GBF family member PfGBF3 and NAC family member PfNAC2 regulate rosmarinic acid biosynthesis under high light.

Rosmarinic acid (RA) is an important medicinal metabolite and a potent food antioxidant. We discovered that exposure to high light intensifies the accumulation of RA in the leaves of perilla (Perilla frutescens (L.) Britt). However, the molecular mechanism underlying RA synthesis in response to high light stress remains poorly understood. To address this knowledge gap, we conducted a comprehensive analysis employing transcriptomic sequencing, transcriptional activation, and genetic transformation techniques. High light treatment for 1 and 48 h resulted in the upregulation of 592 and 1,060 genes, respectively. Among these genes, three structural genes and 93 transcription factors exhibited co-expression. Notably, NAC family member PfNAC2, GBF family member PfGBF3, and cinnamate-4-hydroxylase gene PfC4H demonstrated significant co-expression and upregulation under high light stress. Transcriptional activation analysis revealed that PfGBF3 binds to and activates the PfNAC2 promoter. Additionally, both PfNAC2 and PfGBF3 bind to the PfC4H promoter, thereby positively regulating PfC4H expression. Transient overexpression of PfNAC2, PfGBF3, and PfC4H, as well as stable transgenic expression of PfNAC2, led to a substantial increase in RA accumulation in perilla. Consequently, PfGBF3 acts as a photosensitive factor that positively regulates PfNAC2 and PfC4H, while PfNAC2 also regulates PfC4H to promote RA accumulation under high light stress. The elucidation of the regulatory mechanism governing RA accumulation in perilla under high light conditions provides a foundation for developing a high-yield RA system and a model to understand light-induced metabolic accumulation.

Integrated analysis of miRNA, transcriptome, and degradome sequencing provides new insights into lipid metabolism in perilla seed.

MicroRNAs (miRNA) are small noncoding RNAs that play a crucial as molecular regulators in lipid metabolism in various oil crops. Perilla (Perilla frutescens) is a specific oil crop known for its high alpha-linolenic acid (C18:3n3, ALA) content (>65 %) in their seed oils. In view of the regulatory mechanism of miRNAs in perilla remains unclear, we conducted miRNAs and transcriptome sequencing in two cultivars with distinct lipid compositions. A total of 525 unique miRNAs, including 142 differentially expressed miRNAs was identified in perilla seeds. The 318 miRNAs targeted 7,761 genes. Furthermore, we identified 112 regulated miRNAs and their 610 target genes involved in lipid metabolism. MiR159b and miR167a as the core nodes to regulate the expression of genes in oil biosynthesis (e.g., KAS, FATB, GPAT, FAD, DGK, LPAAT) and key regulatory TFs (e.g., MYB, ARF, DOF, SPL, NAC, TCP, and bHLH). The 1,219 miRNA-mRNA regulation modules were confirmed through degradome sequencing. Notably, pf-miR159b-MYBs and pf-miR167a-ARFs regulation modules were confirmed. They exhibited significantly different expression levels in two cultivars and believed to play important roles in oil biosynthesis in perilla seeds. This provides valuable insights into the functional analysis of miRNA-regulated lipid metabolism in perilla seeds.

Integrated metabolomic and transcriptomic analyses reveal molecular response of anthocyanins biosynthesis in perilla to light intensity.

The perilla anthocyanins have important medicinal and ornamental value, and their contents are significantly affected by light intensity. In view of their molecular mechanisms were not well understood, we integrated the metabolomic and transcriptomic analyses of the light-sensitive perilla variety under different light intensity. The perilla leave color were obviously affected under different treatments. Totally 140 flavonoid metabolites and 2461 genes showed steady change, among which 60 flavonoid metabolites were increased accumulation and 983 genes were upregulated expression under elevated light intensity treatment. Light treatment prominently affected the expression of genes involved in the main anthocyanin metabolites accumulation in perilla leaves. Using WGCNA analysis, we identified 4 key genes in anthocyanin biosynthesis pathway (CHI, DFR, and ANS) and 147 transcription factors (MYB, bHLH, bZIP, ERF, and NAC) involved in malonylshisonin biosynthesis. Among them, 6 MYBs and 4 bZIPs were predicted to play important roles in light regulation of malonylshisonin biosynthesis based on phylogenetic construction, correlation analysis, cis-acting element identification and qPCR verification. The identified key genes and regulatory factors will help us to understand the potential mechanism of photo-regulated anthocyanin accumulation in perilla.