TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat

The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.     

Metabolic profiling and cytological analysis of proanthocyanidins in immature seeds of Arabidopsis thaliana flavonoid accumulation mutants

Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione‐S‐transferase (GST)‐like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar‐like structures, whereas those in tt12, a mutant of the tonoplast‐bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP‐TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent‐insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent‐soluble fraction by LC‐MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP‐TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

Fractionation and chemistry of ethyl acetate-soluble thearubigins from black tea

Sephadex LH-20 chromatography was used to fractionate purified ethyl acetate-soluble thearubigins, prepared from an aqueous ethanolic extract of black tea. Three subfractions were so produced, each having a MW of about 1500 and each being degradable into cyanidin, delphinidin, gallic acid, the same two flavan-3-ols, and the same two flavan-3-ol gallates, though in different yield. Some evidence for the presence of benzotropolone moieties in at least one of the subfractions was obtained. Overall the ethyl acetate-soluble thearubigins are viewed as pentameric flavan-3-ols/flavan-3-ol gallates, containing both hydrolysable and non-hydrolysable interflavanoid links, as well as benzotropolone units, rather than as polymeric proanthocyanidins, a term previously used for all thearubigin subgroups.     

Phytochemistry of the mopane,Colophospermum mopane

The polyphenolic pool of the heartwood of the mopane, Colophospermum mopane Kirk ex J. Leonard, exhibits extreme diversity and complexity. It comprises a variety of monomeric flavonoids, e.g. flavan-3-ols, flavan-3,4-diols including the mopanols and peltogynols, flavonols, dimeric proanthocyanidins, e.g. proguibourtinidins, profisetinidins, promopanidins, propeltogynidins, and a variety of profisetinidin-type triflavanoids. The di- and tri-meric proanthocyanidins are accompanied by several functionalized tetrahydropyrano- and hexahydrodipyrano-chromenes (phlobatannins) that originate from the bi- and tri-flavanoids, respectively, via rearrangement of the pyran heterocycle(s). Owing to the predominance of the 5-deoxy (A-ring) flavan-3-ols, the chain terminating moieties in the biosynthesis of oligo- and poly-meric proanthocyanidins, the di- and tri-meric analogs also exhibit diversity as far as interflavanyl bonding positions are concerned. Such heterogeneity results from the reduced nucleophilicity of the A-rings of 5-deoxy flavan-3-ols, compared to the A-rings of the 5-oxy analogs (catechins), hence permitting alternative centers to participate in proanthocyanidin formation. Biomimetic-type syntheses were extensively utilized to unequivocally establish constitution and absolute stereochemistry of both the conventional and pyran ring rearranged-type di- and tri-meric compounds. Comprehension of the intricate mechanistic and stereochemical course of the pyran ring rearrangement reactions also contributed significantly to unambiguous structure elucidations. The aerial parts of the mopane are rich in essential oils that comprise mainly alpha-pinene and limonene, which are presumably responsible for the strong turpentine odor of the pods. The leaves also contain significant concentrations of beta-sitosterol and stigmasterol which are apparently the source of sterols in various organs of the mopane moth, Gonimbrasia belina. Three diterpenes, dihydrogrindelic acid, labd-13E-en-15-oate and dihydrogrindelaldehyde are present in the bark and seeds, the latter compound exhibiting significant cytotoxicity against a human breast cancer cell line.     

Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.     

Challenges and complexity of functionality evaluation of flavan-3-ol derivatives

Flavan-3-ol derivatives are common plant-derived bioactive compounds. In particular, (–)-epigallocatechin-3-O-gallate shows various moderate biological activities without severe toxicity, and its health-promoting effects have been widely studied because it is a main ingredient in green tea and is commercially available at low cost. Although various biologically active flavan-3-ol derivatives are present as minor constituents in plants as well as in green tea, their biological activities have yet to be revealed, mainly due to their relative unavailability. Here, I outline the major factors contributing to the complexity of functionality studies of flavan-3-ol derivatives, including proanthocyanidins and oligomeric flavan-3-ols. I emphasize the importance of conducting structure-activity relationship studies using synthesized flavan-3-ol derivatives that are difficult to obtain from plant extracts in pure form to overcome this challenge. Further discovery of these minor constituents showing strong biological activities is expected to produce useful information for the development of functional health foods.     

TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis

Flavonoid compounds such as anthocyanins and proanthocyanidins (PAs; so-called condensed tannins) have a multitude of functions in plants. They must be transported from the site of synthesis in the cytosol to their final destination, the vacuoles. Three models have been proposed for sequestering anthocyanins in vacuoles, but the transport machinery for PAs is poorly understood. Novel Arabidopsis mutants, transparent testa 19 (tt19), which were induced by ion beam irradiation, showed a great reduction of anthocyanin pigments in the vegetative parts as well as brown pigments in the seed coat. The TT19 gene was isolated by chromosome walking and a candidate gene approach, and was shown to be a member of the Arabidopsis glutathione S-transferase (GST) gene family. Heterologous expression of a putative ortholog, petunia anthocyanin 9 (AN9), in tt19 complemented the anthocyanin accumulation but not the brown pigmentation in the seed coat. This suggests that the TT19 gene is required for vacuolar uptake of anthocyanins into vacuoles, but that it has also a function different from that of AN9. The depositional pattern of PA precursors in the mutant was different from that in the wild type. These results indicate that TT19 participates in the PA pathway as well as the anthocyanin pathway of Arabidopsis. As involvement of GST in the PA pathway was previously considered unlikely, the function of TT19 in the PA pathway is also discussed in the context of the putative transporter for PA precursors.     

Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles

Mountain birch leaves contain large amounts of structurally variable polymeric proanthocyanidins. Their isolation procedure was enhanced by the addition of liquid-liquid extractions prior to column chromatography over Sephadex LH-20. Isolated polymeric proanthocyanidins were depolymerised by acid-catalysis in the presence of benzyl mercaptan or phloroglucinol in order to study their composition. The resulting degradation products, flavan-3-ols and flavan-3-ol adducts, were analysed with reversed-phase high-performance liquid chromatography using UV photodiode array detection for quantification and electrospray ionisation mass spectrometry for identification. The results showed that polymeric proanthocyanidins contained (epi)gallocatechins and (epi)catechins as the extension units and, mainly, (+)-catechin as the terminal unit. The mean degree of polymerisation was found to be 26 based on thiolysis and 31 based on phloroglucinol degradation.     

Metabolomic Analysis Using Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (UPLC-Q-TOF MS) Uncovers the Effects of Light Intensity and Temperature under Shading Treatments on the Metabolites in Tea

To investigate the effect of light intensity and temperature on the biosynthesis and accumulation of quality-related metabolites, field grown tea plants were shaded by Black Net and Nano-insulating Film (with additional 2–4°C cooling effect) with un-shaded plants as a control. Young shoots were subjected to UPLC-Q-TOF MS followed by multivariate statistical analysis. Most flavonoid metabolites (mainly flavan-3-ols, flavonols and their glycosides) decreased significantly in the shading treatments, while the contents of chlorophyll, β-carotene, neoxanthin and free amino acids, caffeine, benzoic acid derivatives and phenylpropanoids increased. Comparison between two shading treatments indicated that the lower temperature under Nano shading decreased flavonols and their glycosides but increased accumulation of flavan-3-ols and proanthocyanidins. The comparison also showed a greater effect of temperature on galloylation of catechins than light intensity. Taken together, there might be competition for substrates between the up- and down-stream branches of the phenylpropanoid/flavonoid pathway, which was influenced by light intensity and temperature.     

Influence of Maceration Temperature in Red Wine Vinification on Extraction of Phenolics from Berry Skins and Seeds of Grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.     

The Arabidopsis tt19-4 mutant differentially accumulates proanthocyanidin and anthocyanin through a 3' amino acid substitution in glutathione S-transferase

The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3'-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expression differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocyanins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are functional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a 5' GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3' domain affecting proanthocyanidin accumulation by a single amino acid substitution.     

