Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments.

BACKGROUND: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri-or wishbone flower-a popular ornamental plant, has not been attempted. RESULTS: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet."  Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins-mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. CONCLUSIONS: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Straightforward and affordable agroinfiltration with RUBY accelerates RNA silencing research.

Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.

Pitahaya (Hylocereus ocamponis)-Peel and -Flesh Flour Obtained from Fruit Co-Products-Assessment of Chemical, Techno-Functional and In Vitro Antioxidant Properties.

The aim of this work was to assess the chemical composition and physico-chemical, techno-functional, and in vitro antioxidant properties of flours obtained from the peel and flesh of pitahaya (Hylocereus ocamponis) to determine their potential for use as ingredients for food enrichment. The chemical composition, including total betalains, mineral content, and polyphenolic profile, was determined. The techno-functional properties (water holding, oil holding, and swelling capacities) were also evaluated. For the antioxidant capacity, four different methodologies, namely ferrous ion-chelating ability assay, ferric-reducing antioxidant power assay; 1,1-Diphenyl-2-picrylhydrazyl radical scavenging ability assay, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical assay, were used. Pitahaya-peel flour had higher values for protein (6.72 g/100 g), ash (11.63 g/100 g), and dietary fiber 56.56 g/100 g) than pitahaya-flesh flour, with values of 6.06, 3.63, and 8.22 g/100 g for protein, ash, and dietary fiber, respectively. In the same way, pitahaya peel showed a higher content of minerals, betalains, and polyphenolic compounds than pitahaya-flesh flour, with potassium (4.43 g/100 g), catechin (25.85 mg/g), quercetin-3-rhamnoside (11.66 mg/g) and myricetrin (12.10 mg/g) as principal compounds found in the peel. Again, pitahaya-peel flour showed better techno-functional and antioxidant properties than pitahaya-flesh flour. The results obtained suggest that the flours obtained from the peel and pulp of pitahaya (H. ocamponis) constitute a potential material to be utilized as an ingredient in the food industry due to the high content of bioactive compounds such as betalains, phenolic acids, and flavonoids, with notable antioxidant capacity.

Valorization of Red Beetroot (Beta vulgaris L.) Pomace Combined with Golden Linseed (Lini semen) for the Development of Vegetable Crispbreads as Gluten-Free Snacks Rich in Bioactive Compounds.

This work aimed to develop gluten-free snacks such as crispbread based on beetroot pomace (Beta vulgaris L.) and golden linseed (Lini semen). Beetroot is attracting more and more consumer attention because of its nutritional and health properties. The use of beet pomace contributes to waste management. Linseed, known as a superfood with many health-promoting properties, was used to produce crispbreads as an alternative to cereals, which are allergens. Beetroot pomace and whole or ground linseed were used in different proportions to produce crispbread snacks. Chemical and physical analyses were performed including water activity, dry matter, betalains, and polyphenols content, as well as Fourier transform infrared spectroscopy (FTIR). A sensory evaluation and microstructure observations were also performed. The obtained snacks were characterized by low water activity (0.290-0.395) and a high dry matter content (93.43-97.53%), which ensures their microbiological stability and enables longer storage. Beetroot pomace provided betalains-red (14.59-51.44 mg betanin/100 g d.m.) and yellow dyes (50.02-171.12 mg betanin/100 g d.m.)-while using linseed enriched the product with polyphenols (730-948 mg chlorogenic acid/100 g d.m.). FTIR analysis showed the presence of functional groups such as the following: -OH, -C-O, -COOH, and -NH. The most desired overall consumer acceptability was achieved for snacks containing 50% beetroot pomace and 50% linseed seeds. The obtained results confirmed that beetroot pomace combined with linseed can be used in the production of vegetable crispbread snacks.

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.

Traditional medicinal use is linked with apparency, not specialized metabolite profiles in the order Caryophyllales.

PREMISE: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism. METHODS: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF). RESULTS: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use. CONCLUSIONS: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.

pH-dependent stability of major betalains in the encapsulated beetroot extracts (Beta vulgaris L.).

