Jean-Marc Souquet

Polymeric proanthocyanidins from grape skins

Abstract LC-mass spectrometric analysis of the degradation products released by thioacidolysis of a grape ( Vitis vinifera var. Merlot) skin extract showed that catechin, epicatechin, epicatechin gallate and epigallocatechin were the major constitutive units of grape skin tannins. Gallocatechin and epigallocatechin gallate were also detected. Epicatechin represented 60% of the extension units, whereas 67% of the terminal units consisted of catechin. Six fractions were prepared from the skin tannin extract by normal phase and analysed by reverse-phase HPLC after thioacidolysis. The mean degree of polymerization (mDP) calculated for each fraction ranged from three (in fraction I) to 80 (in fraction VI), confirming that proanthocyanidins were eluted from the normal phase column in increasing order of M r . All fractions contained prodelphinidins. The proportion of galloyllated units was low (3% to 6%) and independent of mDP.

Grapevine MATE-Type Proteins Act as Vacuolar H+-Dependent Acylated Anthocyanin Transporters

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1∷ or AM3∷green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H+-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.

Normal-phase high-performance liquid chromatographic separation of procyanidins from cacao beans and grape seeds

Abstract An HPLC method using a normal-phase silica column and a gradient of dichloromethane—methanol—formic acid—water mixtures as the eluent was developed to separate procyanidins on a molecular mass basis, without derivatization. It was successfully applied to the analysis of procyanidin extracts from cacao beans and grape seeds. The monomers and major dimers were resolved as discrete peaks. Oligomeric and polymeric components were eluted in order of increasing degree of polymerization, as confirmed by determining the average molecular mass of successive fractions collected from the normal-phase column using gel permeation chromatography, after acetylation.

Characterization of highly polymerized procyanidins in cider apple (Malus sylvestris var. kermerrien) skin and pulp

Abstract Freeze-dried materials of cider apple pulp and skin were submitted to three successive solid-liquid extractions. The water-acetone extracts contained significant amounts of condensed tannins; thioacidolysis revealed that they were a mixture of highly polymerized procyanidins mainly constituted of (−)-epicatechin units. Electrospray ionization mass spectrometry showed a complete series of polymeric procyanidins with a degree of polymerization up to 17.

Quantitative Genetic Bases of Anthocyanin Variation in Grape ( Vitis vinifera L. ssp. sativa ) Berry: A Quantitative Trait Locus to Quantitative Trait Nucleotide Integrated Study

The combination of QTL mapping studies of synthetic lines and association mapping studies of natural diversity represents an opportunity to throw light on the genetically based variation of quantitative traits. With the positional information provided through quantitative trait locus (QTL) mapping, which often leads to wide intervals encompassing numerous genes, it is now feasible to directly target candidate genes that are likely to be responsible for the observed variation in completely sequenced genomes and to test their effects through association genetics. This approach was performed in grape, a newly sequenced genome, to decipher the genetic architecture of anthocyanin content. Grapes may be either white or colored, ranging from the lightest pink to the darkest purple tones according to the amount of anthocyanin accumulated in the berry skin, which is a crucial trait for both wine quality and human nutrition. Although the determinism of the white phenotype has been fully identified, the genetic bases of the quantitative variation of anthocyanin content in berry skin remain unclear. A single QTL responsible for up to 62% of the variation in the anthocyanin content was mapped on a Syrah × Grenache F1 pseudo-testcross. Among the 68 unigenes identified in the grape genome within the QTL interval, a cluster of four Myb-type genes was selected on the basis of physiological evidence (VvMybA1, VvMybA2, VvMybA3, and VvMybA4). From a core collection of natural resources (141 individuals), 32 polymorphisms revealed significant association, and extended linkage disequilibrium was observed. Using a multivariate regression method, we demonstrated that five polymorphisms in VvMybA genes except VvMybA4 (one retrotransposon, three single nucleotide polymorphisms and one 2-bp insertion/deletion) accounted for 84% of the observed variation. All these polymorphisms led to either structural changes in the MYB proteins or differences in the VvMybAs promoters. We concluded that the continuous variation in anthocyanin content in grape was explained mainly by a single gene cluster of three VvMybA genes. The use of natural diversity helped to reduce one QTL to a set of five quantitative trait nucleotides and gave a clear picture of how isogenes combined their effects to shape grape color. Such analysis also illustrates how isogenes combine their effect to shape a complex quantitative trait and enables the definition of markers directly targeted for upcoming breeding programs.

Interspecific variation in leaf litter tannins drives decomposition in a tropical rain forest of French Guiana

Tannins are believed to be particularly abundant in tropical tree foliage and are mainly associated with plant herbivore defense. Very little is known of the quantity, variation, and potential role of tannins in tropical leaf litter. Here we report on the interspecific variability of litter condensed tannin (CT) concentration among 16 co-occurring tropical rain forest tree species of French Guiana and explore the functional significance of variable litter CT concentration for litter decomposition. We compared some classical methods in the ecological literature to a method based on high-performance liquid chromatography (HPLC), coupled with CT degradation by phloroglucinolysis. The same litter was allowed to decompose in the field in the presence or absence of soil fauna. We found large interspecific differences in the average polymerization degree (2.7 to 21.3, for non-extractable CT) and concentration of litter CT (0-3.7% dry mass, for total CT) determined by HPLC, which did not correlate with Folin total phenolics but correlated reasonably well with acid butanol CT. The concentration and polymerization degree of HPLC-determined CT were the only variables of the multitude of measured initial litter quality parameters that explained a significant amount of variation in litter mass loss among species, irrespective of animal presence. However, animal presence increased mean litter mass loss by a factor of 1.5, and the fauna effect on decomposition was best explained by a negative correlation with total HPLC CT and by a positive correlation with hemicellulose. Our results suggest that the commonly used acid butanol assay yields a reliable estimate of interspecific variation in CT concentration. However, the chemical structure of CTs, such as the polymerization degree, adds important information for the understanding of the functional role of CTs in litter decomposition. We conclude that the wide variation in structure and concentration of leaf litter CTs among tropical tree species is an important driver of decomposition in this nutrient-poor Amazonian rain forest.

