Search for: All records

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure–function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products. 

The evolution ofl‐DOPA4,5‐dioxygenase activity, encoded by the geneDODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed thatl‐DOPA4,5‐dioxygenase activity evolved via a single Caryophyllales‐specific neofunctionalisation event within theDODAgene lineage. However, this neofunctionalisation event has not been confirmed and theDODAgene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear.

To address this, we functionally characterised 23 distinctDODAproteins forl‐DOPA4,5‐dioxygenase activity, from four betalain‐pigmented and five anthocyanin‐pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updatedDODAphylogeny, we then explored the evolution ofl‐DOPA4,5‐dioxygenase activity.

We find that lowl‐DOPA4,5‐dioxygenase activity is distributed across theDODAgene lineage. In this context, repeated gene duplication events within theDODAgene lineage give rise to polyphyletic occurrences of elevatedl‐DOPA4,5‐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 elevatedl‐DOPA4,5‐dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.