The Mechanism Of Regulating MPK10 Activity By H₂S-mediated S-persulfidation In Arabidopsis

The mitogen-activated protein kinases（MAPKs）cascade pathway is a conserved signal transduction pathway in eukaryotes,which can respond to various biotic and abiotic stresses and play a role in plant development and hormone signal transduction（Sheen 2001）.Hydrogen sulfide（H₂S）,as an important gas signaling molecule,is actively involved in regulating plant and development and stress response.H₂S regulates protein activity and transmits signals by S-persulfidation.In recent years,many studies have shown that H₂S signaling is closely related to the plant MAPKs cascade pathway,however,the molecular mechanism by which S-persulfidation regulates the activity of MAPKs family member proteins and realizes the interactive regulation of H₂S signaling and MAPKs cascade pathway is still unclear.Aiming at this scientific problem,this subject proved the existence of S-persulfidation in MAPK10 by liquid chromatography-mass spectrometry.The MPK10modification site was identified by secondary mass spectrometry;the reliability of S-persulfidation modification was verified by the Modified biotin switch method;the in vitro phosphorylation experiments demonstrate that MPK10 has autophosphorylation activity and kinase activity;BES1（Bri1-EMS-suppressor 1）,a substrate interacting with MPK10,was identified by protein interaction research techniques;finally,by constructing transgenic genetic lines,it was demonstrated that H₂S signaling delays leaf senescence through S-persulfidation of MPK10 protein.Ultimately,the mechanism of regulates MPK10 activity through S-persulfidation was elucidated in Arabidopsis.The main conclusions of this study are as follows:1.Demonstrated that the MPK10 protein has S-persulfidation modification and determine the modification site.Cloning the MPK10 gene,expressing it in prokaryotic cells,and purifying the recombinant protein HIS-MPK10-SUMO in vitro.High-resolution LC-MS/MS was used to prove the existence of S-persulfidation modification on MPK10,and the S-persulfidation modification site was identified by secondary mass spectrometry analysis.2.In order to verify the reliability of S-persulfidation modification,in this study,the cysteine residues of MPK10 were mutated,and alanine（ala,A）was used instead of cysteine（cys,C）to simulate the removal of S-persulfidation.S-persulfidation analysis of wild type（WT）and mutant MPK10 proteins by Modified Biotin Switch Method（MBSM）.The results showed that Na HS（H₂S donor）enhanced the level of S-persulfidation of MPK10^WT protein in a dose-dependent manner,which proved that the S-persulfidation of MPK10 protein was authentic and reliable.By detecting the S-persulfidation target site mutein MPK10^CSSH-A and other site muteins(AtMPK10^C1-A,AtMPK10^C2-A and AtMPK10^C3-A),it was found that the modification of MPK10^CSSH-A protein disappeared,and the modification of other site muteins did not,it is proved that the identified S-persulfidation modification site is true and reliable.3.The effect of S-persulfidation modification on the autophosphorylation activity of MPK10 was demonstrated by in vitro phosphorylation experiments.Na HS treatment can inhibit the autophosphorylation activity of MPK10^WT,and there is a dose-dependent inhibitory effect.S-persulfidation modification also inhibited the autophosphorylation activity of mutants such as AtMPK10^C1-A,AtMPK10^C2-A and AtMPK10^C3-A,but the inhibitory effect abolished in the MPK10^CSSH-A mutant.The above results demonstrate that S-persulfidation modification inhibits the autophosphorylation activity of kinase proteins through specific cysteine residues.4.In this study,the universal kinase substrate Myelin Basic Protein（MBP）and the natural substrate transcription factor BES1 of MPK10 were used to examine the effect of S-persulfidation modification on the phosphorylation activity of MPK10.The effect of H₂S on the kinase activity of AtMPK10 was examined by in vitro kinase assay,and it was found that Na HS treatment also inhibited the phosphorylation level of AtMPK10 substrates in a dose-dependent manner,but the inhibition was abolished in MPK10^CSSH-A mutant.In conclusion,it was proved that S-persulfidation also inhibites the phosphorylation activity of MPK10 protein substrate through specific cysteine residues.5.In this study,the mpk10 mutant strains of Arabidopsis thaliana were used as the background to construct MPK10 gene complementary lines(mpk10/35S-MPK10^WT)and S-persulfidation site-directed mutant lines(mpk10/35S-MPK10^CSSH-A),and screened to obtain homozygous genetic material.The senescence rates among experimental groups were compared by in vitro leaf senescence experiments.Compared with WT,leaves of mpk10mutant lines showed delayed senescence.The genetic complementation line mpk10/35S-MPK10^WT complements the senescent phenotype of mpk10.Na HS and Brassinolide（BL）treatment delayed leaf senescence in genetic material.The point mutant genetic line mpk10/35S-MPK10^CSSH-A is attenuated in response to delayed leaf senescence,suggesting that H₂S signaling delays leaf senescence through S-persulfidation of MPK10protein.6.MPK10 acts by phosphorylating target substrates.In this study,the interaction of MPK10 with transcription factor BES1 in vivo was confirmed by Bimolecular Fluorescence Complementation（BiFC）and Luciferase Complementation Assay（LCA）.The interaction between MPK10 and BES1 was localized to the nucleus by confocal laser microscopy.