Gain-of-function mutations in beet DODA2 identify key residues for betalain pigment evolution.

The key enzymatic step in betalain biosynthesis involves conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) to betalamic acid. One class of enzymes capable of this is 3,4-dihydroxyphenylalanine 4,5-dioxygenase (DODA). In betalain-producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain-nonfunctional DODA into a betalain-functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets, Arabidopsis and yeast, and gene silencing was performed by virus-induced gene silencing. Site-directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris and Mirabilis jalapa both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site-directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain-nonfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants.

Bioproduction of a betalain color palette in Saccharomyces cerevisiae.

Betalains are a family of natural pigments found exclusively in the plant order Caryophyllales. All members of this chemical family are biosynthesized through the common intermediate betalamic acid, which is capable of spontaneously condensing with various primary and secondary amines to produce betalains. Of particular interest is the red-violet betanin, most commonly obtained from Beta vulgaris (beet) as a natural food dye. We demonstrate the first complete microbial production of betanin in Saccharomyces cerevisiae from glucose, an early step towards a fermentation process enabling rapid, on-demand production of this natural dye. A titer of 17mg/L was achieved, corresponding to a color intensity obtained from 10g/L of beetroot extract. Further, we expanded the spectrum of betalain colors by condensing betalamic acid with various amines fed to an engineered strain of S. cerevisiae. Our work establishes a platform for microbial production of betalains of various colors as a potential alternative to land- and resource-intensive agricultural production.

Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales.

Diverse natural products are synthesized in plants by specialized metabolic enzymes, which are often lineage-specific and derived from gene duplication followed by functional divergence. However, little is known about the contribution of primary metabolism to the evolution of specialized metabolic pathways. Betalain pigments, uniquely found in the plant order Caryophyllales, are synthesized from the aromatic amino acid l-tyrosine (Tyr) and replaced the otherwise ubiquitous phenylalanine-derived anthocyanins. This study combined biochemical, molecular and phylogenetic analyses, and uncovered coordinated evolution of Tyr and betalain biosynthetic pathways in Caryophyllales. We found that Beta vulgaris, which produces high concentrations of betalains, synthesizes Tyr via plastidic arogenate dehydrogenases (TyrAa /ADH) encoded by two ADH genes (BvADHα and BvADHß). Unlike BvADHß and other plant ADHs that are strongly inhibited by Tyr, BvADHα exhibited relaxed sensitivity to Tyr. Also, Tyr-insensitive BvADHα orthologs arose during the evolution of betalain pigmentation in the core Caryophyllales and later experienced relaxed selection and gene loss in lineages that reverted from betalain to anthocyanin pigmentation, such as Caryophyllaceae. These results suggest that relaxation of Tyr pathway regulation increased Tyr production and contributed to the evolution of betalain pigmentation, highlighting the significance of upstream primary metabolic regulation for the diversification of specialized plant metabolism.

Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals.

Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

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

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.

Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants.

Betalains are tyrosine-derived red-violet and yellow pigments, found in plants only of the Caryophyllales order. Although much progress has been made in recent years in the understanding of the betalain biosynthetic process, many questions remain open with regards to several of the proposed steps in the pathway. Most conspicuous by its absence is the characterization of the first committed step in the pathway, namely the 3-hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine (l-DOPA). We used transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) to identify a novel, betalain-related cytochrome P450-type gene, CYP76AD6, and carried out gene silencing and recombinant expression assays in Nicotiana benthamiana and yeast cells to examine its functionality. l-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1, which was previously thought to only catalyze a succeeding step in the pathway. While CYP76AD1 catalyzes both l-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to metabolically engineer entirely red-pigmented tobacco plants through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway.

Flavonoid Properties in Plant Families Synthesizing Betalain Pigments (Review).

The anthocyanin pigments are contained in the flowers, fruits, leaves and roots of almost plant species. On the other hand, distribution of the betacyanins are limited in eight families of the order Caryophyllales, i.e. Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Didiereaceae, Nyctaginaceae, Phytolaccaceae and Portulacaceae. However, other flavonoids, i.e. flavones, C-glycosylflavones, flavonols, flavanones, dihydroflavonols, chalcones, aurones, and flavan and proanthocyanidins, are synthesized in betalain-containing families. In this review, distribution and properties of the flavonoids in eight betalain-containing families are described.

Swapping one red pigment for another.

Betalains are bright red and yellow pigments, which are produced in only one order of plants, the Caryophyllales, and replace the more familiar anthocyanin pigments. The evolutionary origin of betalain production is a mystery, but a new study has identified the first regulator of betalain production and discovered a previously unknown link between the two pigment pathways.

The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway.

Nearly all flowering plants produce red/violet anthocyanin pigments. Caryophyllales is the only order containing families that replace anthocyanins with unrelated red and yellow betalain pigments. Close biological correlation of pigmentation patterns suggested that betalains might be regulated by a conserved anthocyanin-regulating transcription factor complex consisting of a MYB, a bHLH and a WD repeat-containing protein (the MBW complex). Here we show that a previously uncharacterized anthocyanin MYB-like protein, Beta vulgaris MYB1 (BvMYB1), regulates the betalain pathway in beets. Silencing BvMYB1 downregulates betalain biosynthetic genes and pigmentation, and overexpressing BvMYB1 upregulates them. However, unlike anthocyanin MYBs, BvMYB1 will not interact with bHLH members of heterologous anthocyanin MBW complexes because of identified nonconserved residues. BvMYB1 resides at the historic beet pigment-patterning locus, Y, required for red-fleshed beets. We show that Y and y express different levels of BvMYB1 transcripts. The co-option of a transcription factor regulating anthocyanin biosynthesis would be an important evolutionary event allowing betalains to largely functionally replace anthocyanins.

Metabolite profiling of red and white pitayas (Hylocereus polyrhizus and Hylocereus undatus) for comparing betalain biosynthesis and antioxidant activity.

Metabolite profiling of red and white pitayas (Hylocereus polyrhizus and Hylocereus undatus) was performed using gas chromatography-time-of-flight-mass spectrometry and ultraperformance liquid chromatography-quadrupole-time-of-flight-mass spectrometry with multivariate analysis. Different species and parts of pitayas (red peel, RP; white peel, WP; red flesh, RF; and white flesh, WF) were clearly separated by partial least-squares discriminate analysis. Furthermore, betalain-related metabolites, such as betacyanins and betaxanthins, or their precursors were described on the basis of their metabolites. The results of antioxidant activity tests [1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and ferric reducing ability of plasma (FRAP)], total phenolic contents (TPC), total flavonoid contents (TFC), and total betacyanin contents (TBC) showed the following: RP ≥ WP > RF > WF. TPC, TFC, TBC, and betalain-related metabolites were higher in the peel than in the flesh and suggested to be the main contributors to antioxidant activity in pitayas. Therefore, peels as well as pulp of pitaya could beneficially help in the food industry.