Fig. 3: Functional analysis of BGC4(5D) genes by in vitro enzymatic assays and metabolic analysis of wheat mutant lines.

a LC-MS chromatograms of in vitro enzymatic reactions with naringenin 4’,7-dimethyl ether (3) as substrate, and microsomal fractions from yeast expressing TaCYP71C164 and/or TaCYP71F53. Extracted ion chromatogram (EIC) for molecular [M + H]⁺ ion of the predicted reaction product artocarpanone A (4) (m/z = 317.1010) is shown. Y-axes are linked. Chromatogram of artocarpanone A (4) purified compound is given for reference. b Chromatograms of in vitro enzymatic reactions with artocarpanone A (4) as substrate, and microsomal fractions from yeast expressing TaCYP71C164 and/or TaCYP71F53. Extracted ion chromatogram (EIC) for the [M + H]⁺ ion of the predicted reaction product 2’-O-demethyl-triticein (5) (m/z = 315.0855) is shown. Y-axes are linked. Minor peaks observed in the empty vector and TaCYP71C164 samples originate from a different compound with similar retention time and m/z to compound (5) (Supplementary Fig. S13). Chromatogram of 2’-O-demethyl-triticein (5) purified compound is given for reference. c Extracted LC-MS chromatograms of blade extracts from MeJA-treated plants of TILLING mutants (Cad1684, omt8; Cad1682, omt3; Cad0227/Cad1793, cyp71f53) and ‘Cadenza’ wildtype parental line. Chromatograms for commercial standards (naringenin (1), isosakuranetin (2), naringenin 4’,7-dimethyl ether (3) and purified compounds (artocarpanone A (4), 2’-O-demethyl-triticein (5), and triticein (6) are shown in bottom row, for reference. Digitoxin was used as an internal standard (IS) in all samples. Extracted ion chromatograms (EIC) are for ions representing the compounds specified above. Y-axes are linked for chromatograms of each extracted mass, but not between different masses. d Illustration of proposed triticein biosynthetic pathway, impeded by loss-of-function mutations of TaOMT8, TaOMT3, or TaCYP71F53.