Plant Biotechnology Reports

, Volume 8, Issue 1, pp 37–48

Diversity in plant red pigments: anthocyanins and betacyanins


    • Department of Biological SciencesOchanomizu University
Review Article

DOI: 10.1007/s11816-013-0294-z

Cite this article as:
Sakuta, M. Plant Biotechnol Rep (2014) 8: 37. doi:10.1007/s11816-013-0294-z


Plant pigments are of interest for research into questions of basic biology as well as for purposes of applied biology. Red colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Though anthocyanins are broadly distributed among plants, betacyanins have replaced anthocyanins in the Caryophyllales. Red plant pigments are good indicator metabolites for evolutionary studies of plant diversity as well as for metabolic studies of plant cell growth and differentiation. In this review, we focus on the biosynthesis of anthocyanins and betacyanins and the possible mechanisms underlying the mutual exclusion of betalains and anthocyanins based on the regulation of the biosynthesis of these red pigments.


AnthocyaninsBetacyaninsMolecular mechanism of biosynthesisSecondary metabolismEvolution of metabolism

Some aspects of plant pigments in biological studies

The use of naturally occurring pigments as a quality food colorant has increased because of consumer wariness of synthetic dyes and a clear preference for pigments of natural origin. Besides imparting attractive colors to food products, some red pigments confer free-radical scavenging and associated antioxidant activities (Cai et al. 2003; Stintzing et al. 2005; Tesoriere et al. 2008; Fracassetti et al. 2013; Li et al. 2013). For example, anthocyanins and betalains used as food colorants may provide protection against certain oxidative stress-related disorders in humans (Lee et al. 2005; Allegra et al. 2007; Wallace 2011; Christensen et al. 2012; Hobbs et al. 2012).

Plant pigments are of interest for research into questions of basic biology as well as for purposes of applied biology, including food science and molecular breeding of flower color. Red colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. These pigments are stored in vacuoles and serve important functions in plant reproduction by recruiting pollinators and seed dispersers. Anthocyanins, a type of flavonoid, are responsible for most pinks, reds, mauves, and blues in flowers and fruits. Blue flower color is due to intermolecular co-pigmentation involving anthocyanins and other flavonoids, flavones, or flavonols (Mol et al. 1998; Harborne and Williams 2000). Though anthocyanins are broadly distributed among plants, betacyanins have replaced anthocyanins in the Caryophyllales, excluding the families Caryophyllaceae and Molluginaceae (Strack et al. 2003).

A secondary metabolism is expressed in specific tissues and cells at specific stages of growth in higher plants, implying a close correlation between secondary metabolism and the growth and morphological differentiation of cells (Komamine et al. 1989). For example, anthocyanin accumulation is observed in some epidermal cells of petals, fruits, stems, and leaves. On the other hand, some secondary metabolites are not localized in specific tissues; the accumulation of betacyanins is observed in all tissues of red beet roots. These facts suggest that there are two patterns of accumulation of secondary metabolites in relation to growth and differentiation in plants (Sakuta and Komamine 1987). Regulatory mechanisms of anthocyanin biosynthesis in suspension cultures of Vitis sp. and betacyanin in Phytolacca americana were investigated in relation to cell division activity (Sakuta et al. 1994). Anthocyanin accumulation in Vitis cells showed a negative correlation with cell division. Aphidicolin, an inhibitor of DNA synthesis, or a reduced phosphate concentration in culture medium induced a substantial increase in anthocyanin accumulation as well as the inhibition of cell division. In contrast, betacyanin biosynthesis in Phytolacca cells showed a clear positive correlation with cell division: peak betacyanin accumulation was observed during the log phase of batch cultures (Sakuta et al. 1986). Aphidicolin and propyzamide, an antimicrotubule drug, reduced betacyanin biosynthesis and accumulation at concentrations that were inhibitory to cell division (Hirano and Komamine 1994). These facts suggest that there are two patterns of accumulation in relation to cell division, growth, and proliferation in plants. In suspension cultures of carrot, both anthocyanin synthesis and the induction of embryogenesis were induced by a lack of auxin in the growth medium (Ozeki and Komamine 1981, 1986). Small cell aggregates selected by sieving at every subculture step were fractionated by density gradient centrifugation in Ficoll. Anthocyanin synthesis was induced in fractions with densities less than 14 % Ficoll after the cells were transferred to auxin-free medium, while the cells in fractions with densities greater than 14 % Ficoll differentiated into embryos after transfer to auxin-free medium. Anthocyanin synthesis occurred in the lighter cells when cell division ceased and cell elongation began. The addition of auxin caused division of the elongated cells and the disappearance of anthocyanin. These results indicate that anthocyanin synthesis is closely correlated with morphological differentiation and is negatively correlated with cell division or proliferation.

