Figure 3.

Betalain‐pigmented species have a single DODA that has high activity when heterologously expressed in Nicotiana benthamiana and other DODA that have no or marginal activity. (a) A representative leaf is shown from the agroinfiltration of l‐DOPA 4,5‐dioxygenase (DODA) genes from Stegnosperma halimifolium, Beta vulgaris, Carnegiea gigantea and Mesembryanthemum crystallinum. The DODA coding sequences were cloned into multigene constructs containing the other structural genes necessary for betacyanin production (BvCYP76AD1, MjcDOPA‐5GT). The infiltration spots are labelled according to the DODA variant. For all species, the pBC‐BvDODAα1 multigene vector is included as a positive control (P). (b) The betanin content of the infiltration spots was measured using HPLC and data were represented relative to the BvDODAα1 spot present in each biological replicate. The data were combined after calculating relative amounts (RQ) for each species from individual species‐specific experiments (Supporting Information Figs S8–S11). Bars show means ± SD; n = 5 for S. halimifolium, B. vulgaris, C. gigantea and M. crystallinum, except for BvDODAα2 (n = 4) and BvDODAα3 (n = 4). (c) A representative HPLC trace for the DODA α 1 gene from each species. The left peak is betanin and the right peak is its isomer, isobetanin. The traces are offset for presentation. ‘pLUC’ is a negative control plasmid carrying the firefly luciferase gene and ‘Betanin’ is a commercially available extract from beet hypocotyl which was used to validate the retention time of betanin in these samples.