Cloning And Functional Analysis Of Lesion Mimic Genes LLM1 And LLM3 In Rice

Rice lesion mimic mutants(LMMs)can aid in unraveling the molecular mechanisms underlying hypersensitive response(HR),programmed cell death(PCD),senescence,and disease resistance.The research on these mutants can provide theoretical basis and material support to improve disease resistance and premature senescence in rice though molecular design breeding.In this study,we identified two LMMs,llm1(leaf lesion mimic 1)and llm3(leaf lesion mimic 3),from the Yun Dao32(YD32)mutant library.We cloned the LLM1 and LLM3 genes using a map-based cloning method and conducted functional analysis at the physiological,biochemical,molecular biology,and plant pathology levels.The primary research findings are as follows:Both llm1 and llm3 exhibit lesion mimic at the seedling stage,accumulating a large amount of ROS,which triggered PCD.Genetic analysis and map-based cloning showed that the lesion mimic phenotype of llm1 and llm3 were controlled by a single recessive gene,respectively.Furthermore,their functions were verified by transgenic experiments.Tissue expression pattern analysis and GUS staining showed that LLM1 and LLM3 were constitutively expressed and has the highest expression level in leaves.Subcellular localization showed that LLM1 and LLM3 located in the chloroplast.LLM1 encodes zeaxanthin epoxidase(ZEP),which is involved in the xanthophyll cycle,abscisic acid(ABA),and carotenoid synthesis.Mutation of LLM1 causes abnormal chloroplast development and structure,impaired photoinhibition,disturbed carotenoid composition,and reduced ABA content in llm1.The detection results of carotenoid components showed that 24 carotenoid substances were significantly different between llm1 and wild type(WT);Notably,zeaxanthin content and violaxanthin content,which are involved in the xanthophyll cycle,were increased by66.03-fold and decreased by 76.19-fold,respectively.These results indicate that in the xanthophyll cycle was altered in llm1.q RT-PCR showed that the expression of genes related to carotenoid synthesis was significantly down-regulated in llm1 compared with the WT.Furthermore,treatment with the inhibitor of carotenoid synthesis Fluridone(Flu)showed that llm1 had more reddish-brown spots and accumulated more reactive oxygen species(ROS)on leaves than the WT.These findings suggest that changes in the content of carotenoid substances are critical contributors to the appearance of lesion mimics in llm1.The results of ABA treatment showed that llm1 was reduced dormancy and more sensitive to ABA than the WT.Salt treatment showed that llm1 was more sensitive to salt than the WT.Taken together,the LLM1 mutation impairs chloroplast development and disrupts the xanthophyll cycle,leading to changes in carotenoids,and accumulation of ROS,ultimately resulting in programmed cell death and the formation of lesion mimics.And the mutation also makes llm1 more sensitive to ABA and salt.llm3 was senescence after flowering.Dark treatment of llm3 led to rapid chlorophyll and chloroplast degradation and a significant decrease in the expression levels of photosynthetic proteins,indicating increased susceptibility to senescence.Additionally,llm3 showed a significant increase in ABA content and differential expression of genes related to ABA metabolism and signal transduction.And LLM3 was found to interact with the ABA synthesis-related protein NCED1.These results suggest that LLM3 may regulate rice senescence by negatively regulating ABA synthesis.Furthermore,compared to the WT,llm3 exhibited increased callose deposition,salicylic acid(SA)and jasmonic acid(JA)content,upregulated defense-related gene expression,and enhanced resistance to rice blast disease.Transcriptome analysis also revealed significant differences in gene expression related to defense reactions between the WT and llm3.These findings suggest that LLM3 negatively regulates rice defense reactions.