Plant red pigments are good indicator metabolites for evolutionary studies of plant diversity as well as for metabolic studies of plant cell growth and differentiation. In fact, the distribution across species of secondary metabolites, including red pigments, provides a useful trait for chemotaxonomic studies of higher plants. Although anthocyanins are widely distributed in higher plants in the form of flower and fruit pigments, betacyanins have replaced anthocyanins in the Caryophyllales, excluding the families Caryophyllaceae and Molluginaceae (Mabry 1980; Strack et al. 2003). Anthocyanins have not been reported in these plants. Anthocyanins and betacyanins never occur together in the same plant. Nothing is known about the mutual exclusion of anthocyanins and betacyanins in higher plants. This curious phenomenon has been examined from genetic and evolutionary perspectives (Brockington et al. 2011). While the structural and regulatory genes of anthocyanin biosynthesis are well known, the biosynthesis of betacyanins is poorly understood and the evolutionary mechanism of the mutual exclusion of these two pathways remains a mystery (Stafford 1994). Betalains are also found in some fungi, including the poisonous mushroom fly agaric (Amanita muscaria). The specific occurrence of betalains in phylogenetically distinct plants is another taxonomic mystery. In the next part of this review, we focus on the biosynthesis of anthocyanins and betacyanins and the possible mechanisms underlying the mutual exclusion of betalains and anthocyanins based on the regulation of the biosynthesis of these red pigments.

Anthocyanin biosynthesis

The biosynthetic pathway of flavonoids is one of the best-studied examples of secondary metabolism in higher plants, and most flavonoid biosynthetic genes have been cloned and analyzed (Holton and Cornish 1995; Forkmann and Martens 2001; Shirley 2001; Koes et al. 2005) (Fig. 1). Flavonoids are synthesized from phenylalanine, a product of the shikimate pathway, via the general phenylpropanoid and flavonoid pathway. The activity of the phenylalanine-generating shikimate pathway is closely correlated with flavonoid synthesis (Suzuki et al. 1995). Phenylalanine ammonia-lyase catalyzes the conversion of phenylalanine to trans-cinnamate as the first step of the multi-branched general phenylpropanoid pathway, which supplies substrates for flavonoid and lignin biosynthesis. Chalcone synthase (CHS) catalyzes the first and key regulatory step of flavonoid biosynthesis, which involves the stepwise condensation of three acetyl units from malonyl-coenzyme A (CoA) with the coumaroyl moiety of 4-coumaroyl-CoA derived from the phenylpropanoid pathway to give rise to the C15 flavonoid skeleton, naringenin chalcone (Hahlblock and Scheel 1989). In most angiosperms, CHS is encoded by multiple gene copies, whereas Arabidopsis, parsley, and snapdragon all have a single copy of the gene. The expression of each member of the CHS multigene family is induced by developmental signals and a variety of environmental cues such as light, pathogen attack, mechanical wounding, and nutrient stress (Dixon and Paiva 1995; Lawson et al. 1994; Logemann et al. 2000; Sakuta 2000; Zeier et al. 2004). The organization and differential regulation of individual genes in the CHS multigene family during development and in response to different environmental stimuli have been reported in detail (Christensen et al. 1998; Ryder et al. 1987; Tuteja et al. 2004; Wingender et al. 1989). Further substitutions following isomerization of naringenin chalcone lead to the formation of flavones, flavonols, and anthocyanins (Tanaka et al. 2008; Ozeki et al. 2011) (Fig. 1).
Fig. 1

