Mapping of specialized metabolite terms onto a plant phylogeny using text mining and large language models.

Plants produce a staggering array of chemicals that are the basis for organismal function and important human nutrients and medicines. However, it is poorly defined how these compounds evolved and are distributed across the plant kingdom, hindering a systematic view and understanding of plant chemical diversity. Recent advances in plant genome/transcriptome sequencing have provided a well-defined molecular phylogeny of plants, on which the presence of diverse natural products can be mapped to systematically determine their phylogenetic distribution. Here, we built a proof-of-concept workflow where previously reported diverse tyrosine-derived plant natural products were mapped onto the plant tree of life. Plant chemical-species associations were mined from literature, filtered, evaluated through manual inspection of over 2500 scientific articles, and mapped onto the plant phylogeny. The resulting "phylochemical" map confirmed several highly lineage-specific compound class distributions, such as betalain pigments and Amaryllidaceae alkaloids. The map also highlighted several lineages enriched in dopamine-derived compounds, including the orders Caryophyllales, Liliales, and Fabales. Additionally, the application of large language models, using our manually curated data as a ground truth set, showed that post-mining processing can largely be automated with a low false-positive rate, critical for generating a reliable phylochemical map. Although a high false-negative rate remains a challenge, our study demonstrates that combining text mining with language model-based processing can generate broader phylochemical maps, which will serve as a valuable community resource to uncover key evolutionary events that underlie plant chemical diversity and enable system-level views of nature's millions of years of chemical experimentation.

Dopamine-derived pigments in nature: identification of decarboxybetalains in Amaranthaceae species.

A unique family of decarboxylated betalains derived from dopamine has recently been discovered. Due to the lack of chemical standards, the existence and distribution of decarboxylated betalains in nature remain unknown. Traditional betalains contain L-dihydroxyphenylalanine as the starting point of the biosynthetic pathway and betalamic acid as a structural and functional unit, while the recently discovered betalains rely on dopamine. Here, 30 dopamine-derived betalains were biotechnologically produced, purified, and characterized, creating an unprecedented library to explore their properties and presence in nature. The maximum absorbance wavelengths for the pigments ranged between 461 and 485 nm. HPLC analysis showed retention times between 0.6 and 2.2 min higher than traditional betalains due to their higher hydrophobicity. The presence of decarboxybetalains in nature was screened using HPLC-ESI-Q-TOF mass spectrometry in various species of the Amaranthaceae family: beetroot (Beta vulgaris subsp. vulgaris), Swiss chard (B. vulgaris var. cicla), celosia (Celosia argentea var. plumosa), and quinoa (Chenopodium quinoa). The latter species had the highest content of decarboxybetalains (28 compounds in its POEQ-143 variety). Twenty-nine pigments were found distributed among the different analyzed plant sources. The abundance of decarboxybetalains demonstrated in this work highlights these pigments as an important family of phytochemicals in the order Caryophyllales.

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.

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.

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.

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.

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.

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation.

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

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.

First report of betalain production from endolichenic Bacillus sp. LDAB-1 from Dirinaria aegilita: Insights from novel quantification methodology of image processing.

Pigments are widely used in food supplements envisaging attractive colors along with health benefits. The desired advancements in the nutraceutical and antioxidant properties of pigments utilized in food products necessitate the search for novel additives. The present study is the first in the field to report the pigment-producing endolichenic bacteria, Bacillus sp. LDAB-1 from Dirinaria aegilita. Morphological, biochemical, and molecular characterization of the bacterium emphasizes that ideal pigment production occurs when utilizing sucrose and sodium nitrate. The pigment was salted out and dialyzed for further qualitative characterization using ultraviolet-visible, fluorescence, and Fourier transform infrared spectra and the results corroborated the presence of betalains. The antioxidant activity of betalain is closer to the efficiency of α-tocopherol, which confers the pigment properties for antioxidant and nutraceutical significance. An optimal methodology for pigment affirmation is an issue when using an alternative methodology. Hence, the present assessment employs a comparative analysis of findings from both a spectrophotometric method and image processing technology encompassing RGB, CMYK, YCbCr, and L*a*b* color space models. Amongst these, the L*a*b* model potentially provides an effective modality for determining the pigment concentration. Bland-Altman plot analysis indicates similar consistency levels in betalain quantification by both methods at 95% confidence intervals, affirming the integrity and consistency of color image processing technology. Consequently, the present study represents novelty and innovativeness in reporting endolichenic Bacillus sp. LDAB-1 from D. aegilita and a rational image optimization protocol for pigment elucidation characteristics.

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

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

Beetroot as a novel ingredient for its versatile food applications.

