4D-proteome analysis reveals the role of hydrogen sulfide-based priming in attenuating salinity-induced GABA accumulation in rice

Soil salinity severely constrains rice productivity by inducing ionic imbalance, oxidative damage, and metabolic disruption. Hydrogen sulfide (H₂S) has emerged as an important signaling molecule in plant stress responses, yet its mechanistic role in salinity tolerance remains incompletely understood. Here, we investigated the function of H₂S in modulating salinity stress responses in rice using two cultivars with contrasting salinity tolerance, the sensitive Dongjin and the tolerant IR73. Salinity stress resulted in severe growth inhibition, particularly in Dongjin, accompanied by elevated malondialdehyde and hydrogen peroxide levels. H₂S donor (NaHS) pretreatment significantly alleviated these symptoms and reduced oxidative damage, whereas its scavenger (hypotaurine) exacerbated stress effects. Expression analysis of ion transporter genes revealed cultivar-specific responses, with NaHS selectively stabilizing Na⁺ and K⁺ homeostasis rather than broadly inducing salinity-responsive genes. To further gain a molecular insight into these H₂S responses, we employed data-independent acquisition (DIA) proteomics, which led to the identification of 6710 protein groups and 1635 differentially modulated protein groups. Functional analysis of the H₂S and salinity-responsive proteins revealed coordinated modulation of redox-related enzymes, sulfur metabolism, and regulatory proteins in IR73. In particular, a significant modulation of proteins associated with γ-aminobutyric acid (GABA) metabolism was observed. qRT-PCR-based expression analysis and GABA quantification revealed that H₂S pretreatment suppressed excessive activation of GABA biosynthesis and accumulation, indicating that GABA acts as a marker of stress severity rather than a primary mediator of H₂S-induced tolerance. Collectively, our results demonstrate that H₂S enhances salinity tolerance in rice by reducing stress perception, maintaining redox and ionic homeostasis, and minimizing secondary stress responses, providing new insights into H₂S-mediated stress adaptation mechanisms.