Biosynthetic pathway of flavonoids. PAL phenylalanine ammonia-lyase, CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, FNS flavone synthase, DFR dihydroflavonol 4-reductase, LAR leucoanthocyanidin reductase, ANS anthocyanidin synthase, ANR anthocyanidin reductase, UFGT UDP-glucose: flavonoid 3-O-glucosyltransferase

Dihydroflavonol 4-reductase (DFR) is the first committed enzyme of anthocyanin and proanthocyanidin (PA) biosynthesis in the late flavonoid biosynthetic pathway; it catalyzes the NADPH-dependent reduction of dihydroflavonols into leucoanthocyanidins. Leucoanthocyanidins are converted into colored anthocyanidins by anthocyanidin synthase (ANS). The subsequent stabilization of anthocyanidins by glucosylation and acylation leads to a variety of anthocyanins. The DFR genes of various plant species have been cloned (Beld et al. 1989; Helariutta et al. 1993; Sparvoli et al. 1994; Bernhardt et al. 1998) and the regulation of DFR expression has been studied in several species (Dooner et al. 1991; Martin et al. 1991; Quattrocchio et al. 1993). The role of DFR as a regulatory enzyme in anthocyanin biosynthesis has been well characterized. DFR contributes to the control of the range of flower color (Tanaka et al. 1998). Flower pigmentation patterns involving color flecks and sectors are controlled by the mutation of DFR by transposable elements (Inagaki et al. 1999; Itoh et al. 2002).

Anthocyanidin synthase is responsible for the formation of colored anthocyanidins from colorless leucoanthocyanidins. Genes and cDNAs encoding ANS have been isolated from a number of plant species. The deduced amino acid sequences imply that ANS probably belongs to a family of 2-oxoglutarate-dependent oxygenases, but precise biochemical information about the ANS reaction mechanism is lacking. Saito et al. (1999) provided the first direct evidence that ANS is a 2-oxoglutarate-dependent oxygenase that converts leucoanthocyanidins to 3-flaven-2,3-diol. With glucosylation at the C-3 position of 3-flaven-2,3-diol by UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) followed by its transport into vacuoles, colored anthocyanidin 3-glucoside is formed (Nakajima et al. 2001). Further modification of anthocyanidins, glycosylation, and acylation of aglycons generates a variety of anthocyanins in higher plants. For more detailed descriptions, especially of the biochemical mechanisms and cellular compartmentalization of glycosylation and acylation of aglycones, see Ozeki et al (2011).

Proanthocyanidins are a group of polyphenolic secondary metabolites that are synthesized as oligomers or polymers of flavan-3-ol units via the flavonoid pathway. PAs are derived from the pathway that produces anthocyanins. PAs and anthocyanins share the same biosynthetic precursor, leucoanthocyanidin, which is synthesized from phenylalanine via the general phenylpropanoid and “early” flavonoid biosynthetic pathway (Fig. 1). The “late” biosynthetic pathway for PAs, which produces the building blocks of PAs, involves leucoanthocyanidin reductase (LAR) for the production of (+)-flavan-3-ols such as (+)-catechin (Tanner et al. 2003), ANS, and anthocyanidin reductase (ANR) for the production of (−)-(epi)-flavan-3-ols such as (−)-epicatechin (Fig. 1). However, the steps involved in their transport to the vacuole, hydroxylation, and polymerization of the monomers of (epi)catechin are unknown.

