Network-based identification of hub transcription factors associated with benzylisoquinoline alkaloid biosynthesis in Papaver somniferum

Papaver somniferum L. (Opium poppy) is one of the world's most economically valuable medicinal plants, and it is the industrial source of essential compounds such as painkillers and other pharmecutical drugs. Recently, large amounts of opium poppy transcriptome data have become accessible in public databases, consisting of data on the different tissues and ecotypes. Despite the importance of this plant, there is little information about the regulatory mechanisms involved in secondary metabolites, especially in benzylisoquinoline alkaloids (BIAs) biosynthesis at the omics level in opium poppy. Herein, we employed co-expression and co-regulation network analysis using Weighted Gene Co-expression Network Analysis (WGCNA) to infer and reveal gene interactions in opium poppy by using RNA-Seq data. To validate possible hub transcription factors (TFs), partial least squares regression (PLS) and Receiver operating characteristic (ROC) analyses were conducted. We identified nine significant co-regulated modules (comprising1501 genes) and hub TF genes related to the biosynthesis of BIAs, including WRKY3, WRKY32, MYB3R-5, bZIP, APRR2, MYB43, MYB82, bHLH, and WRKY40. The results suggest that these hub genes can play a vital role in co-regulating genes involved in secondary metabolic pathways in opium poppy. Also, we detected common regulatory motifs related to hub TFs (WRKY and MYB) of important co-regulated BIA modules. We implied their common regulatory role in the biosynthesis of secondary metabolites in the opium poppy. The results illustrated that co-expressed genes of the modules share common regulatory motifs, especially related to hub TFs of each module, and that they may define their common regulation. ROC analysis with high diagnostic value (AUC = 1) identified the possible role of the hub TF involved in the BIAs pathways. PLS analysis showed a considerable effect of hub TFs (WRKY and bZIP) on the related genes. Our WGCNA analysis at the omics level, along with identifying hub TFs, highlights the regulatory potential of these genes and the primary molecular mechanisms involved in the BIA biosynthetic pathway in opium poppy. These findings provide valuable insights for the regulon engineering of candidate hub TFs in expression systems.