ABSTRACT: Nitrogen is a crucial nutrient element for plant growth and productivity, with both excess and deficiency in nitrogen fertilizer application posing adverse effects on plants and the environment. The internal mechanisms by which the medicinal plant Epimedium pubescens (E. pubescens) adapts to varying nitrogen levels remain unclear. This study employed one-year-old E. pubescens as the experimental material to systematically analyze the changes in plant growth traits, carbon metabolites, and Icariin-Flavonoids content under different exogenous nitrogen levels. Furthermore, it examined the transcriptional changes in gene expression within E. pubescens in response to varying nitrogen levels. The results showed that under moderate nitrogen levels (7.5 mmol/L NO3-), E. pubescens exhibited increased biomass accumulation and flavonoid synthesis. However, deficient or excessive nitrogen levels (0, 22.5 mmol/L NO3-) significantly inhibited photosynthesis in E. pubescens, reducing the content of starch, soluble sugars, and Icariin-Flavonoids, leading to decreased biomass and accompanied by changes in leaf color (pale green or browning). Transcriptome analysis revealed the underlying molecular mechanisms of these changes in plants regulated by different nitrogen levels. Nitrogen deficiency and excess triggered distinct transcriptional response patterns, with the number of differentially expressed genes (DEGs) peaking at S2 (36 days) under nitrogen deficiency and significantly declining at S3 (48 days), while the number of DEGs under excess nitrogen continuously increased over time from S1 to S3 (12-48 days). Both conditions significantly affected the expression of genes related to carbon and nitrogen metabolism, flavonoid synthesis, and stress response. Based on the correlation analysis of expression levels of genes related to these pathways, growth traits, and metabolite content indicators, we constructed regulatory network diagrams for carbon-nitrogen metabolism and Icariin-flavonoid metabolism-related genes. Furthermore, we identified hub genes that may be involved in regulating Icariin-flavonoid metabolism in response to nitrogen levels, such as UGT (Ebr06G044290), EpF3H (Ebr04G062950), EpCHS5 (Ebr03G073940) (Fig. 9A), UGT (Ebr06G044660), and EpUGT13_A (Ebr06G044210). Additionally, among the DEGs obtained at the S3 stage, we discovered the transcription factors MYB1_CROXC (Ebr04G001770) and MYB12_ARATH (Ebr01G065030). We constructed their associated gene networks under different nitrogen levels, which may primarily regulate the expression of genes like UGT, CHS, and F3H involved in the flavonoid synthesis stage of E. pubescens under varying nitrogen conditions. In summary, this study has revealed the growth performance and the variation patterns of Icariin-Flavonoid metabolism in E. pubescens in response to different exogenous nitrogen levels, as well as their complex underlying mechanisms. Additionally, key genes involved in regulating the synthesis of flavonol glycosides in E. pubescens by nitrogen levels have been identified. This provides an important basis for a deeper understanding of the mechanisms of nitrogen regulation on the growth and secondary metabolism of medicinal plants. Furthermore, it offers theoretical support and potential genetic resources for the efficient utilization of nitrogen fertilizers in E. pubescens cultivation and the breeding of high-nitrogen-use-efficiency varieties.
