Hydrogen Sulfide Regulates The Root Development Through Mediating S-persulfidation Of Cytokinin Oxidase 2 In Arabidopsis Thaliana

Cytokinin（CTK）regulates plant cell division and differentiation and plays a key role in plant root development.Cytokinin oxidase（CKX）irreversibly degrades CTK,thereby regulating CTK concentration-dependent physiological processes.Hydrogen sulfide（H₂S）is an important gas signaling molecule that actively participates in the regulation of plant growth/development and response to stress.In plants,H₂S can interact with a variety of plant hormones（such as auxin,ethylene,abscisic acid,etc.）to regulate plant metabolism and developmental processes,but the cross-interaction relationship between H₂S and CTK is not yet clear.H₂S-mediated S-persulfidation is a newly discovered type of protein post-translational modification that can directly regulate protein activity and function.In this article,Arabidopsis thaliana was used as the material to deeply explore the molecular mechanism of cross-interaction between S-persulfidation of CKX2 and CTK to regulate plant root development.The main findings are as follows:1.Prove the existence of S-persulfidation modification of CKX2.Cloning of CKX2gene,prokaryotic expression and persulfidation of recombinant protein in vitro.High-resolution liquid chromatography-MS/MS（LC-MS/MS）was used to prove the existence of S-persulfidation on CKX2,and secondary mass spectrometry shows the exact S-persulfidation site.2.Express and purify ^SPCKX2^WT（SP stands for signal peptide）、CKX2^WTand the corresponding single site mutant protein with all Cys residues.Using the modified biotin switch method（MBSM）,it was proved that H₂S mediates the S-persulfidation modification of ^SPCKX2^WTand CKX2^WT,and the S-persulfidation modification site of CKX2 proteinthe is specific.3.The enzyme activity analysis of Arabidopsis total CKX,recombinant ^SPCKX2^WTand CKX2^WTshowed that exogenous Na HS treatment enhanced the degradation ability of Arabidopsis total CKX,^SPCKX2^WTand CKX2^WTto CTK.The enzyme activity analysis of CKX2^CSSHA,CKX2^C1-A,CKX2^C2-A,and CKX2^C3-Afound that the Cys residue mutation at the target site results CKX2 lost response to H₂S,and the promoting effect of enzyme activity disappeared;H₂S can still promote the enzyme activity after Cys mutation at other sites.Therefore,H₂S-mediated S-persulfidation modification positively regulates the enzymatic activity of CKX2.4.Na HS and KT exogenously treated wild-type Arabidopsis thaliana and found that H₂S and CTK play opposite roles in root hair elongation,lateral root development and extension,and auxin distribution at the tip of the main root,which proves the S-persulfidation of CKX2 is a bridge between H₂S and CTK interaction,so that H₂S and CTK play an antagonistic role in root development.5.Using the wild-type Arabidopsis as the genetic background,construct overexpressing CKX2 line.It was observed that the rosette part volume and rosette leaf area of the overexpressing CKX2 line became smaller,the bending degree of the leaf became smaller,indicating that the expression of CKX2 was significantly up-regulated.The angle analysis of the four genetic lines of OE-LCD/oasa1,WT,OE-CKX2 and ckx2 found that the angle between the main root and the lateral root in the OE-CKX2 line and the OE-LCD/oasa1 line was smaller than the wild type.The angle between the main root and the lateral root in the ckx2 line became larger,which further proved that the S-persulfidation of CKX2 inhibited the extension of the lateral root.In conclusion,H₂S-mediated S-persulfidation positively regulates the enzymatic activity of CKX2 and reduces the content of CTK.S-persulfidation of CKX2 acts as a bridge for the interaction between H₂S and CTK,which makes H₂S and CTK antagonize in Arabidopsis root hair elongation,lateral root development and extension,and auxin distribution at the tip of the main root,and jointly regulate Arabidopsis root system development.