Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica.

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l-1 betanin and 55 ± 7 mg l-1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr-1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

The Antioxidant Activity of Betanin protects MRC-5 cells Against Cadmium Induced Toxicity.

Cadmium (Cd) can induce both acute and chronic effects in the lungs depending on the time and the exposure route. Betanin is a component derived from the roots of red beets and it is well-known for its antioxidant and anti-apoptosis effects. The current study aimed to survey the protective effects of betanin on cell toxicity induced by Cd. Different concentration of Cd alone and in combination with betanin was assessed in MRC-5 cells. The viability and oxidative stress were measured using resazurin and DCF-DA methods respectively. Apoptotic cells were assessed by PI staining of the fragmented DNA and western blot analysis detected the activation of caspase 3 and PARP proteins. Cd exposure for 24 h declined viability and increased ROS production in MRC-5 cells compared to the control group (p < 0.001). Also, Cd (35 µM) elevated DNA fragmentation (p < 0.05), and the level of caspase 3-cleaved and cleaved PARP proteins in MRC-5 cells (p < 0.001). Co-treatment of cells with betanin for 24 h significantly enhanced viability in concentrations of 1.25 and 2.5 µM (p < 0.001) and 5 µM (p < 0.05) and declined ROS generation (1.25 and 5 µM p < 0.001, and 2.5 µM p < 0.01). As well as, betanin reduced DNA fragmentation (p < 0.01), and the markers of apoptosis (p < 0.001) compared to the Cd-treated group. In conclusion, betanin protects lung cells against Cd-induced toxicity through antioxidant activity and inhibition of apoptosis.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

Betanin alleviates oxidative stress through the Nrf2 signaling pathway in the liver of STZ-induced diabetic rats.

BACKGROUND: Continuing hyperglycemia causes and exacerbate oxidative stress. Betanin as the principal pigment of red beet root has antioxidant, anti-inflammatory, and anti-diabetic properties. The purpose of this study was to investigate the potency of betanin on antioxidant defense in STZ-induced diabetic rats' livers. METHODS: STZ at a single dose of 60 mg/kg body weight was intraperitoneally injected and betanin (10, 20, and 40 mg/kg/day) was administered orally for 28 days. Malondialdehyde (MDA), total antioxidant capacity (TAC), protein carbonyl (PC) levels, and the enzyme activity of superoxide dismutase (SOD), catalases and glutathione peroxidases (GPx) were evaluated in the liver. Furthermore, gene expression of Nrf2 and mentioned antioxidant enzymes were measured by Real-time PCR. RESULTS: Betanin (10 and 20 mg/kg) significantly reduced PC levels and increased antioxidant enzyme activity in diabetic rats compared to the control diabetic group (P < 0.01). In comparison to the diabetic control group, all studied genes expression in diabetic rats were increased significantly with betanin at doses of 10 and 20 mg/kg (P < 0.02). The increase in gene expression at 20 mg/kg of betanin was significantly stronger than others (P < 0.015) except for the catalase (P = 0.201), that was almost the same. Moreover, treatment of diabetic rats with 20 mg/kg of betanin could significantly increase TAC levels (P < 0.05) and decrease MDA levels (P < 0.001) compared to diabetic control group. CONCLUSIONS: Betanin could increase the antioxidant capacity of liver tissue associated with the Nrf2-mediated pathway in a dose-dependent manner.

Study of the electrochemical betanidin oxidation path using computational methods.

Betalains can be used in the food, drug, and cosmetic industries and have shown their bioactive potential. For these reasons, unraveling their oxidation mechanism is of high importance and demands a systematic and multidisciplinary study. Moreover, the properties mentioned above are drastically influenced by pH and other physicochemical conditions. Betanidin (1) is a relevant molecule of this family and is crucial to elucidating the oxidation mechanism in which its pigment is involved. In the present study, the pKas and oxidation potential values for all protic groups of 1 were analyzed using B3LYP/6-31+G(d,p)/SMD as the computational methodology. Moreover, six explicit water molecules were added to improve the solvation-free energy values. The oxidation mechanism at each pH was constructed and analyzed in depth. On the other hand, cyclic voltammetry simulations allowed obtaining electrochemical data from experiments and support the proposed mechanism. In the present work, the main oxidation path of 1 is described and consists of a concerted electron-proton transfer followed by a sequential electron and proton transfer to obtain the o-quinone product or a quinone methide molecule.

