Distinct Nitrogen Forms Shape Flavonoid Biosynthesis and Gene–Metabolite Networks in Erigeron breviscapus

Nitrogen (N) sources critically influence both agronomic performance and secondary metabolism in medicinal plants. Understanding how different forms of nitrogen affect plant growth and the biosynthesis of valuable secondary metabolites is essential for optimizing cultivation practices and enhancing crop medicinal quality. In this study, Erigeron breviscapus (Vant.) Hand.–Mazz., a medicinal herb renowned in traditional Chinese medicine for its bioactive flavonoids such as scutellarin with neuroprotective and cardiovascular therapeutic effects, was cultivated under various N treatments—nitrate (NO3−–N), ammonium (NH4+–N), and urea [CO(NH2)2]—and compared to an N-free control. All N treatments significantly enhanced plant height, leaf area, biomass, and overall yield, with nitrate-N providing the most pronounced growth benefits. Metabolomic profiling identified 387 known metabolites, primarily flavonoids, exhibiting distinct accumulation patterns under each N form. Transcriptomic analyses revealed substantial differences in gene expression, with nitrate-N inducing the greatest number of differentially expressed genes (DEGs). Integration of metabolomic and transcriptomic data uncovered co-expression modules linking candidate regulatory genes, such as cytochrome P450s, MYB transcription factors, and glycosyltransferases, to specific flavonoids, including quercetin-3-O-glucoside and apigenin. These findings elucidate how different N sources modulate flavonoid biosynthesis in E. breviscapus, revealing molecular mechanisms underlying N-mediated flavonoid biosynthesis, which can contribute to optimized fertilizer strategies. This research enhances both the medicinal quality and yield of this important medicinal plant by revealing key gene–metabolite networks, thereby offering valuable insights for metabolic engineering and sustainable cultivation practices.