Mechanistic analysis of the coordination between MbEGS4 and MbEOMT in methyleugenol synthesis by Melaleuca bracteata F. Muell

Methyleugenol is widely found in essential oils from aromatic plants, attracting insects for pollination and possessing antimicrobial and antioxidant features. Methyleugenol content in the essential oil from Melaleuca bracteata leaves is 90.46 %, making it an excellent material for assessing methyleugenol’s biosynthetic pathway. Eugenol synthase (EGS) and eugenol-o-methyltransferase (EOMT) represent key genes in methyleugenol’s biosynthetic pathway but their respective functions are unclear. In this study, overexpression and virus-induced gene silencing (VIGS) techniques were applied to investigate the major regulatory gene among MbEGS1, MbEGS2, MbEGS3, and MbEGS4 in the biosynthesis pathway of methyleugenol. We also aimed to examine the coordination mechanism between EGS and EOMT in methyleugenol production by M. bracteata. The data revealed MbEGS4 transcription and methyleugenol content were most significantly increased after overexpression of the MbEGS1–4 gene in M. bracteata and Fragaria vesca. Conversely, MbEGS4 transcription and methyleugenol content were most significantly reduced after MbEGS1–4 gene silencing by VIGS in M. bracteata. These results underscore a major regulatory role for the MbEGS4 gene in the biosynthetic pathway of methyleugenol in M. bracteata. Remarkably, upon overexpression and silencing of MbEOMTs, the transcription levels of MbEGSs were notably upregulated, with MbEGS4 showing the highest increase. Collectively, these findings suggested the presence of a specific transcription factor bound to the promoters of MbEGS and MbEOMT, inducing their expression for the synergistic and efficient production of methyleugenol in M. bracteata.