Cloning And Functional Analysis Of Early-senescence Leaf ES2 Gene And Temperature-sensitive White-green Leaf WGL5 Gene In Rice

In higher plants,the chloroplasts in the leaves mainly perform photosynthesis,which provides energy for plant growth and development.When the development of chloroplasts in leaves is blocked,the structure of chloroplasts and the content of chlorophyll change,it will cause mutations in plants,and the leaves will have a phenotype of leaf color,which will affect yield.The discovery of these mutants provides a theoretical basis for the study of molecular mechanisms related to chloroplast synthesis,degradation,and development.It provides more germplasm resources for hybrid seed production,ornamental rice breeding and production practice.In this study,we obtained two rice leaf color-related mutants early senescence 2（es2）and white green leaf 5（wgl5）by EMS mutagenesis of the japonica rice variety Wuyunjing 7（WYJ7）.The phenotype and genetic analysis were performed in the two mutants,and corresponding genes ES2 and WGL5 were cloned.The main findings are introduced as following:1.Early-senescence leaf ES2 gene:（1）Phenotypic analysis:The es2 mutant showed leaf senescence from the seedling stage until the mature stage.At the seedling stage,yellow spots distributed throughout the leaves,and the aging leaves gradually withered.In addition,the es2 mutant showed significant decrease in plant height,internode length,pancile length,number of primary branch,number of secondary branch,grain width and 1000-grain weight,while the number of tillers increased significantly compared to the wild-type plants.（2）The chlorophyll content of the es2 mutant was decreased at seedling,tillering and heading stages,and the photosynthetic rate was decreased at tillering stage.NBT and DAB staining showed a large accumulation of O₂^-and H₂O₂ in the es2 mutant.In es2,the contents of H₂O₂ and MDA,the activities of SOD,POD and APX were increased significantly,while the activities of CAT decreased significantly.And a large number of DNA fragments accumulated and cell apoptosis occurred in the es2 mutant.（3）Transmission electron microscopy（TEM）observation and analysis:TEM analysis showed that stroma lamellae structure in leaves were disorderly arranged and more osmiophilic granules in leaves of the es2 mutant.Therefore,mutation in ES2 led to abnormal chloroplast development.（4）qRT-PCR analysis:The expression of genes related to senescence,ROS,and chlorophyll degradation were significantly up-regulated,and genes related to chlorophyll synthesis,photosynthesis,and chloroplast development were significantly down-regulated in the es2 mutant.（5）Genetic analysis and fine mapping of the ES2 gene:Genetic analysis showed that the es2 mutant was controlled by a recessive nuclear gene.The ES2 gene was fine mapped between ID2-3 and ID2-4 within a 116.73 kb region on chromosome 2 in rice.Sequencing revealed that a single base mutation occurred with G to A,which caused replacement of glycine with glutamic acid,in the coding region of the LOC＿Os02g32370 gene.（6）Complementation and overexpression test:Complementation and overexpression test was conducted and phenotype,gene expression level,chlorophyll contents and photosynthetic parameters of all transgenic plants were restored to those of the wild-type.（7）Subcellular localization:Fluorescence confocal observation revealed that p35S::ES2-GFP fusion protein was co-localized with chloroplast autofluorescence,which indicated that ES2 was localized to nucleus and cytoplasm of cells.（8）Gene expression pattern:GUS staining detected ES2 expressed in various tissues of rice.Consistently,qRT-PCR results showed that ES2 was expressed in roots,stems,leaves,sheaths and panicles,with highest expression level in leaves followed by stems,panicles,roots and sheaths.2.Temperature-sensitive white-green leaf WGL5 gene:（1）Phenotypic analysis:The wgl5 mutant showed a white-green leaf phenotype at the seedling and tillering stage.After the heading stage,the aging leaves color appeared white-green,and the young leaves showed a green phenotype.The chlorophyll content of the wgl5 mutant was decreased at the seedling and tillering stages,and the photosynthetic rate was decreased at tillering stage.（2）Temperature processing analysis:The phenotype of the mutant was not significantly different from that of the wild-type WYJ7 at 25°C.The leaves of mutants showed albino at35°C and the chlorophyll content was significantly decreased compared to WYJ7.TEM observation found that leaves of wild-type WYJ7 had well developed chloroplasts and a dense lamella structure,while chloroplast development was hampered in the wgl5 mutant at high temperature（35°C）.（3）Determination of physiological indicators in the tillering stage:TEM analysis showed that the stroma lamellae structure in leaves were disorderly arranged in the wgl5 mutant.Simultaneously,more osmiophilic granules（OG）were found in wgl5 compared to WYJ7.Therefore,mutation in WGL5 led to abnormal chloroplast development.NBT and DAB staining showed a large accumulation of O₂^-and H₂O₂ in wgl5 mutant.TUNEL assay showed a large number of DNA fragments accumulated and cell apoptosis occurred.（4）Genetic analysis and fine mapping of the WGL5 gene:Genetic analysis showed that the wgl5 mutant was controlled by a recessive nuclear gene.The WGL5 gene was fine mapped between ZL-8 and RM440 within a 51.72 kb region in chromosome 5.Sequencing revealed that a single base mutation occurred in the coding region of the LOC＿Os05g33840gene,G to A,which caused replacement of glycine with Serine acid.（5）Complementation and overexpression test:Complementation and test was conducted and phenotype,gene expression level and chlorophyll contents of all transgenic plants were restored to those of the wild-type.（6）Subcellular localization:Fluorescence confocal observation revealed that p35S::WGL5-GFP fusion protein was co-localized with chloroplast autofluorescence,which indicated that WGL5 is a chloroplast localization protein.（7）Gene expression pattern:GUS staining detected WGL5 expressed in various tissues of rice.Consistently,qRT-PCR results showed that WGL5 was expressed in roots,stems,leaves,sheaths and panicles,with highest expression level in leaves followed by stems,sheaths,roots and panicles.