Dehydrogenation of Betacyanins in Heated Betalain-Rich Extracts of Red Beet (Beta vulgaris L.).

Betacyanins are a group of water-soluble red-violet compounds containing nitrogen in their structure. These are biosynthesized in red beetroot (Beta vulgaris L.), a widely consumed vegetable that contains significant amounts of nutritious and bioactive compounds which are also found in dietary supplements. This contribution presents results of betacyanin thermal oxidation (resulting in dehydrogenation) interrelated with decarboxylation in selected acetate/phosphate buffers at pH 3-8 and at 85 °C, which may be of particular significance for formulation and performance of foods. Most of the reaction products were detected at the highest concentrations in the acidic solutions (pH 3-4). The main dehydrogenation reaction pathways were monitored by LC-DAD-MS/MS and were associated with decarboxylation of the principal extract pigments, betanin/isobetanin and neobetanin, at carbon positions C-2 and C-17. Additional reactions are accompanied by the 2,15-decarboxylation processes at different dehydrogenation levels with 15-decarboxy-betanin and 2,15-bidecarboxy-betanin, structurally elucidated by NMR analysis, as the distinct indicators of this route type. For other novel pigments detected, 2,15-bidecarboxy-xanbetanin, 2,15-bidecarboxy-xanneobetanin and 2,15,17-tridecarboxy-neobetanin, additional high resolution mass spectrometric analyses were performed and confirmed their molecular formulas.

Multi-colored shades of betalains: recent advances in betacyanin chemistry.

Covering: 2001 to 2021Betacyanins cover a class of remarkable natural red-violet plant pigments with prospective chemical and biological properties for wide-ranging applications in food, pharmaceuticals, and the cosmetic industry. Betacyanins, forming the betalain pigment group together with yellow betaxanthins, have gained much attention due to the increasing social awareness of the positive impact of natural products on human health. Betalains are commercially recognized as natural food colorants with preliminarily ascertained, but to be further investigated, health-promoting properties. In addition, they exhibit a remarkable structural diversity based on glycosylated and acylated varieties. The main research directions for natural plant pigments are focused on their structure elucidation, methods of their separation and analysis, biological activities, bioavailability, factors affecting their stability, industrial applications as a plant-based food, natural colorants, drugs, and cosmetics as well as methods for high-yield production and stabilization. This review covers period of the last two decades of betacyanin research. In the first part of the review, we present an updated classification of all known betacyanins and their derivatives identified by chemical means as well as by mass spectrometric and NMR techniques. In the second part, we review the current research reports focused on the chemical properties of the pigments (decarboxylation, oxidation, conjugation, and chlorination reactions as well as the acyl group migration phenomenon) and describe the semi-synthesis of natural and artificial fluorescent betalamic acid conjugates, showing various prospective research directions.

Color attributes, betacyanin, and carotenoid profiles, bioactive components, and radical quenching capacity in selected Amaranthus gangeticus leafy vegetables.

Four selected A. gangeticus accessions were evaluated in terms of color attributes, phytopigments, including betaxanthin, betacyanin, and carotenoid profiles, proximate, minerals, and antioxidant capacity (AC). Color attributes, phytopigments, proximate, minerals, and AC of A. gangeticus significantly varied across the accessions. For the first time, we identified four betacyanin compounds, such as amaranthine, iso-amaranthine, betanin, iso-betanin. We also identified five carotenoid compounds zeaxanthin neoxanthin, violaxanthin, lutein, and pro-vitamin A in A. gangeticus accessions. A. gangeticus contained adequate carbohydrates, protein, moisture, and dietary fiber. We found adequate iron, manganese, copper, zinc, sodium, molybdenum, boron, potassium, calcium, magnesium, phosphorus, sulfur in A. gangeticus accessions. The accessions LS7 and LS9 had considerable color attributes, betacyanin, and carotenoid compounds, proximate, nutraceuticals, betalain, betaxanthin, and AC that could be used as preferable potent antioxidant varieties for consumption as sources of phytopigments, nutraceuticals, and antioxidants. The correlation study revealed that antioxidant constituents of A. gangeticus accession were strongly associated with AC. The identified components of betacyanin and carotenoid in A. gangeticus demands detail pharmacological study. The baseline data on color attributes, betacyanin, and carotenoid profiles, betaxanthins, betalains, and AC obtained in this present study could contribute to the scientific evaluation of pharmacologically active principles in A. gangeticus.

