Contrary to convention: Apoplastic ascorbate oxidation surfaces as a crucial conductor in salt stress

• Apoplastic AO activity is essential, not detrimental, for salt tolerance. • AO knockout increases AsA redox state but triggers ROS over accumulation via NOX. • Novel AO-NOX-ROS signaling module governs salt tolerance in rice. Salinity as a major constraint in rice cultivation under climate change. Ascorbic acid (AsA) is a crucial antioxidant in plants, and its intracellular accumulation generally enhances stress tolerance. In the apoplast, AsA is catalyzed by ascorbate oxidase (AO) to its oxidized form of dehydroascorbate (DHA). However, the relationship between apoplastic AO activity and salt tolerance remains unexplored. This study examined the effects of suppressing apoplastic AO activity on AsA homeostasis, reactive oxygen species (ROS) metabolism, and salt tolerance by knocking out the AO genes, OsAAO2 and OsAAO4 . These results indicated that both OsAAO2 and OsAAO4 responded to salt stress. Under 0.8% NaCl treatment, the survival rates of knockout lines (AO2-KO and AO4-KO) decreased by 0.42-fold and 0.25-fold, respectively, compared with the wild type. Apoplastic AO activity in AO2-KO and AO4-KO was reduced, accompanied by an increased AsA/DHA ratio. Furthermore, upregulation of NOXA expression and enhanced NADPH oxidase activity in the knockout lines led to significant intracellular ROS accumulation and subsequent oxidative damage, ultimately reducing salt tolerance. These findings reveal the pivotal AO–NOX–ROS signaling module in rice salt tolerance, suggesting that apoplastic AO activity could be a novel target for improving crop salt tolerance.