Minor betalains in fruits of Hylocereus species

Betacyanins in peel and flesh of fruits of different Hylocereus species were identified by means of GC/MS, electrospray MS/MS, HPLC as well as (1)H and (13)C NMR techniques. As hitherto unknown pigments: betanidin 5-O-(2'-O-beta-D-apiofuranosyl)-beta-D-glucopyranoside, betanidin 5-O-(4'-O-malonyl)-beta-D-glucopyranoside and betanidin 5-O-[(5'-O-E-sinapoyl)-2'-O-beta-D-apiofuranosyl]-beta-D-glucopyranoside were elucidated. The sinapoyl moiety attachment position in the structure of betacyanins was established for the first time. The peel contained a more complex pattern of betacyanins with apiofuranosyl moiety. Other recently identified pigments were also present in the samples and their (1)H or (13)C NMR spectra were recorded. In the case of phyllocactin and its 4'-isomer the migration of the malonyl group was noticed.     

Variation and genetic parameters of fruit colour and polyphenol composition in an apple seedling population segregating for red leaf

Red flesh colour is a relatively new target for apple breeding programmes and understanding genetic relationships between this trait and other fruit characters, including polyphenol compounds, is important for both breeders and marketers of new red flesh cultivars. In this study, fruit peel and flesh colours and concentrations of up to 20 individual fruit polyphenols within each tissue were examined in fruit harvested from a 14-family apple seedling population segregating for red and green leaf. Red leaf seedlings always produced red flesh fruit that varied from pale red to complete dark red cortical tissue (type 1 red flesh). Some (20 %) of green leaf seedlings also produced fruit with red flesh, albeit at low intensity (type 2 red flesh). Cyanidin 3-O-galactoside was the dominant anthocyanin in both fruit tissues, with concentrations being 1,900 times higher in the flesh and 2.5 times higher in the peel of fruit from red than from green leaf seedlings. Red leaf seedlings also had 59 % more flesh epicatechin and 17 % less total peel flavonols, but other polyphenols were not associated with leaf colour. Heritability estimates for red flesh colour, flesh cyanidin 3-O-galactoside, flesh and peel catechins were high in red leaf and low in green leaf seedlings. Conversely, estimates for red peel coverage and two peel anthocyanins were higher in green compared to those from red leaf seedlings. Other than these, heritability estimates were high only for dihydrochalcones and hydroxycinnamic acids from each tissue for both leaf colours but low for all other flesh and peel flavan-3-ols, procyanidins and most peel flavonols irrespective of leaf colour. Genetic correlations between polyphenol compounds varied considerably, but were broadly similar for red and green leaf seedlings. Genetic correlations were mostly moderate to high between compounds of the same metabolic group, but low between compounds from different groups. These results are discussed in relation to the genetic control of flesh colour and polyphenol accumulation in apple, as well as to implications for breeding red flesh apples with altered polyphenol composition.     

Structural implications on color, fluorescence, and antiradical activity in betalains

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors on flowers and fruits of most plants of the order Caryophyllales. They are classified as betacyanins, exhibiting a violet coloration, and betaxanthins, which exhibit yellow coloration. Traditionally, betalains have been defined as condensation products of betalamic acid with different amines and amino acids, but the implication of the pigment structure for their properties has not been investigated. This paper explores different structural features of the betalains, revealing the clues for the switch from yellow to violet color, and the loss of fluorescence. A relevant series of 15 betalain-related compounds (both natural and novel semisynthetic ones) is obtained and characterized by chromatography, UV-vis spectrophotometry, fluorescence, and electrospray ionization mass spectroscopy. Antiradical properties of individual pure compounds in a broad pH range are studied under the ABTS^•+ radical assay. Relevance of specific bonds is studied, and differences between betaxanthins and betacyanins are used to explore in depth the structure–antiradical activity relationships in betalains.     

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

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

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

Pigments of fly agaric (Amanita muscaria)

The complex pigment pattern of fly agaric (Amanita muscaria) cap skins has been studied by LC-DAD and mass spectrometry. Among the betaxanthins the corresponding derivatives of serine, threonine, ethanolamine, alanine, Dopa, phenylalanine and tryptophan are reported for the first time to contribute to the pigment pattern of fly agarics. Betalamic acid, the chromophoric precursor of betaxanthins and betacyanins, muscaflavin and seco-dopas were also detected. Furthermore, the red-purple muscapurpurin and the red muscarubrin were tentatively assigned while further six betacyanin-like components could not be structurally allocated. Stability studies indicated a high susceptibility of pigment extracts to degradation which led to rapid colour loss thus rendering a complete characterization of betacyanin-like compounds impossible at present. Taking into account these difficulties the presented results may be a starting point for a comprehensive characterization of the pigment composition of fly agarics.     