Regulation of anthocyanin biosynthesis

Two clusters of co-regulated structural genes can generally be distinguished in flavonoid biosynthesis: early biosynthetic genes (i.e., CHS, CHI, F3H, FNS and FLS), which are involved in the synthesis of flavones and flavonols, and late biosynthetic genes (i.e., DFR, ANS, UFGT, LAR and ANR), which are involved in anthocyanin and PA biosynthesis (Mol et al. 1998; Gonzalez et al. 2008) (Fig. 1). CHS catalyzes the first and key regulatory step of flavonoid synthesis, which is stimulated by developmental signals and various environmental cues. Delineation of the cis elements and their trans factors underlying the rapid activation of CHS genes provides a basis for characterizing the terminal stages of the signal transduction pathway involved in the deployment of early transcription-dependent defenses. G- and H-box motifs are essential for the transcriptional activation of CHS in response to developmental and environmental cues (Arias et al. 1993; Loake et al. 1992). Some basic leucine zipper (bZIP) transcription factors responsible for the UV-, pathogen-, or stress-responsive activation of flavonoid biosynthetic genes have been identified, although MYB and basic helix-loop-helix (bHLH) transcriptional activators appear to be responsible for the tissue-specific expression of genes involved in flavonoid biosynthesis. G-box/H-box binding factor1 (G/HBF-1), a bZIP protein that binds to the G-box and adjacent H-box in the proximal region of the CHS15 promoter, is rapidly phosphorylated in elicited soybean cells and binds to the CHS15 promoter (Dröge-Laser et al. 1997). G/HBF-1 was initially reported to have been isolated from soybean, but the isolated cDNA clone was subsequently reported to have originated from tobacco and was found not to be full-length. The full-length cDNA for a tobacco bZIP protein was later isolated and designated BZI-1 (Heinekamp et al. 2002). Soybean bZIP protein1 (SBZ1) was identified as a transcription factor involved in the activation of plant defense responses. Recombinant SBZ1 was shown to bind CHS7 and CHS1 at the promoter region, which confers the elicitor-inducible expression of CHS genes (Yoshida et al. 2008b). BZI-1 and SBZ1 are closely related to common plant regulatory factor2 (CPRF2) from parsley (Kircher et al. 1998). Members of the CPRF bZIP transcription factor family are proposed to be involved in the regulation of CHS genes by light (Weisshaar et al. 1991; Wellmer et al. 1999, 2001). CPRF2 is a phosphoprotein in vivo and its phosphorylation is rapidly increased in response to light. In contrast to G/HBF-1, the phosphorylation of CPRF2 does not alter its DNA-binding activity (Kircher et al. 1999; Wellmer et al. 1999).

The accumulation of anthocyanins and PAs is induced by the expression of late genes of the flavonoid pathway, which are regulated by a ternary transcriptional complex containing an R2R3-MYB-type transcription factor, a bHLH transcription factor, and a WD40 repeat (WDR) protein (Baudry et al. 2004; Broun 2005; Koes et al. 2005; Ramsay and Glover 2005). WDR proteins and bHLH factors show relatively ubiquitous expression during the regulation of distinct developmental processes such as the formation of trichomes, root hairs, and seed coat mucilage (Schiefelbein 2003; Zhang et al. 2003; Broun 2005), as well as the accumulation of anthocyanins and proanthocyanins, which are determined by physical interactions of WDR and bHLH proteins with more specific MYB proteins (Broun 2005; Koes et al. 2005; Ramsay and Glover 2005). The MYB superfamily consists of more than 100 members in higher plants (198 MYB genes in the Arabidopsis genome, 183 in rice [Yanhui et al. 2006], and 158 in Lotus japonicus [Sato et al. 2008]). A dramatically expanded R2R3-MYB family characterized by two imperfectly conserved repeated N-terminal MYB domains, each of which forms a DNA-binding helix-turn-helix structure that is an important protein–protein interaction motif, has been well characterized (Feller et al. 2011).

