Panax quinquefolius is a globally valued medicinal plant rich in bioactive flavonoids, yet the molecular mechanisms underlying their biosynthesis remain poorly understood. In this study, we integrated transcriptomic and metabolomic analyses to investigate tissue-specific flavonoid accumulation and regulatory networks in roots, leaves, and flowers. Metabolomic profiling identified 141 flavonoid metabolites, with flavones, flavonols, and C-glycosylflavones predominantly enriched in aerial tissues (leaves and flowers), while specific glycosides like tricin 7-O-acetylglucoside showed root-specific accumulation. Transcriptome sequencing revealed 15,551–18,946 DEGs across tissues, and the reliability of the transcriptomic data was validated by qRT-PCR. KEGG and GO annotation analyses suggested that these DEGs may play a crucial role in the biosynthesis and metabolism of secondary metabolites. From the DEGs, UGTs and MYB TFs were identified and subjected to correlation analysis. Functional validation through in vitro enzymatic assays confirmed that PqUGT71A1 catalyzes apigenin and naringenin glycosylation at the 7-OH position. Additionally, subcellular localization and yeast one-hybrid assays demonstrated that PqMYB7 and PqMYB13 interact with the PqUGT71A1 promoter and activate its expression.. This study unveils the spatial dynamics of flavonoid metabolism in P. quinquefolius and establishes a MYB-UGT regulatory axis, providing critical insights for metabolic engineering and bioactive compound optimization in medicinal plants.

Panax quinquefolius L. is an important medicinal plant, and flavonoids are among its main secondary metabolites. The R2R3-MYB transcription factor plays an irreplaceable role in plant growth, development, and secondary metabolism. In our study, we identified 159 R2R3-MYBs and analyzed their physical and chemical properties in P. quinquefolius. The protein length of 159 PqMYBs varied from 107 to 1050 amino acids. The molecular weight ranged from 12.21 to 116.44 kDa. The isoelectric point was between 4.57 and 10.34. We constructed a phylogenetic tree of P. quinquefolius and Arabidopsis thaliana R2R3-MYB family members, and PqMYB members were divided into 33 subgroups. Transcriptome data analysis showed that the expression patterns of PqMYBs in root, leaf, and flower were significantly different. Following the MeJA treatment of seedlings, five candidate PqMYB genes demonstrated a response. A correlation analysis of PqMYBs and candidate flavonoid pathway genes showed that PqMYB2, PqMYB46, and PqMYB72 had correlation coefficients that were higher than 0.8 with PqCHS, PqANS4, and PqCCoAMT10, respectively. Furthermore, a transient expression assay confirmed that the three PqMYBs were localized in the nucleus. We speculated that these three PqMYBs were related to flavonoid biosynthesis in P. quinquefolius. These results provided a theoretical basis and a new perspective for further understanding the R2R3-MYB gene family and the biosynthesis mechanism of secondary metabolites in P. quinquefolius.

Perilla frutescens (L.) Britt. is a traditional medicinal and culinary plant with a long history of cultivation and significant potential for broader utilization. The basic helix-loop-helix (bHLH) gene family is essential for regulating plant growth, development, stress responses, and secondary metabolism. However, the bHLH gene family in P. frutescens has not yet been characterized. In this study, a total of 205 bHLH genes were identified in P. frutescens through genome mining and analysis. Phylogenetic analysis classified these PfbHLH genes into 23 distinct subfamilies. Promoter analysis revealed an enrichment of cis-acting elements linked to plant hormone signaling and stress responses, suggesting their potential regulatory roles in development, growth, and stress adaptation. Expression profiling based on publicly available RNA-seq data demonstrated tissue-specific expression patterns of PfbHLH genes in roots, stems, and leaves. Four PfbHLH genes (PfbHLH66, PfbHLH45, PfbHLH13, and PfbHLH5) showed significant responses to methyl jasmonate (MeJA) induction. Yeast one-hybrid assays confirmed that these PfbHLH proteins could bind to the cis-acting G-box (CACGTG) element. This study offers new perspectives on the evolution, regulatory mechanisms, and functional roles of the bHLH gene family in P. frutescens. The findings deepen our understanding of the functional diversity within this gene family and establish a foundation for genetic enhancement and the biosynthesis of medicinal compounds in the species.

Lobetyolin is a key bioactive compound in Codonopsis pilosula, but the existing literature has not well explored the regulatory mechanisms governing its biosynthesis. This study performed a genome-wide identification of 175 AP2/ERF transcription factors (TFs), categorizing them into the AP2, ERF, and RAV subfamilies. Promoter analysis revealed numerous hormone- and stress-responsive cis-elements, with a notable prevalence of methyl jasmonate (MeJA)-related motifs. In MeJA-treated hairy root cultures, we observed a significant increase in lobetyolin accumulation. Integrated transcriptomic and metabolomic analyses demonstrated the activation of jasmonate signaling and secondary metabolism pathways. Two MeJA-induced TFs, CpERF140 and CpERF138, were identified as key regulatory candidates. Subsequent experiments confirmed their nuclear localization and specific binding affinity for GCC-box motifs. This study constitutes the inaugural genome-wide characterization of the AP2/ERF family in C. pilosula, employing hairy root culture as an effective platform for functional investigation. Our findings offer novel perspectives on the transcriptional regulation of lobetyolin biosynthesis, providing a foundation for the future metabolic engineering of this medicinal plant.

Phenolic acids are the major bioactive compounds in Perilla frutescens (L.) Britt; however, the regulatory roles of NAC transcription factors (TFs) in their biosynthesis remain unclear. Here, we performed a genome-wide identification and characterization of the NAC family in P. frutescens and explored their involvement in phenolic acid production. A total of 108 PfNAC genes were identified and classified into 17 subfamilies. Expression and promoter analyses suggested potential roles in secondary metabolism. PfNAC29 is located in the plasma membrane and necleus, while PfNAC40 and PfNAC80 are located in the nucleus.Yeast one-hybrid and dual-luciferase assays demonstrated that these TFs bind to the CATGTG motif in the PfC4H promoter and activate its transcription. Overexpression in transgenic hairy roots significantly increased rosmarinic acid, caffeic acid, and ferulic acid accumulation, accompanied by upregulation of key biosynthetic genes. These results indicate that PfNAC29, PfNAC40, and PfNAC80 act as positive regulators of phenolic acid biosynthesis and provide promising targets for metabolic engineering in medicinal plants.