A high-density integrated map for grapevine based on three mapping populations genotyped by the Vitis18K SNP chip.

KEY MESSAGE: We present a high-density integrated map for grapevine, allowing refinement and improved understanding of the grapevine genome, while demonstrating the applicability of the Vitis18K SNP chip for linkage mapping. The improvement of grapevine through biotechnology requires identification of the molecular bases of target traits by studying marker-trait associations. The Vitis18K SNP chip provides a useful genotyping tool for genome-wide marker analysis. Most linkage maps are based on single mapping populations, but an integrated map can increase marker density and show order conservation. Here we present an integrated map based on three mapping populations. The parents consist of the well-known wine cultivars 'Cabernet Sauvignon', 'Corvina' and 'Rhine Riesling', the lesser-known wine variety 'Deckrot', and a table grape selection, G1-7720. Three high-density population maps with an average inter-locus gap ranging from 0.74 to 0.99 cM were developed. These maps show high correlations (0.9965-0.9971) with the reference assembly, containing only 93 markers with large order discrepancies compared to expected physical positions, of which a third is consistent across multiple populations. Moreover, the genetic data aid the further refinement of the grapevine genome assembly, by anchoring 104 yet unanchored scaffolds. From these population maps, an integrated map was constructed which includes 6697 molecular markers and reduces the inter-locus gap distance to 0.60 cM, resulting in the densest integrated map for grapevine thus far. A small number of discrepancies, mainly of short distance, involve 88 markers that remain conflictual across maps. The integrated map shows similar collinearity to the reference assembly (0.9974) as the single maps. This high-density map increases our understanding of the grapevine genome and provides a useful tool for its further characterization and the dissection of complex traits.

Commercial Yeast Strains Expressing Polygalacturonase and Glucanase Unravel the Cell Walls of Chardonnay Grape Pomace.

Industrial wine yeast strains expressing hydrolytic enzymes were fermented on Chardonnay pomace and were shown to unravel the cell walls of the berry tissues according to the enzyme activities. The yeasts produced a native endo-polygalacturonase (Saccharomyces cerevisiae × Saccharomyces paradoxus hybrid, named PR7) and/or a recombinant endo-glucanase (S. cerevisiae strains named VIN13 END1 and PR7 END1). The impact of the enzymes during the fermentations was evaluated by directly studying the cell wall changes in the berry tissues using a Comprehensive Microarray Polymer Profiling technique. By the end of the fermentation, the endo-glucanase did not substantially modify the berry tissue cell walls, whereas the endo-polygalacturonase removed some homogalacturonan. The recombinant yeast strain producing both enzymes (PR7 END1) unravelled the cell walls more fully, enabling polymers, such as rhamnogalacturonan-I, ß-1,4-D-galactan and α-1,5-L-arabinan, as well as cell wall proteins to be extracted in a pectin solvent. This enzyme synergism led to the enrichment of rhamnogalacturonan-type polymers in the subsequent NaOH fractions. This study illustrated the potential utilisation of a recombinant yeast in pomace valorisation processes and simulated consolidated bioprocessing. Furthermore, the cell wall profiling techniques were confirmed as valuable tools to evaluate and optimise enzyme producing yeasts for grape and plant cell wall degradation.

Seeing the Forest through the (Phylogenetic) Trees: Functional Characterisation of Grapevine Terpene Synthase (VviTPS) Paralogues and Orthologues.

Gene families involved in specialised metabolism play a key role in a myriad of ecophysiological and biochemical functions. The Vitis vinifera sesquiterpene synthases represent the largest subfamily of grapevine terpene synthase (VviTPS) genes and are important volatile metabolites for wine flavour and aroma, as well as ecophysiological interactions. The functional characterisation of VviTPS genes is complicated by a reliance on a single reference genome that greatly underrepresents this large gene family, exacerbated by extensive duplications and paralogy. The recent release of multiple phased diploid grapevine genomes, as well as extensive whole-genome resequencing efforts, provide a wealth of new sequence information that can be utilised to overcome the limitations of the reference genome. A large cluster of sesquiterpene synthases, localised to chromosome 18, was explored by means of comparative sequence analyses using the publicly available grapevine reference genome, three PacBio phased diploid genomes and whole-genome resequencing data from multiple genotypes. Two genes, VviTPS04 and -10, were identified as putative paralogues and/or allelic variants. Subsequent gene isolation from multiple grapevine genotypes and characterisation by means of a heterologous in planta expression and volatile analysis resulted in the identification of genotype-specific structural variations and polymorphisms that impact the gene function. These results present novel insight into how grapevine domestication likely shaped the VviTPS landscape to result in genotype-specific functions.

