Ethylene response factors (ERFs) are pivotal regulators in mediating plant stress adaptation; however, the roles of osmotic stress-responsive ERFs in tomato remain poorly characterized. Here, we comprehensively investigate the function of SlERF.D2, a member of the ERF transcription factor family, in modulating osmotic stress adaptation. Expression profiling indicated that SlERF.D2 responded to diverse abiotic stimuli, such as drought and salt, as well as ethylene and abscisic acid (ABA). Combined physiological and metabolomic analyses of SlERF.D2 overexpression and knockout lines revealed a negative regulatory role of SlERF.D2 in tomato's osmotic stress adaptation. Biochemical and molecular assays further revealed that SlERF.D2 directly targets the promoter of SlPP2C1, an ABA signaling suppressor, to activate its expression, thereby impairing ABA-dependent stomatal closure and accelerating water loss. Notably, ethylene-induced SlERF.D2 expression required the direct binding of SlEIL1/2/3/4 to the SlERF.D2 promoter. Furthermore, ethylene activated SlPP2C1 transcription in an SlERF.D2-dependent manner through direct transcriptional regulation by SlERF.D2. Thus, the ethylene-SlEIL1/2/3/4-SlERF.D2-SlPP2C1 transcriptional cascade module is involved in the antagonism of ABA-induced stomatal closure. Concurrently, transcriptomic profiling and metabolic analyses further demonstrated that SlERF.D2 repressed the anthocyanin biosynthetic pathway, leading to a reduced anthocyanin content and increased reactive oxygen species (ROS) levels. Our findings delineate a novel regulatory module wherein SlERF.D2 coordinates stomatal closure and ROS homeostasis to modulate the sensitivity of tomato plant to osmotic stresses, providing an applicable target for improving osmotic stress adaptation in tomato.

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