OsSPL4 negatively regulates drought tolerance in rice by directly activating OsSTPK

Drought stress is a major constraint on global rice ( Oryza sativa L.) production. The SQUAMOSA promoter-binding protein-like (SPL) family of transcription factors regulates diverse aspects of plant development and stress responses; however, the roles of many SPL members, including OsSPL4, remain poorly understood in rice. Here, we characterized the function of OsSPL4 in drought tolerance using CRISPR-Cas9 knockout mutants and overexpression lines in the japonica variety ZH11. Under drought stress, knockout mutants exhibited significantly enhanced tolerance compared with wild-type plants, with higher survival rates (approximately 46–53%), greater biomass accumulation, reduced malondialdehyde (MDA) content, and increased activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). In contrast, overexpression lines showed heightened sensitivity to drought stress, displaying severe wilting and exacerbated oxidative damage. OsSPL4 is predominantly expressed in leaves and is rapidly downregulated upon drought exposure. Subcellular localization and transcriptional activation assays revealed that OsSPL4 is a nuclear-localized protein with strong transcriptional activation activity. Mechanistically, we demonstrated that OsSPL4 directly binds to the promoter of OsSTPK , which encodes a serine/threonine protein kinase implicated in abiotic stress signaling, and positively regulates its transcription. Notably, the expression pattern of OsSTPK closely parallels that of OsSPL4 . Collectively, our findings establish that OsSPL4 functions as a negative regulator of drought tolerance by directly activating OsSTPK transcription and modulating reactive oxygen species (ROS) scavenging capacity. This study uncovers a novel OsSPL4–OsSTPK regulatory module in the drought stress response network and identifies OsSPL4 as a promising genetic target for enhancing drought resistance in rice through genome editing.