Characterization And Map-based Cloning Of Two Early Senescence Genes In Rice

Leaf senescence is a key component of plant growth and development,which refers to the process of a series of programmed cell death in plant-specific cells,tissues and whole plants.Elary senescence is one of the main factors affecting the stability of rice production.Early senescence has a positive effect on plant production,and delaying the senescence of rice can increase its yield.But its molecular regulation is not yet fully understood,so it is of great significance to study rice premature senescence to improve rice yield.In this study,we analysis the phenotype and gene mapping on two early senescence mutants.1.es4 mutant first appeared yellow phenotype at the maximal tillering stage.The es4 mutants exhibited a significantly increased MDA and H₂O₂ but decreased chlorophyll content,soluble protein content,enzymatic activity and photosynthetic rate in the leaves,which are indicative of an early senescence phenotype.Genetic analysis showed that the mutant trait was controlled by a single recessive nuclear gene.The target gene was then anchored on the long arm of chromosome 4,and then fine mapped to a 33.5Kb region using a mapping population consisting of 1061 mutant individuals,harboring 7 open reading frames?ORFs?.It is found by sequencing analysis that there is eight bases deletion in first exon of the calcium-dependent protein kinase?OsCDPK12?,resulting in a frameshift mutation in the mutant.In addition,the OsCDPK12 was proved to be the candidiate gene by functional complementation and CRISPR/Cas9.OsCDPK12 was transcribed in various tissues and was mainly expressed in leaves.Expression of ES4-GFP fusion protein indicated that OsCDPK12 was located in the whole cell.The expression levels of some key genes involved in senescence and photosynthesis was significantly changed in the es4 mutant.Overexpression of OsCDPK12 plants increased the chlorophyll content and photosynthetic ability.Based on our results,it is showed OsCDPK12 was required for the normal development of photosynthesis in rice,while the impaired function of OsCDPK12 was responsible for the early senescence phenotype.2.The early senescence mutant lmm326 was obtained from an EMS-induced ZH11 mutant library.At 5-leaf stage,there are brown lesions on lower leaves of lmm326 initially.Compared with the wild type,the photosynthetic pigment content,net photosynthetic rate,plant height,seed setting rate,tiller number,and 1000-grain weight of lmm326 were significantly reduced.Evans blue and DAB staining assay indicated that leaves of lmm326 accumulated more dead cells and H₂O₂.Additionally,lmm326plants displayed higher resistance to 4 races of rice blast compared with wild type.According to genetic analysis,the phenotype of mutant was controlled by a single recessive gene.Here,we employed map-based cloning approach to finely map the LMM326 gene through F₂ population derived from a cross between lmm326 and Dular,and the gene was narrowed down to a 38-Kb region on the long arm of chromosome 1,which harbored 6 ORFs.Sequence analysis revealed that a single base substitution at position C433T in Os01g0919900 CDS resulted in F145L after translation,and the target gene is allelic to OsSSI2.Real-time PCR analysis showed that expression level of defense-related genes were significantly higher in lmm326 than in wild type plant.These results demonstrate that the LMM326 is likely to participate in the signaling pathway of SA,and its mutant activates the defense response in rice.