Melatonin alleviates salt stress partially through the VvRBOHA-dependent H2O2 signaling pathway in grapes

• Melatonin (MT) alleviates salt stress in grapes and alters the abundance of multiple signaling molecules. • H₂O₂ signaling may regulate the upstream of salt-tolerant ET and IAA pathways. • Overexpression of VvRBOHA markedly enhances salt tolerance in transgenic plants. Salt stress represents a major abiotic constraint impairing grapevine growth and productivity. While our previous work demonstrated that melatonin (MT) alleviates salt stress in grapes, the synergistic crosstalk between MT and diverse signaling pathways—particularly the hydrogen peroxide (H₂O₂)—remains poorly characterized. To address this, the present study combined MT with inhibitors of key signaling pathways to elucidate the mechanism underlying MT-mediated salt tolerance via H₂O₂ signaling, and further explored the role of endogenous H₂O₂ in this process through RBOHA overexpression. Results showed that MT significantly mitigated salt-induced impairments. Among inhibitor treatments, the auxin (IAA) transport inhibitor TIBA (Triiodobenzoic Acid) resulted in the lowest salt tolerance score, followed by the H₂O₂ inhibitor DPI (diphenyleneiodonium). Promoter analysis revealed that VvRBOH genes contain hormone and stress-responsive cis-elements. Overexpression of VvRBOHA enhanced salt tolerance in Arabidopsis thaliana and tomato, concomitant with increased endogenous hormone biosynthesis. In conclusion, MT alleviates salt stress in grapes partially through a VvRBOHA -dependent H₂O₂ signaling pathway that promotes hormone synthesis. Additionally, H₂O₂ may act upstream of MT to regulate salt tolerance-related ethylene (ET) and IAA pathways. This study provides valuable insights into dissecting plant adaptive responses to abiotic stress.