Are seven amino acid substitutions sufficient to explain the evolution of high l-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018).

This work revisits a publication by Bean et al. (2018) that reports seven amino acid substitutions are essential for the evolution of l-DOPA 4,5-dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Bean et al. (2018). Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Bean et al. (2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Bean et al. (2018) to re-observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2-mut3 variant. Multiple in vivo assays, in both Saccharomyces cerevisiae and Nicotiana benthamiana, did not result in visible DODA activity in BvDODAα2-mut3, with betalain production always 10-fold below BvDODAα1. In vitro assays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2-mut3 proteins, explaining their differing performance in vivo. In summary, we were unable to replicate the in vivo analyses of Bean et al. (2018), and our quantitative in vivo and in vitro analyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Bean et al. (2018).

Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales.

The evolution of l-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. To address this, we functionally characterised 23 distinct DODA proteins for l-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l-DOPA 4,5-dioxygenase activity. We find that low l-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny. In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.