PA accumulation in the Arabidopsis seed coat is regulated by a ternary transcription complex containing transparent testa 2 (TT2), an R2R3-MYB-type transcription factor, TT8, a bHLH transcription factor, and TTG1, a WDR protein (Baudry et al. 2004). Production of anthocyanin pigment1 (PAP1) and production of anthocyanin pigment2 (PAP2) in Arabidopsis (Gonzalez et al. 2008) and anthocyanin2 (AN2) in Petunia (Quattrocchio et al. 1999) are redundant R2R3-MYB transcription factors and positive regulators of the genes involved in anthocyanin biosynthesis. In L. japonicas, the DFR, ANS, and ANR promoters were tested as potential targets of the transcription factors LjTT2 and LjPAP (Yoshida et al. 2008a, 2010b). When LjTT8 and LjTTG1 were co-expressed, LjTT2 regulated the expression of DFR, ANS, and ANR. The combination of LjPAP with LjTT8 and LjTTG1 also showed significant activation of the DFR and ANS promoters, but relatively weak activation of the ANR promoter, suggesting that LjPAP is specifically responsible for anthocyanin production by regulating the expression of DFR and ANS (Yoshida et al. 2010a).

Betacyanin biosynthesis

The anthocyanin biosynthetic pathway is one of the best-studied examples of secondary metabolism in higher plants, but the biochemistry and genetics of the betalain biosynthetic pathway remain relatively uncharacterized (Tanaka et al. 2008; Ozeki et al. 2011; Sakuta and Ohmiya 2011). Radioactive feeding experiments revealed that betalains are synthesized from tyrosine via DOPA (Garay and Towers 1966; Nassif-Makki and Constabel 1972), and that DOPA is converted to the betalain intermediates betalamic acid and cyclo-DOPA (Miller et al. 1968). Feeding experiments using doubly-labeled tyrosine showed that extradiol cleavage, a step in common with the synthesis of stezolobic acid (Saito and Komamine 1976, 1978), and subsequent closure by the binding of nitrogen to carbon 3 gives rise to betalamic acid (Fischer and Dreiding 1972; Impellizzeri and Piattelli 1972). The conjugation of betalamic acid and amino acids or amines leads to the formation of yellow betaxanthins, while the conjugation of betalamic acid and cyclo-DOPA results in the formation of betanidin, the aglycone of red betacyanin (Fig. 2). These condensation reactions occur spontaneously (Schliemann et al. 1999; Strack et al. 2003). Betacyanins are normally stored as glycosides in vacuoles after two types of glucosylation reactions at either the betanidin step (Sciuto et al. 1972; Heuer and Strack 1992) or the cyclo-DOPA step (Wyler et al. 1984; Sasaki et al. 2004).
Fig. 2

Biosynthetic pathway of betalains. I tyrosine hydroxylase, II DOPA 4,5-dioxygenase, III cytochrome P450 (CYP76AD1), IVcyclo-DOPA 5-O-glucosyltransferase, V spontaneous reaction, VI betanidin 5-O-glucosyltransferase

Extensive studies of the biosynthetic reactions of betalains led to the discovery of several enzymes that function in early betalain biosynthesis, including polyphenol oxidase-type tyrosinase (Joy et al. 1995; Steiner et al. 1996, 1999) and extradiol DOPA dioxygenase (DOD) (Gandía-Herrero and García-Carmona 2013). Tyrosinase is a copper-type bifunctional enzyme that catalyzes the hydroxylation of tyrosine to DOPA and the subsequent oxidation reaction that converts DOPA to O-quinone (Strack and Schliemann 2001). The cyclization of O-quinone to cyclo-DOPA is assumed to be spontaneous (Strack et al. 2003). However, a recent study suggested that separate enzymes are required to produce l-DOPA from tyrosine and to produce the cyclo-DOPA moiety. CYP76AD1 encoding a novel cytochrome P450 is involved in cyclo-DOPA moiety production in betacyanin biosynthesis in beets (Hatlestad et al. 2012). Tyrosine hydroxylation activity, which converts tyrosine to DOPA, is separated from the oxidation of DOPA to DOPA O-quinone in Portulaca grandiflora (Yamamoto et al. 2001), but the corresponding gene is as yet unknown.

