Hydrogen sulfide antagonizes cytokinin to change root system architecture through persulfidation of <scp>CKX2</scp> in Arabidopsis

Hydrogen sulfide (H₂S) is an endogenous gaseous signaling molecule, which has been shown to play an important role in plant growth and development by coupling with various phytohormones. However, the relationship between H₂S and cytokinin (CTK) and the mechanisms by which H₂S and CTK affect root growth remain poorly understood. Endogenous CTK was analyzed by UHPLC-ESI-MS/MS. Persulfidation of cytokinin oxidase/dehydrogenases (CKXs) was analyzed by mass spectrometry (MS). ckx2/CKX2^{wild-type (WT)}, OE CKX2 and ckx2/CKX2^{Cys(C)62alanine(A)} transgenic lines were isolated with the ckx2 background. H₂S is linked to CTK content by CKX2, which regulates root system architecture (RSA). Persulfidation at cysteine (Cys)62 residue of CKX2 enhances CKX2 activity, resulting in reduced CTK content. We utilized 35S-LCD/oasa1 transgenic lines to investigate the effect of endogenous H₂S on RSA, indicating that H₂S reduces the gravitropic set-point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2^Cys62 changes the elongation of cells on the upper and lower flanks of LR elongation zone, confirming that Cys62 of CKX2 is the specificity target of H₂S to regulate RSA in vivo. In conclusion, this study demonstrated that H₂S negatively regulates CTK content and affects RSA by persulfidation of CKX2^Cys62 in Arabidopsis thaliana.