Persulfidation And Function Regulation Of PHD Protein AL7 Mediated By H₂S In Arabidopsis

Hydrogen sulfide（H₂S）is an endogenous gas signal molecule,which can help plants resist abiotic and biological stresses at different levels.At present,the main way for H₂S to play its physiological function in plants is to induce S-sulfhydrylation of protein cysteine residues.PHD Finger（plant home domain,PHD）protein contains a large amount of cysteine,which plays an important role in regulating plant cell cycle,growth and development and environmental adaptation.No research has shown whether H₂S can regulate the function of PHD Finger protein through S-sulfhydrylation modification.In this study,Arabidopsis thaliana was used as the research material to preliminarily analyze the regulatory relationship between H₂S and PHD Finger protein AL7（Alfin1 like 7）,which provides some new clues for further research on H₂S action targets.The main results of the study are as follows:（1）According to literature and database information,three PHD protein family members EBS（Early bolting in short days,EBS）,SHL（short life,SHL）and AL7 with potential S-sulfhydrylation targets and associated with existing reported functions of H₂S were screened out;The genes EBS（735bp）,SHL（717bp）,AL7（789bp）including the restriction site were cloned.Then we used homologous recombination to construct three prokaryotic expression vectors for subsequent protein expression and purification.（2）EBS,SHL and AL7 proteins were purified by Ni NTA affinity chromatography column.The S-sulfhydrylation level of EBS,SHL and AL7 proteins in vitro was detected by biotin switch method.The results showed that H₂S could thiothiolate AL7 protein.（3）It has been reported that RING1a can interact with AL7.We cloned the RING1a gene（1599 bp）containing the restriction site,and constructed the prokaryotic expression vector of GST-RING1a.However,it is very difficult to express the full length protein of RING1a in vitro;Therefore,we constructed the RING1a C,and expressed the RING1a C protein for pull down detection.The RING1a C protein is a RING1a truncated protein containing the RAWUL domain.（4）We purified a large amount of His-AL7 protein in vitro,and divided the protein into CK group and H₂S treatment group on average.They were incubated with the same amount of GST-RING1a C protein,and the strength of the binding ability between the proteins was analyzed by pull down test.The experimental results showed that the interaction between AL7 protein and RING1a protein was weakened under the action of H₂S,which indicated that H₂S could affect the binding ability of AL7 protein to RING1a protein by S-sulfhydrylation.（5）Homozygous al7 Arabidopsis mutants were identified by various methods.We observed the germination rate of WT,lcd and al7 seeds under normal growth conditions and cold stress conditions.The results showed that under normal conditions,there was no difference in seed germination rate among different genotypes.Under cold stress,the germination rate of al7 was significantly higher than that of WT,and the germination rate of lcd was significantly lower than that of WT.The germination rate of WT was significantly increased when the physiological concentration H₂S was applied,the phenotype of lcd was significantly restored when the physiological concentration H₂S was applied,and the germination rate of al7 did not change significantly.This indicates that AL7 negatively regulates Arabidopsis resistance to cold stress,and H₂S is upstream of AL7 and is involved in this process.To sum up,on the one hand,H₂S can S-sulfhydrylation modify AL7 protein and affect its interaction with RING1a protein;On the other hand,AL7 plays a negative role in the process of Arabidopsis resistance to cold stress,and H₂S participates in this process through AL7.