FIGURE 8.9 Examples of glycosylation (A) and acylation (B) reactions of betacyanins. The enzymes are uridine 5'-diphosphoglucose:betanidin 5-0- and 6-O-glucosyltransferases, 5-GT and 6-GT, respectively. For the acylation, HCA is the 1-O-hydroxycinnamoyl-transferase. (Adapted from Vogt et al.96 and Delgado-Vargas et al.17)

between betalamic acid and an amine group inside the vacuole, and new betaxanthins (vulgaxanthin III and IV) are formed when hairy root cultures of B. vulgaris var. Lutea are supplemented with the corresponding L-amino acids.99 Consequently, scientific evidence exists supporting that condensation of betalamic acid with amino acids is a spontaneous process (Figure 8.8C).

Betalain-decoloring enzymes have been found in different plants such as B. vulgaris,100'101 A. tricolor,90 and Phytolacca americana.102 These enzymes have a metal ion in the active site. In addition, it has been suggested that they are similar to peroxidases,103 and indeed horseradish peroxidase catalyzes the oxidation of betanin with betalamic acid, one of the final products.104 Betalain-oxidase enzymes have been also described in Amaranthus spp., and betalain extraction must be carefully controlled to avoid degradation.105

b. Biosynthesis Regulation

Betalain production is complex, as observed with other secondary metabolites; different physiological and environmental factors are involved. Supplementation of seedlings with l-DOPA or kinetin as well as light exposition induces a significant increase in betalain production.106

Interestingly, free betalamic acid, which is absent in plants that produce only betacyanins, is detected in betaxanthin-producing plants, suggesting a regulatory mechanism during its biosynthesis.84 It appears that in plants producing betacyanins exclusively, the biosynthesis of betalamic acid and cyclo-DOPA is coordinated with their condensation, with the accumulation of betalamic acid arrested.87

Four color genotypes have been isolated from cell lines of red beet producing betalains; betaxanthin and betacyanin appear to occur through a limited number of discrete, stable, and differentiated states. It has been proposed that a range of colors is observed in cell lines by effect of DNA modifications such as transpositions, translocations, inversions, breakages, and fusions, resulting in the relocation of genes within the same or another chromosome. Additionally, betalain synthesis could be tightly coupled to cellular morphology.94 With suspension cultures of P. americana, inhibitors of cell division induce the accumulation of betacyanins, probably as a result of the lack of DNA synthesis or because the cell cycle is arrested.107

c. Molecular Biology of Betalain Biosynthesis

As previously mentioned, most of the work on betalain biosynthesis is related to purification of enzymes. Particularly, enzyme activities involved in glycosylation and degradation of betalains have been isolated, but molecular biology information is scarce.88 Two cDNA clones encoding for polyphenol oxidase have been isolated from P. americana producing betalains. It is suggested that betalain production is regulated at the transcriptional level because substantial levels of mRNA are observed only in the ripening of betalain-containing fruits.108 From A. muscaria comes the gene dodA, which encodes for a DOPA dioxygenase,88 for transformation of P. grandiflora petals to produce muscaflavin pigment, a betalain not common in plants.109 In brief, it is necessary to develop much more research work to unravel the biosynthesis and regulation of betalains.

5. Functions a. Taxonomic Markers

As discussed above, betalain production is restricted to the order Caryophyllales and only two families have anthocyanins instead of betalains, Caryophyllaceae and Molluginaceae, suggesting an early differentiation of this order into groups with different kinds of pigments.84 It has been proposed that the order Centrospermae, including Cactaceae, must be reserved for betalain-containing families.83

As can be observed, betalains and anthocyanins have very different structures and their distribution is mutually exclusive. Thus, the use of betalains as taxonomic markers is warranted. Moreover, the appearance of betalains in higher fungi, clearly not related to flowering plants, could be a case of chemical convergence under an evolutionary phase.83

b. Ecological and Physiological Aspects

As with anthocyanins, betalains are present in flowers or fruits and may play a role as attractants for vectors (insects or birds) in the pollination process and in seed dispersal by animals.83110 Their appearance in other plant structures such as leaves, stem, and root is not functionally easy to explain. However, betalain accumulation in red beet root has been related to the storage of carbohydrates as a physiological response under stress conditions.111 Betalains have been proposed as a defense mechanism because they are accumulated when tissues are injured. Moreover, their appearance occurs in association with antifungal proteins in some plants.112

Interestingly, betanin and vulgaxanthin are effective inhibitors of indoleacetic acid (IAA) oxidase. It has been observed that the inhibitory effect of IAA on wheat root elongation is counteracted by betanin. Thus, betalains could modulate the effect and metabolism of processes involving auxins; however, this role has not been corroborated directly in plants.83

6. Methodological Aspects a. Extraction

The betalain extraction process is carried out as described in Figure 8.10.17 To achieve complete extraction, methanol or ethanol solutions are preferred.83 It is also recommended that the extraction be carried out at low temperature and in darkness. The extract obtained by this procedure contains a large quantity of sugars; thus, tinctoreal power is low. Consequently, a fermentation process reduces the sugar content and improves the coloring agent. Sometimes, it is desirable to inactivate degradative enzymes by heating (70°C, 2 min), although this may destroy some of the pigments. By their structural nature, betalains are basic compounds and greater purification can be achieved by a slight acidification with hydrochloric acid or with acidified ethanol (0.4 to 1% HCl); subsequently, the addition of 95% aqueous ethanol yields betaxanthins.85113

b. Separation and Purification

Common methodologies used in betalain analysis are shown in Table 8.17.17 Analytical separations include electrophoresis and TLC. By using electrophoresis, betalain isomers and aglycones have been separated, which makes it a powerful methodology for analysis.114 Electrophoretic mobility for betaxanthins has been related to indicaxanthin and for betacyanins with betanin.85 Reports on TLC are scarce because of the high polarity of betalains; however, introduction of acids improves the separation (Table 8.17). Betalains are directly observed after chromatographic

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