Integrative transcriptomic-physiological analysis deciphers nitrogen-mediated carbon reallocation balancing growth and flavonoid metabolism in Epimedium pubescens

Nitrogen availability critically shapes medicinal plant quality by coordinating the "growth-secondary metabolism" trade-off, yet its regulatory mechanisms remain elusive in the non-model species <i>Epimedium pubescens</i>. Through physiological-transcriptomic integration under five nitrogen levels (0, 3.5, 7.5,15, 22.5 mM NO₃ ^-), we demonstrated that moderate nitrogen (MN: 7.5 mM NO₃ ^-) optimally balanced biomass accumulation (22%-53% higher than low nitrogen [LN: 0 mM NO₃ ^-] and high nitrogen [HN: 22.5 mM NO₃ ^-]) with maximal Icariin-type flavonoid production (19%-34% higher than LN/HN). Extreme nitrogen stresses (LN/HN) impaired photosynthetic efficiency (18%-20% reduction), disrupted carbon-nitrogen homeostasis, and restricted flavonoid biosynthesis by hindering carbon reallocation (soluble sugars reduced by 26%-27%, starch by 30%-43%). Time-series transcriptomics revealed distinct response dynamics: LN triggered active transcriptional reprogramming at mid-stage (36 days after treatment, DAT), whereas HN responses were delayed to late-stage (48 DAT). Weighted gene co-expression network analysis (WGCNA) identified the grey60 module as a hub coordinating carbon-nitrogen metabolism and mRNA processing. A tripartite regulatory network linking nitrogen-responsive genes (e.g., <i>EpF3H</i>, <i>UGT</i>), Icariin-type flavonoid/carbon metabolism (e.g., icariin, soluble sugars), and growth phenotypes (e.g., biomass, photosynthesis) elucidated how nitrogen optimizes the trade-off between medicinal quality and yield in <i>E. pubescens</i>. This study provides molecular targets for precision nitrogen management to enhance both medicinal quality and yield, while establishing an integrative framework combining physiological and transcriptomic analyses to investigate metabolic trade-offs in non-model plants.