The extradiolic 4,5-cleavage of DOPA is required for the formation of betalamic acid (Fig. 2) and is catalyzed by extradiol DOPA-4,5-dioxygenase. DOD was first detected in the betalain-producing fungus Amanita muscaria (Girod and Zrÿd 1991) and later characterized as extradiol DOPA-2,3- and -4,5-dioxygenase (Hinz et al. 1997; Mueller et al. 1997b). Genetic complementation of white petals of P. grandiflora by particle bombardment transformation indicated that Amanita DOD catalyzed both 4,5- and 2,3-aromatic ring cleavage. This result showed that the formation of not only betalains, but also muscaflavin, a pigment that is not found in higher plants, is catalyzed by Amanita DOD (Mueller et al. 1997a). A novel plant DOD, distinct from Amanita DOD, was originally isolated as a gene that encodes the DOPA-4,5-dioxygenase from P. grandiflora (Christinet et al. 2004). The role of Portulaca DOD in the betalain biosynthetic pathway was determined by genetic complementation in white petals of P. grandiflora, in which the set of genes for color formation is missing (Christinet et al. 2004). Recently, the enzymatic activity of recombinant DOD was shown to catalyze the conversion of DOPA to betalamic acid in vitro (Sasaki et al. 2009; Gandía-Herrero and García-Carmona 2012).

Betacyanins are normally stored as glycosides in vacuoles. Two different forms of glucosylation reactions occur in the betacyanin biosynthetic pathway. The first is at the cyclo-DOPA step and the second is at the betanidin step, similar to the biosynthetic processes of other plant secondary products such as anthocyanins. Two regiospecific betanidin glucosylating enzymes, betanidin-5-O-glucosyltransferase (5-GT) and betanidin-6-O-glucosyltransferase (6-GT), were partially purified from Livingstone daisy (Dorotheanthus bellidiformis) cell cultures (Heuer and Strack 1992). Although 5-GT and 6-GT discriminate between individual hydroxyl groups of their respective substrates, they catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, and anthocyanidins (Vogt et al. 1997, 2002).

On the other hand, cyclo-DOPA-5-O-glucoside, rather than betanidin or betanin, was shown to be an efficient precursor of amaranthin in Celosia cristata (Sciuto et al. 1974). In addition, cyclo-DOPA-5-O-glucoside accumulates in young beet plants (Wyler et al. 1984) and root peels of red beet (Kujala et al. 2001). cyclo-DOPA5GT activity has been detected in crude extracts prepared from red petals of Mirabilis jalapa and several betacyanin-producing plants (Sasaki et al. 2004). Furthermore, cDNAs encoding cyclo-DOPA5GT were isolated from the petals of M. jalapa and the inflorescences of C. cristata (Sasaki et al. 2005b). The expression profile of cyclo-DOPA5GT agreed with cyclo-DOPA5GT activity during the development of M. jalapa petals. cyclo-DOPA-5-glucoside glucuronosyltransferase activity was detected in C. cristata (Sasaki et al. 2005a), indicating that modification with the glucuronic acid moiety occurs at cyclo-DOPA. These facts indicate the presence of dual pathways for glycosylation in betacyanin biosynthesis. Additional studies of the glycosylation and subsequent acylation in betalain biosynthesis may elucidate which pathway is the main route for betalain production, or if the step at which glycosylation and acylation occurs is species-dependent.

