Yellow Betaxanthins Research Articles

THE INFLUENCE OF RED BEET VARIETIES AND CULTIVATION ON THEIR BIOCHEMICAL AND SENSORY PROFILE

Modern pharmacology shows that beetroot extracts exhibit antihypertensive and hypoglycaemic activity as well as excellent antioxidant activity. The promising results of the phytochemicals contained in the extracts suggest the opportunity for their use in functional foods.This study aimed to evaluate the biochemical profile (total ash and crude protein content, total polyphenols and betalains content) and sensory properties (5 points hedonic scale) of beetroot varieties from bio-B (B Taunus, B Boro, B Gesche) and traditional production-TP (TP Taunus, TP Boro, TP Rocket). The beetroots were obtained from a private producer from Austria. The crude protein content in evaluated red beetroots ranged from 1.54 % (B Taunus) to 5.32 % of fresh weight (TP Boro). The ash content range was 0.53 % (TP Rocket and TP Boro) to 0.63 % of fresh weight (TP Taunus). The total polyphenol content was the highest in sample of B Taunus – 3.29 mg GAE kg-1 of fresh weight (GAE – gallic acid equivalent). Betalains were determined separately as the red – violet betacyanins and the yellow betaxanthins. The concentration of betacyanins ranged from 5.7 mg.kg-1 (TP Boro) to 8.07 mg.kg-1 of fresh weight (B Gesche), while that of betaxanthins – from 2.77 mg.kg-1 (TP Boro) to 5.39 mg.kg-1 of fresh weight (B Gesche). Sensory properties (appearance, color, consistency, aroma, taste) of all the beetroot varieties were evaluated overall as good with the best score in a taste and overall appearance noted for TP Boro. The results improve our knowledge on beetroot as a valuable vegetable and indicate that further studies on the properties of different varieties of red beet are justified.

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 μmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

A Genome-Wide Identification Study Reveals That HmoCYP76AD1, HmoDODAα1 and HmocDOPA5GT Involved in Betalain Biosynthesis in Hylocereus.

Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitayas are the only at large-scale commercially grown fruit containing abundant betalains for consumers. Currently, the key genes involved in betalain biosynthesis remain to be fully elucidated. Moreover, genome-wide analyses of these genes in betalain biosynthesis are not available in betalain-producing plant species. In this study, totally 53 genes related to betalain biosynthesis were identified from the genome data of Hylocereus undatus. Four candidate genes i.e., one cytochrome P-450 R gene (HmoCYP76AD1), two L-DOPA 4,5-dioxygenase genes (HmoDODAα1 and HmoDODAα2), and one cyclo-DOPA 5-O glucosyltransferase gene (HmocDOPA5GT) were initially screened according to bioinformatics and qRT-PCR analyses. Silencing HmoCYP76AD1, HmoDODAα1, HmoDODAα2 or HmocDOPA5GT resulted in loss of red pigment. HmoDODAα1 displayed a high level of L-DOPA 4,5-dioxygenase activity to produce betalamic acid and formed yellow betaxanthin. Co-expression of HmoCYP76AD1, HmoDODAα1 and HmocDOPA5GT in Nicotiana benthamiana and yeast resulted in high abundance of betalain pigments with a red color. These results suggested that HmoCYP76AD1, HmoDODAα1, and HmocDOPA5GT play key roles in betalain biosynthesis in Hylocereus. The results of the present study provide novel genes for molecular breeding programs of pitaya.

Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum).

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T1 plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.

Defining Color Change in Pitaya: A Close Look at Betacyanin Synthesis Genes in Stenocereus queretaroensis

Betalains are tyrosine-derived plant pigments present in several species of the Caryophyllales order. Betalains are classified in red betacyanins and yellow betaxanthins and are implicated in plant stress tolerance and visual attraction for pollinators. The compounds are used as natural colorants in many industries. Today, there is little information on betalain biosynthesis with several key enzymes that remain unknown on plants of the Caryophyllales order. Omic tools have proven to be very useful in gaining insights into various molecular mechanisms. In this study, we used suspension cells from fruits of the cactus Stenocereus queretaroensis. Two growing conditions were used to perform RNA-seq and differential expression analysis to help identify betalain biosynthesis-related genes. We found 98 differential expressed genes related to aromatic amino acids and betalain biosynthesis pathways. Interestingly, we found that only one gene of the betalain synthesis pathway was differentially expressed. The rest of the genes belong to the aromatic amino acid pathway, including hydroxy phenylpyruvate-related genes, suggesting the possibility of an alternative biosynthetic pathway similar to that observed in legumes.

