Nucleobase-ascorbate transporter OsNAT9 regulates seed vigor and drought tolerance by modulating ascorbic acid homeostasis in rice.

Drought and seed aging severely impact crop yield and seed vigor, respectively. Here, we identified the rice protein OsNAT9, a nucleobase-ascorbate transporter, as being crucial for seed vigor and drought tolerance. Knockout of OsNAT9 resulted in a significant reduction in seed vigor; however, the application of exogenous ascorbic acid (AsA) and the breaking of seed dormancy restored this phenotype, suggesting that OsNAT9 regulates seed vigor by modulating seed dormancy. Furthermore, the Osnat9 mutants exhibited decreased AsA concentration in the endosperm, impairing the scavenging of reactive oxygen species (ROS) in aged seeds, which disrupted starch structure and seed vigor. During the aging process, both the knockout and overexpression of OsNAT9 affected AsA efflux, disrupting the redox homeostasis of AsA pools, increasing ROS accumulation, and ultimately reducing embryo vigor. In addition, the Osnat9 mutants displayed reduced drought tolerance, accompanied by decreased AsA concentration and increased ROS accumulation, whereas OsNAT9-overexpressed lines showed the opposite phenotypes. The OsNAT9 protein exhibited either a uniform or punctate distribution on the cytomembrane. Protoplast secretion assays and microscale thermophoresis experiments further confirmed that OsNAT9 functions as a cytomembrane-localized efflux transporter responsible for AsA secretion. This study highlights the dual role of OsNAT9 in regulating seed vigor and drought tolerance by maintaining the homeostasis of AsA pools and reducing ROS accumulation. These findings provide novel insights into AsA efflux transport and its implications for seed vigor and stress adaptation. Furthermore, this study identifies OsNAT9 as a potential target for enhancing crop stress tolerance and seed longevity.