Extended Data Figure 4. Characterization of GsOMT2.

a) Addition of GsOMT2 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of putative 4 (m/z 330.1700) and production of a new compound corresponding to both a methylation and a hydroxylation (m/z 360.1805), as shown here via LC-MS chromatograms. EIC = extracted ion chromatogram. This activity was confirmed >3 independent experiments. b) MS/MS fragmentation spectrum of the generated m/z 360.1805 product (*) at a collision energy of 20V. MS/MS fragmentation of this peak was performed twice, with similar results each time. c) Tabulated list and putative structures for ion fragments generated from MS/MS analysis of the m/z 360.1805 product. See Supplementary Information for a detailed analysis of MS/MS results. d) Untargeted metabolite analysis (XCMS) comparing the presence vs. absence of GsOMT2 within the transient co-expression system (n=6 independent replicates for each experimental condition). Shown are unique mass signatures (P < 0.1 between samples, as determined via XCMS) in ranked order based upon their increasing (top panel) or decreasing (bottom panel) fold-change in abundance between the two compared conditions. The mass isotopologues (M₀, M₁, and M₂) for the presumed product (m/z 360.1805) are highlighted in red, while the mass isotopologues (M₀, M₁) of the presumed substrate (m/z 330.1700) are highlighted in blue. r.t. = retention time. e) Proposed reaction catalyzed by GsOMT2, and tentatively GsCYP75A109, as supported by MS/MS fragmentation and prior labeling studies. Note that compound 5 is not observed within our co-expression system, presumably due to its consumption to 6.