide staining and determination of sugar content in leavesThe results of I₂-KI staining indicated that starch accumulates gradually before leaf yellowing,since the amount of starch accumulated at the four-leaf stage was higher than that at the three-leaf stage.The high level of starch ultimately led to yellowing.Analysis of the concentrations of sugar in leaves of Esl10 showed that many starch granules are present in the yellowing portions of Esl10 leaves and that the concentrations of sucrose and starch in Esl10 leaves were significantly higher than those of the wild type.5 Physiological indexes related to senecenceCompared with the wild type,the concentrations of O₂^-,H₂O₂,and?OH in the first,second,and third leaves of Esl10 were significantly higher than those in the wild type,leading to excessive MDA production,and the activities of CAT,POD,and SOD were significantly reduced at the tillering stage,aggravation of membrane lipid peroxidation,and induced the early senescence of the leaves.6 Expression analysis of genes associated with pigment synthesis and photosynthesisCompared with the wild type,the transcription levels of pigment synthesis related genes,the expression levels of carotenoids synthesis pathway related genes,and the relative expression levels of genes associated with photosynthesis were all changed.Among these pathways,the expression levels of these genes were reduced to a lower level,indicating that the chloroplast development and photosynthetic capacity of Esl10 leaves were limited.7 Expression analysis of starch metabolism and sucrose metabolism related genesThe relative expression levels of genes associated with starch metabolism in leaves of Esl10 changed to varying degrees compared with those of the wild type.In the sucrose transport pathway,the relative expression levels of OsSWEET11 and OsSWEET14 were downregulated.In the sucrose synthesis pathway,the relative expression levels of genes OsCFBP1,OsCFBP2,OsSPS1,and OsSPS11 showed different degrees of downregulation.In the transport pathway of phosphotransferase,the relative expression levels of OsTPT1 and OsTPT2 were significantly lower.In the starch synthesis pathway,the relative expression levels of OsAGPL1 and OsAGPL2were upregulated.In addition,the expression level of OsISA3 was downregulated.Therefore,it is speculated that the mutated gene of Esl10 affects directly or indirectly the expression of genes associated with metabolism or transport pathway of sugar in the leaves,resulting in the disruption of the sucrose transport pathway.The TP reduction required for sucrose synthesis leads to a change in TP distribution pathways.Thus,TP is increasingly used for starch synthesis in chloroplasts.A high concentration of sucrose inhibited starch decomposition,resulting in a lower extent of starch decomposition and ultimately starch accumulated in the chloroplast.8 Genetic analysisThe Esl10 mutant was crossed with the indica-type,low-temperature-sensitive,two-line sterile line 56S.Premature senescence were observed in the F₁ progeny.Plants showed character segregation in F₂ generation.The F₂ progeny comprised 4116Esl10-like mutant plants and 1314 normal plants.The segregation of mutant plants and normal plants perfectly fitted a ratio of 3:1(?2=1.859<?2_0.05,1=3.84).This finding indicated that the phenotype of Esl10 is controlled by a dominant nuclear gene.9 Gene mapping and candidate genesThe ESL10 was mapped between the SNP markers SNP585 and SNP498 on chromosome 7 with a physical distance of 416.29 kb,an interval in which no senescence gene has been cloned and which contains 56 annotated genes.Of all the annotated genes,one encodes a salt tolerance protein,one encodes a glycosyl hydrolase family 10 protein,one encodes an ELMO/CED-12 family protein,one encodes a regulator of chromosome condensation domain containing protein,one encodes a HAD superfamily phosphatase,one encodes a WRKY transcription factor,one encodes a glutamate-cysteine ligase,two encode no apical meristem proteins,and the other genes mostly encode transposons,retrotransposons,hypothetical proteins,and expressed proteins.The WRKY family transcription factors are involved in the regulation of various biological processes,including senescence,and responses to abiotic stresses and abscisic acid in plants.However,there was no differential locis between the wild type and the Esl10 mutant.No SAGs have been reported in this interval,so ESL10 may be a new SAG.The results provide a foundation for gene cloning and functional analysis of ESL10.