Evaluation of Cytotoxic Activity of Betanin Against U87MG Human Glioma Cells and Normal Human Lymphocytes and Its Anticancer Potential Through Mitochondrial Pathway.

Recent studies revealed an antioxidant activity and anticancer efficiency of betanin. In this study, we investigated the cytotoxic effects and the possible mechanisms of betanin-induced apoptosis against U87MG human glioma cells and compared the results to those of human normal lymphocytes. MTT assay, caspase-3 activation assays in cells and succinate dehydrogenases (SDH), mitochondrial swelling, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and cytochrome C release assays in isolated mitochondria were obtained from U87MG human glioma cells and noncancerous human lymphocytes The results illustrated the significant cytotoxic effect of betanin on U87MG human glioma cells, with a concentration value that inhibits 50% of the cell growth of 7 µg/ml after 12 h of treatment. MTT assay demonstrated that the betanin is selectively toxic to U87MG human glioma cells, and betanin induced cell apoptosis via activation of caspase-3 along with modulation of apoptosis-related mitochondria. Meanwhile, betanin selectively increased ROS formation, mitochondria swelling, MMP decrease, and cytochrome c release in cancerous mitochondria but in normal mitochondria. Based on the evidence obtained from this study, it is concluded that the betanin is a promising natural compound to fight U87MG human glioma cells via induction of apoptosis through activation of intrinsic pathways.

Betalains: Potential Drugs with Versatile Phytochemistry.

Currently, the demand for natural colorants is increasing instead of synthetic colorants for foodstuff, because they are harmless to human health. Betalain is group of compounds containing nitrogen and water soluble pigment. Betalain is classified into two main classes, betacyanin which is the condensation of betalamic acid with cyclo-DOPA and betaxanthin which is the conjugation of amino acid or amines with betalamic acid. They are used to color various foods and medicines. Betalain is different from anthocyanin because betalains contain nitrogen in their structures. It is interesting to hear that betalains and anthocyanins are individually significant but they have not seen together in the same plant. Their stability influenced by various factors such as, temperature, pH, water activity and light. In this review basic chemistry of betalains, classes, subclasses, their sources and biosynthesis, factors affecting their stability, health and food industry applications are discussed. Moreover, mentioned work signifies the potent anticancer, antioxidant and antimalarial activities of betalains, furthermore provides a help to do more scientific work on it.

Advances and future directions in betalain metabolic engineering.

Betalains are nitrogenous red and yellow pigments found in a single order of plants, the Caryophyllales, and in some higher fungi. They are responsible for the colors observed in many ornamental plants, as well as in various food products, where they are used as natural colorants. Their nutritional properties and attractive colors make them an appealing target for metabolic engineering. This is further heightened by the limited availability of natural betalain sources, arising from their relative scarcity in the plant kingdom, particularly in edible plants. Recent progress in decoding their biosynthetic pathway has facilitated stable heterologous production of betalains in several plant and microbial systems. Here, we provide a brief review of recent advances and discuss current approaches and possible future directions in betalain metabolic engineering, including expanding the chemical diversity of betalains and increasing their yield, exploring new host organisms for their heterologous production, and engineering their secretion from the cell.

Thermoprotective properties of Opuntia ficus-indica f. inermis cladodes and mesocarps on sheep lymphocytes.

This study aims to investigate the thermoprotective properties of Opuntia ficus-indica f. inermis. Extracts were prepared from cladodes (CE) and mesocarps (ME), then subjected to a spectrophotometric and LC-MS analyses. Lymphocytes were isolated from peripheral blood of non-stressed sheep, supplemented with CE, ME, betanin or α-tocopherol, and subjected to two thermal treatments: 40 and 41 °C, for 6 h. Viable lymphocytes and H2O2 production were evaluated. The antioxidant activity of ME was 3.43 folds higher than CE. The LC-MS analysis of CE and ME allowed identifying 11 phenolic acids, 2 flavanones, 6 flavones, 3 flavonols and 1 betanin type betacyanin. Lymphocytes mortality increased linearly as function of the severity and the duration of heat stress. This mortality was correlated with H2O2 production. At 41 °C, only ME allowed maintaining lymphocytes viability. Moreover, ME was more efficient than CE in reducing H2O2 production. This thermoprotection was ensured by betaxanthin and betacyanin pigments. Interestingly, betanin was more efficient than α-tocopherol in preventing hyperthermia-induced lymphocytes' mortality. We report here for the first time the thermoprotective properties of cladodes and mesocarps of Opuntia ficus-indica f. inermis. Betanin was able to maintain lymphocyte viability through reducing H2O2 production, and therefore the oxidative-induced heat stress.

