Elucidating the molecular biosynthetic mechanism of phenylethanoid glycosides in two new varieties of Forsythiae Fructus via a multi-omics analysis combined with WGCNA

Forsythiae Fructus, the fruit of Forsythia suspensa (Thunb. ) Vahl, is a traditional Chinese medicine with broad-spectrum pharmacological properties. However, the highly variable quality of Forsythiae Fructus in the market significantly hinders industry development. There is an urgent need to elucidate the quality formation mechanisms of high-quality varieties. Based on the "Trait-Component-Activity" comprehensive evaluation system, we first conducted a comparative analysis of quality differences between high-quality cultivars (JQ-4 and JQ-5) and conventional cultivar. The anti-inflammatory activities of JQ-4 and JQ-5 were proved to be stronger than conventional cultivar using an LPS-induced RAW264. 7 cell inflammatory mode. Then, transcriptomic and metabolomic profiling combined with WGCNA analysis was performed to preliminarily explore the molecular mechanisms underlying high-quality varieties. JQ-4 and JQ-5 exhibited significantly higher concentrations of forsythoside A, poliumoside, and salidroside. The biosynthesis pathway of PhGs exhibited distinct regulatory profiles, as evidenced by the analysis of gene expression and metabolite abundance. WGCNA identified pal, 4cl, hct, cse, and aadc/tydc as key structural genes involved in PhGs accumulation, while WRKY family transcription factors potentially play regulatory roles in PhGs biosynthesis. PCR validation revealed pal (Fsu₀21970), 4cl (Fsu₀20387 and Fsu₀04861), hct (Fsu₀03224 and Fsu₀23483), cse (Fsu₀13786), and aadc/tydc (Fsu₀00625) play a critical role in quality formation. Overall, this study elucidated the molecular mechanisms underlying the high-quality formation and provided novel insights into the biosynthesis of PhGs and the development of superior genetic resources of Forsythia suspensa cultivars. • A “Trait-Component-Activity” system supports quality evaluation and breeding of Forsythia suspensa. • Enhanced anti-inflammatory activity and higher Phenylpropanoid glycosides (PhGs) in new Forsythia suspensa cultivars. • Multi-omics strategy combined with WGCNA identified key genes and WRKY TFs in PhGs biosynthesis. • WRKY TFs (e. g. , WRKY65/75) directly regulate PhGs-related genes. • Fruit width and epicarp spots correlate with high PhGs, aiding selection.