Molecular Mechanism Of Deacetylated Glutathione Peroxidase OsGPX1 Regulated By Histone Deacetylase OsHDAC15 In Response To Water Stress In Oryza Sativa

Post translational modification(PTM)is considered to be the covalent and generally enzymatic modification of proteins after syntheses of proteins.Among those modifications,acetylation modification occurring in lysine residues plays a key role in regulating function of proteins.The acetylation levels of proteins are modulated by histone acetyltransferases(HATs)and histone deacetylase(HDACs).GPXs(Glutathione peroxides;EC 1.11.1.12)generally reduce reactive oxygen species(ROS)by the thioredoxin pathway in plants to maintain redox state in plants and prevent the cellular component from oxide damage.On the other hand,GPXs were involved in maintaining redox state in plants and have interaction with transcription factors.Our lab previous research showed OsHDA15 have interaction with OsGPX1.To further explore the function of OsGPX1 and mechanism how OsGPX1 responds to water stress,we explore this topic by molecular and genetic evidence,and results as follows.(1).Functional analysis of OsGPX1.To explore the function and active sites of OsGPX1,we first constructed prokaryotic expression vectors of 42,71 and 90 site cysteine residue mutation,then we expressed and purified recombinant proteins.The enzyme activity of recombinant OsGPX1 protein and mutants showed mutation in 71 and 90 site cysteine residues leaded the loss of peroxidase enzyme activity and mutant of 42 site cysteine residues kept 75%of normal OsGPX1’s peroxidase activity.Some research showed most GPXs in plants act as monomer and some of them exist two different redox state in vivo or vitro.OsGPX1 is a monomer proved by yeast two-hybrid system.We treat OsGPX1 and its cystine mutant proteins with H2O2 and DTT.Result showed that OsGPX1 exists reduced and oxidized state,while mutation of 71 or 90 site cysteine residues caused the loss of oxidized state.We can conclude that disulfide bond between 71 and 90 site cysteine residues affects redox state of OsGPX1.The results of redox state,consistent with the enzyme activity,showing that 71 and 90 site cysteine residues play a key role in the function of OsGPX1.(2).OsGPX1 enhances tolerance for water stress by modulating redox homeostasis in rice.In order to further explore its role in water stress response in rice,we used PEG to process the simulation of water stress.The expression of OsGPX1 persistently increased after rice was exposed to PEG stress for 6 h.35S:OsGPX1 rice showed higher tolerance for PEG stress compared with wild type.Compared with wild type,35S:OsGPX1 rice reduced the MDA content and the percent leakage of electrolyte under PEG treatment.Results also showed that overexpression of OsGPX1 enhances rice glutathione peroxides activity and reduces H2O2 content under PEG treatment.Further testing expression of water deprivation responsive genes,it was found that expression of OsLEA3,OsNCED1,OsbZIP23,OsbZIP46 and OsARAG1 genes was significantly enhanced in 35S:OsGPX1.Those results show that OsGPX1 enhances the tolerance for water stress by modulating redox homeostasis and increasing expression of water deprivation responsive genes.(3).OsHDA15 deacetylate OsGPX1 to regulate subcellular localization of OsGPX1.The results of yeast two-hybrid system and protoplasts transient showed that OsHDA15 have interaction with OsGPX1 and regulate the acetylation level of OsGPX1.Under PEG treatment,OsGPX1 proteins were transmitted into the nucleus,with the acetylation level of OsGPX1 markedly raising.Experiments of transient expression in rice protoplasts showed that acetylation level of OsGPX1 proteins in the protoplasts of 35S:OsHDA15 is lower than that in wild-type protoplasts,and PEG induced nuclear accumulation of OsGPX1 is decreased in the 35S:OsHDA15’s protoplasts.We can conclude that the higher acetylation modification of OsGPXl and its nuclear accumulation induced by water stress are negatively regulated by OsHDA15.Based on our lab’s pervious mass spectrometry results of OsGPX1,we found that acetylation of OsGPX1 94,121,159,162 and 163 sites lysine residues were regulated by OsHDA15.In order to verify the effect of these site acetylation modifications on OsGPX1 function,we built the prokaryotic expression and rice protoplasts transient expression vectors of lysine residues mutant.Results of non-reduced SDS-PAGE showed that deacetylation of OsGPX1 mediated by OsHDA15 do not affect redox state of OsGPX1 protein.According to results in transient expression rice protoplasm we found that PEG treatment induced the nuclear accumulation of normal OsGPX1,but did not induce nuclear accumulation of OsGPX1159,162,163K/R proteins(159,162 and 163 site lysine were mutant to arginine),indicating that 159,162 and 163 site lysine residues were involved in acetylatemediated nuclear accumulation of OsGPX1 protein.We conclude that OsHDA15 control nuclear accumulation of OsGPX1 by deacetylating 159,162 and 163 site lysine residues.(4).35S:OsHDA15 rice is more sensitive to water stress.To explore the role of OsHDA15 in water stress response,we first tested the expression of OsHDA15 gene under PEG,NaCl and H2O2 stress.The results of qRT-PCR showed that OsHDA15 gene expression constantly declined under PEG treatment.We constructed transgenic rice plants overexpressing OsGPX1 under the control of 35S promoter.The phenotypes showed that 35S:OsHDA15 rice,compared with wild type,was more sensitive to PEG treatment,accompanying with accumulation of MDA and rise of electrolyte leakage.Further,we tested expression of water deprivation responsive gene under PEG treatment in 35S:OsHDA15 rice.And results showed that compared with wild type,OsLEA3,OsNAC6,NECD1 and OsbZIP23 genes expression was lower in 35S:OsHDA15,while the expression of OsARAG1 and OsbZIP46 genes were basically unchanged.Above results suggested that OsHDA15 negatively regulate water stress in rice.In conclusion,OsGPX1 as glutathione peroxides can enhance tolerance for water stress in rice and OsHDA15 deacetylate OsGPX1 to control subcellular localization of OsGPX1.