Betalain is a water-soluble pigment contained in Caryophyllales plants. It not only holds potential as a natural food colorant but also offers various health benefits, acting as an antioxidant. This study focused on analyzing the pH-dependent stability of encapsulated betalain pigments extracted from red beetroot (Beta vulgaris L.) using methods such as absorption spectroscopy, HPLC, and LC-MS. The major pigments identified were vulgaxanthin I, betanin, isobetanin, and neobetanin, alongside minor components, including three betaxanthin species and a degradation product known as betalamic acid. Spectrophotometric analyses revealed that above pH 8, the betalain peak at 435 nm decreased and red-shifted to a peak at 549 nm, a shift that could be reversed through neutral pH treatment. At pH 11, a new broad peak appeared at 410 nm and was identified as betalamic acid. To assess the pH-dependency of each betalain, the targeted betalains were separated and quantified through HPLC after incubation across a wide pH range of 2-11 and during storage. After 3 days of storage in highly alkaline conditions (pH 10-11), major betalains, with the exception of neobetanin, underwent significant degradation. Conversely, these pigments displayed relative stability in acidic conditions. In contrast, neobetanin showed vulnerability to acidic conditions but exhibited tolerance to alkaline pH levels of 10-11. The degradation product, betalamic acid, demonstrated a similar susceptibility to alkaline pH as betanins. In conclusion, the significant stability decrease under highly alkaline conditions results not only from the hydrolytic reaction of betalains but also from the degradation of betalamic acid itself. PRACTICAL APPLICATION: Encapsulation methods are used to enhance the stability of betalains against temperature variations; however, the effects of pH, especially when considering individual betalain species, are not well understood. Despite betalains exhibiting similar features and being suitable for a wide pH range from acid to alkaline conditions, they are significantly affected by alkaline pH levels exceeding 10, as well as by storage duration. This study demonstrated the application of encapsulation to pH-dependent stability, and the findings offer valuable insights and a fresh perspective on betalains as red biocolorants, extending their potential application to a wide range of pH-controlled food products.

Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana.

Synthetic biology provides emerging tools to produce valuable compounds in plant hosts as sustainable chemical production platforms. However, little is known about how supply and utilization of precursors is coordinated at the interface of plant primary and specialized metabolism, limiting our ability to efficiently produce high levels of target specialized metabolites in plants. L-Tyrosine is an aromatic amino acid precursor of diverse plant natural products including betalain pigments, which are used as the major natural food red colorants and more recently a visual marker for plant transformation. Here, we studied the impact of enhanced L-tyrosine supply on the production of betalain pigments by expressing arogenate dehydrogenase (TyrA) from table beet (Beta vulgaris, BvTyrAα), which has relaxed feedback inhibition by L-tyrosine. Unexpectedly, betalain levels were reduced when BvTyrAα was coexpressed with the betalain pathway genes in Nicotiana benthamiana leaves; L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA) levels were drastically elevated but not efficiently converted to betalains. An additional expression of L-DOPA 4,5-dioxygenase (DODA), but not CYP76AD1 or cyclo-DOPA 5-O-glucosyltransferase, together with BvTyrAα and the betalain pathway, drastically enhanced betalain production, indicating that DODA is a major rate-limiting step of betalain biosynthesis in this system. Learning from this initial test and further debottlenecking the DODA step maximized betalain yield to an equivalent or higher level than that in table beet. Our data suggest that balancing between enhanced supply ("push") and effective utilization ("pull") of precursor by alleviating a bottleneck step is critical in successful plant synthetic biology to produce high levels of target compounds.

Anti-Obesity Effect of Different Opuntia stricta var. dillenii's Prickly Pear Tissues and Industrial By-Product Extracts in 3T3-L1 Mature Adipocytes.