Factors controlling betacyanin biosynthesis

Betacyanin accumulation in the Caryophyllales is markedly affected by environmental factors. In the last quarter of 20th century, the effects of various environmental factors on betacyanin accumulation were investigated. Light-stimulated betacyanin synthesis is mediated by the red/far-red reversible action of phytochromes (Nicola et al. 1973a, 1973b, 1974; Elliott 1979c; Spasic et al. 1985). Plant growth regulators also affect betacyanin accumulation. In particular, cytokinins markedly promote betacyanin accumulation (Piatteri 1976). The action of light and kinetin on betacyanin synthesis was analyzed (Kochhar 1972a, 1972b; Kochhar et al. 1981). In etiolated Amaranthus seedlings, exogenously supplied cytokinins strongly promote betacyanin accumulation in the dark (Bauberger and Mayer 1960). This occurs mainly in two specific tissues, the lower epidermal cells of cotyledons, excluding guard cells, and hypocotyl endodermis (Elliott 1983). A bioassay for cytokinins using betacyanin accumulation as a marker in Amaranthus seedlings was reported (Biddington and Thomas 1973) and the variability of this method was discussed (Elliott 1979a, b, c, d). It was also reported that betacyanin accumulation is affected by various growth regulators other than cytokinins. In suspension cultures of P. americana, betacyanin accumulation was strongly stimulated by 2,4-D at a concentration of 5 μM (Sakuta et al. 1991), but betacyanin accumulation in Beta vulgaris callus cultures was suppressed by 2,4-D (Constabel and Nassif-Makki 1971). Betacyanin accumulation in P. grandiflora callus cultures was increased by NAA, another synthetic auxin, at a concentration of 1 ppm (Endress 1976). Inhibitory effects of gibberellic acid and abscisic acid on betacyanin accumulation were shown in seedlings of Amaranthus (Biddington and Thomas 1977; Stobert and Kinsman 1977; Guruprasad and Laloraya 1980) and suspension cultures of P. americana (Hirano et al. 1996). However details of the mode of action of growth regulators on betacyanin accumulation are poorly understood.

Molecular mechanism of the mutual exclusion of anthocyanins and betacyanins

Anthocyanins and betacyanins never occur together in the same plant. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales, except in the families Caryophyllaceae and Molluginaceae (Mabry 1980; Strack et al. 2003). Anthocyanins have not been found in the betacyanin-producing Caryophyllales (Harborne 1996). This curious mutual exclusion has been examined from genetic and evolutionary perspectives (Koes et al. 1994; Stafford 1990, 1994; Brockington et al. 2011), but little is known about the molecular basis.

Although the molecular mechanism of betacyanin biosynthesis is still relatively poorly understood, the biosynthetic pathway of flavonoids is one of the best-studied examples of secondary metabolism in higher plants. Most flavonoid biosynthetic genes have been cloned and analyzed, and factors that control transcription of the genes have been isolated by genetic means. Whereas anthocyanins are absent in almost all members of the Caryophyllales, species of this family do contain other flavonoids, especially flavonols (Iwashina 2001). For example, the yellow tepals of the Astrophytum species contain the flavonol glycosides quercetin-3-O-galactoside and quercetin-3-O-rhamnosylglucoside together with the aglycones quercetin, kaempferol, and isorhamnetin in the form of spherical crystals (Iwashina et al. 1988). Dihydroflavonols occur at the branch point in the flavonoid biosynthetic pathway leading to flavonols and anthocyanins (Fig. 1). This suggests that anthocyanin biosynthesis from dihydroflavonols may be blocked in the Caryophyllales. Some insight can be gained from DFR and ANS, which are involved in the conversion of dihydroflavonols to anthocyanins. The isolation and functional identification of DFR and ANS from spinach (Spinacia oleracea) and pokeweed (P. americana), which are non-anthocyanin-producing plants of the Caryophyllales, was reported (Shimada et al. 2004, 2005). Expression profiling revealed that DFR and ANS were not expressed in most tissues and organs other than seeds in S. oleracea.