Therapeutic Application of Betalains: A Review.

Anthocyanins, betalains, riboflavin, carotenoids, chlorophylls and caramel are the basic natural food colorants used in modern food manufacture. Betalains, which are composed of red–violet betacyanin and yellow betaxanthins, are water-soluble pigments that color flowers and fruits. Betalains are pigments primarily produced by plants of the order Caryophyllales. Because of their anti-inflammatory, cognitive impairment, anticancer and anti-hepatitis properties, betalains are useful as pharmaceutical agents and dietary supplements. Betalains also exhibit antimicrobial and antimalarial effects, and as an example, betalain-rich Amaranthus spinosus displays prominent antimalarial activity. Studies also confirmed the antidiabetic effect of betalains, which reduced glycemia by 40% without causing weight loss or liver impairment. These findings show that betalain colorants may be a promising alternative to the synthetic dyes currently used as food additives.

The establishment of callus and cell suspension cultures of Hylocereus costaricensis for the production of betalain pigments with antioxidant potential

Hylocereus costaricensis, commonly known as red-purple dragon fruit or pitaya, is a climbing vine cactus that is currently widely grown as a fruit crop and has gained popularity as a potential source of both natural colourants and antioxidants contributed by betalain compounds. Betalains comprised of red-purple betacyanins and yellow betaxanthins. This study aimed to establish callus and cell suspension cultures of H. costaricensis from in vitro germinated seedlings and to evaluate their betalain content and antioxidant profiles. Epicotyl and cotyledon segments of 60-day-old axenic seedlings were examined for their callus induction potential on half-strength Murashige and Skoog (MS) medium supplemented with various concentrations of naphthaleneacetic acid (NAA) and Thidiazuron (TDZ) under different illumination (16-h photoperiod and darkness). Different combinations of picloram and 2,4-dichlorophenoxyacetic acid (2,4-D) were subsequently tested for the induction of friable callus. The betalains, total phenolic content, total flavonoid content and antioxidant activity DFRSA in cell suspension cultures were quantified using spectrophotometric analysis. Generally, cotyledon explants cultured under 16-h photoperiod was optimum for callus induction. Compact callus with roots was formed on medium supplemented with NAA while granular callus was formed in TDZ supplemented medium. Among the 36 treatments tested, supplementation of 10 μM picloram induced friable callus with average fresh mass of 0.817 g (10.2-fold as compared to the control treatment). Picloram at 10 μM concentration was also found to be effective for callus proliferation and initiation of cell suspension culture. Yellow and red callus were produced and proliferated on the same medium through repetitive clonal selection. The higher concentration of sucrose was more effective for cell mass accumulation while lower sucrose concentration stimulated the accumulation of betalain compounds and antioxidants in the cell suspension cultures of H. costaricensis. This study indicates that callus and cell suspension culture could be used as an efficient system for sustainable production of betalain compounds which can contribute to functional food colouring and pharmaceutical industries.

Metabolic Engineering of Escherichia coli for de Novo Production of Betaxanthins.

Betalains are emerging natural pigments with high tinctorial strength and stability, physiological activities, and fluorescent properties for potential application in food, cosmetic, and pharmaceutical industries. Betalains including yellow betaxanthins and red betacyanins are mainly restricted in the Caryophyllales plants. To expand the availability of individual betaxanthins, here, we constructed an Escherichia coli BTA6 for de novo biosynthesis of betalamic acid. Using this strain as a monoculture platform, 14 yellow and 2 red betaxanthins were produced by feeding amino acids and amines. Furthermore, we constructed an l-histidine overproducing strain using chromosome engineering to deattenuate regulation and established a coculture system. After optimization of the initial inoculation ratios and fermentation conditions, the compatible and robust coculture system produced 287.69 mg/L of histidine-betaxanthin. This is the first report on de novo production of betaxanthins in engineered E. coli using glucose as a carbon source. Our work highlights the feasibility of microbial cell factories to produce individual betalains.

