The ArMAPK5-ArbHLH3-Ar4CL3 module is associated with salicylic acid-induced phenolic biosynthesis in Agastache rugosa

Agastache rugosa is a medicinal and aromatic plant that accumulates phenolic compounds with important pharmacological and antioxidant activities. However, the physiological and molecular mechanisms underlying elicitor-induced phenolic accumulation remain insufficiently understood. In this study, an Agrobacterium rhizogenes C58C1-mediated hairy root system was established in A. rugosa and used to investigate the effects of salicylic acid (SA, 100 μM) on phenolic metabolism. Integrated HPLC analysis, enzyme activity assays, transcriptome profiling, and functional validation showed that SA markedly promoted phenolic accumulation in hairy roots. After 36 h of SA treatment, rosmarinic acid content reached 3.44 mg/g DW, accompanied by increases in caffeic acid, protocatechuicaldehyde, and chlorogenic acid contents. SA treatment also enhanced peroxidase activity, which increased to 4.94-fold that of the control, indicating an active physiological response associated with phenolic metabolism. RNA-seq analysis identified 13,356 differentially expressed genes, with significant enrichment in phenylpropanoid biosynthesis, plant hormone signal transduction, and MAPK signaling pathways. Further functional analyses revealed that ArMAPK5 interacts with ArbHLH3, while ArbHLH3 directly activates Ar4CL3 expression by binding to the G-box motif (CACGTG) in its promoter. Together, these results suggest that the ArMAPK5-ArbHLH3-Ar4CL3 module contributes to SA-induced activation of phenylpropanoid metabolism in A. rugosa hairy roots. This study provides new insight into the physiological and transcriptional regulation of phenolic metabolism in medicinal plants.