H₂S‐Mediated GH3.1 Persulfidation Regulates IAA Homeostasis to Enhance Nodulation Formation and Nitrogen Fixation in Robinia pseudoacacia

Hydrogen sulphide (H 2 S), a gaseous signalling molecule, plays a multifaceted role in plant physiology by enhancing adaptability to environmental stresses. However, the regulatory mechanism of symbiotic nitrogen (N) fixation by H 2 S in indeterminate nodules of woody legumes remains unclear. In this study, we investigated the mechanism by which H 2 S promotes nodulation and N fixation in the woody legume Robinia pseudoacacia . Exogenous H 2 S significantly enhanced rhizobium infection, nodule formation and nitrogenase activity, demonstrating its positive role in the symbiotic process. Transcriptomic analysis of roots and nodules revealed that H 2 S signalling modulates auxin metabolism, particularly through the regulation of indole‐3‐acetic acid (IAA) homeostasis. H 2 S was found to promote free IAA accumulation and reduce IAA conjugation (IAA‐Asp and IAA‐Glu). Further investigation revealed that H 2 S directly targets GH3.1, a key IAA‐amido synthetase responsible for IAA conjugation. Specifically, H 2 S mediated persulfidation at Cys304 of GH3.1, inhibiting its enzymatic activity and preventing IAA inactivation. This modification was confirmed by LC–MS/MS, UPLC‐ESI‐MS/MS and site‐directed mutagenesis. This post‐translational modification maintained active IAA levels, facilitating early nodule development. These findings highlight the active role of H 2 S in regulating IAA homeostasis, thereby enhancing indeterminate nodule formation and N fixation through persulfidation of the Cys304 residue of GH3.1 in R. pseudoacacia .