Differential Regulation of Pre-Harvest Sprouting by OsERF1 and OsERF94 Through Hormone Signaling and Metabolic Reprogramming in Rice

Pre-harvest sprouting (PHS), the premature germination of grains on the mother plant, causes substantial yield loss and grain-quality deterioration in rice under humid conditions. Although seed dormancy and germination are largely controlled by hormonal balance, the transcriptional mechanisms linking hormone signaling with metabolic adaptation during PHS remain unclear. In this study, we investigated the roles of two ethylene-responsive factor transcription factors, OsERF1 and OsERF94, in rice PHS regulation using CRISPR/Cas9-mediated knockout lines, together with physiological, gene-expression, and metabolite analyses. The oserf1-KO mutant showed reduced seed dormancy and increased germination under PHS-inducing conditions, accompanied by altered expression of abscisic acid- and gibberellin-related genes. In contrast, the oserf94-KO mutant exhibited enhanced dormancy and reduced germination, with decreased expression of hypoxia-responsive fermentation genes and impaired carbohydrate mobilization, as indicated by reduced soluble sugar and ethanol accumulation and increased starch content. These results suggest that OsERF1 contributes primarily to hormone-mediated dormancy maintenance, whereas OsERF94 supports metabolic activation required for germination under high-moisture conditions. Collectively, this study proposes a dual regulatory framework in which hormonal control and hypoxia-associated carbon metabolism coordinately determine rice PHS susceptibility.