Mechanisms Of The Interaction Between Melatonin And Salicylic Acid Signals In Plant Immunity Response In Arabidopsis

Plant diseases severely limit crop growth and yield,which causes a great challenge to food security.Salicylic acid is an important hormone in plants that plays an important role in plant’s local defence and systemic acquired resistance(SAR).Melatonin is an important signaling molecule in plants that regulates stomatal closure and expression of disease resistance genes using its receptor PMTR1(Phytomelatonin Receptor 1)via the GPA1/NADPH oxidase mediated ROS signaling pathway and via mitogen-activated protein kinase(MAPK)cascades.However,the regulatory mechanisms underlying interactions between melatonin and salicylate signaling is not clear.In this study,mechanisms underlying interactions between melatonin and salicylate signaling is analysed at the molecular and physiological levels using Arabidopsis thaliana as the material.The results obtained are summarized as follows:1.Salicylic acid regulates stomatal closure and local defense in plants through melatonin receptor PMTR1.The sensitivity of the npr1-1 mutants to melatonin induced stomatal closure was decreased when compared to wild-type plants(Col-0),indicating that melatonin induced stomatal closure may be partially dependent on the salicylic acid receptor NPR1.However,salicylic acid fails to induce stomatal closure and ROS production in pmtr1 mutants,indicating that salicylic acid induced stomatal closure and ROS production are PMTR1-dependent.Exogenous melatonin treatment reduced the numbers of pathogenic Pst DC3000 colonies in leaves of Col-0,snat1 and npr1-1,however neither melatonin nor salicylic acid reduced the pathogen content in pmtr1 mutants.Pharmacological and genetic results have shown that the NADPH oxidase inhibitor DPI and the cell wall peroxidase inhibitor SHAM inhibit stomatal closure induced by both melatonin and salicylic acid,respectively.Melatonin induces the stomatal closure of prx33 and prx34 mutants,which are not sensitive to salicylic acid,whereas salicylic acid induces the stomatal closure of rboh D/F,which is not sensitive to melatonin,indicating that melatonin and salicylic acid induce stomatal closure through NADPH oxidase and cell wall peroxidase-mediated ROS production,respectively.RT-q PCR analysis revealed that neither melatonin nor salicylic acid induces the expression of PRX33 and PRX34 in pmtr1 mutant,indicating that melatonin mediated signaling regulates salicylic acid induced stomatal closure by regulating the expression of cell wall peroxidase genes,such as PRX33 and PRX34.Taken together,these results suggest that PMTR1-mediated signaling pathway is involved in salicylic acid-induced stomatal closure and leaf defense by regulating the expression of PRX33 and PRX34 genes.2.PMTR1 and NPR1 integrate melatonin and salicylic acid signaling to regulate expression of genes encoding salicylic acid pathway,and melatonin induces the translocation of NPR1 into the nucleus through the NO signaling pathway.Both 10μM exogenous melatonin and 100 μM exogenous salicylic acid treatments significantly induced the expression of genes associated with salicylic acid synthesis(including ICS1,EPS1,PBS3),salicylic acid receptor gene NPR1,and genes of downstream of salicylic acid signaling pathway(such as PR1,PR2,and PR5)in Col-0within 2 hours,with the induction levels peaking at 8 h.By contrast,the expression of these genes was significantly lower in pmtr1 and npr1-1 mutants than that in Col-0,and melatonin failed to induce the expression of genes encoding salicylic acid synthesis and its receptor gene NPR1 in pmtr1 and npr1-1 mutants,indicating that PMTR1 and NPR1 integrate melatonin and salicylic acid signals to regulate genes expression in the salicylic acid signaling pathway.Further analysis revealed that it takes 8 hours after melatonin treatments to induce the entry of NPR1 into the nucleus,and the NO scavenger c PTIO inhibits the melatonin-induced entry of NPR1 into the nucleus.In contrast,SA induces the entry of NPR1 into the nucleus after only 2 hours treatments,and the NO scavenger c PTIO did not inhibit the SA-induced entry of NPR1 into the nucleus.These findings suggest that the entry of NPR1 induced by melatonin is mediated by the NO signal,and that SA acts downstream of NO to induce the entry of NPR1 into the nucleus.These results suggest that melatonin may regulate the expression of genes related to SA synthesis through the signaling pathway mediated by PMTR1 to increase SA levels,which in turn further promotes the entry of NPR1 into the nucleus where it binds to transcription factors and activate the downstream PRs genes expression.3.Melatonin and salicylic acid regulate expression of PMTR1 through NPR1 and TGA1.RT-q PCR results showed that salicylic acid failed to induce the expression of melatonin biosynthetic genes either in Arabidopsis plants deficient either in melatonin or salicylic acid pathways,but it could induce the expression of the melatonin receptor gene PMTR1 in Col-0 and that neither melatonin nor salicylic acid failed to induce the expression of PMTR1 in npr1-1 and tga1 mutants.In addition,analysis using a dual luciferase reporter gene showed that TGA1 can interact with the PMTR1 promoter,and NPR1 promotes the regulation of TGA1 on PMTR1 expression.