Opuntia stricta var. dillenii fruit is a source of phytochemicals, such as betalains and phenolic compounds, which may play essential roles in health promotion. The aim of this research was to study the triglyceride-lowering effect of green extracts, obtained from Opuntia stricta var. dillenii fruit (whole fruit, pulp, peel, and industrial by-products (bagasse)) in 3T3-L1 mature adipocytes. The cells were treated on day 12, for 24 h, after the induction of differentiation with the extracts, at doses of 10, 25, 50, or 100 µg/mL. The expression of genes (PCR-RT) and proteins (Western blot) involved in fatty acid synthesis, fatty acid uptake, triglyceride assembly, and triglyceride mobilisation was determined. The fruit pulp extraction yielded the highest levels of betalains, whereas the peel displayed the greatest concentration of phenolic compounds. The extracts from whole fruit, peel and pulp were effective in reducing triglyceride accumulation at doses of 50 µg/mL or higher. Bagasse did not show this effect. The main mechanisms of action underpinning this outcome encompass a reduction in fatty acids synthesis (de novo lipogenesis), thus limiting their availability for triglyceride formation, alongside an increase in triglyceride mobilisation. However, their reliance is contingent upon the specific Opuntia extract.

A critical review on the stability of natural food pigments and stabilization techniques.

This comprehensive review article delves into the complex world of natural edible pigments, with a primary focus on their stability and the factors that influence them. The study primarily explores four classes of pigments: anthocyanins, betalains, chlorophylls and carotenoids by investigating both their intrinsic and extrinsic stability factors. The review examines factors affecting the stability of anthocyanins which act as intrinsic factors like their structure, intermolecular and intramolecular interactions, copigmentation, and self-association as well as extrinsic factors such as temperature, light exposure, metal ions, and enzymatic activities. The scrutiny extends to betalains which are nitrogen-based pigments, and delves into intrinsic factors like chemical composition and glycosylation, as well as extrinsic factors like temperature, light exposure, and oxygen levels affecting for their stability. Carotenoids are analyzed concerning their intrinsic and extrinsic stability factors. The article emphasizes the role of chemical structure, isomerization, and copigmentation as intrinsic factors and discusses how light, temperature, oxygen, and moisture levels influence carotenoid stability. The impacts of food processing methods on carotenoid preservation are explored by offering guidance on maximizing retention and nutritional value. Chlorophyll is examined for its sensitivity to external factors like light, temperature, oxygen exposure, pH, metal ions, enzymatic actions, and the food matrix composition. In conclusion, this review article provides a comprehensive exploration of the stability of natural edible pigments, highlighting the intricate interplay of intrinsic and extrinsic factors. In addition, it is important to note that all the references cited in this review article are within the past five years, ensuring the most up-to-date and relevant sources have been considered in the analysis.

Supercritical extraction of betalains from the peel of different pitaya species.

BACKGROUND: Pitaya is a fruit with high consumer acceptance and health benefits. Pitaya peel is a waste product with potential in the food industry, as an antioxidant enrichment and natural colouring. Therefore, there is an interest in recovering its constituents and searching for pitaya species with greater potential. This work aimed to obtain bioactive extracts from the dried peel of pitaya fruits of the species Selenicereus monacanthus (Lem.), S. costaricensis W. and S. undatus H. using supercritical fluids at different pressures (100, 250 and 400 bar) and ethanol-water 15% v/v or ethanol 100% as co-solvents. The extraction yield, antioxidant activity, colour and total betalain content were evaluated. RESULTS: The extract obtained from S. monacanthus showed the highest extraction yield (49.6 g kg-1), followed by S. costaricensis (27.5 g kg-1) and S. undatus (17.7 g kg-1) at 400 bar and 35 °C using ethanol 15%, v/v. The antioxidant capacity was strongly influenced by pressure, favouring the obtaining of betalain-rich extracts at higher pressures, especially in the species S. costaricensis (0.6 g kg-1) and S. monacanthus (0.3 g kg-1). To improve the extraction of S. undatus (the most cultivated species), the procedure of subsequential extractions was applied. This procedure considerably increased the extraction yield, antioxidant activity and total content of betalains. The use of ethanol 100% provided more bioactive fractions and achieved a good separation of betalains. CONCLUSION: The supercritical extraction method can overcome the challenge of efficiently extracting compounds from pitaya peel, due to the presence of bioactive compounds of great polarity. © 2024 Society of Chemical Industry.

Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

Productos alimenticios etiquetados como “orgánicos” a basede cultivos andinos: un estudio de sus bio-componentes y actividad antioxidante in vitro / Food products labeled as “organic” based on Andean crops: a study of their biocomponents and antioxidant activity in vitro

Objetivo: En este estudio se plantea la evaluación de sus propiedades antioxidantes de varios productos con etiquetado “orgánico” elaborados con matrices alimentarias de cultivos andinos.Materiales y métodos: Las diferentes muestras con etiquetado “orgánico” fueron evaluadas en función a sus propiedades antioxidantes y evaluadas mediante métodos espectrofotométricos. Resultados: Los resultados de este estudio muestran que las semillas seguido de las mezclas de harinas (PM1 y PM3) presentan altos contenidos de proteínas, mientras que la semilla de chía (S2) fueron ricos en lípidos y cenizas. Mejores contenidos de polifenoles, flavonoides, flavanoles, flavonoles y antocianinas fueron observados en las mezclas de harinas (PM1 a PM5), estas mezclas fueron obtenidas a partir de maca, quinua, maíz morado, cacao, y lúcuma, mientras que las semillas como quinua perlada roja, amaranto, quinua roja, quinua negra, y quinua tricolor contribuyeron con betaxantinas y betacianinas. El orden de clasificación de los productos alimenticios en función del valor antioxidante y composición nutricional ubicó en primer lugar a PM2 (quinua, cacao criollo, maíz morado, algarrobo, canela), segundo lugar para maíz morado y en tercer lugar para flakes.Conclusiones: Los hallazgos de este estudio permitieron establecer que las mezclas de harinas con etiquetado “orgánico” mostraron un alto potencial antioxidante.(AU)

Objective: In this study, the evaluation of the antioxidantproperties of several products labeled “organic” made withfood matrices of Andean crops is proposed.Material and methods: A total of 23 samples with “or-ganic” labelling based on Andean crops were evaluated withrespect to their nutritional composition, antioxidant properties(total polyphenols, total flavonoids, anthocyanins, betalains,and DPPH radical scavenging activity).Result: The results of this study show that the seeds (S1to S7) followed by the flour mixtures (PM1 and PM3) presenthigh protein contents, while the chia seed (S2) was rich inlipids and ashes. Regarding polyphenols, flavonoids, fla-vanols, flavonols and anthocyanins, it was observed that theflour mixtures (PM1 to PM5) presented better contents, these mixtures were obtained from maca, quinoa, purple corn, co-coa, and lucuma, while the seeds as red pearl quinoa, ama-ranth, red quinoa, black quinoa, and tri-color quinoa con-tributed betaxanthins and betacyanins. The order ofclassification of the food products with the “organic” labelbased on the antioxidant value and nutritional compositionplaced the PM2 sample in first place (quinoa, Criollo cocoa,purple corn, carob powder, cinnamon), followed by purplecorn flour (P1) and in third place for flakes (FL). Conclusion: The findings of this study allowed us to es-tablish that the flour mixtures labeled “organic” showedgreater DPPH radical scavenging activity.(AU)

In Vitro Screening and Lipid-Lowering Effect of Prickly Pear (Opuntia Ficus-Indica L. Mill.) Fruit Extracts in 3T3-L1 Pre-Adipocytes and Mature Adipocytes.

Opuntia ficus-indica fruits have been widely used due to their nutritional composition and beneficial effects on health, particularly against chronic diseases such as diabetes, obesity, cardiovascular diseases and cancer, among others. In recent years, prickly pear peel and pulp extracts have been characterised, and a high number of bioactive compounds have been identified. This study aimed to analyse the triglyceride-lowering effect of prickly pear peel and pulp extracts obtained from fruits of three varieties (Pelota, Sanguinos, and Colorada) in 3T3-L1 maturing and mature adipocytes. At a concentration of 50 µg/mL, peel extracts from Colorada reduced triglyceride accumulation in pre-adipocytes and mature adipocytes. Additionally, at 25 µg/mL, Pelota peel extract decreased triglyceride content in mature adipocytes. Moreover, maturing pre-adipocytes treated with 50 and 25 µg/mL of Sanguinos pulp extract showed a reduction of triglyceride accumulation. In addition, the lipid-lowering effect of the main individual betalain and phenolic compounds standards were assayed. Piscidic acid and isorhamnetin glycoside (IG2), found in Colorada peel extract, were identified as the bioactive compounds that could contribute more notably to the triglyceride-lowering effect of the extract. Thus, the betalain and phenolic-rich extracts from Opuntia ficus indica fruits may serve as an effective tool in obesity management.

