Time-series transcriptome provides insights into the gene regulation network involved in the icariin-flavonoid metabolism during the leaf development of Epimedium pubescens

Herba Epimedii (Epimedium) leaves are rich in prenylated flavonol glycosides (PFGs) with high medicinal value. However, the dynamics and regulatory network of PFG biosynthesis remain largely unclear. Here, we combined metabolite profiling (targeted to PFGs) and a high-temporal-resolution transcriptome to elucidate PFGs' regulatory network in Epimedium pubescens and identified key candidate structural genes and transcription factors (TFs) involved in PFG accumulation. Chemical profile analysis revealed that PFG content was quite different between buds and leaves and displayed a continuous decline with leaf development. The structural genes are the determinant reasons, and they are strictly regulated by TFs under temporal cues. We further constructed seven time-ordered gene co-expression networks (TO-GCNs) of PFG biosynthesis genes (including EpPAL2, EpC4H, EpCHS2, EpCHI2, EpF3H, EpFLS3, and EpPT8), and three flavonol biosynthesis routines were then predicted. The TFs involved in TO-GCNs were further confirmed by WGCNA analysis. Fourteen hub genes, comprising 5 MYBs, 1 bHLH, 1 WD40, 2 bZIPs, 1 BES1, 1 C2H2, 1 Trihelix, 1 HD-ZIP, and 1 GATA were identified as candidate key TFs. The results were further validated by TF binding site (TFBS) analysis and qRT-PCR. Overall, these findings provide valuable information for understanding the molecular regulatory mechanism of PFGs biosynthesis, enriching the gene resources, which will guide further research on PFG accumulation in Epimedium.