Fig. 5: Identification of residues involved in the product specificity of TXSSs.

A Peak area analysis of triterpenoids detected in the floral, leaf and root tissues of pot marigold and Russian dandelion plants. Error bars indicate the mean and standard error of 3 biological replicates. B Phylogenetic tree of TXSSs in which the foreground branch containing taxa that predominantly produce taraxasterol is highlighted in light red. The Log likelihood ratio test at the foreground branch node indicates that this branch is likely under positive selection (p < 0.01). Cc Cynara cardunculus; Tpr Tragopogon pratensis; Td Tragopogon dubius; Ls Lactuca sativa; Ce Cichorium endivia; Tc Taraxacum coreanum; Tm Taraxacum mongolicum; Tk Taraxacum kok-saghyz; Co Calendula officinalis; Ca Calendula arvensis; Hn Helianthus niveus; Ha Helianthus annuus; Tpa Tanacetum parthemium; Cse Chrysanthemum seticuspe. Accession numbers are provided in Supplementary Table 4. C Structural models illustrating the predicted position of the taraxasteryl cation in the active site of CoTXSS and TkTXSS. Residues that differ between them are highlighted in orange (CoTXSS) and red (TkTXSS). Asterisks indicate that residues are under positive selection in non-basal Cichorieae D Total ion chromatograms of extracts of N. benthamiana leaves transiently expressing wild type and mutated CoTXSS and TkTXSS; E Quantification of triterpenes produced by transiently expressing wild type and mutated CoTXSS and TkTXSS; Error bars indicate the mean and standard error of 6 biological replicates (independent infiltrations). Significant differences in Ψ-taraxasterol (orange lowercase letters) and taraxasterol (red lowercase letters) were analysed using a Kruskal-Wallis test followed by post-hoc Wilcoxon rank sum test with a Benjamini-Hochberg correction. Samples that do not share the same lower-case letter are significantly different from each other (p < 0.05). P values are provided in Supplementary Data 8.