Extended Data Fig. 3: Functional characterization of cinchonamine oxidase (CpCiO).
a. Extracted ion chromatograms (EIC) of the extract from the transient expression of CpCiS in N. benthamiana with infiltration of cinchonaminal (13), showing that CpCiO converts 13 into quinoline compounds cinchonidinone (8) and cinchoninone (9), which exist in a tautomeric equilibrium. This experiment was repeated five times with similar results. b. MS/MS (20 to 50 eV) spectra of the CpCiO product and standard 8. c. VIGS-treated C. pubescens plantlets transformed with a vector designed to silence CpCiO and the marker gene CpMgChl. The phenotypic characteristic resulting from CpMgChl silencing (leaf bleaching) is indicated by an arrow and the relative expression of CpCiO is shown. d. Representative total ion chromatogram (TIC) of leaf tissue extracts from VIGS-CpCiO-transformed Cinchona plantlets and control plantlets, along with standards cinchonamine and cyclocinchonaminal (10 and 13); EIC of cinchonidinone (8) and cinchoninone (9), and bar graphs for metabolite levels of the CpCiO knockdown tissue in comparison to controls are shown, corroborating the effective silencing of CpCiO. In c and d, the values represent mean ± s.e.m. (n = 5 biological replicates) and statistical analyses were performed using a two-sided, unpaired Welch’s t-test. e. Reaction catalyzed by CpCiO.
