Elucidating the Mechanisms Underlying Differential Anthocyanin Biosynthesis and Its Link to Stem Color and Root Isoflavonoid Levels in <i>Astragalus membranaceus</i> var. <i>mongholicus</i>

Abstract Astragalus membranaceus var. mongholicus (AMM) is the principal botanical source of Huangqi, a traditional medicinal herb whose therapeutic value primarily stems from the accumulation of isoflavones and other bioactive compounds in the roots. In this study, field surveys across major AMM production regions revealed pronounced natural variation in stem coloration. Chemical analysis showed that the roots of the red-stemmed type contained significantly higher levels of four bioactive isoflavones and volatile organic compounds than those in green-stemmed plants. Metabolomic profiling further revealed a specific enrichment of cyanidin-based anthocyanins in the red stems, establishing the metabolic basis of the red stem phenotype. Both transcriptomic and metabolomic analyses indicated an overall upregulation of the flavonoid and phenylpropanoid biosynthetic pathways in the stem and root tissues of red-stemmed AMM. Weighted gene co-expression network analysis (WGCNA) identified six key genes (AmC4H, AmCHS, AmCHI, AmF3H, AmF3′H, and AmBZ1) that were strongly associated with the red stem phenotype, all of which were specifically highly expressed in red stems. Functional assays confirmed their roles in anthocyanin biosynthesis. Molecular modeling provided further insights into the substrate specificity of AmBZ1. This study proposes stem color as a visible phenotypic reference for early-stage germplasm selection in AMM, and characterizes the molecular basis underlying red stem formation, providing a foundation for elite germplasm development and molecular breeding.