OsCSA Acts as an Upstream Activator of OsCOMT12 to Positively Regulate Copper Stress Tolerance in Rice

Melatonin (MT) is a secondary metabolite that plays a role in environmental stress response in plants. However, the physiological and molecular mechanisms by which MT alleviates excessive copper (Cu) toxicity in rice (Oryza sativa) remain unknown. Herein, exogenous MT application increased root and shoot biomass and inhibited the expression of Cu uptake-associated genes, thereby reducing the Cu uptake capacity of the root and Cu concentration in vivo and effectively enhancing Cu stress tolerance compared with Cu treatment. Transcriptome analysis revealed the essential transcription factor CARBON STARVED ANTHER (OsCSA) as responsive to Cu stress. Compared with the wild type, OsCSA overexpression lines exhibited significantly enhanced Cu tolerance, which was manifested by increased MT content, up-regulated expression of the reactive oxygen species (ROS) scavenging genes CATALASE C and ASCORBATE PEROXIDASE, elevated activities of catalase, superoxide dismutase, and ascorbate peroxidase, and decreased hydrogen peroxide and malondialdehyde content. OsCSA-knockout plants showed the opposite results, and exogenous MT rescued its excessive Cu-sensitive phenotype. Further experiments established that OsCSA directly binds to the GTTA/TAAC element of CAFFEIC ACID O-METHYLTRANSFERASE 12 (OsCOMT12) promoter, up-regulating its expression. Under Cu stress, significant increases in Cu concentration and ROS accumulation were also observed in loss-of-function oscomt12 mutants, consistent with the results observed in OsCSA-knockout lines. Additionally, knockout of OsCOMT12 in the OsCSA-overexpression background abolished the positive effect of OsCSA on Cu stress tolerance. These findings establish the OsCSA-OsCOMT12 regulatory module as a new mechanism for MT-mediated Cu stress response in rice, providing a target for genetic improvement of Cu-tolerant crops.