Identification And Gene Mapping Of An Early Senescent Leaf Mutant Esl11 In Rice

Leaf is the most important photosynthesis organ of green plants,so the early senescence of leaf will seriously affect the crop yield and quality.Studies show that if the senescence of functional leaf could delay one day,the yield of rice can increase 2%theoretically and the actual increasion is about 1%.So,it is effective to improve the yield and quality of rice by studying the regulatory mechanism of aging on the molecular and genetic level,cloning the early senescence associated genes and studying its function,then making the early senescence better in agricultural production.In this study,a premature decay mutant esl11?early senescence leaf 11?was obtained by EMS?ethyl methane sulfonate?mutagenesis,and phenotypic identification,agronomic traits analysis,physiological index analysis,histochemical observation,cytological observation,sugar content analysis,aging-related gene expression analysis and gene mapping were performed on mutant esl11.The main conclusions are as follows:1.The wild type leaves is normal growth and remain green until the physiological aging of rice.But,the mutant leaves began to yellow aging in the outside and the tip leaves after three-leaf stage,not including the heart leaf.Then,the yellow aging gradually extended to the lower middle leaves and finally result in the entire leaf aging death.The phenomenon exist until rice mature.Compared with the wild type,the plant height of mutant es111 is significantly decreased,while the secondary branch length,the spike length,the number of grains per panicle,the seed setting rate and the1000-grain weight are also significantly reduced.2.At tillering stage,compared with the wild type,the contents of chlorophyll a,chlorophyll b,total chlorophyll and carotenoid in esl11 at the tip of the leaves were reduced by 83.56%?84.82%?83.76%?17.93%,respectively.Chlorophyll a,chlorophyll b and total chlorophyll change are all reaching extremely significant level.And,the content of photosynthetic pigment in base of esl11 leaves have the same change as in the apex.At the same time,the net photosynthetic rate of both the tip and base of the mutant esl11 leaves was significantly lower than that of the wild type.From the cytological observation,it can be concluded that the red fluorescence of the mutant esl11 is weaker than that of the wild type,indicating that the chloroplast of esl11 is damaged.Transmission electron microscopy shows that there were obvious starch grains in the chloroplast of the mutant esl11,which destroyed the structure of the chloroplast.3.I₂-KI staining shows that the esl11 leaves are dyed blue when the wild type leaves are not dyed any color,indicating that the starch in esl11 leaves was significantly higher than in the wild type.The analysis of leaf sugar content shows that the sucrose,glucose and starch of the mutant esl11 is significantly higher than the wild type at the base and tip of leaves.The expression analysis of sugar associated genes shows that the expression level of starch synthesis genes in esl11 is significantly higher than wild type.And the triosephosphate transport genes,sucrose synthesis genes and sucrose transport genes is significantly down-regulated in esl11.4.Though the analysis of aging-related content,we find that H₂O₂,O₂^-,·OH content in the tip and base of esl11 leaves were significantly higher than the wild type.And the CAT and SOD activities in the tip and base of esl11 leaves were significantly decreasing.At the same time,the content of MDA in the tip and base of esl11 leaves is higher than the wild type,reaching extremely significant and significant level,respectively.Trypan blue staining shows that the tip of esl11 leaves have been dyed dark blue,when the wild type leaves is light blue,indicating cell death occur in the tip of esl11 leaves.The esl11 leaves present bronzing in the yellow aging zone though DAB staining,when the wild type leaves present normal color,indicating the H₂O₂accumulation in the yellow aging zone of esl11 leaves.5.Compared with the wild type,the expression of chlorophyll-related genes DVR,CAO1,NYC1 and NYC4 in the mutant esl11 are down-regulated,and the expression levels of SGR and NYC3 are up-regulated,indicating that the chlorophyll content of the mutant esl11 have changed,which is related to the determination of photosynthetic pigment content.The results are consistent with the destruction of the chloroplast of the mutant esl11;the expression of the OsNAP and Osh69 genes are significantly up-regulated and the OsDOS expression is significantly down-regulated,indicating that the mutant esl11 leaves accelerate aging.The expression levels of reactive oxygen species related genes NOE1,OsCATA and OsCATB are significantly up-regulated,indicating that the CAT activity in the mutant leaves increase and the H₂O₂ content increase.The expression levels of PCD-related genes RLS1 and PDCD5 are up-regulated,indicating that the mutant esl11 leaf cells accelerate the process of programmed cell death.6.The mutant esl11 is controlled by a pair of recessive nuclear genes,and the esl11 is finally mapped between the markers SNP580 and SNP830 on chromosome 7,with a physical distance of 143.0 kb.There are 22 annotated genes in this interval,including two encoding transposon proteins,two encoding retrotransposon proteins,eight encoding expression proteins,and 10 functional genes,including two encoding peroxidase precursors,two encoding ankyrin repeat domain-containing protein,others encoding a NUC153 domain containing protein,harpin-induced protein 1 domain containing protein,pyruvate Pi dikinase regulatory protein,oral cancer overexpressed protein 1,tyrosyl-DNA phosphodiesterase 1.