Figure 6. GC-MS analysis of miltiradiene-derived diterpenoids obtained by transient expression of CfCYP76AH15 in N. benthamiana leaves.
(A) Total ion chromatograms (TIC) of extracts transiently expressing CfCXS, CfGGPPS, CfTPS1 and CfTPS3 (miltiradiene biosynthesis genes) in combination with water (-) or CfCYP76AH15. Dehydroabietadiene (DE) and miltiradiene (MT) were observed in the (-) extract, whereas ferruginol was observed in extracts from tissue expressing the miltiradiene biosynthesis genes together with CfCYP76AH15. In root cork extracts, ferruginol was detected together with dehydroabietadiene. Presence of ferruginol was confirmed by comparison to an authentic standard (Ignea et al., 2016a), while presence of miltiradiene (B) and dehydroabietadiene (C) were confirmed by comparison of m/z spectra with previously characterized compounds (Andersen-Ranberg et al., 2016). For every combination, extracts from leaves of three different N. benthamiana plants have been analyzed and representative chromatograms are shown.