Transcriptome–Metabolome Integration Reveals Key Regulators of Isoflavone Biosynthesis in Soybean for Enhanced Nutritional Quality

Isoflavones are flavonoids with antioxidant and health-promoting properties; however, the genetic basis underlying their accumulation remains unclear. Here, we integrate metabolomic and transcriptomic analyses to investigate isoflavone biosynthesis in two sister soybean lines differing in seed isoflavone content. UPLC-MS/MS reveals that GD10 consistently accumulates higher levels of isoflavone glycosides during late seed maturation. Transcriptomic profiling shows that isoflavone 7-O-glucosyltransferase ( IF7GT3 ) and chalcone isomerase ( CHI4 ) are upregulated in GD10, whereas GD12 exhibits higher expression of flavonol synthase ( FLS1 ) and flavonoid 3′-monooxygenase ( CYP75B1 ), redirecting flux toward competing flavonoid branches. Co-expression network analysis identified 20 transcription factors potentially involved in isoflavone regulation, of which colocalize with previously identified flavonoid QTLs. Hairy-root assays demonstrate that IF7GT3 promotes daidzin accumulation, whereas Y1H and dual-luciferase assays demonstrate that TF_NAC_2 directly binds to the IF7GT3 promoter and activates its expression. These findings provide molecular targets for breeding soybean cultivars with enhanced isoflavone content.