Beta vulgaris, also known as Beetroot, is a member of a family of Chenopodiaceae and is widely used as a natural food colorant. It gets its distinctive color due to nitrogen-containing water-soluble pigments betalains. Beetroot is an exquisite cradle of nutrients, including proteins, sucrose, carbohydrates, vitamins (B complex and vitamin C), minerals, fiber. They also contain an appreciable amount of phenolic compounds and antioxidants such as coumarins, carotenoids, sesquiterpenoids, triterpenes, flavonoids (astragalin, tiliroside, rhamnocitrin, kaempferol, rhamnetin). Recent studies evidenced that beetroot consumption had favorable physiological benefits, leading to improved cardiovascular diseases, hypertension, diabetes, cancer, hepatic steatosis, liver damage, etc. This review gives insights into developing beetroot as a potential and novel ingredient for versatile food applications and the latest research conducted worldwide. The phytochemical diversity of beetroot makes them potential sources of nutraceutical compounds from which functional foods can be obtained. The article aimed to comprehensively collate some of the vital information published on beetroot incurred in the agri-food sector and a comprehensive review detailing the potentiality of tapping bioactive compounds in the entire agriculture-based food sector.

The evidence for anthocyanins in the betalain-pigmented genus Hylocereus is weak.

Here we respond to Zhou (BMC Genomics 21:734, 2020) "Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying peel and pulp color formation" published in BMC Genomics. Given the evolutionary conserved anthocyanin biosynthesis pathway in betalain-pigmented species, we are open to the idea that species with both anthocyanins and betalains might exist. However, in absence of LC-MS/MS spectra, apparent lack of biological replicates, and no comparison to authentic standards, the findings of Zhou (BMC Genomics 21:734, 2020) are not a strong basis to propose the presence of anthocyanins in betalain-pigmented pitaya. In addition, our re-analysis of the datasets indicates the misidentification of important genes and the omission of key flavonoid and anthocyanin synthesis genes ANS and DFR. Finally, our re-analysis of the RNA-Seq dataset reveals no correlation between anthocyanin biosynthesis gene expression and pigment status.

Formation of carboxylated and decarboxylated betalains in ripening grains of Chenopodium quinoa by a dual dioxygenase.

Chenopodium quinoa (quinoa) is a pseudo-cereal that forms part of the cultural heritage of Andean countries, and its grains have high nutritional value and potential health benefits. Betalains are nitrogenous water-soluble pigments and bioactive molecules that contribute to these health-promoting properties. Betalains are restricted to plants of the order Caryophyllales, to which quinoa belongs. A new family of betalains has been discovered in the form of unconventional decarboxylated pigments. Here, we show that these pigments accumulate in ripening quinoa grains of fluorescent nature, and are putatively based on a dopamine-cleaving activity. This study describes for the first time the purification and molecular and functional characterization of a 4,5-dopamine extradiol dioxygenase enzyme from plants. It is a monomeric protein with a molecular mass of 34.5 kDa characterized by chromatography, electrophoresis, and time-of-flight mass spectrometry. We demonstrate that this key enzyme has a dual function in a square-shaped biosynthetic pathway towards the formation of both carboxylated and decarboxylated pigments. Enzyme kinetic properties are characterized for the production of 6-decarboxy-betalamic acid and 3,4-dihydroxy-l-phenylalanine-derived betalamic acid, the two structural units of plant pigment in nature. The profile of multiple betalains present in quinoa grains has been reproduced in one-pot bioreactors containing the novel enzyme and two competing substrates.

Evolution and function of red pigmentation in land plants.

BACKGROUND: Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. SCOPE: In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure-function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. CONCLUSIONS: The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

Phytonutrients, Colorant Pigments, Phytochemicals, and Antioxidant Potential of Orphan Leafy Amaranthus Species.

