Molecular Mechanism Of Melatonin In Promoting Rice Seed Germination Under Salt Stress,and In Regulating Leaf Senescence And Yield Related Traits

Rice(Oryza sativa)is one of the most important food crops,and improving rice yield and stress tolerance is an important task of modern rice breeding.Melatonin,a kind of indole hormone which is ubiquitous in animals,has been shown to regulate immune function,improve the quality of sleep,and alleviate neurasthenia of human.Melatonin is also found to be ubiquitous in plants,and studies have revealed that the functions of melatonin include in promoting root growth and development,regulating other phytohormones,and improving resistance to biotic and abiotic stresses.Although many studies have revealed the functions of melatonin in rice salt response and leaf development,the effects of melatonin on rice seed germination under salinity are currently unclea,and the role of melatonin in regulating rice leaf senescence and yield traits remains to be investigated.This dissertation contains two major parts:first,we comprehensively integrated physiological,metabolomic and transcriptomic analyses to provide insights into the roles of exogenous melatonin in promoting rice seed germination under salt stress;second,we constructed the transgenic lines of rice melatonin biosynthesis enzyme gene,OsCOMT,and analyzed its functions and working mechanisms in regulating endogenous melatonin content as well as rice leaf senescence and yield traits.Part ? Molecular mechanisms of exogenous melatonin in promoting rice seed germination under salt stressSalinity is a major abiotic stress in rice production,which affects many aspects of plant growth,especially seed germination.In this study,we performed germination test of rice seeds under different concentrations of NaCl and melatonin,and analyzed the potential mechanisms of exogenous melatonin in rice seed germination under salinity through comprehensive transcriptome sequencing,metabolome profiling,and physiological assays.The main results are as follows:1.Based on the germination rate analysis of three different treatment conditions(control,salt stress,melatonin pretreatment followed by salt stress),we found proper concentration of melatonin pretreatment significantly increased rice seed germination rate under salt stress.The germination rate of rice seeds under 100 mM NaCl increased by 35%after 5?M melatonin pretreatment.2.Based on the transcriptome analysis of different treatments,we identified 4794 differentially expressed genes(DEGs)that were affected by melatonin pretreatment under salinity,including 2843 upregulated and 1951 downregulated genes.3.Functional annotation showed that many DEGs were associated with antioxidative processes,phytohormone biosynthesis and signal transduction pathways.4.Physiological analysis revealed that melatonin pretreatment enhanced the activity of peroxidase(POD),superoxide dismutase(SOD)and catalase(CAT)under salinity.In addition,exogenous melatonin significantly increased the levels of zeatin(ZT)and indole-3-acetic acid(IAA),and reduced the levels of jasmonic acid(JA)and abscisic acid(ABA)under salinity.5.Through metabolome analysis,230 differential metabolites were identified among three treatments.Exogenous melatonin significantly promoted the accumulation of palmitic acid,mevalonic acid and arachidic acid,and reduced the level of glycosides.Taken together,the comprehensive analysis of multi-omics revealed that exogenous melatonin can enhance rice seed tolerance to salinity during germination through transcription regulation,activation of antioxidants and modulation of phytohormones.These results contribute to the understanding of the mechanisms of melatonin promoting rice germination under salinity,and may have application value in alleviating salinity damages during rice production.Part ? Functional analysis of rice melatonin synthase gene OsCOMT in regulating leaf senescence and yieldIn order to understand the biological roles of endogenous melatonin in rice development and yield production,this study investigated the biological roles and regulatory mechanisms of rice melatonin biosynthesis enzyme gene OsCOMT in rice growth and development using its overexpression and CRISPR-Cas9 lines.The main results are as follows:1.OsCOMT is constitutively expressed in all tested tissues,and its expression was higher in roots and leaves.Both GUS staining and GUS activity test revealed that the expression of OsCOMT in leaves gradually increased as the development progressed,indicating that OsCOMT may be involved in leaf senescence regulation.2.Subcellular localization assay of OsCOMT in rice protoplast and tobacco epidermal cells both showed that OsCOMT-GFP fusion protein was predominantly localized in the cytoplasm and plasma membrane.3.OsCOMT has acetylserotonin O-methyltransferase activity both in vitro and in vivo.oscomt mutant lines showed a 57.9%decrease in melatonin content compared with wild type,and the OsCOMT-OE lines increased melatonin content by 48.4%.Thus,OsCOMT is involved in rice endogenous melatonin biosynthesis.4.Compared with wild type,oscomt plants showed significant decrease in grain number per panicle,thousand grain weight,grain size,and grain yield per plant,while OsCOMT-OE lines showed significant increase in grain number per panicle,thousand grain weight,grain size,and the grain yield per plant increased by 17.5%.5.After flowering,oscomt plants exhibited presenile phenotypes,including decreased chlorophyll content,decreased number and abnormal structure of chloroplast,reduced photochemical efficiency,accumulation of ROS and increased cell death.In contrast,OsCOMT-OE plants showed delayed leaf senescence at ripening stage compared with WT,and extended the duration of high photosynthetic efficiency.6.OsCOMT has potential functions in vascular bundle patterning.The number and size of vascular bundles in OsCOMT-OE lines were increased compared with WT,while oscomt mutant showed abnormal development of vascular tissue in leaf and stem.7.Application of 100uM exogenous melatonin at 85dps for 10 consecutive days can partly rescue the leaf presenile phenotype and yield decrease of oscomt,proving that these phenotypes were connected with decreased endogenous melatonin content.Taken together,our results show that OsCOMT is involved in rice endogenous melatonin synthesis,and significantly regulate the endogenous melatonin content,and further affect rice grain yield through regulating ROS accumulation,leaf senescence,and vascular tissue patterning.These results indicate that OsCOMT is a promising target gene for rice genetic improvement.