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.     

Isolation of catechin-converting human intestinal bacteria

Aims: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. Methods and Results: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin‐converting human faecal suspension. The isolates catalysed individual steps in the degradation of (?)‐epicatechin and (+)‐catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C‐ring of both (?)‐epicatechin and (+)‐catechin giving rise to 1‐(3,4‐dihydroxyphenyl)‐3‐(2,4,6‐trihydroxyphenyl)propan‐2‐ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1‐(3,4‐dihydroxyphenyl)‐3‐(2,4,6‐trihydroxyphenyl)propan‐2‐ol to 5‐(3,4‐dihydroxyphenyl)‐γ‐valerolactone and 4‐hydroxy‐5‐(3,4‐dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. Conclusions: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. Significance and Impact of the Study: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin‐converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.     

Medicago glucosyltransferase UGT72L1: potential roles in proanthocyanidin biosynthesis

In the first reaction specific for proanthocyanidin (PA) biosynthesis in Arabidopsis thaliana and Medicago truncatula, anthocyanidin reductase (ANR) converts cyanidin to (?)-epicatechin. The glucosyltransferase UGT72L1 catalyzes formation of epicatechin 3′-O-glucoside (E3′OG), the preferred substrate for MATE transporters implicated in PA biosynthesis in both species. The mechanism of PA polymerization is still unclear, but may involve the laccase-like polyphenol oxidase TRANSPARENT TESTA 10 (TT10). We have employed a combination of cell biological, biochemical and genetic approaches to evaluate this PA pathway model. The promoter regions of UGT72L1 and MtANR share common cis-acting elements and direct overlapping, but partially distinct, expression patterns. UGT72L1 and MtANR are localized in the cytosol, whereas TT10 is localized to the vacuole. Over-expression of UGT72L1 in M. truncatula hairy roots results in increased accumulation of PA-like compounds, and loss of function of UGT72L1 partially reduces epicatechin, E3′OG and extractable PA levels in M. truncatula seeds. Expression of UGT72L1 in A. thaliana leads to a massive increase in E3′OG in immature seed, but reduced levels of extractable PAs. However, when UGT72L1 was expressed in the Arabidopsis tt10 mutant, extractable PA levels increased and seed coat browning was delayed. Our results suggest that glycosylation of epicatechin is important for both PA precursor transport and assembly, but that additional redundant pathways may exist.     

Phylloflavan,a characteristic constituent of Phyllocladus species

Selective visualization of 2D TLC of extracts of Phyllocladus species with vanillin—hydrochloric acid showed a characteristic pattern of constituents attributable to catechin, epicatechin and phylloflavan, a new flavanoid compound. Optical rotation measurements showed that while catechin and epicatechin are of the normal type with the 2R configurations, the flavan moiety in phylloflavan is of the opposite 2S configuration. Spectroscopic data of the new compound and its hydrolysis products showed phylloflavan to be ent-epicatechin-3-δ-(3,4-dihydroxyphenyl)-β-hydroxypentanoate.     

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon

Proanthocyanidins(PAs) are specialized metabolites that infuence persimmon fruit quality.Normal astringent(A)-type and non-astringent(NA)-type mutants show significant variation in PA accumulation, but the infuencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by Dk DTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pa...     

Multigene synergism increases the isoflavone and proanthocyanidin contents of Medicago truncatula

Isoflavones and proanthocyanidins (PAs), which are flavonoid derivatives, possess many health benefits and play important roles in forage‐based livestock production. However, the foliage of Medicago species accumulates limited levels of both isoflavones and PAs. In this study, biosynthesis of isoflavone and PA in Medicago truncatula was enhanced via synergy between soya bean isoflavone synthase (IFS1); two upstream enzymes, chalcone synthase (CHS) and chalcone isomerase (CHI); and the endogenous flavanone 3‐hydroxylase (F3H). Constitutive expression of GmIFS1 alone resulted in ectopic accumulation of the isoflavone daidzein and large increases in the levels of the isoflavones formononetin, genistein and biochanin A in the leaves. Furthermore, coexpression of GmIFS1 with GmCHS7 and GmCHI1A generally increased the available flux to flavonoid biosynthesis and resulted in elevated isoflavone, flavone and PA contents. In addition, down‐regulation of MtF3H combined with coexpression of GmIFS1, GmCHS7 and GmCHI1A led to the highest isoflavone levels (up to 2 μmol/g fresh weight in total). Taken together, our results demonstrate that multigene synergism is a powerful means to enhance the biosynthesis of particular flavonoids and can be more broadly applied to the metabolic engineering of forage species.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The influence of mycorrhizal colonization on growth in the greenhouse and on catechin, epicatechin and procyanidin in roots of Fagus sylvatica L.

