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.