Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta.

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure-function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.

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.

Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor.

Amaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

Integrated sRNAome and RNA-Seq analysis reveals miRNA effects on betalain biosynthesis in pitaya.

BACKGROUND: MicroRNAs (miRNAs) and their regulatory functions in anthocyanin, carotenoid, and chlorophyll accumulation have been extensively characterized in many plant species. However, the miRNA regulatory mechanism in betalain biosynthesis remains mostly unknown. RESULTS: In this study, 126 conserved miRNAs and 41 novel miRNAs were first isolated from Hylocereus monacanthus, among which 95 conserved miRNAs belonged to 53 miRNA families. Thirty-four candidate miRNAs related to betalain biosynthesis were differentially expressed. The expression patterns of those differential expressed miRNAs were analyzed in various pitaya tissues by RT-qPCR. A significantly negative correlation was detected between the expression levels of half those miRNAs and corresponding target genes. Target genes of miRNAs i.e. Hmo-miR157b-HmSPL6-like, Hmo-miR160a-Hpcyt P450-like3, Hmo-miR6020-HmCYP71A8-like, Hmo-novel-2-HmCYP83B1-like, Hmo-novel-15-HmTPST-like, Hmo-miR828a-HmTT2-like, Hmo-miR858-HmMYB12-like, Hmo-miR858-HmMYBC1-like and Hmo-miR858-HmMYB2-like were verified by 5'RACE and transient expression system in tobacco. CONCLUSIONS: Hmo-miR157b, Hmo-miR160a, Hmo-miR6020 Hmo-novel-2, Hmo-novel-15, Hmo-miR828a and Hmo-miR858 play important roles in pitaya fruit coloration and betalain accumulation. Our findings provide new insights into the roles of miRNAs and their target genes of regulatory functions involved in betalain biosynthesis of pitaya.

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.

Isolation and characterization of the betalain biosynthesis gene involved in hypocotyl pigmentation of the allotetraploid Chenopodium quinoa.

In quinoa seedlings, the pigment betalain accumulates in the hypocotyl. To isolate the genes involved in betalain biosynthesis in the hypocotyl, we performed ethyl methanesulfonate (EMS) mutagenesis on the CQ127 variety of quinoa seedlings. While putative amaranthin and celosianin II primarily accumulate in the hypocotyls, this process produced a green hypocotyl mutant (ghy). This MutMap+ method using the quinoa draft genome revealed that the causative gene of the mutant is CqCYP76AD1-1. Our results indicated that the expression of CqCYP76AD1-1 was light-dependent. In addition, the transient expression of CqCYP76AD1-1 in Nicotiana benthamiana leaves resulted in the accumulation of betanin but not isobetanin, and the presence of a polymorphism in CqCYP76A1-2 in the CQ127 variety was shown to have resulted in its loss of function. These findings suggested that CqCYP76AD1-1 is involved in betalain biosynthesis during the hypocotyl pigmentation process in quinoa. To our knowledge, CqCYP76AD1-1 is the first quinoa gene identified by EMS mutagenesis using a draft gene sequence.

Evaluación de la capacidad antioxidante, características fisicoquímicas y perfil sensorial de Opuntia robusta y O. ficus-indica / Evaluation of antioxidant capacity, physicochemical characteristics and sensory profile of Opuntia robusta and O. ficus-indica

Las Opuntia spp. son un recurso fitogenético Mexicano de gran valor nutritivo y alto contenido de betalaínas, compuestos conocidos por sus propiedades antioxidantes. Este estudio evaluó las características fisicoquímicas, el contenido de betalaínas y su capacidad antioxidante (CA), así como el perfil sensorial de frutos de O. robusta y O. ficusindica. Esta última presentó mayor acidez y contenido de sólidos solubles (F= 769,2; P= 0,0001), (F= 360,4; P ≤0,0001), que O. robusta. En humedad y contenido de cenizas no hubo diferencias significativas entre ambas especies. La concentración de betalaínas fue superior en Opuntia robusta (F=529,1; P= ≤0,0001) betacianinas (0,114 mg/mL pulpa) y betaxantinas (0,073 mg/ mL de pulpa), en O. ficus-indica (0,023 mg/ mL de pulpa y 0,0198 mg/ mL de pulpa). Se encontraron diferencias significativas en la CA (F=545,9; P ≤0,0001), en O. ficus-indica hasta 195,38 µmol equivalente Trolox/ mL por el método Ácido2, 2´-azino-bis-(3-etilbenzotiazolina)6-sulfónico (ABTS) y 22% de inhibición de radicales libres por el método 2,2 difenil-1-pricrilhidrazilo (DPPH), para O. robusta 165,6 µmol equivalente Trolox/ mL y más del 36% de inhibición de radicales libres. Los resultados mostraron que la CA está directamente relacionada con la concentración de betacianinas y betaxantinas. Ambas variedades de Opuntia exhiben una tendencia a lo dulce y ácido, con aromas, sabores y resabios con notas frutales y vegetales. Estos resultados sugieren que estas especies pueden ser empleadas para la extracción de betalaínas debido a su gran potencial para utilizarse en la industria como fuente de pigmentos naturales con propiedades antioxidantes y agradables características sensoriales(AU)