Differences in berry skin and pulp cell wall polysaccharides from ripe and overripe Shiraz grapes evaluated using glycan profiling reveals extensin-rich flesh.

Shiraz is a widely planted cultivar in many of the world's top wine regions where it is used for the production of top-quality single varietal or blended red wines. Cell wall changes during grape ripening and over-ripening have been investigated, particularly in the context of understanding berry deconstruction thereby facilitating the release of favorable compounds during winemaking. However, no information is available on cell wall changes during berry shrinkage in Shiraz. Glycan microarray technology was used to directly profile Shiraz berries for cell wall polysaccharide and glycoprotein epitopes. Skins and pulp tissues were profiled separately and revealed that whereas the skin was rich in pectins and xyloglucans, the pulp tissues were mainly composed of extensin glycoproteins. Overripe (26-28°B) berries, particularly those from the warmer region site, revealed degradation of their pectin and extensin epitopes.

Tracking cell wall changes in wine and table grapes undergoing Botrytis cinerea infection using glycan microarrays.

BACKGROUND AND AIMS: The necrotrophic fungus Botrytis cinerea infects a broad range of fruit crops including domesticated grapevine Vitis vinifera cultivars. Damage caused by this pathogen is severely detrimental to the table and wine grape industries and results in substantial crop losses worldwide. The apoplast and cell wall interface is an important setting where many plant-pathogen interactions take place and where some defence-related messenger molecules are generated. Limited studies have investigated changes in grape cell wall composition upon infection with B. cinerea, with much being inferred from studies on other fruit crops. METHODS: In this study, comprehensive microarray polymer profiling in combination with monosaccharide compositional analysis was applied for the first time to investigate cell wall compositional changes in the berries of wine (Sauvignon Blanc and Cabernet Sauvignon) and table (Dauphine and Barlinka) grape cultivars during Botrytis infection and tissue maceration. This was used in conjunction with scanning electron microscopy (SEM) and X-ray computed tomography (CT) to characterize infection progression. KEY RESULTS: Grapes infected at veraison did not develop visible infection symptoms, whereas grapes inoculated at the post-veraison and ripe stages showed evidence of significant tissue degradation. The latter was characterized by a reduction in signals for pectin epitopes in the berry cell walls, implying the degradation of pectin polymers. The table grape cultivars showed more severe infection symptoms, and corresponding pectin depolymerization, compared with wine grape cultivars. In both grape types, hemicellulose layers were largely unaffected, as was the arabinogalactan protein content, whereas in moderate to severely infected table grape cultivars, evidence of extensin epitope deposition was present. CONCLUSIONS: Specific changes in the grape cell wall compositional profiles appear to correlate with fungal disease susceptibility. Cell wall factors important in influencing resistance may include pectin methylesterification profiles, as well as extensin reorganization.

Analysis of Plant Cell Walls Using High-Throughput Profiling Techniques with Multivariate Methods.

Plant cell walls are composed of a number of coextensive polysaccharide-rich networks (i.e., pectin, hemicellulose, protein). Polysaccharide-rich cell walls are important in a number of biological processes including fruit ripening, plant-pathogen interactions (e.g., pathogenic fungi), fermentations (e.g., winemaking), and tissue differentiation (e.g., secondary cell walls). Applying appropriate methods is necessary to assess biological roles as for example in putative plant gene functional characterization (e.g., experimental evaluation of transgenic plants). Obtaining datasets is relatively easy, using for example gas chromatography-mass spectrometry (GC-MS) methods for monosaccharide composition, Fourier transform infrared spectroscopy (FT-IR) and comprehensive microarray polymer profiling (CoMPP); however, analyzing the data requires implementing statistical tools for large-scale datasets. We have validated and implemented a range of multivariate data analysis methods on datasets from tobacco, grapevine, and wine polysaccharide studies. Here we present the workflow from processing samples to acquiring data to performing data analysis (particularly principal component analysis (PCA) and orthogonal projection to latent structure (OPLS) methods).

