<scp>H</scp> ₂ <scp>S</scp> ‐Mediated <scp>GH3</scp> .1 Persulfidation Regulates <scp>IAA</scp> Homeostasis to Enhance Nodulation Formation and Nitrogen Fixation in <i>Robinia pseudoacacia</i>

Hydrogen sulphide (H₂S), 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 H₂S in indeterminate nodules of woody legumes remains unclear. In this study, we investigated the mechanism by which H₂S promotes nodulation and N fixation in the woody legume Robinia pseudoacacia. Exogenous H₂S 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 H₂S signalling modulates auxin metabolism, particularly through the regulation of indole-3-acetic acid (IAA) homeostasis. H₂S was found to promote free IAA accumulation and reduce IAA conjugation (IAA-Asp and IAA-Glu). Further investigation revealed that H₂S directly targets GH3.1, a key IAA-amido synthetase responsible for IAA conjugation. Specifically, H₂S 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 H₂S in regulating IAA homeostasis, thereby enhancing indeterminate nodule formation and N fixation through persulfidation of the Cys304 residue of GH3.1 in R. pseudoacacia.