Red Betacyanins Research Articles

Isoform-resolved genome annotation enables mapping of tissue-specific betalain regulation in amaranth.

Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.

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.

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.

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...

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

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.

Impact of storage conditions and variety on quality attributes and aroma volatiles of pitahaya (Hylocereus spp.)

Pitahaya are increasing in popularity but knowledge on the effects of storage and the potential impact of variety on subsequent quality following storage is incomplete, particularly in terms of the potential effects on sensory acceptability. In this study six varieties of pitahaya, having white, pink, and red internal flesh coloration, were harvested and evaluated for sensory and quality attributes at harvest and following storage for two weeks at either 5°C or 10°C. Storage did not influence overall visual liking or color of the external portion of the fruit as discerned by the panelists. Alterations in the internal flesh color, such as a slight darkening, were noted to occur by instrumental measurement, but these differences were not noticeable to the panelists. Losses in sugars and acids occurred in the flesh during storage at both storage temperatures and were related to declines in fructose, glucose and malic acid. In contrast, antioxidant activity was reduced by storage at 5°C but was unchanged at 10°C, with betacyanin concentration not differing from that determined at harvest. Aldehydes were the most abundant aroma volatiles detected in juice from the flesh, with storage increasing total aldehyde amount, particularly at 10°C. Total alcohols, on the other hand, were reduced by storage, the amount of reduction not dependent on storage temperature. Regardless of the storage-induced changes in the various components measured, panelists did not report any significant differences in overall liking, flavor, sweetness, tartness or texture. There were substantial varietal differences in sensory and quality attributes, regardless of the impact of storage. ‘Cebra’ had very low sweetness relative to the other varieties and had low flavor and overall liking scores. Panelist perception of flavor quality was most closely linked with varietal differences in SSC and TA, although there were clear differences in aroma volatiles as well. Mexicana, a white-fleshed variety, had the lowest antioxidant activity, corresponding to low amounts of the red betacyanin pigments. The study indicated that storage, and particularly that at 10°C, caused a variety of measureable changes in a range of pitahaya quality parameters but that these changes did not substantially alter the sensory quality of the fruit. Further research is needed to determine whether or not varietal or storage-induced differences in aroma volatile content have an impact on pitahaya flavor.

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.

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.

Chemical stability and colorant properties of betaxanthin pigments from Celosia argentea.

The chemical stability and colorant properties of three betaxanthins recently identified from Celosia argentea varieties were evaluated. Lyophilized betaxanthin powders from yellow inflorescences of Celosia exhibited bright yellow color and high color purity with strong hygroscopicity. The aqueous solutions containing these betaxanthins were bright yellow in the pH range 2.2-7.0, and they were most stable at pH 5.5. The betaxanthins in a model system (buffer) were susceptible to heat, and found to be as unstable as red betacyanins (betanin and amaranthine) at high temperatures (>40 degrees C), but more stable at 40 degrees C with the exclusion of light and air. The three betaxanthins had slightly higher pigment retention than amaranthine/isoamaranthine in crude extracts at 22 degrees C, as verified by HPLC analysis. Lyophilized betaxanthins had much better storage stability (mean 95.0% pigment retention) than corresponding aqueous solutions (14.8%) at 22 degrees C after 20 weeks. Refrigeration (4 degrees C) significantly increased pigment retention of aqueous betaxanthins to 75.5%.

<i>tert</i>.-Butylhydroperoxide 処理による赤ビート切片からのベタシアニン リーケージに及ぼす各種の添加物質の影響

With the purpose of getting much information about the mechanism of leakage observed at the onset of plant senescence or other physiological disorders, a model system using red beet discs has been investigated in our study. The present paper describes the effects of various chemicals added to the incubation medium, in which red beet discs are exposed or pre-exposed to tert. -butylhydroperoxide (BHP), on the leakage of red betacyanin pigment from the cells. The mechanism of betacyanin leakage induced by the BHP treatment is suggested from these results.1. Under the same conditions reported in our previous paper, the treatment with BHP (10mM and 8hrs) evidently caused the abrupt increase of OD 540nm (leakage of red betacyanin pigment), and the optical density reached about 1.0 from 5hrs to 7hrs after the treatment.2. The addition of 5mM sodium diethyldithiocarba mate, 20mM thiourea, 5.0mM (+)-catechin and 1mM n-propyl gallate, separately, to the incubation medium effectively inhibited the increase in OD 540nm, when each chemical and BHP were added simultaneously.3. L-Cysteine (5mM) and D-ascorbic acid (5mM), which are well known as endogenous reducing agents, also showed the inhibitory effect on Betacyanin leakage of red beet discs.4. When (+)-catechin (5mM), cysteine (10mM) and thiourea (20mM) wereadded, separately, to the incubation medium in the absence of BHP after the BHP treatment (10mM and 8hrs), they also showed significant effects on inhibition of betacyanin leakage.5. The presence of 5mM EDTA or 2.5mM FeCl3 in the incubation medium containing 10mM BHP enhanced the increase in OD 540nm considerably.These results suggest that BHP taken into the discs of red beet, as indicated in the red cells of human blood, may be transformed by either free Fe3+, chelated Fe3+ or other endogenous unknown factors to the tert. -butoxyl radical, and consequently will attack various biomembranes in the cells to cause the lakage of red betacyanin pigment. Further biochemical investigation will be required to confirm the mechanism.