The plasma membrane H⁺-ATPase OSA2 negatively regulates salt tolerance in rice seedlings

Plasma membrane (PM) H⁺-ATPases are pivotal for plant adaptation to abiotic stresses; however, the specific function of the rice (Oryza sativa L.) PM H⁺-ATPase isoform OSA2 in salt stress responses remains elusive. In this study, using the Zhonghua 11 (ZH11) cultivar, we created an OSA2 knockout mutant (osa2-9) via CRISPR/Cas9 technology and generated OSA2 overexpression lines (OSA2OE). Subcellular localization assays confirmed that OSA2 protein is specifically localized to the cell plasma membrane. Physiological and biochemical analyses under 150 mM NaCl revealed that the survival rate of osa2-9 was significantly higher than that of OSA2OE, albeit slightly lower than the wild type (WT). Salt stress induced a marked elevation in catalase (CAT) and peroxidase (POD) activities in WT plants, effectively mitigating malondialdehyde (MDA) accumulation. Conversely, OSA2OE plants exhibited suppressed CAT activity, excessive MDA accumulation, and exacerbated oxidative damage. Furthermore, quantitative real-time PCR (qRT-PCR) demonstrated that OSA2 overexpression significantly repressed the transcription of salt tolerance- and reactive oxygen species (ROS) metabolism-related genes, such as OsHKT1 and OsodCc1, in the roots. Collectively, our findings identify OSA2 as a negative regulator that compromises salt tolerance in rice seedlings, likely by perturbing PM H⁺ transport and disrupting antioxidant homeostasis. This study provides a theoretical framework for elucidating the molecular mechanisms underlying rice salt tolerance and for the breeding of salt-resilient cultivars.