Integrated transcriptomic and metabolomic analysis unveils biosynthetic mechanisms of key bioactive isoflavonoid accumulation in Astragalus mongholicus under different light treatments

Abstract Astragali Radix, the dried root of Astragalus mongholicus, is a prominent traditional Chinese medicinal material, with isoflavonoids serving as its primary bioactive components. Light serves as a pivotal environmental cue modulating isoflavonoid biosynthesis, its regulatory influence on A. mongholicus remains poorly understood, and the underlying biosynthetic pathways have yet to be fully elucidated. This study investigated the impacts of different light on isoflavonoid accumulation through integrated transcriptome and metabolome analyses. Compared to white light, red and blue light treatments significantly altered the expression of 93 genes within the isoflavonoid biosynthetic pathway, 52 of which were up-regulated. Metabolomic analysis revealed 35 differential isoflavonoid metabolites, with 23 compounds, including the primary medicinal components calycosin (CA) and formononetin (FO), showing increased accumulation. Notably, blue light exhibited a significantly stronger promoting effect on CA and FO accumulation than red light. Through integrated multi-omics analysis, we revealed the molecular network underlying this light quality–specific regulation. Correlation analysis revealed that multiple structural genes were significantly positively correlated with these key active components, among which AmCHR, AmCHS, AmCHI, and AmIFS were identified as core regulatory genes. The biochemical roles of candidate genes AmCHR, AmCHS, AmCHI, and AmIFS were validated via in vitro enzymatic assays. Furthermore, utilizing a newly established nonsterile hairy root transformation system, functional validation through overexpression and RNA interference experiments demonstrated that these candidate genes positively regulate the accumulation of key isoflavonoids, including CA, calycosin-7-O-β-D-glucoside, FO, and ononin. These findings were further corroborated by transient gene silencing using antisense oligodeoxynucleotides. This study elucidates the regulatory network of light-induced isoflavonoid biosynthesis in A. mongholicus and provides essential genetic resources for the metabolic engineering and sustainable production of these medicinal compounds.