One possible explanation for the lack of anthocyanin synthesis in the Caryophyllales may be the suppression or limited expression of DFR and ANS (Shimada et al. 2005). The evolution of cis-regulatory elements was proposed to be a major source of morphological diversification, as mutations in cis-regulatory elements often lead to dramatic tissue-specific pattern changes while preserving the essential roles of these genes in other processes (Shapiro et al. 2004; Gompel et al. 2005). The modification of DFR and ANScis-regulatory elements may have led to limited expression of DFR and ANS, resulting in defective anthocyanin synthesis in the Caryophyllales (Shimada et al. 2007). Another possibility is a loss of function of transcriptional regulator(s) of anthocyanin biosynthetic genes in the Caryophyllales. The presence of abundant ANS and DFR transcripts in seeds is likely to contribute to PA synthesis in the seed coat in which TT2 homologs might regulate the expression of the biosynthetic genes involved. The characterization and functional analysis of anthocyanin regulators such as PAP-like homologs may provide further insights into the lack of anthocyanin synthesis in the Caryophyllales. A more detailed analysis of the promoters and characterization of regulators of flavonoid biosynthesis will provide further understanding of the mechanisms that regulate flavonoid biosynthesis in the Caryophyllales and may reveal why these plants do not produce anthocyanins.

Recent extensive biochemical and molecular studies of betacyanin biosynthesis have provided insight into the evolution of betacyanin synthesis. The identification and characterization of plant DOD in P. grandiflora (Christinet et al. 2004), M. jalapa (Sasaki et al. 2009), P. americana (Takahashi et al. 2009), and B. vulgaris (Gandía-Herrero and García-Carmona 2012) advanced the molecular biological analysis of betalain biosynthesis in the Caryophyllales. In addition, some DODs from non-betalain-producing plants possess DOD activity in vitro (Tanaka et al. 2008), although the functions of these homologs in vivo remain elusive. The analyses of DOD homologs in species of both betalain-producing and non-betalain-producing plants should provide a better understanding of the evolution of DOD genes in betalain biosynthesis in the Caryophyllales. The amino acid sequences following His in the conserved H-P-S/A-N/D-x-T-P motif found in the DODs of betalain-producing plants were suggested to be involved in the substrate specificity of DOD (Christinet et al. 2004). Therefore, it would be interesting to clarify the contribution of the conserved motif to the substrate affinity of DOD and to see if DODs in non-betalain-producing plants have different substrate specificities and functions compared to DOD in betalain-producing plants. However, little is known about naturally occurring products of extradiol aromatic ring-cleaving activities in higher plants with the exception of stizolobic acid and stizolobinic acid in Stizolobium hassjoo (Saito and Komamine 1976, 1978). This makes it difficult to perform experimental studies on the substrate specificity of DODs. It is possible that DOD in the Caryophyllales was duplicated early in evolution and that the accumulation of base substitutions led to different regulatory systems or novel functional roles in betalain-producing plants. Therefore, further analysis of DOD homologs in other species of both betalain-producing and non-betalain-producing plants should provide a better understanding of the evolution of DOD genes involved in betalain biosynthesis in the Caryophyllales.

Betalain accumulation was observed in transgenic Arabidopsis overexpressing fungal or plant DOD when supplied with DOPA (Harris et al. 2012). These results indicate that in addition to the expression of active DOD, the presence of DOPA might act as a regulatory factor for betalain biosynthesis. These results support the hypothesis that the lack of betalain in these plants is due to the absence of DOPA. In the plant kingdom, DOPA has only been found in the betalain-producing Caryophyllales and some legume species (Komamine 1962) in which betalains have not been found. DOPA in the Caryophyllales is thought to be synthesized from tyrosine by tyrosine hydroxylase or tyrosinase (Steiner et al. 1996, 1999; Yamamoto et al. 2001). However, the biological function and regulatory mechanisms of DOPA metabolism in higher plants have not been elucidated. Further analysis of DOD homologs in various plants such as legumes might reveal the contribution of DOD to betalain biosynthesis in the Caryophyllales. Recent advances in bioinformatics will provide effective tools for gaining insight into differences in catalytic activity and substrate specificity of enzymes. We can now combine experimental and computational approaches to study the biological properties of enzymes. Such methods are sure to effectively guide and shorten the path to determining the functionally important residues of the enzymes involved in betalain synthesis.

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