Characterization of underlying transcription factors that regulate betalain pigment formation in beets

The plant kingdom is a colorful place with most vascular plants producing phenylalanine-based red/violet anthocyanin pigment. Only a single order of flowering plants, the Caryophyllales, is known to produce an unusual pigment known as Betalain. Betalains encompasses an entire range of colors between yellow to red, are nitrogen-containing water-soluble compounds derived from tyrosine, and are acidic in nature due to the presence of several carboxyl groups. The betalain and the anthocyanin pigments are mutually exclusive. The pathway and enzymes for betalain biosynthesis, from tyrosine to the end products, red/violet betacyanins and yellow betaxanthins, has largely been determined. Little however is known about the regulation of the biosnynthetic genes. The strong biological correlation between the anthocyanin and the betalains prompted the suggestion that the molecular regulation of betalains and anthocyanins uses the same MYB and bHLH and WD-repeat regulators, the MBW complex. The work described here strives to understand the regulatory mechanisms controlling betalain pigmentation in the Caryophyllales and how they can be controlled and influenced. To understand the pathway, there was a pressing need for analysis at the biochemical, molecular, and genetic levels. Before the work reported here, two pigment biosynthetic genes were identified. The gene/ enzyme responsible for step two was identified as DOPA 4, 5-dioxygenase (DODA) functioning to produce betalamic acid (BA) from LDOPA (Christinet et al., 2004), and later we showed that a novel cytochrome P450, CYP76AD1, is absolutely required for the red pigment in beets by catalyzing the step producing cyclo-DOPA from the LDOPA substrate (Hatlestad et al., 2012). Through this project I have: (1) discovered and characterized Beet MYB1 (BvMYB1), a MYB evolved from the anthocyanin regulating MYBs, and analyzed how BvMYB1 regulates betalain production by its interaction with DNA and other proteins; (2) determined overlapping functional redundancies of BvMYB1 with two other R2R3 BvMYBs, BvMYB2 and BvMYB3; (3) identified a beet bHLH protein, BvbHLH1, and a beet WD-repeat protein, BvTTG1, that function similar to the Arabidopsis thaliana proteins; and finally (4) I worked towards characterizing a novel BvMYB1 Response Element (MRE) that BvMYB1 directly binds to activate betalain biosynthetic genes. By identifying members of the regulatory complex for the betalain pathway I hope to contribute toward understanding the evolutionary replacement of anthocyanins by betalains within a single flowering order, and fill a lack of knowledge about producing, controlling, and influencing this valuable natural pigment.