Identification Of Melatonin Synthesis Gene And The Signal Transduction Mechanism Of Melatonin-regulated Cold Tolerance In Watermelon

Cold stress is a major environmental factor that detrimentally affects plant growth and development.Watermelon(Citrullus lanatus L.)is an economically important horticulture crop globally.Its origin can be traced to tropical and subtropical regions of Africa.Watermelon production is threatened by its susceptibility to low temperatures.In recent years,with the continuous expansion of protected cultivation area of watermelon in off season,low temperatures in winter and early spring have seriously affected the yield and quanlity of watermelon.Therefore,it will provide a great practical significance for improving the cold tolerance of existing watermelon cultivars by exploring the ways to alleviate the cold damage and mining the tolerance genes in watermelon.Melatonin is a pleiotropic signaling molecule that regulates plant growth and responses to various abiotic stresses.The COMT genes involved in the last step of melatonin synthesis in plants were cloned in model plants,including Arabidopsis,tomato,and rice;however,COMT has not yet been characterized in nonmodel plants such as watermelon,which limits the research of melatonin function in other plant species.Furthermore,there are few reports about the signal transduction pathway of melatonin regulating cold tolerance in watermelon.In this study,we cloned the first melatonin biosynthetic gene(caffeic acid O-methyltransferase,Cl COMT1)from a species in the Cucurbitaceae,and clarified that H₂O₂ and Ca²⁺signal mediated the melatonin-inducing cold tolerance in watermelon.It provides a theoretical basis and technical support for investivating the signal pathway and molecular mechanism of melatonin-induced cold tolerance in watermelon.The main results are as follows:1.The role of watermelon caffeic acid O-methyltransferase(Cl COMT1)in melatonin biosynthesis and abiotic stress tolerance.a total of 16 putative O-methyltransferase(Cl OMT)genes were identified in watermelon.Among them,Cl OMT03(Cla97C07G144540)was considered a potential COMT gene(renamed Cl COMT1)based on its high identities(60.00%–74.93%)to known COMT genes involved in melatonin biosynthesis,expression in almost all tissues,and upregulation under abiotic stresses.The Cl COMT1 protein was localized in the cytoplasm.Overexpression of Cl COMT1 significantly increased melatonin contents,while Cl COMT1 knockout using the CRISPR/Cas-9 system decreased melatonin contents in watermelon calli.In addition,Cl COMT1 expression in watermelon was upregulated by cold,drought,and salt stress,accompanied by increases in melatonin contents.Overexpression of Cl COMT1 enhanced transgenic Arabidopsis tolerance against such abiotic stresses.These results indicate that Cl COMT1 plays an essential role in melatonin biosynthesis in watermelon is a positive regulator of plant tolerance to abiotic stresses.2.Melatonin confers watermelon tolerance against cold stress by Cl CNGC20 controlling the Ca²⁺influx.Application of melatonin or Ca²⁺at appropriate concentrations conferred watermelon tolerance against cold stress;however,blocking of Ca²⁺signal with EGTA(a calcium chelator)or La Cl₃(a Ca²⁺channel inhibitor)abolished melatonin-induced cold tolerance.Melatonin stimulated Ca²⁺influx and the accumulation of cytoplasmic free Ca²⁺([Ca²⁺]_cyt).The q RT-PCR analysis of 13 cyclic nucleotide-gate ion channel genes(Cl CNGCs)in watermelon genome showed that four Cl CNGCs(Cl CNGC2,Cl CNGC10,Cl CNGC17,and Cl CNGC20)expression was upregulated more than five folds by a combinant treatment of melatonin and cold.Then,Cl CNGC2,Cl CNGC10,Cl CNGC17,or Cl CNGC20 was knocked out in watermelon calli to measure their Ca²⁺flow.Gene Cl CNGC2 mutantion resulted in a slight decrease in Ca²⁺influx rate stimulated by cold stress and/or melatonin.Gene Cl CNGC10knockout had no effect on Ca²⁺influx.Gene Cl CNGC17 participated in melatonin-regulated Ca²⁺influx but not responded to cold stress.Gene Cl CNGC20 knockout significantly compromised melatonin-induced Ca²⁺influx both under normal and cold conditions.Thus,Cl CNGC20 plays a key role in melatonin-and cold-regulated Ca²⁺influx.By yeast two hybrid screen,bimolecular fluorescence complementation and luciferase complementation test,we confirmed the interactions between Cl CNGC20 and Ca M2,Ca M5,or Ca M7.In addition,melatonin positively regulated Cl CNGC20 expression while negatively regulated the expression of three Ca Ms in the early response to cold stress,in which the trend of Ca M7expression was completely opposite to that of Cl CNGC20.These results indicate that melatonin stimulates the Ca²⁺influx by regulating the activity of Cl CNGC20,which may be negatively regulated by Ca M7.3.Positive interaction between H₂O₂ and Ca²⁺mediates melatonin-induced C-REPEAT BINDING FACTOR(CBF)pathway and cold tolerance.According to the results,melatonin induced H2O2 accumulation,which was associated with the upregulation of Respiratory Burst Oxidase Homolog D(Cl RBOHD)during the early response to cold stress in watermelon.Besides,melatonin and H₂O₂ induced[Ca²⁺]_cyt accumulation in response to cold.This was associated with the upregulation of cyclic nucleotide-gated ion channel 2(Cl CNGC2)in watermelon.However,blocking of Ca²⁺influx channels abolished melatonin-or H2O2-induced CBF pathway and cold tolerance.Ca²⁺also induced Cl RBOHD expression and H2O2accumulation in early response to cold stress in watermelon.Inhibition of H2O2 production in watermelon by RBOH inhibitor or in Arabidopsis by At RBOHD knockout compromised melatonin-induced[Ca²⁺]_cyt accumulation and melatonin-or Ca²⁺-induced CBF pathway and cold tolerance.Overall,these findings indicate that melatonin induces RBOHD-dependent H2O2 generation in early response to cold stress.Increased H2O2 promotes[Ca²⁺]_cytaccumulation,which in turn induces H2O2 accumulation via RBOHD,forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.