Genomic basis of seed colour in quinoa inferred from variant patterns using extreme gradient boosting.

Quinoa is an agriculturally important crop species originally domesticated in the Andes of central South America. One of its most important phenotypic traits is seed colour. Seed colour variation is determined by contrasting abundance of betalains, a class of strong antioxidant and free radicals scavenging colour pigments only found in plants of the order Caryophyllales. However, the genetic basis for these pigments in seeds remains to be identified. Here we demonstrate the application of machine learning (extreme gradient boosting) to identify genetic variants predictive of seed colour. We show that extreme gradient boosting outperforms the classical genome-wide association approach. We provide re-sequencing and phenotypic data for 156 South American quinoa accessions and identify candidate genes potentially controlling betalain content in quinoa seeds. Genes identified include novel cytochrome P450 genes and known members of the betalain synthesis pathway, as well as genes annotated as being involved in seed development. Our work showcases the power of modern machine learning methods to extract biologically meaningful information from large sequencing data sets.

Multiple mechanisms explain loss of anthocyanins from betalain-pigmented Caryophyllales, including repeated wholesale loss of a key anthocyanidin synthesis enzyme.

In this study, we investigate the genetic mechanisms responsible for the loss of anthocyanins in betalain-pigmented Caryophyllales, considering our hypothesis of multiple transitions to betalain pigmentation. Utilizing transcriptomic and genomic datasets across 357 species and 31 families, we scrutinize 18 flavonoid pathway genes and six regulatory genes spanning four transitions to betalain pigmentation. We examined evidence for hypotheses of wholesale gene loss, modified gene function, altered gene expression, and degeneration of the MBW (MYB-bHLH-WD40) trasnscription factor complex, within betalain-pigmented lineages. Our analyses reveal that most flavonoid synthesis genes remain conserved in betalain-pigmented lineages, with the notable exception of TT19 orthologs, essential for the final step in anthocyanidin synthesis, which appear to have been repeatedly and entirely lost. Additional late-stage flavonoid pathway genes upstream of TT19 also manifest strikingly reduced expression in betalain-pigmented species. Additionally, we find repeated loss and alteration in the MBW transcription complex essential for canonical anthocyanin synthesis. Consequently, the loss and exclusion of anthocyanins in betalain-pigmented species appear to be orchestrated through several mechanisms: loss of a key enzyme, downregulation of synthesis genes, and degeneration of regulatory complexes. These changes have occurred iteratively in Caryophyllales, often coinciding with evolutionary transitions to betalain pigmentation.

Improving red pitaya fruit quality by nano-selenium biofortification to enhance phenylpropanoid and betalain biosynthesis.

Red pitaya, the representative tropical and subtropical fruit, is vulnerable to quality deterioration due to climate or agronomic measures. Nano-selenium (Nano-Se) has shown positive effects on crop biofortification in favour of reversing this situation. In this study, Se could be enriched efficiently in red pitayas via root and foliar application by Nano-Se, which induced higher phenolic acids (16.9-94.2%), total phenols (15.7%), total flavonoids (29.5%) and betacyanins (34.1%) accumulation in flesh. Richer antioxidative features including activities of SOD (25.2%), CAT (33.8%), POD (77.2%), and levels of AsA (25.7%) and DPPH (14.7%) were obtained in Nano-Se-treated pitayas as well as in their 4-8 days shelf-life. The non-targeted metabolomics indicated a boost in amino acids, resulting in the stimulation of phenylpropanoid and betalain biosynthesis. In conclusion, the mechanism of Nano-Se biofortification for red pitaya might be fortifying pigment, as well as the enzymatic and non-enzymatic antioxidant substances formation by regulating primary and secondary metabolism facilitated by Se accumulation.

