Spatial distribution of isoprenoid enzymes and MpABCG1 transporter influences sesquiterpene accumulation in Marchantia polymorpha oil bodies

Marchantia polymorpha oil bodies (OBs) are specialized cell structures housing a diverse array of C15-terpenes, called sesquiterpenes. These compounds act as herbivore repellents, yet the enzymes responsible for the biosynthesis of their precursors remain poorly characterized. We investigated the localization of isoprenoid biosynthetic enzymes using translational and transcriptional reporters, coupled with confocal microscopy. Most enzymes localized as predicted (e.g., cytosol, plastid and the endoplasmic reticulum), and OB cells were identified as the primary sites of sesquiterpene biosynthesis. To explore OBs as potential storage sites for terpenes, we attempted to produce exogenous but easily identifiable compounds in Marchantia, such as the diterpene taxadiene and the triterpene β-amyrin. Targeting to OB cells resulted in measurable amounts of these compounds, but their yields remained unaffected by the overexpression of key precursor genes, underscoring challenges in redirecting metabolic flux. To further investigate terpene accumulation in OBs, we focused on MpABCG1, an ABC transporter previously reported to localize at the OB membrane. While MpABCG1 overexpression mildly increased endogenous sesquiterpene levels, its disruption via CRISPR dramatically reduced sesquiterpene accumulation. These findings establish that MpABCG1 is necessary for sesquiterpene accumulation in OBs and add to current knowledge of terpene synthesis compartmentalisation in Marchantia polymorpha.