Characterization And Fine Mapping Of Lesion Mimic And Premature Leaf Senescence 9 Mutant In Rice(Oryza Sativa L.)

“Lesion mimic”refers to necrotic spots produced by rice without obvious stress,mechanical damage or pathogen attack,similar to necrotic spots produced by hypersensitivity reactions（HR）.Lesion mimic mutants are ideal materials for studying defense response mechanisms and programmed cell death（PCD）.In addition,rice leaf senescence is a complex and delicate process,which is the final stage of leaf development.Numerous factors,including external environmental factors such as temperature and light,are involved in the regulation of lesion mimic and leaf senescence in rice.In order to further reveal the molecular mechanism of rice lesion mimic and leaf senescence,Wuyujing 3（WYJ3）was irradiated with ⁶⁰Coγ-ray to obtain a rice lesion mimic and premature senescence mutant,which we named lmpls9（lesion mimic and premature leaf senescence 9）.Based on the phenotypic observation of mutants,this paper constructs genetic populations,performs fine mapping,and obtains the following research results:（1）High temperature can promote the occurrence of lesion mimics and premature leaf senescence of lmpls9.The lmpls9 did not show lesion mimics and leaf senescence before 30 days after seeding（DAS）,which was indistinguishable from the wild-type phenotype.Under field planting conditions,the mutants began to appear the lesion mimic phenotype from about 30 DAS,and then the leaves around the lesions gradually turned chlorotic and leaf senescence occurred.These phenotypes persist throughout subsequent developmental cycles.At the mature stage,the leaf tips of the mutant lmpls9 were completely senescent and dead,while the wild-type remained green at the same stage.In addition,we also found that the appearance of“lesion mimic”phenotypes of lmpls9 at 35°C was significantly earlier than that at 25°C.At the same time,the mutants grown in Harbin did not show obvious lesion mimics and leaf senescence until heading,while the leaves of the mutants grown in Jinhua at the same period had severe lesion mimics and senescence.（2）LMPLS9 regulates chloroplast development and photosynthetic capacity in leaves.The chloroplast ultrastructure of rice leaves was observed by transmission electron microscope,and it was found that a large number of chloroplasts with disordered grana stacking were found in the leaves of mutant lmpls9,and the starch granules and osmium-philic granules were significantly increased compared with WYJ3.At the same time,we found that the chlorophyll content and maximum light conversion efficiency（Fv/Fm）of lmpls9 were also significantly reduced.Correspondingly,the main agronomic traits of lmpls9,including effective tiller number,grain number per ear,and 1000-grain weight,were significantly lower than those of the wild-type.Furthermore,detached flag leaves of lmpls9 were more yellowish than that of WYJ3 after dark incubation.Through quantitative real-time PCR（q RT-PCR）,it was found that the expressions of senescence-associated genes（SAGs）SGR,RCCR1,and Os I57 were up-regulated by 6.2-fold,2.2-fold,and 2.3-fold in lmpls9,respectively.These results indicate that LMPLS9 mutation affects chloroplast development and leaf photosynthetic capacity,resulting in premature leaf senescence and reduced yield of lmpls9.（3）Reactive oxygen species（ROS）accumulate in lmpls9 leaves.ROS are critical signaling substances in rice senescence.Through histochemical staining and quantitative detection,it was found that the content of ROS in lmpls9 was significantly higher than that in WYJ3.Meanwhile,the activities of peroxidase（POD）,superoxide dismutase（SOD）,and glutathione reductase（GR）were increased in mutant lmpls9 by 7,1,4,and 1.8 times,respectively,compared with wild-type WYJ3.These results indicate that the mutant lmpls9 actively responded to the accumulation of ROS in leaves,but was still insufficient to clear ROS.The accumulated ROS may play an important role in the leaf senescence of lmpls9.（4）lmpls9 enhances rice defense.WYJ3 and lmpls9 were inoculated with bacterial blight P6,and the length of the lesions was counted after 21 days.It was found that the disease resistance of lmpls9 plants was significantly stronger than that of WYJ3.q RT-PCR detection of defense-related genes showed that the expression levels of defense-related genes PBZ1 and PO-C1 were increased by 8.9 times and1.94 times in lmpls9 leaves,respectively.These results suggest that the LMPLS9mutation activates defense signals and enhances the resistance of rice plants to bacterial blight.（5）The map-based cloning of lmpls9.lmpls9 was hybridized with Taichung Native 1（TN1）.Genetic analysis showed that none of the F1 produced by the cross showed lesion mimics and senescence,indicating that this trait is recessive.There was a typical 3:1 ratio between the wild-type and lmpls9 phenotypes in the F2 population,indicating that the lmpls9 phenotype is controlled by a recessive nuclear gene.After preliminary mapping and fine mapping,the gene locus was finally limited to an interval of about 128 kb between molecular markers ZR 667 and ZR 659 on rice chromosome 9.According to the rice genome annotation database website（http://rice.plantbiology.msu.edu/index.shtml）,it was found that there are 23 possible open reading frames（ORFs）in this genomic region.The expression of these genes was analyzed by Genevestigator software,and several genes highly expressed in the leaves or senescence stage were selected as candidate genes for subsequent verification.