Abstract

Stomatal closure regulated by abscisic acid (ABA) is a critical plant response to drought, with reactive oxygen species (ROS), playing a central signaling role. However, the involvement of antioxidant enzymes and their regulators in this process remains insufficiently understood. Here, we investigate the role of miR398 and its target gene SlCSD1, which encodes a Cu/Zn superoxide dismutase, in ABA-induced stomatal closure and drought tolerance in tomato (Solanum lycopersicum). Overexpression of sly-miR398b or knockout of SlCSD1 resulted in elevated O₂•⁻ levels, enhanced stomatal closure, and improved drought tolerance. In contrast, sly-miR398b mutants showed diminished O₂•⁻ accumulation, reduced stomatal closure, and increased sensitivity to drought. Pharmacological assays confirmed that miR398b-mediated stomatal responses depend on O₂•⁻ signaling. Further analysis revealed that ABA-responsive element binding factor (SlABF2) directly binds to an ABA-responsive element (ABRE) in the sly-miR398b promoter and activates its expression. Overexpression of SlABF2 upregulated expression of sly-miR398b, increased O₂•⁻ production, and promoted stomatal closure and drought tolerance. Moreover, SlABF2 activity is regulated by Sucrose Nonfermenting Related Kinase 2.6 (SlSnRK2.6), a core component of the ABA signaling pathway. Together, these findings identify a regulatory cascade in which the SnRK2.6–ABF2–miR398b–CSD1 module fine-tunes O₂•⁻ levels to control stomatal aperture, thereby enhancing drought tolerance in tomato.