Hydrogen sulfide antagonizes cytokinin to change root system architecture through persulfidation of CKX2 in Arabidopsis.

Hydrogen sulfide (HS) 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 HS and cytokinin (CTK) and the mechanisms by which HS 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, OE CKX2 and ckx2/CKX2 transgenic lines were isolated with the ckx2 background. HS 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 HS on RSA, indicating that HS reduces the gravitropic set-point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2 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 HS to regulate RSA in vivo. In conclusion, this study demonstrated that HS negatively regulates CTK content and affects RSA by persulfidation of CKX2 in Arabidopsis thaliana.