ABA is involved in OsDSR3-mediated regulation of alkali tolerance in rice

Alkali stress is one of the most damaging abiotic stresses that affect rice growth and yield. The Domain of unknown function (DUF) protein family and abscisic acid (ABA) are critical for abiotic stress tolerance in plants. We previously identified OsDSR3, a novel stress-responsive gene from the DUF966 family that positively regulates rice tolerance to alkali stress. However, it remains unclear whether OsDSR3 relies on the ABA signaling to modulate the molecular mechanisms underlying rice alkali resistance. Using RNA sequencing and RT-qPCR, we found differential expression of ABA-related genes in OsDSR3 overexpression lines under alkaline stress. Further analysis revealed that OsDSR3 overexpression lines exhibited increased sensitivity to ABA treatment, whereas osdsr3 mutants exhibited the opposite phenotype. Consistent with these findings, the ABA content was significantly higher in OsDSR3 overexpression lines and lower in osdsr3 mutants compared to wild type. In addition, the exogenous ABA application enhanced the alkali tolerance of OsDSR3 overexpression lines. This enhancement was attributed to the increased activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), elevated levels of osmotic regulating substances (proline, soluble proteins), reduced levels of reactive oxygen species (ROS including superoxide anion and hydrogen peroxide), maintenance of membrane integrity, accumulation of endogenous ABA levels, and activation of gene expression related to the ABA signaling. These effects were significantly more pronounced in OsDSR3 overexpression lines than in the osdsr3 mutant. Furthermore, using a yeast two-hybrid assay, we demonstrated that OsDSR3 interacts with OsMT-3a. This interaction enhances ROS scavenging via the ABA pathway, thereby positively regulating alkaline tolerance in rice. This study provides deeper insight into the mechanism of OsDSR3 regulating alkali stress.