Hydrogen sulfide (H₂S) is an important signaling molecule in plants. Our previous report suggested that H₂S signaling affects the actin cytoskeleton and root hair growth. However, the underlying mechanisms of its effects are not understood. S-Sulfhydration of proteins is regulated directly by H₂S, which converts the thiol groups of cysteine (Cys) residues to persulfides and alters protein function. In this work, we studied the effects of S-sulfhydration on actin dynamics in Arabidopsis (Arabidopsis thaliana). We generated transgenic plants overexpressing the H₂S biosynthesis-related genes l-CYSTEINE DESULFHYDRASE (LCD) and d-CYSTEINE DESULFHYDRASE in the O-acetylserine(thiol)lyase isoform a1 (oasa1) mutant and Columbia-0 backgrounds. The H₂S content increased significantly in overexpressing LCD/oasa1 plants. The density of filamentous actin (F-actin) bundles and the F-actin/globular actin ratio decreased in overexpressing LCD/oasa1 plants. S-Sulfhydration also was enhanced in overexpressing LCD/oasa1 plants. An analysis of actin dynamics suggested that S-sulfhydration inhibited actin polymerization. We also found that ACTIN2 (ACT2) was S-sulfhydrated at Cys-287. Cys-287 is adjacent to the D-loop, which acts as a central region for hydrophobic and electrostatic interactions and stabilizes F-actin filaments. Overaccumulation of H₂S caused the depolymerization of F-actin bundles and inhibited root hair growth. Introduction of ACT2 carrying a Cys-287-to-Ser mutation into an act2-1 mutant partially suppressed H₂S-dependent inhibition of root hair growth. We conclude that H₂S regulates actin dynamics and affects root hair growth.

Hydrogen sulfide (H₂S) displays a positive physiological function in plant growth. Limited data are available on the influence of H₂S on cell wall growth in plants. Here, we outline a novel mechanism to show how H₂S regulates cell wall cellulose biosynthesis and deposition in Alfalfa. NaHS treatment increased the length of epidermal cells in the hypocotyl of Alfalfa. Transcriptome analysis revealed that NaHS treatment caused the differential expression of a mass of cell wall-related genes. These differentially expressed genes (DEGs) directly associated with cellulose and hemicellulose biosynthesis, and pectin degradation. Cell wall composition analysis found that NaHS treatment increased the contents of cellulose and hemicellulose, but decreased pectin content. Atomic force microscopy (AFM) was used to observe cellulose microfibrils. The diameter of cellulose fibril was decreased, the arrangement of fibrillar bundles was altered, and NaHS increased the interspace of fibrillar bundles. Cellulose synthase complexes (CSCs) dynamics closely related to cellulose synthesis. We found that NaHS increased the mobile rates of CSC particles. These data suggest that the H₂S signal enhanced the plasticity of the cell wall by regulating cellulose fibrils deposition and decreasing the pectin content. The increase of cellulose and hemicellulose content resulted in cell wall expansion and cell elongation.