Integrated metabolome and transcriptome analyses reveal the seasonal dynamic accumulation and regulation network of benzylisoquinoline alkaloids in Stephania tetrandra roots

Stephania tetrandra is a medicinal plant that contains four main types of benzylisoquinoline alkaloids (BIAs): mono-BIAs, bis-BIAs, aporphine-type BIAs (APT-BIA), and tetrahydroprotoberberines (THBs). S. tetrandra is particularly rich in the bis-BIAs, tetrandrine and fangchinoline, which are critical quality markers and major components of the traditional Chinese medicine “Fangji”. However, changes in their BIA profiles and associated metabolic regulatory networks over time remain unclear. In this study, metabolomics and transcriptomics were integrated to investigate the dynamic accumulation patterns and underlying mechanisms of these alkaloids across three developmental stages: Autumnal Equinox (AuEq), Winter Solstice (WinSo), and Vernal Equinox (VeEq). Quantification revealed that both alkaloids reached their highest concentrations at VeEq, with tetrandrine levels more than doubling from WinSo. Untargeted metabolomics identified 61 differentially accumulated BIAs, displaying stage-specific accumulation patterns. Hierarchical clustering grouped these BIAs into four structural categories, predominantly enriched at VeEq. Transcriptome profiling showed significant upregulation of genes involved in isoquinoline alkaloid biosynthesis during VeEq, especially key enzymes such as TyrAT, TYDC, 4HPPDC, and NCS. Co-expression and protein interaction network analyses further identified abscisic acid (ABA) signaling pathways as major hormonal regulators. Notably, ABA levels and exogenous ABA application confirmed its pivotal role in promoting bis-BIA biosynthesis, acting in concert with reactive oxygen species (ROS) to fine-tune alkaloid production. Collectively, this study establishes a hormone–ROS–BIA regulatory framework that explains seasonal fluctuations in alkaloid content and provides new entry points for improving the medicinal quality and cultivation strategies of S. tetrandra .