Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)

Abstract

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.

The evolution of betalain biosynthesis in Caryophyllales

“…Sunnadeniya et al . () confirmed these findings and identified yet another CYP76AD homologue, BvCYP76AD5 from B. vulgaris , also with tyrosine hydroxylase activity. Expression of both BvCYP76AD5 and BvCYP76AD6 homologues in yeast led to the production of l ‐DOPA (Sunnadeniya et al ., ).…”

“…() confirmed these findings and identified yet another CYP76AD homologue, BvCYP76AD5 from B. vulgaris , also with tyrosine hydroxylase activity. Expression of both BvCYP76AD5 and BvCYP76AD6 homologues in yeast led to the production of l ‐DOPA (Sunnadeniya et al ., ). Unlike BvCYP76AD1 , the newly discovered enzymes BvCYP76AD5 and BvCYP76AD6 only exhibit tyrosine hydroxylase activity and cannot perform the l ‐DOPA oxidase step.…”

“…Unlike BvCYP76AD1, the newly discovered enzymes BvCYP76AD5 and BvCYP76AD6 only exhibit tyrosine hydroxylase activity and cannot perform the L-DOPA oxidase step. However, betanidin was detected by LC-MS at very low intensities, suggesting that these enzymes may have a weak ability to produce cyclo-DOPA (Sunnadeniya et al, 2016). The differential ability to produce cyclo-DOPA between BvCYP76AD1, BvCYP76AD5 and BvCYP76AD6 provides the genetic architecture for selective production of yellow or red betalains, as is commonly seen in agricultural cultivars (Sunnadeniya et al, 2016).…”

“…It was argued that in these betaxanthin-pigmented taxa, the tyrosine hydroxylase step must still be intact, with the L-DOPA oxidase step disrupted, preventing betacyanin formation. As outlined in the following, the prediction that PPOs could be involved in betalain biosynthesis has been borne out, and on the basis of comparative transcriptomic analyses, cytochrome P450-type enzymes are responsible for the catalysis of these two steps (Hatlestad et al, 2012;Polturak et al, 2016;Sunnadeniya et al, 2016).…”

“…Further to the discovery of the tyrosine hydroxylase activity of BvCYP76AD1 (DeLoache et al, 2015;Polturak et al, 2016), subsequent efforts uncovered additional enzymes responsible for catalysing the first step of the betalain pathway. Polturak et al (2016) showed that tyrosine hydroxylation is also catalysed by a New Phytologist (2019) (Sunnadeniya et al, 2016). Unlike BvCYP76AD1, the newly discovered enzymes BvCYP76AD5 and BvCYP76AD6 only exhibit tyrosine hydroxylase activity and cannot perform the L-DOPA oxidase step.…”