Isolation of amaranthin synthetase from Chenopodium quinoa and construction of an amaranthin production system using suspension-cultured tobacco BY-2 cells.

Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti-inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow-2 (BY-2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 µm (mean ± SEM) amaranthin and 26.60 ± 1.53 µm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV-1 protease activity, whereas betanin did not.

Plant betalains: Chemistry and biochemistry.

Betalains are vacuolar pigments composed of a nitrogenous core structure, betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid]. Betalamic acid condenses with imino compounds (cyclo-l-3,4-dihydroxy-phenylalanine/its glucosyl derivatives), or amino acids/derivatives to form variety of betacyanins (violet) and betaxanthins (yellow), respectively. About 75 betalains have been structurally unambiguously identified from plants of about 17 families (known till date) out of 34 families under the order Caryophyllales, wherein they serve as chemosystematic markers. In this review, all the identified betalain structures are presented with relevant discussion. Also, an estimated annual production potential of betalains has been computed for the first time. In addition, mutual exclusiveness of anthocyanins and betalains has been discussed in the wake of new evidences. An inclusive list of betalain-accumulating plants reported so far has been presented here to highlight pigment occurrence and accumulation pattern. Betalain synthesis starts with hydroxylation of tyrosine to DOPA, and subsequent cleavage of aromatic ring of DOPA resulting to betalamic acid formation. This pathway consists of two key enzymes namely, bifunctional tyrosinase (hydroxylation and oxidation) and DOPA dioxygenase (O2-dependent aromatic ring cleavage). Various spontaneous cyclisation, condensation and glucosylation steps complement the extended pathway, which has been presented here comprehensively. The biosynthesis is affected by various ecophysiological factors including biotic and abiotic elicitors that can be manipulated to increase pigment production for commercial scale extraction. Betalains are completely safe to consume, and contribute to health.

Transposon-mediated mutation of CYP76AD3 affects betalain synthesis and produces variegated flowers in four o'clock (Mirabilis jalapa).

The variegated flower colors of many plant species have been shown to result from the insertion or excision of transposable elements into genes that encode enzymes involved in anthocyanin synthesis. To date, however, it has not been established whether this phenomenon is responsible for the variegation produced by other pigments such as betalains. During betalain synthesis in red beet, the enzyme CYP76AD1 catalyzes the conversion of L-dihydroxyphenylalanine (DOPA) to cyclo-DOPA. RNA sequencing (RNA-seq) analysis indicated that the homologous gene in four o'clock (Mirabilis jalapa) is CYP76AD3. Here, we show that in four o'clock with red perianths, the CYP76AD3 gene consists of one intron and two exons; however, in a mutant with a perianth showing red variegation on a yellow background, a transposable element, dTmj1, had been excised from the intron. This is the first report that a transposition event affecting a gene encoding an enzyme for betalain synthesis can result in a variegated flower phenotype.

Purification and characterization of a betanidin glucosyltransferase from Amaranthus tricolor L catalyzing non-specific biotransformation of flavonoids.

Betacyanins are the major pigments present in Amaranthus tricolor, a leafy vegetable consumed globally. The terminal glycosylation of the aglycone betanidin is an important step in the biosynthesis of this natural red antioxidant pigment. A betanidin 5-O-glucosyltransferase (BGT) was fully purified to 134 folds (specific activity, 265.2 nkat mg(-1)) from the red amaranth by ammonium sulfate precipitation followed by hydrophobic interaction, anion exchange and size exclusion chromatography. Homogeneity of the purified protein was confirmed by 2-dimensional polyacrylamide gel electrophoresis (2D PAGE). The molecular weight of the enzyme determined by liquid chromatography-mass spectrometry (LC-MS) was found to be 62.8 kDa. Furthermore, the enzyme glycosylated flavonoids (kaempferol and quercetin) but not anthocyanidins, presence of which is mutually exclusive to betacyanin accumulating plants. The apparent Km (344±2.34 µM) and Vmax (17.24 µM min(-1)) of the enzyme were determined by LC-MS/MS. Peptide mass fingerprinting of the purified protein showed 38.4% coverage of peptide masses with anthocyanidin 3-O-glucosyltransferase from Zea mays. Study on this purified enzyme, for the first time, revealed its role of glycosylation in biosynthesis of betacyanin in A. tricolor and indicates promiscuous substrate-specificity.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

