Abstract
Astragalus membranaceus is a well-known medicinal plant rich in bioactive compounds, particularly the isoflavonoid calycosin, which exhibits diverse pharmacological properties. However, there is limited research on the influence of light conditions on calycosin biosynthesis and the underlying molecular mechanisms. In this study, we exposed in vitro plantlets of A. membranaceus to different LED light sources (white, red, and blue) and found that red light treatment significantly enhanced both biomass accumulation and calycosin content. Through transcriptome analysis combined with real-time PCR we identified several key genes involved in calycosin biosynthesis, among which AmI3'H showed the most significant upregulation under red light. Structural analysis and molecular docking indicated that AmI3'H, a member of the CYP81 clan of cytochrome P450 enzymes, interacts with formononetin, a precursor of calycosin. To verify its functional role, transient overexpression of AmI3'H was performed in Nicotiana benthamiana using Agrobacterium-mediated infiltration along with exogenous formononetin. The co-infiltration resulted in calycosin production exclusively at 24 h post-infiltration, accompanied by elevated antioxidant activity and total flavonoid content. Additionally, transcriptome-based prediction identified AmbHLH30 as a potential transcription factor (TF) regulating calycosin biosynthesis, showing the highest expression under red light conditions. These results suggest that red light not only promotes calycosin accumulation but also modulates the expression of biosynthetic genes and transcriptional regulators, providing valuable insights for the metabolic engineering of isoflavonoid biosynthesis in A. membranaceus.