The underutilized Amaranthus leafy vegetables are a unique basis of pigments such as ß-cyanins, ß-xanthins, and betalains with radical scavenging capacity (RSC). They have abundant phytonutrients and antioxidant components, such as pigments, vitamins, phenolics, and flavonoids. Eight selected genotypes (four genotypes from each species) of underutilized Amaranthus leafy vegetables were evaluated for phytonutrients, pigments, vitamins, phenolics, flavonoids, and antioxidants in a randomized complete block design under ambient field conditions with three replicates. The studied traits showed a wide range of variations across eight genotypes of two species of Amaranthus leafy vegetables. The highest fat, ß-xanthins, K, dietary fiber, Mg, ß-cyanins, Mn, chlorophyll ab, Zn, TP, TF, betalains, chlorophyll a content, and (RSC) (DPPH) and RSC (ABTS+) were obtained from A. tricolor accessions. Conversely, the highest protein, Cu, carbohydrates, Ca, and chlorophyll b content were obtained from A. lividus accessions. The highest dry matter, carotenoids, Fe, energy, and ash were obtained from A. tricolor and A. lividus. The accession AT2 confirmed the highest vit. C and RSC (DPPH) and RSC (ABTS+); AT5 had the highest TP content; and AT12 had the highest TF content. A. tricolor accessions had high phytochemicals across the two species, such as phytopigments, vitamins, phenolics, antioxidants, and flavonoids, with considerable nutrients and protein. Hence, A. tricolor accessions can be used as high-yielding cultivars comprising ample antioxidants. The correlation study revealed that vitamin C, pigments, flavonoids, ß-carotene, and phenolics demonstrated a strong RSC, and showed a substantial contribution to the antioxidant potential (AP) of A. tricolor. The investigation exposed that the accessions displayed a plentiful origin of nutritional values, phytochemicals, and AP with good quenching ability of reactive oxygen species (ROS) that provide enormous prospects for nourishing the mineral-, antioxidant-, and vitamin-threatened community.

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

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

Comparative Analysis of the Extradiol Ring-Cleavage Dioxygenase LigB from Arabidopsis and 3,4-Dihydroxyphenylalanine Dioxygenase from Betalain-Producing Plants.

Diverse arrays of naturally occurring compounds in plants are synthesized by specialized metabolic enzymes, many of which are distributed taxonomically. Although anthocyanin pigments are widely distributed and ubiquitous, betalains have replaced anthocyanins in most families in Caryophyllales. Anthocyanins and betalains never occur together in the same plant. The formation of betalamic acid, catalyzed by 3,4-dihydroxyphenylalanine (DOPA) 4,5-extradiol dioxygenase (DOD), is a key step in betalain biosynthesis. DODs in betalain-producing plants are coded by LigB genes, homologs of which have been identified in a wide range of higher plant orders, as well as in certain fungi and bacteria. Two classes of LigB homologs have been reported: those found in anthocyanin-producing species and those found in betalain-producing species, which contain DOD. To gain insight into the evolution of specialized metabolic enzymes involved in betalain biosynthesis, we performed a comparative biochemical analysis of Arabidopsis LigB, an extradiol ring-cleavage dioxygenase in anthocyanin-producing Arabidopsis and Phytolacca DOD1 of betalain-producing Phytolacca americana. We show that Arabidopsis LigB catalyzes 2,3-extradiol cleavage of DOPA to synthesize muscaflavin, whereas Phytolacca DOD1 converts DOPA to betalamic acid via 4,5-extradiol cleavage. Arabidopsis LigB also converts caffeic acid, a ubiquitous phenolic compound in higher plants, to iso-arabidopic acid in vitro via 2,3-extradiol cleavage of the aromatic ring. Amino-acid substitution in Arabidopsis LigB and Phytolacca DOD1 led to variable extradiol ring-cleavage function, supporting the suggestion that catalytic promiscuity serves as a starting point for the divergence of new enzymatic activities.

The report of anthocyanins in the betalain-pigmented genus Hylocereus is not well evidenced and is not a strong basis to refute the mutual exclusion paradigm.

Here we respond to the paper entitled "Contribution of anthocyanin pathways to fruit flesh coloration in pitayas" (Fan et al., BMC Plant Biol 20:361, 2020). In this paper Fan et al. 2020 propose that the anthocyanins can be detected in the betalain-pigmented genus Hylocereus, and suggest they are responsible for the colouration of the fruit flesh. We are open to the idea that, given the evolutionary maintenance of fully functional anthocyanin synthesis genes in betalain-pigmented species, anthocyanin pigmentation might co-occur with betalain pigments, as yet undetected, in some species. However, in absence of the LC-MS/MS spectra and co-elution/fragmentation of the authentic standard comparison, the findings of Fan et al. 2020 are not credible. Furthermore, our close examination of the paper, and re-analysis of datasets that have been made available, indicate numerous additional problems. Namely, the failure to detect betalains in an untargeted metabolite analysis, accumulation of reported anthocyanins that does not correlate with the colour of the fruit, absence of key anthocyanin synthesis genes from qPCR data, likely mis-identification of key anthocyanin genes, unreproducible patterns of correlated RNAseq data, lack of gene expression correlation with pigmentation accumulation, and putative transcription factors that are weak candidates for transcriptional up-regulation of the anthocyanin pathway.

Anthocyanin synthesis potential in betalain-producing Caryophyllales plants.

Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3-MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.