Molecular Mechanism Of Hydrogen Sulfide Enhancing Salt Tolerance Via S-sulfhydration Of CAT2 In Arabidopsis

Salt stress can lead to metabolic disorders in plant cells and induce the accumulation of reactive oxygen species（ROS）.In order to reduce the oxidative damage caused by ROS,plants have formed an effective antioxidant system to remove excess ROS.Catalase（CAT）can directly decompose H₂O₂ into H₂O and O₂.It is an essential enzyme for ROS scavenging.It can effectively regulate the dynamic balance of ROS and play an important role in resisting salt stress.There are three genes encoding CAT in Arabidopsis:CAT1,CAT2 and CAT3.When CAT2 is mutated,CAT activity is lost by 80%,indicating that CAT2 plays a major role in the response of CAT to salt stress.However,how CAT2 activity regulated is not clear.As a new type of gas signal molecule,Hydrogen sulfide（H₂S）participates in the response process of plants to various stresses and enhances stress resistance by scavenging ROS,but the molecular mechanism is not clear.It has been proved that H₂S can S-sulfhydrate Cys residues of target proteins to change the activity and function of proteins,and then participate various physiological processes.In this experiment,through the proteomic analysis of Arabidopsis S-sulfhydrylation,it was found that CAT2 is one of the target proteins that may be modified by S-sulfhydrylation.So whether H₂S mediates the S-sulfhydrylation of CAT2,thereby regulating CAT activity and regulating ROS balance to improve plant salt tolerance?In this paper,biological techniques and methods such as molecular biology,biochemistry and genetics were used to explore the molecular mechanism of H₂S-mediated CAT2 S-sulfhydrylation in regulating salt stress response with WT,LCD,DCD,and CAT2-related deletion mutants and overexpressing lines were used as experimental materials.The following conclusions are initially drawn:1.Both H₂S and CAT2 are involved in the response of salt stress in Arabidopsis.The expression levels of H₂S synthase genes LCD and DCD and the content of H₂S in WT were significantly increased under salt stress.And the seed germination rates and seedling growth of H₂S synthase deletion mutants lcd and dcd were significantly inhibited.LCD and DCD overexpressing lines were less inhibited.NBT and DAB staining showed more ROS accumulation in lcd and dcd,which proved that endogenous H₂S could improve the salt tolerance of Arabidopsis by scavenging ROS.At the same time,the seed germination rate and seedling growth of cat2 deletion mutant were significantly weaker than WT under salt stress,while CAT2overexpressing lines showed higher salt tolerance,indicating that both H₂S and CAT2 can improve salt tolerance;2.CAT2 is involved in H₂S-regulated response to salt stress in Arabidopsis.In order to explore the relationship between H₂S and CAT2 in response to salt stress,we used pharmacological experiments to prove that the external application of H₂S donor sodium hydrosulfide（Na HS）could partially alleviate the effect of germination rate,root growth and seedling growth of WT and CAT2-overexpressing lines under salt stress.But it has no obvious effect on cat2 deletion mutant.The accumulation of ROS in WT,CAT2-overexpressing lines and deletion mutants increased significantly after the external application of the H₂S scavenger Hypotaurine（HT）.Suggesting that CAT2 is located at H₂S downstream to response salt stress.3.H₂S-mediated S-sulfhydrylation of CAT2 enhances salt tolerance in Arabidopsis.The total protein was extracted from seedlings of WT,LCD and DCD-related materials treated with Na Cl,and detected the level of S-sulfhydrylation.The lcd and dcd mutant material total protein S-sulfhydrylation level did not change significantly,LCD and DCD overexpressing lines significantly enhanced S-sulfhydrylation level,indicating that S-sulfhydrylation involved salt stress response process in Arabidopsis thaliana.The prokaryotic expression of CAT2 was treated with Na HS,and it was found that the level of S-sulfhydrylation of CAT2 also increased with the increase of the concentration of Na HS treatment.The S-sulfhydrylation level of CAT2 was lower in the lcd and dcd mutants under salt stress.After treatment,the level of S-sulfhydrylation of CAT2 increased;the level of S-sulfhydrylation of CAT2 increased in LCD and DCD overexpressing materials under salt stress,and the level of S-sulfhydrylation of CAT2 was reduced by exogenous HT,indicating that CAT2 can be modified by H₂S S-sulfhydrylation both in vivo and in vitro.The CAT2 enzyme activity is enhanced after Na HS treatment.Through point mutation and S-sulfhydrylation detection,it was found that the S-sulfhydrylation site of CAT2 was 420th Cys,and the enzyme activity of the mutated protein after Na HS treatment showed no significant change in the activity of CAT2^C420S,while the protease activity of other site mutations was increased after Na HS treatment,which proved that H₂S enhanced the activity of CAT2 enzyme by S-sulfhydrylation modifying the 420th Cys of CAT2 to improve the salt tolerance of Arabidopsis thaliana.This study proves that H₂S can precisely regulate CAT2 enzyme activity to enhance salt tolerance via S-sulfhydrylation of CAT2 in Arabidopsis.It provides a new theoretical basis for analysing deeply of the mechanism of H₂S involved in responding to salt stress in Arabidopsis.