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

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.