Overexpression of VviPGIP1 and NtCAD14 in Tobacco Screened Using Glycan Microarrays Reveals Cell Wall Reorganisation in the Absence of Fungal Infection.

The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC-MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.

Deconstructing Wine Grape Cell Walls with Enzymes During Winemaking: New Insights from Glycan Microarray Technology.

Enzyme-aid maceration is carried out in most modern winemaking industries with a range of positive impacts on wine production. However, inconsistencies in enzyme efficiency are an issue complicated by unclear targets (limited information available on berry cell wall architecture of different cultivars) and the complex wine environment (i.e., fermenting must). Recent studies have been performed to develop a clearer picture of grape cell wall structures, maceration effects, and interactions between important wine compounds and grape-derived polysaccharides. This review highlights critically important recent studies on grape berry cell wall changes during ripening, the importance of enzymes during maceration (skin contact phase) and deconstruction processes that occur during alcoholic fermentation. The novelty of the Comprehensive Microarray Polymer Profiling (CoMPP) technique using cell wall probes (e.g., antibodies) as a method for following cell wall derived polymers during different biological and biotechnological processes is discussed. Recent studies, using CoMPP together with classical analytical methods, confirmed the developmental pattern of berry cell wall changes (at the polymer level) during grape ripening. This innovative technique were also used to track enzyme-assisted depectination of grape skins during wine fermentation and determine how this influence the release of wine favourable compounds. Furthermore, polysaccharides (e.g., arabinogalactan proteins) present in the final wine could be identified. Overall, CoMPP provides a much more enriched series of datasets compared to traditional approaches. Novel insights and future studies investigating grape cell wall and polyphenol interactions, and the tailoring of enzyme cocktails for consistent, effective and "customized" winemaking is advanced and discussed.

The Transcriptional Responses and Metabolic Consequences of Acclimation to Elevated Light Exposure in Grapevine Berries.

An increasing number of field studies that focus on grapevine berry development and ripening implement systems biology approaches; the results are highlighting not only the intricacies of the developmental programming/reprogramming that occurs, but also the complexity of how profoundly the microclimate influences the metabolism of the berry throughout the different stages of development. In a previous study we confirmed that a leaf removal treatment to Sauvignon Blanc grapes, grown in a highly characterized vineyard, primarily affected the level of light exposure to the berries throughout their development. A full transcriptomic analysis of berries from this model vineyard details the underlying molecular responses of the berries in reaction to the exposure and show how the berries acclimated to the imposing light stress. Gene expression involved in the protection of the photosynthetic machinery through rapid protein-turnover and the expression of photoprotective flavonoid compounds were most significantly affected in green berries. Overall, the transcriptome analysis showed that the berries implemented multiple stress-mitigation strategies in parallel and metabolite analysis was used to support the main findings. Combining the transcriptome data and amino acid profiling provided evidence that amino acid catabolism probably contributed to the mitigation of a likely energetic deficit created by the upregulation of (energetically) costly stress defense mechanisms. Furthermore, the rapid turnover of essential proteins involved in the maintenance of primary metabolism and growth in the photosynthetically active grapes appeared to provide precursors for the production of protective secondary metabolites such as apocarotenoids and flavonols in the ripening stages of the berries. Taken together, these results confirmed that the green grape berries responded to light stress much like other vegetative organs and were able to acclimate to the increased exposure, managing their metabolism and energy requirements to sustain the developmental cycle toward ripening. The typical metabolic consequences of leaf removal on grape berries can therefore now be linked to increased light exposure through mechanisms of photoprotection in green berries that leads toward acclimation responses that remain intact until ripening.

Combining hydrothermal pretreatment with enzymes de-pectinates and exposes the innermost xyloglucan-rich hemicellulose layers of wine grape pomace.