The hidden face of food phenolic composition

Plant polyphenols are extremely diverse, due to the occurrence of several basic structures, numerous substitutions and, for some groups, of polymers (tannins). Plant polyphenol composition depends on the plant species and organ, with some molecules specific of particular plant families while others are ubiquitous. The polyphenol content is classically assessed by global analysis methods, which lack specificity and accuracy. These methods have been replaced with high performance liquid chromatography (HPLC), that enables accurate determination of individual molecules, provided they can be unambiguously identified and calibration curves can be established. However, HPLC analysis is restricted to simple compounds and difficult to apply in the case of complex extracts. Further difficulties encountered in the case of polymers include irreversible adsorption on the stationary phases. Proanthocyanidin analysis by HPLC after acid-catalysed depolymerisation in the presence of a nucleophile permits to overcome these problems and shows that proanthocyanidins predominate in the polyphenol composition of most plants. Large varietal differences in tannin quantitative and qualitative composition were observed for all plant species studied. Moreover, analysis is usually performed after extraction, which may lead to significant underestimation of the polyphenol content, since a large proportion is not extracted by usual solvents. This may be due to covalent binding to other plant constituents and to non-covalent adsorption on plant solids. Such matrix effect also influences the taste perception of polyphenols and their fate in the digestive tract, from in-mouth interactions with salivary proteins to their metabolism by colon microflora, with potential influence on bioavailability.

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis. Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.

Characterization of microbial metabolism of Syrah grape products in an in vitro colon model using targeted and non-targeted analytical approaches

PurposeSyrah red grapes are used in the production of tannin-rich red wines. Tannins are high molecular weight molecules, proanthocyanidins (PAs), and poorly absorbed in the upper intestine. In this study, gut microbial metabolism of Syrah grape phenolic compounds was investigated.MethodsSyrah grape pericarp was subjected to an enzymatic in vitro digestion model, and red wine and grape skin PA fraction were prepared. Microbial conversion was screened using an in vitro colon model with faecal microbiota, by measurement of short-chain fatty acids by gas chromatography (GC) and microbial phenolic metabolites using GC with mass detection (GC–MS). Red wine metabolites were further profiled using two-dimensional GC mass spectrometry (GCxGC-TOFMS). In addition, the effect of PA structure and dose on conversion efficiency was investigated by GC–MS.ResultsRed wine exhibited a higher degree of C1–C3 phenolic acid formation than PA fraction or grape pericarp powders. Hydroxyphenyl valeric acid (flavanols and PAs as precursors) and 3,5-dimethoxy-4-hydroxybenzoic acid (anthocyanin as a precursor) were identified from the red wine metabolite profile. In the absence of native grape pericarp or red wine matrix, the isolated PAs were found to be effective in the dose-dependent inhibition of microbial conversions and short-chain fatty acid formation.ConclusionsMetabolite profiling was complementary to targeted analysis. The identified metabolites had biological relevance, because the structures of the metabolites resembled fragments of their grape phenolic precursors or were in agreement with literature data.

Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping

BackgroundProanthocyanidins (PAs), or condensed tannins, are flavonoid polymers, widespread throughout the plant kingdom, which provide protection against herbivores while conferring organoleptic and nutritive values to plant-derived foods, such as wine. However, the genetic basis of qualitative and quantitative PA composition variation is still poorly understood. To elucidate the genetic architecture of the complex grape PA composition, we first carried out quantitative trait locus (QTL) analysis on a 191-individual pseudo-F1 progeny. Three categories of PA variables were assessed: total content, percentages of constitutive subunits and composite ratio variables. For nine functional candidate genes, among which eight co-located with QTLs, we performed association analyses using a diversity panel of 141 grapevine cultivars in order to identify causal SNPs.ResultsMultiple QTL analysis revealed a total of 103 and 43 QTLs, respectively for seed and skin PA variables. Loci were mainly of additive effect while some loci were primarily of dominant effect. Results also showed a large involvement of pairwise epistatic interactions in shaping PA composition. QTLs for PA variables in skin and seeds differed in number, position, involvement of epistatic interaction and allelic effect, thus revealing different genetic determinisms for grape PA composition in seeds and skin. Association results were consistent with QTL analyses in most cases: four out of nine tested candidate genes (VvLAR1, VvMYBPA2, VvCHI1, VvMYBPA1) showed at least one significant association with PA variables, especially VvLAR1 revealed as of great interest for further functional investigation. Some SNP-phenotype associations were observed only in the diversity panel.ConclusionsThis study presents the first QTL analysis on grape berry PA composition with a comparison between skin and seeds, together with an association study. Our results suggest a complex genetic control for PA traits and different genetic architectures for grape PA composition between berry skin and seeds. This work also uncovers novel genomic regions for further investigation in order to increase our knowledge of the genetic basis of PA composition.