Molecular Mechanism Of OsGPX1 And OsSRT2 In Response To Salt Stress In Oryza Sativa

Protein post-translational modifications(PTMs)refers to the covalent and generally enzymatic modification of proteins following protein biosynthesis.PTMs is a dynamic,reversible protein processing process,which occurring in prokaryotic and eukaryotic cells,and extensively mediates important physiological processes.such as cell growth,development,and stress response.Among these PTMs,acylation modifications occurring on lysine residues are the one of most important regulatory mechanisms in regulating protein function.However,the mechanism of lysine acylation modifications in response to abiotic stress in plants is currently unclear.To solve the above questions,we identified acetylatedand succinylated-proteins in rice under oxidative stress by proteomics and studied the function and regulatory mechanism of acetylation in response to salt stress by reverse genetics,and molecular biological evidences,and acquired results as follows.(1)In this study,we first used specific antibodies to detect the acetylation and succinylation levels of rice protein,which extracted from different growth stages and tissues.It was found that acetylation and succinylation were commonly happened in rice proteins.Subsequently,we performed lysine succinyl-and acety-proteomes of rice leaves by using antibody affinity enrichment,high resolution liquid chromatography mass spectrometry,and bioinformatics analysis.We successfully identified 2,593 succinylated proteins and 1,024 acetylated proteins.KEGG enrichment analysis and subcellular localization analysis of the identified succinylated proteins revealed that these proteins were mainly enriched in the carbon metabolism pathway and chloroplast;Further domain and motif analysis of the identified succinylated proteins showed that Thioredoxin-like domain is the most frequently modified domain,and the main types of succinylated motifs including R-9Ksu,K-10Ksu,etc.For acetylated protein,and the acetylated peptides were found mainly in the histoneassociated domain,and the acetylation-modified motif types are KacR+1,KacK+1,etc.Further analysis revealed that 723 rice proteins were simultaneously acetylated and succinylated.These proteins are mainly enriched in pathways involved in photosynthetic carbon assimilation(eg,the subunit of Ribulose bisphosphate carboxylase oxygenase;RuBisCO),glycolysis,TCA cycle,and pentose phosphate.KEGG enrichment analysis of differentially expressed succinylated and acetylated proteins under oxidative stress revealed that the level of protein succinylation in the ribosomal,glyoxylate and dicarboxylate metabolism pathway were significantly up-regulated.And the proteins enriched in glyoxylate and dicarboxylate metabolism,endoplasmic reticulum protein processing and photosynthetic carbon fixation pathways were down-regulated;while the differentially expressed acetylated proteins are mainly down-regulated,and these proteins are mainly enriched in the glyoxylate and dicarboxylate metabolism,glutathione metabolism and amino acid metabolism pathways.These results indicate that the acetylated proteins have a certain similarity with the succinylated protein,but the modified sites are quite different.Therefore,the above results not only greatly explored the acetylation and succinylation-modified proteins and sites in rice,but also demonstrated the biological role of post-translational modifications in regulating protein function in various proteins.(2)Furthermore,we selected two succinylation modified proteins,catalase A(OsCATA;Q0E4K1)and glutathione-S-transferase(OsGSTU6;Q8S718)for further validation.In vitro desuccinylation and activity assay showed that the level of succinylation of the OsCATA recombinant protein was decreased and the activity was up-regulated by 50%after addition of the mammalian desuccinylase SIRT5.The addition of SIRT5 also resulted in a decrease of succinylation level of OsGSTU6,but the activity of GSTU6 was reduced nearly 50%.These results suggested that succinylation could regulate protein activity.(3)Based on above data,we choose an oxidative stress triggered-acetylated protein,OsGPXl(Glutathione Peroxidase),for further study.Our results showed that OsGPX1 is a peroxidase that relies on thioredoxin as an electron donor.Acetylated modification increases the activity of OsGPXl,which is mainly regulated by the K94.Compared with wild type,rice seedlings overexpressing OsGPXl showed higher salt tolerance by maintaining redox homeostasis.And the acetylation level of OsGPX1 was increased after salt stress.Subcellular localization and western blotting results indicated that OsGPX1 localizes in the cytoplasm and nucleus.OsGPX1 protein could transfer to the nucleus under NaCl and MV treatment,which is regulated by the acetylation level of OsGPXl.The increase in the acetylation level promotes the nuclear shuttling of OsGPX1.Subsequently,we screened the interacting protein for acetylation of OsGPXl by yeast two-hybrid.Finally,we found that histone deacetylase OsHDA15 and the histone acetyltransferase OsHAC701 interact with OsGPX1 in yeast.The interaction between them was further verified by bimolecular fluorescence complementation(BiFC),yeast two-hybrid,GST pulldown and co-immunoprecipitation(Co-IP).In vitro and in vivo experiments indicated that OsHDA15 could reduce OsGPXl activity by deacetylation,whereas OsHDA15-mediated deacetylation inhibits the nuclear shuttling of OsGPX1.These results indicate that deacetylation by OsHDA15 negatively regulates OsGPX1-mediated salt stress response.(4)Our results showed that many proteins involved in primary metabolism could be succinylated.However,the source and regulatory mechanism of succinylation in plant are poorly understood.In this study,we first obtained the OsSRT2 recombinant protein by prokaryotic expression and purification.The enzyme activity assay showed that OsSRT2 is a NAD+-dependent deacetylase and also has a variety of deacylase activities.Among these,deformylase,deacetylase and depropionylase activities are relatively high,and the devalerylase,deisovalerylase and depalmitoylase activities are relatively weak.Further salt treatment results showed that OsSRT2 overexpressing transgentic rice and Arabidopsis were sensitive to salt stress,while srt2 mutant was more tolerant to salt stress.Knockout of OsSRT2 intensify the increased antioxidant defence under salt stress,these results were conducted by eliminating the excessive production of ROS,and alleviating lipid peroxidation.It indicated that OsSRT2 may involve in redox homeostasis maintenance under salt stress.Subcellular localization results showed that OsSRT2 localizes in the Mitochondria.However,yeast two-hybrid results showed that OsSRT2 has no interaction with the proteins involved in redox homeostasis maintenance.Compared with the wild type,the levels of deacetylation,depropionylation,debutyrylation and desuccinylation in the total protein and nuclear proteins of the srt2 mutant were significantly enhanced,especially for histone modification.Further western blotting results showed that,except for propionylation,the levels of acetylation,succinylation and butyrylation on histone H3 were upregulated in wild type seedlings under salt stress.especially acetylation and butyrylation.The up-regulation of H3 acetylation by salt stress shows no difference in the srt2 mutant,but the up-regulation of H3 butyrylation by salt stress was further enhanced in the srt2 mutant.The above results were further confirmed in OsSRT2 overexpressing line.The up-regulation of H3 acetylation caused by salt stress was also up-regulated in the over-expressed material,while the level of butyrylation of H3 did not change significantly.These results suggest that OsSRT2 negatively regulates salt resistance by disturbing redox homeostasis and deactylating histone.In summary,our study first identified a large number of succinylated and acetylated modified proteins and sites in rice,and then initially explored the function of acetylation modified-OsGPX1 in response to oxidative stress in rice,especially during salt stress and the regulatory mechanism of OsSRT2,function as a multiple deacylase in response to salt stress.The relevant research results will help to expand our understanding of the function of posttranslational modification of proteins in plant stress response and provide theoretical and experimental basis for further application of molecular biological methods to improve crop resilience.