Effect of light quality on Betalain content of red beet (Beta vulgaris L.) cultured in vitro

Background Red beet (Beta vulgaris L.) is the most important edible crop that belongs to the family Amaranthaceae. It is considered as an excellent source of foliate and manganese. It contains betalains, which are considered as natural pigments consisting of two major components, the yellow betaxanthin and violet betacyanin. Objectives In this paper, the effect of different light qualities [white, blue, green, red, and ultraviolet (UV)] on the red beet extract content was examined. The main aim of this study was to choose the light quality which will elevate betalains. Materials and methods Red beet seeds were well sterilized and germinated on Murashigue and Skoog basal medium. After 4 weeks, the germinated seedlings (explants) were exposed to different wave lengths. Three replicates for each wavelength treatment were collected after 10, 20, and 30 days. Regarding UV treatment, cultures were exposed to UV rays type C for 10, 20, or 30 min, and three replicates for each time period were collected. Fresh weight of each explants was measured and stored at −20°C till further usage. Overall, 0.2 g of fresh weight was used for extraction using 2 ml extraction solvent (80 ml methanol+20-ml sterilized distilled water+100 μl phosphoric acid). The antioxidant activity, total phenols, and betalains were determined using spectrophotometer. Results and conclusion The highest values of both fresh weight and free radical scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl percentage were recorded with exposing the cultures to the blue light for 30 days or the exposure to UV rays for 30 min (1.285 and 0.746 g and 42.27 and 43.88%, respectively). It was obviously recorded that exposing the cultures to the red light for 10 days or exposing them to the UV rays for 10 min gave the highest values of the total phenol (1.54 and 0.88 mg GAE/g FW, respectively). The highest value of betalains (Betacyanin and Betaxanthin) was recorded with exposing the cultures to the red light for 30 days (0.12 and 0.077 mg/g FW, respectively).

Light Emission in Betalains: From Fluorescent Flowers to Biotechnological Applications.

The discovery of visible fluorescence in the plant pigments betalains revealed the existence of fluorescent patterns in flowers of plants of the order Caryophyllales, where betalains substitute anthocyanins. The serendipitous initial discovery led to a systemized characterization of the role of different substructures on the photophysical phenomenon. Strong fluorescence is general to all members of the family of betaxanthins linked to the structural property that the betalamic acid moiety is connected to an amine group. This property has led to bioinspired tailor-made probes and to the development of novel biotechnological applications in screening techniques or microscopy labeling. Here, we comprehensively review the photophysics, photochemistry, and photobiology of betalain fluorescence and describe all current applications.

The push and pull of plant specialized metabolism underlies a long-standing, colorful mystery.

This article is a Commentary on Lopez‐Nieves et al., 217: 896–908.

Limited Tyrosine Utilization Explains Lower Betalain Contents in Yellow than in Red Table Beet Genotypes

Betalains are tyrosine-derived pigments that consist of red-violet betacyanins and yellow betaxanthins. These pigments are major sources of natural food dyes in the United States. Decades of table beet breeding efforts have increased betalain pigmentation, but yellow betaxanthin accumulation has been lower than that of red betacyanins. To identify possible bottlenecks in betalain production, here we conducted comparative analyses of betalains and their precursor, tyrosine, in various beet genotypes. Consistent with previous studies, red beets had much higher betalain concentrations than yellow beets. Conversely, tyrosine levels were higher in yellow than in red genotypes in both table beet and Swiss chard. Interestingly, increased tyrosine levels were positively correlated with elevated betalain accumulation among red but not yellow genotypes, especially at a later developmental stage, suggesting that yellow beets are not efficiently converting tyrosine into betalain pigments. On the basis of these result...

Chemical and Antioxidant Properties of Betalains.

Betalains are vacuolar pigments composed of a nitrogenous core structure, betalamic acid. Betalamic acid condenses with imino compounds (cyclo-DOPA/its glucosyl derivates) or amino acids/derivates to form violet betacyanins and yellow betaxanthins. These pigments have gained the curiosity of scientific researchers in recent decades. Their importance was increased not only by market orientation toward natural colorants and antioxidants but also by their safety and health promoting properties. To date, about 78 betalains have been identified from plants of about 17 families. In this review, all of the identified pigments are presented, followed by a comprehensive discussion of their structure-activity relationship.

The effect of thermal treatment on antioxidant capacity and pigment contents in separated betalain fractions.