 The influence of mycorrhizal colonization on beech (Fagus sylvatica L.) root tannin (procyanidin polymer) and its putative precursors catechin and epicatechin was investigated by high performance liquid chromatography. Seedlings planted in a sterile mixture of litter, compost, soil and sand were inoculated with brown beech ectomycorrhizas collected from a woodland (Lactarius subdulcis Bull ex Fr. ×  F. sylvatica). The seedlings were not fertilized during the first year of growth. Nonmycorrhizal control plants showed severe nutrient-deficiency symptoms on their leaves and grew less well than mycorrhizal plants. Mycorrhizal roots contained significantly less catechin, epicatechin and procyanidin polymer than nonmycorrhizal roots. In the second year of growth, the plants were fertilized and procyanidin formation in roots was investigated. None of the fertilized plants showed mineral-deficiency symptoms. Fertilized mycorrhizal roots consistently contained significantly less catechin and epicatechin than nonmycorrhizal controls, but procyanidin polymer content varied between replicate experiments. The possible function of catechin and epicatechin in ectomycorrhizal formation is discussed. Accepted: 11 July 1997     

Decreased anthocyanidin reductase expression strongly decreases silver birch (Betula pendula) growth and alters accumulation of phenolics

Phenolics, formed via a complex phenylpropanoid pathway, are important defensive agents in plants and are strongly affected by nitrogen (N) fertilization. Proanthocyanidins (PAs) are one possible endpoint of the phenylpropanoid pathway, and anthocyanidin reductase (ANR) represents a key enzyme in PA biosynthesis. In this study, the expression of silver birch (Betula pendula) anthocyanidin reductase BpANR was inhibited using the RNA interference (RNAi) method, in three consequent BpANR RNAi (ANRi birches) lines. The growth, the metabolites of the phenylpropanoid pathway, and the number of resin glands of the ANRi birches were studied when grown at two N levels. ANRi birches showed decreased growth and reduction in PA content, while the accumulation of total phenolics in both stems and leaves increased. Moreover, ANRi birches produced more resin glands than did wild‐type (WT) birches. The response of ANRi birches to N depletion varied compared with that of WT birches, and in particular, the concentrations of some phenolics in stems increased in WT birches and decreased in ANRi birches. Because the inhibition of PAs biosynthesis via ANR seriously affected birch growth and resulted in accumulation of the precursors, the native level of PAs in plant tissues is assumed to be the prerequisite for normal plant growth. This draws attention to the real plant developmental importance of PAs in plant tissues.     

Up‐regulation of GhTT2‐3A in cotton fibres during secondary wall thickening results in brown fibres with improved quality

Brown cotton fibres are the most widely used naturally coloured raw materials for the eco‐friendly textile industry. Previous studies have indicated that brown fibre pigments belong to proanthocyanidins (PAs) or their derivatives, and fibre coloration is negatively associated with cotton productivity and fibre quality. To date, the molecular basis controlling the biosynthesis and accumulation of brown pigments in cotton fibres is largely unknown. In this study, based on expressional and transgenic analyses of cotton homologs of ArabidopsisPA regulator TRANSPARENT TESTA 2 (TT2) and fine‐mapping of the cotton dark‐brown fibre gene (Lc1), we show that a TT2 homolog, GhTT2‐3A, controls PA biosynthesis and brown pigmentation in cotton fibres. We observed that GhTT2‐3A activated GhbHLH130D, a homolog of ArabidopsisTT8, which in turn synergistically acted with GhTT2‐3A to activate downstream PA structural genes and PA synthesis and accumulation in cotton fibres. Furthermore, the up‐regulation of GhTT2‐3A in fibres at the secondary wall‐thickening stage resulted in brown mature fibres, and fibre quality and lint percentage were comparable to that of the white‐fibre control. The findings of this study reveal the regulatory mechanism controlling brown pigmentation in cotton fibres and demonstrate a promising biotechnological strategy to break the negative linkage between coloration and fibre quality and/or productivity.     

The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods

Introduction – Pyrrolizidine alkaloids (PAs) serve an important function in plant defence. Objective – To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC‐NPD) and liquid chromatography tandem mass spectrometry (LC‐MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. Methodology – Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC‐NPD, reduction is required to transform PA N‐oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. Results – The lowest PA concentration measured with GC‐NPD was approximately 0.03 mg/g and with LC‐MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC‐NPD. With the LC‐MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC‐NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC‐MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC‐NPD analysis with sulfuric acid extraction and PA N‐oxide reduction with zinc and purification. Conclusions – In terms of sensitivity and discrimination, formic acid extraction in combination with LC‐MS/MS detection is the method of choice for analysing PAs (both free and N‐oxides forms) in plant material. Copyright © 2009 John Wiley & Sons, Ltd.     

TRANSPARENT TESTA 13 is a tonoplast P3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P‐type H⁺‐ATPases in the plasma membrane, and multimeric vacuolar‐type H⁺‐ATPases (V‐ATPases) and vacuolar H⁺‐pyrophosphatases (H⁺‐PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P_3A‐ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA‐filled central vacuoles as observed in the wild‐type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P‐type H⁺‐ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H⁺‐PPase VHP1. Our findings indicate that the P_3A‐ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12‐mediated transport of PA precursors to the vacuole.     

TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP‐type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA‐specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development.