Dihydroflavonol 4-reductase cDNA from non-anthocyanin-producing species in the Caryophyllales

Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plant. Although anthocyanins are widely distributed in higher plants as flower and fruit pigments, betacyanins have replaced anthocyanins in the Caryophyllales. We isolated cDNAs encoding dihydroflavonol 4-reductase (DFR), which is the first enzyme committed to anthocyanin biosynthesis in the flavonoid pathway, from Spinacia oleracea and Phytolacca americana, plants that belong to the Caryophyllales. The deduced amino acid sequence of Spinacia DFR and Phytolacca DFR revealed a high degree of homology with DFRs of anthocyanin-producing plants. The DFR of carnation, an exception in the Caryophyllales that synthesizes anthocyanin, showed the highest level of identity. In the phylogenetic tree, Spinacia DFR and Phytolacca DFR clustered with the DFRs of anthocyanin-synthesizing dicots. Recombinant Spinacia and Phytolacca DFRs expressed in Escherichia coli convert dihydroflavonol to leucoanthocyanidin. The expression and function of DFR in spinach and pokeweed are discussed in relation to the molecular evolution of red pigment biosynthesis in higher plants.     

The role of anthocyanin in photoprotection and its relationship with the xanthophyll cycle and the antioxidant system in apple peel depends on the light conditions

The synthesis of anthocyanin, the xanthophyll cycle, the antioxidant system and the production of active oxygen species (AOS) were compared between red and non‐red apple cultivars, in response to either long‐term sunlight exposure (high light intensity) during fruit development, or to exposure of bagged fruits to lower light intensity late in fruit development. During fruit development of red and non‐red apples, the xanthophyll cycle pool size decreased much more in red apple peel late in development. With accumulation of AOS induced by long‐term sunlight exposure, enhancement of the antioxidant system was found. However, this change became significantly lower in red apple than non‐red apple as fruit developed, which might serve to accelerate the anthocyanin synthesis in red apple peel. When, late in fruit development, bagged fruits were exposed to sunlight, the accumulation of AOS was lower in red apple peel than in non‐red peel. This could be due to the higher anthocyanin concentration in the red peels. Meanwhile, compared with that in non‐red cultivar, the xanthophyll cycle and the antioxidant system in red apple peel were protected first but then down‐regulated by its higher anthocyanin concentration during sunlight exposure. In conclusions, red and non‐red apples peel possess different photoprotective mechanisms under high light conditions. The relationship between anthocyanin synthesis and the xanthophyll cycle, and the antioxidant system, depends on the light conditions that fruit undergoes.     

Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora

Cell cultures derived from three yellow flowering Portulaca grandiflora genotypes contained betacyanins rather than betaxanthins. A betaxanthin-producing cell culture was obtained by subculturing orange cell clusters isolated from the red-violet cell culture of a violet flowering P. grandiflora genotype. Selection of the most strongly yellow coloured cell material reduced the portion of betacyanins considerably and resulted in a P. grandiflora cell culture characterized by a high concentration of betaxanthins and the occurrence of free betalamic acid. Vulgaxanthin I was the main compound. Besides these pigments carotenoids and flavonoids were detectable.Betaxanthin biosynthesis strongly dependend on light. Product accumulation reached its maximum during the stationary phase of the growth cycle. Excretion of pigments, especially of betalains, could not be detected. Vulgaxanthin I as found in the cell culture was identical with one of two main betaxanthins in the yellow petals of P. grandiflora.The yellow P. grandiflora cells grew well on solid modified Murashige and Skoog medium but failed to grow in liquid medium after a few subcultures. In contrast, white P. grandiflora suspension cultures could be established repeatedly.Abbreviations A absorbance - BX I and BX II betaxanthin fractions - DOPA dihydroxyphenylalanin - DW cell dry weight - molar extinction coefficient - SS solvent system     

Carbon starvation reduces carbohydrate and anthocyanin accumulation in red-fleshed fruit via trehalose 6-phosphate and MYB27

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel-coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red-fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6-phosphate. RNA-Seq analysis showed down-regulation of both gene-encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6-phosphate synthase gene TPS1.1a was down-regulated, whereas putative regulatory TPS7 and TPS11 were strongly up-regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up-regulated, whereas MYB27 was also up-regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6-phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits.     