Betalains Alleviate Exercise-Induced Oxidative Stress, Inflammation, and Fatigue and Improve Sports Performance: an Update on Recent Advancement.

PURPOSE OF REVIEW: Beetroot juice is a popular natural food supplement commonly consumed for its health and ergogenic benefits. It contains an abundance of phytochemical compounds, which have been shown to enhance sports endurance and recovery. Among them, nitrate is well-studied and known for improving performance during exercise. On the other hand, betalains, the bioactive pigment, have shown various biological activities including antioxidant, anti-inflammatory, and anti-hypertensive, which may improve exercise performance and post-exercise recovery. Additionally, free radical scavenging activities of betalains could increase nitric oxide availability in the blood, thereby improving blood flow and oxygen supply during strenuous exercise. This review article provides a critical discussion of the non-pathological conditions induced by prolonged or strenuous exercise and betalains' potential in reducing such conditions including muscle damage, inflammation, and fatigue. Additionally, the real-time application of betalains as an ergogenic compound in competitive athletes has been discussed. Finally, future directions and conclusions on the potential of betalains as a natural ergogenic aid in sport endurance are outlined. RECENT FINDINGS: Betalains in beetroot are the major water-soluble nitrogen-containing pigment possessing high antioxidant, anti-inflammatory, and anti-fatigue activities. Betalain supplementation could alleviate exercise-induced oxidative stress, inflammation, and fatigue in competitive athletes. Betalains have the potential to become a natural ergogenic aid or nutraceutical compound for sports people during exercise and competitive performance.

Isolation of High-Purity Betanin from Red Beet and Elucidation of Its Antioxidant Activity against Peroxynitrite: An in vitro Study.

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO-) is the most toxic molecular species among RNS. Betalains are known to possess ONOO- scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO- reaction, ONOO--dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO- cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO- toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.

A Comparative Study of Ethanol and Citric Acid Solutions for Extracting Betalains and Total Phenolic Content from Freeze-Dried Beetroot Powder.

This research compares the extraction of betalains (betacyanin and betaxanthin) and total phenolic content using citric acid and aqueous-ethanol solutions. The aim is to find an environmentally sustainable alternative solvent for extracting these compounds from dried beetroot powder. Using citric acid solution as a solvent offers several benefits over ethanol. Citric acid is a weak organic acid found naturally in citrus fruits, making it a safe and environmentally friendly choice for certain extraction processes. Moreover, the use of citric acid as solvent offers biodegradability, non-toxicity, non-flammability, and is cost effective. A full factorial design and response surface methodology (RSM) were employed to assess the effects of extraction parameters (extraction time (5-30 min), extraction temperature (20, 30, 40 °C), pH of citric acid solution (3, 4, 5) and ethanol concentration (10, 20, 30% v/v)). The yield was determined spectrophotometrically and expressed as mg/g of dry powder. The results showed that citric acid solution yielded 85-90% of the ethanolic extract under identical conditions. The maximum yields of betacyanin, betaxanthin, and total phenolic content in citric acid solution were 3.98 ± 0.21 mg/g dry powder, 3.64 ± 0.26 mg/g dry powder, and 8.28 ± 0.34 mg/g dry powder, respectively, while aqueous-ethanol yielded 4.38 ± 0.17 mg/g dry powder, 3.95 ± 0.22 mg/g dry powder, and 8.45 ± 0.45 mg/g dry powder. Optimisation resulted in maximum extraction yields of 90% for betalains and 85% for total phenolic content. The study demonstrates the potential of citric acid as a viable alternative to polar organic solvents for extracting phytochemicals from plant material, providing comparable results to aqueous-ethanol. Artificial Neural Network (ANN) models outperformed RSM in predicting extraction yields. Overall, this research highlights the importance of exploring bio-solvents to enhance the environmental sustainability of phytochemical extraction.