Chardonnay grape pomace was treated with pressurized heat followed by enzymatic hydrolysis, with commercial or pure enzymes, in buffered conditions. The pomace was unfermented as commonly found for white winemaking wastes and treatments aimed to simulate biovalorization processing. Cell wall profiling techniques showed that the pretreatment led to depectination of the outer layers thereby exposing xylan polymers and increasing the extractability of arabinans, galactans, arabinogalactan proteins and mannans. This higher extractability is believed to be linked with partial degradation and opening-up of cell wall networks. Pectinase-rich enzyme preparations were presumably able to access the inner rhamnogalacturonan I dominant coating layers due to the hydrothermal pretreatment. Patterns of epitope abundance and the sequential release of cell wall polymers with specific combinations of enzymes led to a working model of the hitherto, poorly understood innermost xyloglucan-rich hemicellulose layers of unfermented grape pomace.

The Brassicaceae species Heliophila coronopifolia produces root border-like cells that protect the root tip and secrete defensin peptides.

Background and Aims: Root border cells and border-like cells (BLCs), the latter originally described in Arabidopsis thaliana , have been described as cells released at the root tips of the species in which they occur. BLCs are thought to provide protection to root meristems similar to classical root border cells. In addition, four defensin peptides (Hc-AFP1-4) have previously been characterized from Heliophila coronopifolia , a South African semi-desert flower, and found to be strongly antifungal. This provided an opportunity to evaluate if the BLCs of H. coronopifolia indeed produce these defensins, which would provide evidence towards a defence role for BLCs. Methods: Fluorescence microscopy, using live-cell-imaging technology, was used to characterize the BLCs of H. coronopifolia . Quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence microscopy was used to characterize these defensin peptides. Key Results: BLCs originated at the root apical meristem and formed a protective sheath at the tip and along the sides as the root elongated in solid medium. BLCs have a cellulose-enriched cell wall, intact nuclei and are embedded in a layer of pectin-rich mucilage. Pectinase treatments led to the dissolution of the sheath and dissociation of the root BLCs. Hc-AFP1-4 genes were all expressed in root tissues, but Hc-AFP3 transcripts were the most abundant in these tissues as measured by qRT-PCR. A polyclonal antibody that was cross-reactive with all four defensins, and probably recognizing a general plant defensin epitope, was used in fluorescence microscopy analysis to examine the presence of the peptides in the root tip and BLCs. Data confirmed the peptides present in the root tip tissues, the mucilage sheath and the BLCs. Conclusions: This study provides a link between defensin peptides and BLCs, both embedded in a protective pectin mucilage sheath, during normal plant growth and development. The presence of the Hc-AFP3 defensin peptides in the BLCs suggests a role for these cells in root protection.

Dissecting the polysaccharide-rich grape cell wall matrix using recombinant pectinases during winemaking.

The effectiveness of enzyme-mediated-maceration in red winemaking relies on the use of an optimum combination of specific enzymes. A lack of information on the relevant enzyme activities and the corresponding polysaccharide-rich berry cell wall structure is a major limitation. This study used different combinations of purified recombinant pectinases with cell wall profiling tools to follow the deconstruction process during winemaking. Multivariate data analysis of the glycan microarray (CoMPP) and gas chromatography (GC) results revealed that pectin lyase performed almost as effectively in de-pectination as certain commercial enzyme mixtures. Surprisingly the combination of endo-polygalacturonase and pectin-methyl-esterase only unraveled the cell walls without de-pectination. Datasets from the various combinations used confirmed pectin-rich and xyloglucan-rich layers within the grape pomace. These data support a proposed grape cell wall model which can serve as a foundation to evaluate testable hypotheses in future studies aimed at developing tailor-made enzymes for winemaking scenarios.

Field-Grown Grapevine Berries Use Carotenoids and the Associated Xanthophyll Cycles to Acclimate to UV Exposure Differentially in High and Low Light (Shade) Conditions.

