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.

The Influence of Hydrolytic Enzymes on Tannin Adsorption-Desorption onto Grape Cell Walls in a Wine-Like Matrix.

This study evaluates the capacity of four hydrolytic enzymes to limit the interactions between grape cell-walls and tannins and/or to favor tannin desorption. Adsorption and desorption tests were conducted by mixing a commercial seed tannin with purified skin cell-walls from Syrah grapes, in the presence or absence of hydrolytic enzymes, in a model-wine solution. The effects of the enzymes were evaluated by measuring the tannins in solution by High Performance Liquid Chromatography (HPLC) and the changes in the cell wall polysaccharide network by Comprehensive Microarray Polymer Profiling (COMPP) while the polysaccharides liberated from cell walls were analyzed by Size Exclusion Chromatography (SEC). The results showed that the enzymes limited the interaction between tannins and cell walls, especially cellulase, pectinase and xylanase, an effect associated with the cell wall structural modifications caused by the enzymes, which reduced their capacity to bind tannins. With regards to the tannin desorption process, enzymes did not play a significant role in liberating bound tannins. Those enzymes that showed the highest effect in limiting the adsorption of tannins and in disorganizing the cell wall structure, cellulase and pectinase, did not lead to a desorption of bound tannins, although they still showed a capacity of affecting cell wall structure. The results indicate that enzymes are not able to access those polysaccharides where tannins are bound, thus, they are not a useful tool for desorbing tannins from cell walls. The practical importance implications of these findings are discussed in the manuscript.

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.

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.

The impact of carbohydrate-active enzymes on mediating cell wall polysaccharide-tannin interactions in a wine-like matrix.

Tannins are present in grape skins and seeds from where they are transferred into the must-wine matrix during the maceration stages of winemaking. However, tannin transfer is often incomplete. This could be due, among other reasons, to tannins becoming bound to grape cell wall polysaccharides, including soluble polymers, which are released during vinification and are present in high concentrations in the must/wine. The use of cell wall deconstructing enzymes offers the possibility of reducing these interactions, releasing more tannins into the final wine. The main aim of this study was to evaluate the optimal addition (individually, in combination or sequentially) of hydrolytic enzymes that would prevent tight polysaccharide-tannin associations. The use of comprehensive microarray polymer profiling (CoMPP) methodology provided key insights into how the enzyme treatments impacted the grape cell wall matrix and tannin binding. The results demonstrated that polygalacturonase + pectin-lyase promoted the highest release of tannins into solution.