Abstract: Soybean is a major crop that produces high-quality seed oil for global consumption. However, the regulatory mechanisms underlying seed oil content remain poorly understood. In this study, GmGT-2F, a trihelix transcription factor, was identified as a positive regulator of seed oil content. It was observed that GmGT-2F expression gradually increased during seed development. Furthermore, GmGT-2F overexpression elevated seed oil content and increased the proportion of oleic and linoleic acids in fatty acid composition. However, knocking out GmGT-2F improved seed protein content and seed size. We demonstrated that GmGT-2F binds to the GmAGAL promoter and activates its transcription. Moreover, knockout of GmAGAL and GmGT-2F/GmAGAL reduced α-galactosidase activity and decreased seed oil content. The metabolomic and seed sucrose content analyses of the gmagal and wild-type (WT) plants showed that GmGT-2F affects the transcription of GmAGAL to regulate the activity of α-galactosidase and may control the oil content by influencing the generation and distribution of sucrose in the seed. In addition, GmCYP2 interacts with GmGT-2F, reducing its promoter-binding activity and inhibiting GmAGAL transcription. Haplotype diversity analyses of GmGT-2F, GmCYP2, and GmAGAL revealed combinations associated with increased oil or protein content. This study elucidates the regulatory mechanism by which GmGT-2F regulates seed oil content, expands understanding of trihelix transcription factor function in seed quality trait regulation, and provides new insights for high-quality soybean breeding.

Key words: α-galactosidase, GmAGAL, GmGT-2F, seed oil content, soybean, trihelix transcription factor