Opuntia spp. are a Mexican phytogenetic resource with great nutritive value and high betalains (compounds known for their antioxidant properties) content. Our main goal was to evaluate the physicochemical characteristics, betalains concentration and antioxidant capacity (AC), as well as sensory profiles of Opuntia robusta and O. ficus-indica, where the later one showed higher acidity and soluble solids content (F= 769.2; P= 0.0001 and F= 360.4; P ≤0.0001 respectively) than O. robusta. There was no significant difference between the species in terms of humidity and ash content. Betalains concentrations were higher in Opuntia robusta (F=529.1; P= ≤0.0001), while betacyanins (0.114 mg/ mL pulp) and betaxantins (0.073 mg/ mL de pulp) were higher in O. ficus-indica (0.023 mg/ mL pulp and 0.0198 mg/ mL de pulp). Significant differences for AC were found (F=545.9, P ≤0.0001), with O. ficus-indica showing up to 195.38 µmol Trolox equivalent / mL by the method 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 22% of free radicals inhibition by the method 2, 2 diphenyl-1-pricrylhydrazyl (DPPH), while 165.6 µmol Trolox equivalent / mL and more than 36% free radicals inhibition were found for O. robusta. Results showed that the antioxidant capacity is directly related with betacyanins and betaxantines concentration. Both Opuntia varieties exhibit a tendency to sweetness and acidity, with aromas, flavors and scents within fruity and vegetable notes. These results suggest that both species could be used in the extraction of betalains due to their great industrial potential as a source of natural pigments with antioxidant properties and pleasant sensorial characteristics(AU)

Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals.

Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants.

Betalains are tyrosine-derived red-violet and yellow pigments, found in plants only of the Caryophyllales order. Although much progress has been made in recent years in the understanding of the betalain biosynthetic process, many questions remain open with regards to several of the proposed steps in the pathway. Most conspicuous by its absence is the characterization of the first committed step in the pathway, namely the 3-hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine (l-DOPA). We used transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) to identify a novel, betalain-related cytochrome P450-type gene, CYP76AD6, and carried out gene silencing and recombinant expression assays in Nicotiana benthamiana and yeast cells to examine its functionality. l-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1, which was previously thought to only catalyze a succeeding step in the pathway. While CYP76AD1 catalyzes both l-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to metabolically engineer entirely red-pigmented tobacco plants through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway.

Metabolite profiling of red and white pitayas (Hylocereus polyrhizus and Hylocereus undatus) for comparing betalain biosynthesis and antioxidant activity.

Metabolite profiling of red and white pitayas (Hylocereus polyrhizus and Hylocereus undatus) was performed using gas chromatography-time-of-flight-mass spectrometry and ultraperformance liquid chromatography-quadrupole-time-of-flight-mass spectrometry with multivariate analysis. Different species and parts of pitayas (red peel, RP; white peel, WP; red flesh, RF; and white flesh, WF) were clearly separated by partial least-squares discriminate analysis. Furthermore, betalain-related metabolites, such as betacyanins and betaxanthins, or their precursors were described on the basis of their metabolites. The results of antioxidant activity tests [1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and ferric reducing ability of plasma (FRAP)], total phenolic contents (TPC), total flavonoid contents (TFC), and total betacyanin contents (TBC) showed the following: RP ≥ WP > RF > WF. TPC, TFC, TBC, and betalain-related metabolites were higher in the peel than in the flesh and suggested to be the main contributors to antioxidant activity in pitayas. Therefore, peels as well as pulp of pitaya could beneficially help in the food industry.

Biosynthesis of betalains: yellow and violet plant pigments.

Betalains are the yellow and violet pigments that substitute anthocyanins in plants belonging to the order Caryophyllales. These pigments have attracted much attention because of their bioactivities, which range from an antioxidant capacity to the chemoprevention of cancer. However, the biosynthetic pathway of betalains remains under discussion; the main steps have been characterized in recent years, but multiple side reactions are possible. The key enzymes involved have only recently been described, providing clues about the regulation of betalain biosynthesis. In this review, we provide a comprehensive view of the biosynthetic scheme of betalains and discuss the different reactions that have been demonstrated experimentally or proposed in the literature.