Identification of key O-methyltransferases involved in phytoalexin scoparone biosynthesis in Euphorbia lathyris

Euphorbia lathyris , a medicinal and bioenergy plant, suffers significant post-harvest losses due to fruit decay, which reduces biomass yield. Scoparone, a coumarin-type phytoalexin, is widely recognized as an indicator of increased resistance to post-harvest decay in plants. Scoparone acts as both a phytoalexin and a therapeutic compound for liver disorders. However, the O -methyltransferase from the E. lathyris (ElCOMT ) gene family, key enzymes responsible for catalyzing the final methylation step in scoparone formation, remain poorly characterized. In this study, 34 ElCOMT genes were identified and classified into two groups: Type I and Type II. Chromosomal localization analysis revealed a non-uniform distribution of ElCOMTs across the E. lathyris genome. Tissue-specific expression profiling and metabolomic analysis in E. lathyris indicated that ElCOMT33 and ElCOMT34 are potentially involved in scoparone biosynthesis. In vitro enzyme activity assays demonstrated that ElCOMT33 functions as a multifunctional enzyme capable of catalyzing two successive O -methylation reactions, converting esculetin to scoparone via scopoletin as an intermediate. ElCOMT34 exhibits a more limited substrate specificity, mediating only the O -methylation of esculetin to yield isoscopoletin. Furthermore, ElCOMT33 demonstrated broad catalytic activity, effectively methylating coumarins, phenylpropanes, and flavonoids. Transgenic assays confirmed that overexpression of ElCOMT33 and ElCOMT34 increased scopoletin and isoscopoletin biosynthesis in E. lathyris hairy roots ( in vivo ), respectively. This study systematically characterized the OMT gene family in E. lathyris and revealed multiple O -methylation steps involved in the biosynthesis of the phytoalexin scoparone. The findings provided a solid theoretical framework for further research on increasing endogenous phytoalexin biosynthesis for breeding disease-resistant plants and reducing post-harvest loss. • Genome-wide identification of 34 O -methyltransferases in Euphorbia lathyris . • ElCOMT33/34 catalyze esculetin methylation in vitro/vivo to enhance phytoalexins. • ElCOMT33 mediates two-step methylation to produce scoparone, a key phytoalexin and drug.