Identification of key MYB transcription factors mediating MeJA-induced eupatilin accumulation by regulating AYFOMT2 expression in Artemisia argyi

Methoxylated flavones in Artemisia argyi, especially eupatilin, are important bioactive components with antibacterial, anti-inflammatory, and anti-tumor activities. Exogenous treatment with methyl jasmonate (MeJA) significantly promoted the contents of methoxylated flavones in A. argyi. However, the molecular regulatory mechanism underlying MeJA-induced methoxylated flavone biosynthesis remains largely unclear in A. argyi. In this study, transcriptional analysis following MeJA treatment revealed that key genes involved in the flavonoid biosynthetic pathway, including PAL, CHI, and CHS, as well as the critical flavonoid O-methyltransferase gene AYFOMT2, were significantly induced. Meanwhile, the MYB transcription factor family exhibited the most pronounced response to MeJA induction. Based on expression profiling, three key candidate genes, AYMYB13, AYMYB15, and AYMYB16, were screened and identified. To further characterize their regulatory mechanism in methoxylated flavones biosynthesis, yeast one-hybrid and dual-luciferase assays confirmed that AYMYB13, AYMYB15, and AYMYB16 directly bind to the promoter of AYFOMT2, thereby promoting the accumulation of eupatilin. Futhermore, overexpression of these three MYB transcription factors in A. argyi calli significantly increased the content of eupatilin. Our study demonstrated that AYMYB13, AYMYB15, and AYMYB16 were key regulators for activating the expression of AYFOMT2 in MeJA-induced methoxylated flavones accumulation. These findings will provide a theoretical foundation for improving eupatilin content and biosynthetic efficiency in A. argyi.