PURPOSE: This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. METHODS: Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. RESULTS: Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻6 and (3.2 ± 0.3) × 10⁻6 cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 µL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 µM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. CONCLUSIONS: Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Betacyanin and other antioxidants production during growth of Opuntia stricta (Haw.) fruits.

Mature cactus pears from Opuntia stricta have a dark purple color due to high betacyanin concentration, whose biosynthesis is initiated with the amino acid L-tyrosine as a primary precursor. This study followed the maturation and ripening processes of Opuntia stricta fruits to harvest them at high betacyanin and other antioxidant concentrations. Fruits lasted 9 months for final ripening. Physical and compositional changes at different maturation and ripening stages have been determined. Thus, ripe fruits were around 4.72 ± 0.10 cm length, 2.94 ± 0.05 cm diameter and 22.71 ± 0.20 g weight; moisture and pH were maintained at 87.05 ± 0.19 % and 3.37 ± 0.12, respectively. Purple pigment production started in the ovary of immature fruits four months after anthesis (MAA). Concentration of all analyzed metabolites increased from immature (4 MAA) until ripe (9 MAA) stage. In ripe fruits, reducing sugars were 4.72 ± 0.54 g/100 g ff and total phenols 135.17 ± 0.68 mg gallic acid/100 g ff. Metabolites identified by HPLC were the betacyanins: betanin (60.17 ± 1.08 mg/100 g ff), isobetanin (7.58 ± 0.94 mg/100 g ff) and betanidin (13.48 ± 0.87 mg/100 g ff). Also, L-ascorbic acid (35.03 ± 1.06 mg/100 g ff) and L-tyrosine (4.43 ± 0.73 mg/100 g ff) were determined. Furthermore, the addition of L-tyrosine or L-dopa to fruit pulp of moderately ripe fruits, increased betacyanin concentrations 17 (103.3 ± 3.8 mg/100 g) and 32 % (114.3 ± 4.1 mg/100 g), respectively.

Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA.

BACKGROUND: Carotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding. RESULTS: The biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of Solanum tuberosum (potato) and petals of Antirrhinum majus. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from Amanita muscaria) or a plant DOD (from Portulaca grandiflora), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed Arabidopsis thaliana (arabidopsis) lines containing 35S: AmDOD produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production. CONCLUSIONS: The fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to cyclo-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an enzyme capable of converting tyrosine to L-DOPA should be sufficient to confer both betaxanthin and betacyanin production to anthocyanin-producing species. The requirement for few novel biosynthetic steps may have assisted in the evolution of the betalain biosynthetic pathway in the Caryophyllales, and facilitated multiple origins of the pathway in this order and in fungi. The stably transformed 35S: AmDOD arabidopsis plants provide material to study, for the first time, the physiological effects of having both betalains and anthocyanins in the same plant tissues.

The effects of host defence elicitors on betacyanin accumulation in Amaranthus mangostanus seedlings.

The effect of elicitors associated with host defence on betacyanin accumulation in Amaranthus mangostanus seedlings was investigated. Under the conditions of the experiments, betacyanin accumulation was generally enhanced by light. Methyl jasmonate (MeJA) treatment increased betacyanin synthesis in a concentration-dependent response. Seedlings treated with ethylene as 5mM Ethephon also had elevated levels of betacyanin. In contrast, salicylic acid (SA) and H(2)O(2) treatments had no influence on betacyanin contents in light or dark. Combined MeJA with Ethephon or H(2)O(2) had an additive effect on betacyanin accumulation in dark-grown seedlings. However, a decline was recorded in light-grown seedlings. Moreover, an antagonistic effect on betacyanin synthesis was found when MeJA and SA were added simultaneously. Our results indicate that betacyanin content in A. mangostanus seedlings can be upregulated by MeJA and ethylene. Both additive and antagonistic effects in regulating betacyanin synthesis in A. mangostanus seedlings were observed between MeJA and other elicitors.