Identification Of The First Phytomelatonin Receptor And Its Regulatory Roles In Stomatal Closure And Drought Resistance

Melatonin is widespread in living organisms on our planet.Melatonin is an important neurohormone in vertebrates,influencing circadian clock.Phytomelatonin was first discovered in plants in 1995.During the past two decades,much progress has been achevied in the role of melatonin in plant growth,development and stresses response.However,it is still controversial whether phytomelatonin is a hormoe due to lacking the basic information about its signaling pathway and receptor.In this study,Vicia faba and Arabidopsis were used to investigate the signaling pathway underlying melatonin in the regulation of stomatal aperture.Additionally,the first phytomelatonin receptor was identified in Arabidopsis and was named as Phytomelatonin Receptor 1?PMTR1?.The role of PMTR1-mediated signaling in the regulation of stomatal closure and dought resistance was finally characterized using transgenic Arabidopsis plants.The main results were shown as below.1.A broad range of melatonin concentrations was used to investigate the role of melatonin in the regulationof stomatal aperture.The results showed that 5-20?mol/L significantly induced stomatal closure and H₂O₂ production in Vicia faba guard cells.However,the 10?mol/L melatonin-induced stomatal closure and H₂O₂ production were abolished by exogenous application of CAT?the H₂O₂ scavenger?and diphenyleneiodonium chloride?DPI,an inhibitor of NADPH oxidase?,but not salicylhydroxamic acid?SHAM,an inhibitor of cell wall peroxidase?,indicating that melatonin-induced stomatal closure is dependent on NADPH oxidase-mediated H₂O₂production.It is well known that the heterotrimeric GTP-binding protein?G protein?regulates stomatal aperture via NADPH oxidase-dependent H₂O₂ production;we,therefore,further analyzed whether G protein is involved in melatonin-induced stomatal closure and H₂O₂ production in faba bean epidermis.The application of GDP-?s?an inhibitor of G?subunit?inhibited stomatal closure and H₂O₂ production induced by melatonin;in constrast,the treatment comprising GTP-?s?an activator of G?subunit?with or without melatonin significantly enhanced H₂O₂ fluorescence intensity and stomatal closure.Additionally,the application of H₂O₂ induced the closure of stomata that had been open in the treatment with GDP-?s only,and the application of DPI abolished the stomatal closure induced by GTP-?s.These results indicated that G protein is a key component in the melatonin signaling by regulating NADPH oxidase-mediated H₂O₂ production.2.Several Arabidopsis mutant lines were used as plant material to further explore the signal transduction pathway and identify the first phytomelatonin receptor underlying melatonin-induced stomatal closure.Similar to Vicia faba,melatonin induced H₂O₂ production and stomatal closure in Arabidopsis,while CAT and DPI inhibited this process.The NADPH oxidase mutants rbohC and rbohD/F were insensitive to melatonin-induced stomatal closure compared to Col-0.The above results further confirmedthat NADPH oxidase-mediated H₂O₂ production acts as a downstream signal of melatonin and induces stomatal closure in Arabidopsis.The G-protein?subunit mutants gp?1-1 and gp?1-2 were found to be insensitive to melatonin-induced H₂O₂ production and Ca²⁺influx.This suggests that melatonin-induced stomatal closure is dependent on the G protein alpha subunit-mediated H₂O₂ and Ca²⁺signaling pathways.By testing the sensitivity of Arabidopsis G protein-coupled receptor mutants such as cand2,cand7,gcr1-1 to melatonin-induced stomatal closure,we found that melatonin induced the stomatal closure of cand7 and gcr1-1,and only cand2 was not sensitive to melatonin-induced stomatal closure.Amino acid sequence analysis indicated that CAND2 is a membrane protein with putative seven transmembrane?-helical structures.Using bimolecular fluorescence complementation analysis,we found that CAND2 specifically interacts with the only G protein alpha subunit?GPA1?in Arabidopsis.GUS staining results and real-time PCR showed that many plant tissues such as guard cells,cotyledons and roots were able to express CAND2,and the expression level was regulated by melatonin levels.These results indicate that CAND2 is the first putative phytomelatonin receptor found in Arabidopsis,and was named Phytomelatonin Receptor 1?PMTR1?.3.Stomata is the main channel for stomatal exchange of photosynthesis in plants,and it is also the main way to lose water in transpiration.95%of the water absorbed by plants diffuses into the atmosphere through the stomata.Therefore,the adjustment of the stomatal aperture degree is an effective way to reduce water dispersion loss and improve water utilization efficiency.In order to further investigate the mechanism of action of melatonin receptor-dependent signaling pathways in stomatal opening and drought stress.In this research,the melatonin receptor PMTR1 plant expression vector was constructed and the overexpressed transgenic ArabidopsisPMTR1-OX was obtained.The results showed that only 0.1-1?mol/L melatonin could induce PMTR1-OX stomatal closure and H₂O₂ production compared with Col-0.Moreover,neither CAT nor DPI failed to restore the stomatal opening in PMTR1-OX to the level of the control group.It indicates that PMTR1-OX is more sensitive to melatonin and has a stronger degree of action.Drought stress can induce the increase of endogenous melatonin levels in plants,so this study then examined the role of PMTR1 in drought resistance.After 14 days of drought treatment,the water content of PMTR1-OX was about 20%higher than Col-0,the dry weight was 1.2 times that of Col-0,and the biomass reduction was only 38%of Col-0.It shows that PMTR1-OX has stronger drought resistance than Col-0.These results are helpful to understand the regulation mechanism of melatonin and its receptor under drought stress,and provide a theoretical basis for its application in the efficient use of crop water and drought stress.