Figure 1. Schematic of deuterium incorporation into secondary metabolites in plants and analysis workflow.

Deuterium from heavy water (D₂O) is incorporated into plant metabolism via several routes. Initial conversion of D₂O to labeled plastoquinol occurs in photosystem II; subsequent steps from photosystem I and related enzymes result in the fixation of solvent-exchangeable atoms (red) to relatively stable carbon-bound hydrogen atoms (blue) during the formation of NADPH. These labeled atoms are incorporated into primary metabolites by central metabolic enzymes. Eventually, all non-exchangeable hydrogen atoms would become labeled, but on the timescales studied in this paper, several routes may contribute to the biosynthesis of secondary metabolites, including direct incorporation of hydrogen from water, reductive incorporation of hydrogen from labeled NAD(P)D, and recycling or turnover of incompletely labeled primary metabolites, resulting in partially deuterated secondary metabolites. In this study, we use LC-MS-based metabolomics approaches to quantify label incorporation into plant metabolomes by finding mass isotopologue distributions (MIDs) using both targeted and untargeted approaches, and using these distributions to estimate relative deuterium abundance in a metabolite by comparison to the MID of an unlabeled control sample.