Transcriptome Responses to Different Nitrogen Levels Reveals Regulatory Networks for Icariin-Flavonoid Metabolism in Epimedium pubescens

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. Overall design: These seedlings were transplanted into cultivation pots filled with a substrate mixture of river sand and perlite (3:2), and then cultured in a greenhouse. The light intensity was maintained at 2000 luX for 14 hours per day, and the temperature was controlled at 22 ± 2°C. After 20 days of irrigation with 12.5% Hoagland nutrient solution for seedling recovery, seedlings with newly emerged roots and having resumed normal growth were subjected to different nitrogen levels: LN (nitrogen deficiency treatment, 0 mmol/L NO3-), MN (moderate nitrogen treatment, 7.5 mmol/L NO3-), and HN (excessive nitrogen treatment, 22.5 mmol/L NO3-).The microelements H3BO3, MnCl2, ZnSO4, CuSO4, and H2MoO4 were added at concentrations of 2.86, 1.81, 0.22, 0.08, and 0.02 g/L, respectively, while Fe-EDTA was added at a concentration of 0.02 mM. The pH of the nutrient solution was adjusted to 6.5, and the seedlings were irrigated with the nutrient solution every two weeks. Each treatment involved 36 seedlings. On the 24d (day), 36d, and 48d of the experiment, fresh leaves from plants under different treatments were collected, rapidly frozen in liquid nitrogen, and then stored at -80°C for later measurement. On the 48d, plants in the LN (nitrogen deficiency) and HN (excessive nitrogen) treatment groups showed obvious symptoms of damage, and subsequently, all treated plants were measured and harvested.