Increased consumption of fruits and vegetables significantly reduces the risk of cardio-vascular disease. This beneficial effect on the human organism is ascribed to the antioxidant compounds these foods contain. Unfortunately, many products, particularly vegetables, need to be subjected to thermal processing before consumption. The aim of this study was to determine the effect of such thermal treatment on the antioxidant capacity and pigment contents in separated fractions of violet pigments (betacyanins) and yellow pigments (betaxanthins and betacyanins). Fractions of violet and yellow pigments were obtained by separation of betalain pigments from fresh roots of 3 red beet cultivars using column chromatography and solid phase extraction (SPE). The betalain pigment content was determined in all samples before and after thermal treatment (90°C/30 min) by spectrophotometry, according to Nilsson's method [1970] and antioxidant capacity was assessed based on ABTS. Betalain pigments in the separated fractions were identified using HPLC-MS. After thermal treatment of betacyanin fractions a slight, but statistically significant degradation of pigments was observed, while the antioxidant capacity of these fractions did not change markedly. Losses of betacyanin content amounted to 13-15% depending on the cultivar, while losses of antioxidant capacity were approx. 7%. HPLC/MS analyses showed that before heating, betanin was the dominant pigment in the betacyanin fraction, while after heating it was additionally 15-decarboxy-betanin. Isolated fractions of yellow pigments in red beets are three times less heat-resistant than betacyanin fractions. At losses of yellow pigment contents in the course of thermal treatment reaching 47%, antioxidant capacity did not change markedly (a decrease by approx. 5%). In the yellow pigment fractions neobetanin was the dominant peak in the HPLC chromatogram, while vulgaxanthin was found in a much smaller area, whereas after heating additionally 2-decarboxy-2,3-dehydro-neobetanin was detected. Both groups of betalain pigments (betacyanins and betaxanthins) exhibit antioxidant capacity before and after heating. Violet beatacyjanins are 3 times more stable when heated than yellow betaxanthins.

Plant Betalains: Safety, Antioxidant Activity, Clinical Efficacy, and Bioavailability.

Betalains are accepted food additives derived from vacuoles of plants belonging to about 17 families in the order Caryophyllales. These pigments are 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-DOPA and/or its glucosyl derivatives) or amines and/or their derivatives to form violet betacyanins (for example, betanin) and yellow betaxanthins (for example, indicaxanthin), respectively. Till date, structures of 75 betalains have been elucidated from plants under the order Caryophyllales. The extracted betalains are safe to consume and they act as micronutrients in the body. In vitro studies to highlight radical-scavenging activity, cell culture studies to assess cytotoxicity and absorption of betalains, and proven clinical efficacies are compiled in this review. The literature on biological activity has not been analyzed for a synthesis of safety, clinical efficacy, and bioavailability to arrive at the concentrations required for the purported health benefits. Most betalains are under-utilized in pharmaceutical and cosmetic preparations due to poor stability and lack of scientific reports highlighting their superior tinctorial strength including flourescence, water solubility, and functional value alongside their bioavailability. This is the first comprehensive review on the dietary safety, biological activity and bioavailability of betalains. Based on this review, for future debate and input from health professionals, a human daily intake of betanin and indicaxanthin can be proposed at 100 and 50 mg, respectively.

To betalains and back again: a tale of two pigments.

To betalains and back again: a tale of two pigments.

Characterisation of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species.

Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae). RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD). In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II). PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene family evolution.

Differential partitioning of betacyanins and betaxanthins employing aqueous two phase extraction

Yellow betaxanthins and red purple betacyanins are genetically and structurally closely related pigments, hence making their separation from each other difficult and tedious. Fractionation of betalains into betacyanin and betaxanthin could be achieved by employing aqueous two-phase extraction (ATPE). Prior to fractionation, purification of betalains (devoid of sugars) was achieved employing ATPE comprising PEG 6000/ammonium sulphate. At a high tie line length (TLL) of 50.65%, phase volume ratio 1.96 and pH around 5, maximum differential partitioning was observed, wherein 92.62% of the betalains partitioned to the PEG-rich (top) phase while 72.38% of sugars partitioned to the salt-rich (bottom) phase. Fractionation of betalains into betacyanin and betaxanthin was achieved by multistage ATPE. At the end of the third stage ATPE, the bottom phase had only betaxanthin without any residual betacyanin. The fractions of betacyanin and betaxanthin were analyzed by spectrophotometric method and confirmed by HPLC.

Analysis of betalains from fruits of Opuntia species

Betalains are of great taxonomic significance in higher plants and occur only in 10 families of the order Caryophyllales (Centrospermae). They are water-soluble nitrogenous pigments. They can be divided into two major structural groups, the red to red-violet betacyanins and the yellow betaxanthins. Betalains are widely used as natural red food colorant as well as antioxidant potentials. Several methods have been published for the determination of betalain in fruits of Opuntia species. The purpose of the current review is to provide a systematic survey of the analytical techniques for the determination of betalain from fruits of Opuntia species.

The beet R locus encodes a new cytochrome P450 required for red betalain production

Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species.