Light quantity and quality modulate grapevine development and influence berry metabolic processes. Here we studied light as an information signal for developing and ripening grape berries. A Vitis vinifera Sauvignon Blanc field experiment was used to identify the impacts of UVB on core metabolic processes in the berries under both high light (HL) and low light (LL) microclimates. The primary objective was therefore to identify UVB-specific responses on berry processes and metabolites and distinguish them from those responses elicited by variations in light incidence. Canopy manipulation at the bunch zone via early leaf removal, combined with UVB-excluding acrylic sheets installed over the bunch zones resulted in four bunch microclimates: (1) HL (control); (2) LL (control); (3) HL with UVB attenuation and (4) LL with UVB attenuation. Metabolite profiles of three berry developmental stages showed predictable changes to known UV-responsive compound classes in a typical UV acclimation (versus UV damage) response. Interestingly, the berries employed carotenoids and the associated xanthophyll cycles to acclimate to UV exposure and the berry responses differed between HL and LL conditions, particularly in the developmental stages where berries are still photosynthetically active. The developmental stage of the berries was an important factor to consider in interpreting the data. The green berries responded to the different exposure and/or UVB attenuation signals with metabolites that indicate that the berries actively managed its metabolism in relation to the exposure levels, displaying metabolic plasticity in the photosynthesis-related metabolites. Core processes such as photosynthesis, photo-inhibition and acclimation were maintained by differentially modulating metabolites under the four treatments. Ripe berries also responded metabolically to the light quality and quantity, but mostly formed compounds (volatiles and polyphenols) that have direct antioxidant and/or "sunscreening" abilities. The data presented for the green berries and those for the ripe berries conform to what is known for UVB and/or light stress in young, active leaves and older, senescing tissues respectively and provide scope for further evaluation of the sink/source status of fruits in relation to photosignalling and/or stress management.

Effect of Commercial Enzymes on Berry Cell Wall Deconstruction in the Context of Intravineyard Ripeness Variation under Winemaking Conditions.

Significant intravineyard variation in grape berry ripening occurs within vines and between vines. However, no cell wall data are available on such variation. Here we used a checkerboard panel design to investigate ripening variation in pooled grape bunches for enzyme-assisted winemaking. The vineyard was dissected into defined panels, which were selected for winemaking with or without enzyme addition. Cell wall material was prepared and subjected to high-throughput profiling combined with multivariate data analysis. The study showed that significant ripening-related variation was present at the berry cell wall polymer level and occurred within the experimental vineyard block. Furthemore, all enzyme treatments reduced cell wall variation via depectination. Interestingly, cell wall esterification levels were unaffected by enzyme treatments. This study provides clear evidence that enzymes can positively influence the consistency of winemaking and provides a foundation for further research into the relationship between grape berry cell wall architecture and enzyme formulations.

Inactive dry yeast application on grapes modify Sauvignon Blanc wine aroma.

This study investigated the potential to improve wine aroma by applying two inactive dry yeast products (IDYs) at the onset of ripening on Sauvignon Blanc grapes. Both products led to increased reduced glutathione concentrations in the grape juice and corresponding wines, as well as differences in individual higher alcohol acetates (HAAs) and ethyl esters of straight chain fatty acids (EEFAs) at the end of fermentation. After two months of storage, a significantly slower decrease of EEFAs and to a lesser extent of HAAs was found for wines made from grapes with IDY applications. These wines also resulted in significantly slower synthesis of ethyl esters of branched acids, whereas varietal thiols were altered in a product-specific manner. The modifications in the wine chemical composition were also sensorially corroborated. This study showed that vineyard additions of IDY products directly on the grapes at the onset of ripening have a subsequent benefit to the production and preservation of aroma in wines.

Grapevine Plasticity in Response to an Altered Microclimate: Sauvignon Blanc Modulates Specific Metabolites in Response to Increased Berry Exposure.

