Identification and characterization of Iberis amara cucurbitadienol synthase. (A) Total ion current (TIC) GC-MS chromatograms of extracts from an empty vector (EV) control yeast strain (black) and a strain expressing IaCPQ (red), and of purified cucurbitadienol as a standard (blue). The peak indicated with a black arrow at 21.9 min corresponds to cucurbitadienol. (B) TIC GC-MS chromatograms of extracts from Nicotiana benthamiana leaves agro-infiltrated with an EV control, IaCPQ, ClCPQ, CpCPQ, and CpSE2 with IaCPQ, and the chromatogram of the cucurbitadienol standard. Black arrows indicate the cucurbitadienol peak. (C) TIC GC-MS chromatograms of extracts from an EV control yeast strain (black) and a strain expressing IaCAS (red), and the chromatogram of a cycloartenol standard (blue). The peak indicated with a black arrow eluting at 26.2 min corresponds to cycloartenol. The second peak indicated with a green arrow eluting at 24.5 min likely corresponds to 31-norcycloartenol. Note, the GC-MS analysis program for yeast samples differs slightly from the one used for GC-MS analysis for plant extracts, giving rise to differences in retention times.