Plant aging and excess light enhance flavan-3-ol content in Cistus clusii

Physiological studies on aging in perennials are mainly focused either on the primary metabolism or the hormonal regulation of the process. However, to our knowledge, the involvement of the secondary metabolism in this process has not yet been explored. Cistus clusii, a Mediterranean sclerophyllous evergreen bush, shows considerable amounts of flavan-3-ols in leaves. In the present study, we aimed at determining the impact of environmental conditions and plant aging in the flavan-3-ol content in C. clusii plants grown in field conditions, which included summer drought and recovery periods. Six-year-old plants suffered more from photo-oxidative stress, especially during excess light periods, and showed lower maximum photosynthetic rates than 1-year-old plants. C. clusii leaves accumulated (−)-epigallocatechin gallate in early summer, in a strong positive correlation with both the photon flux density and the photoperiod, but not with the plant water status. Moreover, C. clusii plants accumulated proanthocyanidins (polymeric flavan-3-ols) in leaves during summer. Older plants showed higher levels of proanthocyanidins and (−)-epicatechin, but only during late spring and summer. From the result of the present study, we conclude that excess light enhances flavan-3-ol content in C. clusii, a process enhanced as plants age due to increased excess light stress.     

Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids

Circular dichroism is a powerful tool for establishing the absolute configuration of flavonoids and proanthocyanidin analogues. It has been utilized to study the configuration of flavanones, dihydroflavonols (3-hydroxyflavanones), flavan-3-ols, flavan-4-ols, flavan-3,4-diols, flavans, isoflavans, isoflavanones, pterocarpans, 6a-hydroxypterocarpans, rotenoids, 12a-hydroxyrotenoids, neoflavonoids, 3,4-dihydro-4-arylcoumarins, 4-arylflavan-3-ols, auronols, homoisoflavanones, proanthocyanidins, and various classes of biflavonoids. Results relevant to the correlation of circular dichroic data and the absolute configuration of the diastereoisomers of some of the above classes of compounds will be discussed.     

Characterization and structures of anthocyanin pigments generated in rosé cider during vinification.

Anthocyanin pigments, which are not found in apple juice, were detected in rosé cider. We confirmed by HPLC/DAD and LC/ESI-MS analyses that some of these anthocyanin pigments generated in rosé cider during vinification corresponded to those formed in model cider containing anthocyanin and flavan-3-ol in the presence of acetaldehyde. To confirm their structures, two anthocyanin pigments formed in a model cider containing cyanidin-3-galactoside and (-)-epicatechin in the presence of acetaldehyde were isolated and purified, and their structures were elucidated by high resolution FAB-MS and (1)H and (13)C NMR analyses. These two pigments were found to consist of cyanidin-3-galactoside and (-)-epicatechin linked by a CH(3)-CH bridge at the 8-position. They were diastereomers that differed in the configuration of the asymmetric methine carbon.     

Cinnamoyl glucosides of catechin and dimeric procyanidins from young leaves of Inga umbellifera (Fabaceae)

The rapidly growing, nearly achlorophyllous, young leaves of Inga umbellifera express high concentrations of mono and dimeric 3-O-gluco-cinnamoyl catechin/epicatechin, rare forms of substituted flavan-3-ols. Here we present structures for five novel compounds in this class: three monomers [catechin-3-O-beta-D-gluco(2-cinnamoyl)pyranoside, catechin-3-O-beta-D-gluco(6-cinnamoyl) pyranoside, catechin-3-O-beta-D-gluco(2,6-biscinnamoyl)pyranoside] and two dimeric procyanidins [catechin-3-O-beta-D-glucopyrano-(4alpha-->8)-catechin-3-O-beta-D-gluco(2-cinnamoyl)pyranoside and catechin-3-O-beta-D-glucopyrano-(4alpha-->8)-epicatechin-3-O-beta-D-gluco(6-cinnamoyl)pyranoside]. The young leaves of Inga umbellifera express high concentrations of 3-O-(cinnamoyl)glucosides of catechin and epicatechin.     

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation

Summary A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.     

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation.

A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.