In this study, the metabolic and physiological impacts of an altered microclimate on quality-associated primary and secondary metabolites in grape (Vitis vinifera) 'Sauvignon Blanc' berries was determined in a high-altitude vineyard. The leaf and lateral shoot removal in the bunch zones altered the microclimate by increasing the exposure of the berries. The physical parameters (berry diameter and weight), primary metabolites (sugars and organic acids), as well as bunch temperature and leaf water potential were predominantly not affected by the treatment. The increased exposure led to higher levels of specific carotenoids and volatile terpenoids in the exposed berries, with earlier berry stages reacting distinctly from the later developmental stages. Plastic/nonplastic metabolite responses could be further classified to identify metabolites that were developmentally controlled and/or responded to the treatment in a predictable fashion (assessed over two consecutive vintages). The study demonstrates that grapevine berries exhibit a degree of plasticity within their secondary metabolites and respond physiologically to the increased exposure by increasing metabolites with potential antioxidant activity. Taken together, the data provide evidence that the underlying physiological responses relate to the maintenance of stress pathways by modulating antioxidant molecules in the berries.

A rapid qualitative and quantitative evaluation of grape berries at various stages of development using Fourier-transform infrared spectroscopy and multivariate data analysis.

Fourier transform (FT) near-infrared (NIR) and attenuated total reflection (ATR) FT mid-infrared (MIR) spectroscopy were used to qualitatively and quantitatively analyse Vitis vinifera L. cv Sauvignon blanc grape berries. FT-NIR and ATR FT-MIR spectroscopy, coupled with spectral preprocessing and multivariate data analysis (MVDA), provided reliable methods to qualitatively assess berry samples at five distinct developmental stages: green, pre-véraison, véraison, post-véraison and ripe (harvest), without any prior metabolite extraction. Compared to NIR spectra, MIR spectra provided more reliable discrimination between the berry samples from the different developmental stages. Interestingly, ATR FT-MIR spectra from fresh homogenized berry samples proved more discriminatory than spectra from frozen homogenized berry samples. Different developmental stages were discriminated by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). In order to generate partial least squares (PLS) models from the MIR/NIR spectral datasets; the major sugars (glucose and fructose) and organic acids (malic acid, succinic acid and tartaric acid) were separated and quantified by high performance liquid chromatography (HPLC) and the data used as a reference dataset. PLS regression was used to develop calibration models to predict the concentration of the major sugars and organic acids in the berry samples from different developmental stages. Our data show that infrared (IR) spectroscopy could provide a rapid, reproducible and cost-effective alternative to the chromatographic analysis of the sugar and organic acid composition of grape berries at various developmental stages, using small sample volumes and requiring limited sample preparation. This provides scope and support for the possible development of hand-held devices to assess quality parameters in field-settings in real-time and non-destructively using IR technologies.

Dissecting the polysaccharide-rich grape cell wall changes during winemaking using combined high-throughput and fractionation methods.

Limited information is available on grape wall-derived polymeric structure/composition and how this changes during fermentation. Commercial winemaking operations use enzymes that target the polysaccharide-rich polymers of the cell walls of grape tissues to clarify musts and extract pigments during the fermentations. In this study, we have assessed changes in polysaccharide composition/turnover throughout the winemaking process by applying recently developed cell wall profiling approaches for monosaccharide composition (GC-MS), infra-red (IR) spectroscopy and comprehensive microarray polymer profiling (CoMPP). CoMPP performed on the concentrated soluble wine polysaccharides showed a fraction rich in rhamnogalacturonan I (RGI), homogalacturonan (HG) and arabinogalactan proteins (AGPs). We also used chemical and enzymatic fractionation techniques in addition to CoMPP to understand the berry deconstruction process more in-depth. CoMPP and gravimetric analysis of the fractionated pomace used aqueous buffers and CDTA solutions to obtain a pectin-rich fraction (pulp tightly-bound to skins) containing HG, RGI and AGPs; and then alkali (sodium carbonate and potassium hydroxide), liberating a xyloglucan-rich fraction (mainly skins). Interestingly this fraction was found to include pectins consisting of tightly associated and highly methyl-esterified HG and RGI networks. This was supported by enzymatic fractionation targeting pectin and xyloglucan polymers. A unique aspect is datasets suggesting that enzyme-resistant pectin polymers 'coat' the inner xyloglucan-rich skin cells. This data has important implications for developing effective strategies for efficient release of favorable compounds (pigments, tannins, aromatics, etc.) from the berry tissues during winemaking. This study provides a framework to understand the complex interactions between the grape matrix and carbohydrate-active enzymes to produce wine of desired quality and consistency.

Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.