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

Abstract

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 4,5dioxygenase 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.

The scaffold-forming steps of plant alkaloid biosynthesis

“…31 Seven amino acid residues have been identied that are sufficient to switch a marginally active DODAa into an active DODAa, 113 and there is evidence that the polyphyletic highly active DODAs converged on similar residues at these key positions. 114 26,27 and the cryptostylines (Cryptostylis fulva). 115 The aldehyde precursor to the Amaryllidaceae alkaloids is 3,4-dihydrobenzaldehyde.…”

The Reuse of Public Datasets in the Life Sciences: Potential Risks and Rewards

“…the eGFP browser (75) Only genes in the annotation included. Only based on the available structural annotation thus alternative transcripts would be missed Analysis of non-canonical splice sites based on genome sequences, annotations, and RNA-Seq datasets (19,55) Batch effects if large sample groups come from the same source and annotation errors will impact analysis results Extraction of new sequences for phylogenetic analysis (18,76) Reliability of source is crucial, transcriptome assemblies are inherently incomplete as not all genes are expressed at the same time Proteome Identification of antimicrobial peptides (22) Prediction, correct modelling and structural analysis are not completely accurate due to e.g. the presence of precursors; validation is required Phospho-proteomics, e.g.…”

Elucidation of the Core Betalain Biosynthesis Pathway in Amaranthus tricolor

“…The function of DODAb is unknown, but the evolution of L-DOPA 4,5dioxygenase activity in betalain-producing plants was proposed to be led by DODAa 9 . Nevertheless, only one paralogous gene from the DODAa clade shows high L-DOPA 4,5-dioxygenase activity in each species, and others exhibit barely detectable activity 12,21 .…”

“…Although the molecular basis for the mutual exclusion of betalains and anthocyanins is still unclear, breakthroughs in the identi cation of genes involved in betalain biosynthesis have shed light on the evolution of betalain pigmentation in Caryophyllales 9,10,11,12 . Unlike anthocyanins, which are derived from L-phenylalanine, betalains are synthesized from L-tyrosine 13,14 .…”

“…Initially, L-tyrosine is hydroxylated to produce L-DOPA by tyrosinases encoded by CYP76AD1 and its orthologs 15,16 . L-DOPA can be converted into betalamic acid by L-DOPA 4,5-dioxygenase encoded by DODA, and betalamic acid can then spontaneously condense with amino acids to form betaxanthins 1,12,17 . Alternatively, L-DOPA can be converted into cyclo-DOPA through the oxidase activity of CYP76AD1, and cyclo-DOPA can then spontaneously condense with betalamic acid to form betanidin 9,18 .…”