The ABCG1 transporter facilitates sesquiterpene accumulation in Marchantia polymorpha oil bodies.

Marchantia polymorpha oil bodies (OBs) are specialised cell structures housing a diverse array of C15-terpenes, called sesquiterpenes. These compounds are known for their roles as herbivore repellents, yet the enzymes responsible for the biosynthesis of their precursors (C5 isoprenoid units) remain poorly characterized. Discrepancies remain between enzyme localizations suggested by computational predictions and those observed in earlier experimental studies, complicating our understanding of terpene biosynthesis. 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, chloroplast and the endoplasmic reticulum), and OB cells were identified as the primary sites of terpene 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 {beta}-amyrin. Targeting to OB cells resulted in measurable amounts of these compounds, but their yields remained unaffected by the over-expression of key precursor genes, underscoring challenges in redirecting metabolic flux. To further investigate terpene accumulation in OBs, we focused on ABCG1, an ABC transporter previously reported to localize at the OB membrane. Overexpression of ABCG1 in OB cells, alongside an exogenous sesquiterpene synthase, only increased the levels of endogenous sesquiterpenes, while CRISPR-mediated disruption of ABCG1 resulted in a dramatic reduction in sesquiterpene accumulation. These findings establish ABCG1 as a critical factor for sesquiterpene retention within OBs and provide new insights into the mechanisms governing terpene metabolism and storage in Marchantia polymorpha.