What weta want: colour preferences of a frugivorous insect

Plants use colours as signals to attract mutualists and repel antagonists. Fleshy-fruits are often conspicuously coloured to signal different types of information including fruit maturity and spatial location. Previous work on fruit colour selection focus on large diurnal vertebrates, yet fruit colours are perceived differently by frugivores with different types of visual systems. Here, we tested whether a nocturnal, frugivorous, seed-dispersing insect selects fruits based on their pigmentation and whether different lighting conditions affect fruit colour selection. We captured 20 Wellington tree weta (Hemideina crassidens) from a forest reserve on the North Island of New Zealand and brought them into laboratory conditions to test their fruit colour preferences. The fruits of Coprosma acerosa, a native shrub species that naturally produces translucent, blue-streaked fruits, were dyed either red or blue. Fruits were then offered to weta in a binary (y-maze) choice test in two light conditions, either at night during a full moon or under artificial light conditions in the lab. Weta preferred unmanipulated, naturally blue-streaked fruits and artificially-blue coloured fruits over those dyed red. Furthermore, their colour preferences were unaffected by light environment. Our results therefore suggest that weta can discriminate between colours (using colour vision) in both light and dark light environments. Their consistent preferences for colours other than red indicate that weta might be responsible for the unusual colours of fleshy-fruits in New Zealand.     

Betalains of Celosia argentea

The betalains of yellow, orange and red inflorescences of common cockscomb (Celosia argentea var. cristata) were compared and proved to be qualitatively identical to those of feathered amaranth (Celosia argentea var. plumosa). In addition to the known compounds amaranthin and betalamic acid, the structures of three yellow pigments were elucidated to be immonium conjugates of betalamic acid with dopamine, 3-methoxytyramine and (S)-tryptophan by various spectroscopic techniques and comparison to synthesized reference compounds; the latter two are new to plants. Among the betacyanins occurring in yellow inflorescences in trace amounts, the presence of 2-descarboxy-betanidin, a dopamine-derived betacyanin, has been ascertained. The detection of high dopamine concentration may be of toxicological relevance in use of yellow inflorescences as a vegetable and in traditional Chinese medicine, common uses for the red inflorescences of common cockscomb.     

Formation and occurrence of dopamine-derived betacyanins

In light of the fact that the main betaxanthin (miraxanthin V) and the major betacyanin (2-descarboxy-betanidin) in hairy root cultures of yellow beet (Beta vulgaris L.) are both dopamine-derived, the occurrence of similar structures for the minor betacyanins was also suggested. By HPLC comparison with the betacyanins obtained by dopamine administration to beet seedlings, enzymatic hydrolysis, LCMS and 1H NMR analyses, the minor betacyanins from hairy roots were identified as 2-descarboxy-betanin and its 6'-O-malonyl derivative. A short-term dopamine administration experiment with fodder beet seedlings revealed that the condensation step between 2-descarboxy-cyclo-Dopa and betalamic acid is the decisive reaction, followed by glucosylation and acylation. From these data a pathway for the biosynthesis of dopamine-derived betalains is proposed. Furthermore, the occurrence of these compounds in various cell and hairy root cultures as well as beet plants (Fodder and Garden Beet Group) is shown.     

植物中甜菜色素的研究进展

甜菜色素是一类主要存在于石竹目植物中的天然植物色素，分为甜菜红素和甜菜黄素。甜菜色素不仅具有吸引昆虫授粉的作用，还可以作为一种重要的渗透调节物质和非酶促抗氧化剂来帮助植物抵抗逆境，维持植物体内正常的生理活动。甜菜色素生物合成途径独特，具有重要的医疗保健价值，目前已被广泛用于食品、药物和化妆品中。本文结合国内外最新研究进展，从理化性质、合成途径、生物学功能和应用价值等方面对甜菜色素进行了介绍，重点阐述了甜菜色素和花青素的关系以及光照对甜菜色素生物合成的影响，以期为进一步深入了解甜菜色素和开发利用甜菜色素提供参考依据。     

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.     

Development of a protocol for the semi-synthesis and purification of betaxanthins

A method for the analytical and semi-preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi-synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed-phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV-vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments.