The Crosstalk Between Hydrogen Sulfide And Melatonin In Response To Drought Stress In Arabidopsis

Hydrogen sulfide（H₂S）as the third gasotransmitter,it plays a very important physiological function in plants.A large number of literatures have reported that H₂S plays an active role in the process of plant stress resistance.The research on H₂S stress resistance mainly focuses on the response to abiotic stress,such as drought stress,heavy metals stress,and salt stress.H₂S regulates target proteins through S-sulfhydration to regulate protein structure and activity.Melatonin（MEL）is a kind of small indole compound widely existing in animals and plants,and it is a strong endogenous free radical scavenger in antioxidants.With the in-depth study of MEL in plants,its physiological functions have gradually attracted the attention of researchers.Studies have shown that MEL played an important role in stress response and regulating plant growth and development.The mechanism of H₂S and MEL in plant response to drought stress has been gradually revealed.The previous research results showed that H₂S induced stomatal closure to enhance plant drought resistance by affecting the activity of ion channels in stomatal guard cells.It has been reported that exogenous application of MEL has a similar regulatory effect.So,do H₂S and MEL interact in the process of closing stomata by regulating the ion channel activity?This study was aimed to reveal the molecular mechanism of H₂S and MEL through closing stomata in response to drought stress,and to understand the interaction between them in the regulation of K⁺channel activity in stomatal guard cells in the process of drought stress resistance.The main results are as follows:1.Exogenous H₂S and MEL could close stomata and increase the relative water content of plants under osmotic stress.Meanwhile,exogenous H₂S and MEL responded to osmotic stress by increasing proline and soluble sugar content,decreasing malondialdehyde（MDA）content and relative electrical conductivity.Using mutant strains of SNAT（serotonin N-acetyltransferase,snat）,COMT1（caffeate O-methyltransferase comt1）,and ASMT（N-acetyl serotonin methyltransferase,asmt）associated with MEL production,we determined that closure stomata by H₂S was partially dependent on MEL.In addition,overexpression of ASMT could promote stomatal closure.Exogenous H₂S increased SNAT,ASMT and COMT1 transcript levels and endogenous MEL content.Furthermore,H₂S induced the sulfhydrylation level of KCO1 and AKT1.Thus,H₂S appeared to promote stomatal closure in response to osmotic stress by increasing the transcriptional levels of MEL synthesis-related genes and S-sulfhydration level of the encoded enzymes.2.Exogenous MEL increased the transcription levels of H₂S production-related enzymes encoding genes LCD and DES1,and increased the H₂S content and production rate under both normal conditions and drought stress.MEL and H₂S supplement could upregulate the drought-related transcription factors CBF2,CBF3,RD29A,DREB2A and DREB2B in response to drought stress.Loss of endogenous H₂S content in mutant lines lcd,des1 and lcd/des1 resulted in suppressed stomatal chosure of MEL,reducing plant survival under stress.The ability of MEL to improve drought stress tolerance was also inhibited,that is,H₂S and MEL were interdependent in response to drought stress.3.Exogenous H₂S and MEL increased the transcription level of K⁺_out channel related genes（KCO1 and GORK）and reduced the transcription level of K⁺_in channel related genes（AKT1,AKT2,KAT1 and KC1）under drought stress.The transcript levels of K⁺_out channel-related genes were decreased in H₂S and MEL-related mutants,while K⁺_inchannel-related genes were increased.H₂S increased the S-sulfhydration level and protein stability of KCO1 and AKT1.Studies of mutants（kco1,kat1,akt1 and gork）showed that lake of KCO1,KAT1,AKT1 and GORK had no effect on endogenous H₂S and MEL content.These results showed that H₂S mediated the MEL induced stoma closure by regulating the K⁺channel.4.This study discovered a novel endogenous H₂S-producing enzyme,cystathionine beta lyase（CBL）.We found that the H₂S content in cbl was lower than that in wild type（Col-0）.Under osmotic stress,the H₂S content of CBL-overexpressing（OE-CBL）plants was significantly increased compared with Col-0.In vitro recombinant CBL activity showed that CBL was able to produce H₂S using L-Cysteine as a substrate.In addition,OE-CBL increased the tolerance of plants to osmotic stress by increasing the transcription levels of drought-related genes（CBF2,CBF3,RD29A,DREB2A and DREB2B）and the relative water loss rate.Compared with Col-0,OE-CBL plants responded to drought stress by significantly closing stomata and improving the survival rate of plants.Root bending experiments showed that OE-CBL alleviated heavy metal,cold,osmotic and salt stress in Arabidopsis.5.We compared the differences between the chemical method and the electrode method for the determination of H₂S content in plants.The results showed that the electrode method was more sensitive and accurate.With the development of plants,the content of H₂S in rosette leaves gradually decreased,and the H₂S content in flowers of reproductive organs was higher than that in rosette leaves of vegetative organs.In summary,we preliminarily studied the roles of H₂S and MEL in response to drought stress,and further explored their relationship with K⁺channels in closing stomata.We found that H₂S enhances drought resistance of Arabidopsis plants by mediating MEL-induced stomatal closure by regulating K⁺channels.Moreover,we also conducted a comprehensive analysis of CBL,a new H₂S producing enzyme,and analyzed its response mechanism in response to drought stress.