PRJNA280277
Stranded transcriptome sequencing from 14 species across the plant order Caryophyllales
Betalain pigments are unique to the Caryophyllales, and are structurally and biosynthetically distinct from the otherwise ubiquitous anthocyanin pigments. Recently two of the genes that encode key enzymes within the betalain synthesis pathway have been identified: 4,5-dioxygenase (DODA) that catalyzes the formation of betalamic acid and CYP76AD1, a cytochrome P450 gene that catalyzes the formation of cyclo-DOPA. Here we describe the molecular evolution of these genes within Caryophyllales. We demonstrate that coincident duplications within the CYP761A and DODA lineages are intimately tied to the origin and evolution of betalain pigmentation. The duplications gave rise to isoforms (DODA-a and CYP761A-a) that are specific to betalain synthesis, and these duplication events arose just prior to the earliest inferred origin of betalain pigmentation in the core Caryophyllales. Both betalain-specific isoforms were mostly lost or down-regulated in the anthocyanic Molluginaceae and Caryophyllaceae, indicating an early single origin of betalain-specific function that correlates with these duplication events, prior to the divergence of Microtea. A likely single origin of the key enzymes in betalain-synthesis pathway and their loss in anthocyanic taxa undermines competing hypotheses that the betalain pigmentation may have evolved more than once, and indicates that the long-term exclusion of betalain pigments from anthocyanic taxa is likely mediated through gene loss. We reveal that the betalain-specific isoforms of CYP76AD1 and DODA in Beta vulgaris are physically located ~50 kb apart on chromosome 2, supporting the idea of a coincident duplication of the DODA and CYP76AD1 lineages, and suggesting the possibility of a metabolic operon in association with the betalain synthesis pathway. We analyze patterns of positive selection among the clades in these radiating DODA and CYP76AD1 lineages and implicate key residues in the neo-functionalization of the betalain-specific clades. Finally, we report the asymmetric diversification of the betalain-specific DODA genes that may be implicated in the evolution of color within the betalain pigment system.