Skin Cell Walls Research Articles

Interactions between grape skin cell wall material and commercial enological tannins. Practical implications

Commercial enological tannins were used to investigate the role that cell wall material plays in proanthocyanidin adsorption. Insoluble cell wall material, prepared from the skin of Vitis vinifera L. cv. Monastrell berries, was combined with solutions containing six different commercial enological tannins (proanthocyanidin-type tannins). Analysis of the proanthocyanidins in the solution, after fining with cell wall material, using phloroglucinolysis and size exclusion chromatography, provided quantitative and qualitative information on the non-adsorbed compounds. Cell wall material showed strong affinity for the proanthocyanidins, one of the commercial tannins being bound up to 61% in the experiment. Comparison of the molecular mass distribution of the commercial enological tannins in solution, before and after fining, suggested that cell walls affinity for proanthocyanidins was more related with the proanthocyanidin molecular mass than with their percentage of galloylation. These interactions may have some enological implications, especially as regards the time of commercial tannins addition to the must/wine.

Relationship between skin cell wall composition and anthocyanin extractability of Vitis vinifera L. cv. Tempranillo at different grape ripeness degree

The relationship between cell wall composition and extractability of anthocyanins from red grape skins was assessed in Tempranillo grape samples harvested at three stages of ripening (pre-harvest, harvest and over-ripening) and three different contents of soluble solids (22, 24 and 26 °Brix) within each stage. Cell wall material was isolated and analysed in order to determine cellulose, lignin, non-cellulosic polysaccharides, protein, total polyphenols index and the degree of esterification of pectins. Results showed the influence of ripeness degree and contents of soluble solids on cell wall composition. Furthermore, principal components analysis was applied to the obtained data set in order to establish relationships between cell wall composition and extractability of anthocyanins. Total insoluble material exhibits the biggest opposition to anthocyanin extraction, while the highest amounts of cellulose, rhamnogalacturonans-II and polyphenols were positively correlated with anthocyanin extraction. Moreover, multiple linear regression was performed to assess the influence of the cell wall composition on the extraction of anthocyanin compounds. A model connecting cell wall composition and anthocyanin extractabilities was built, explaining 96.2% of the observed variability.

Development of a method for the extraction and analysis of grape skin proteins strongly bound to cell walls

<p style="text-align: justify;"><strong>Aim</strong>: To better understand the protein composition of grape skin cell walls, we have developed a method to analyse the strongly bound cell wall proteins.</p><p style="text-align: justify;"><strong>Methods and results</strong>: The protocol was developed with grape skins at full maturity. The critical steps of this protocol were : (i) the elimination of cellular aggregates, (ii) the elimination of soluble proteins, and (iii) the localization of the identified proteins within the cell wall. To verify whether these three conditions were met, the decrease in the quantity of cellular aggregates was followed by optical microscopy, the removal of soluble proteins was measured by chemical assay, and the presence of proteins located in cell walls was demonstrated by extensive bioinformatic analysis. The process made it possible to obtain a four-fold reduction in the amount of cellular aggregates, a reduction in the concentration of soluble proteins below the method detection limit, and a high proportion of proteins predicted to be secreted (79 %).</p><p style="text-align: justify;"><strong>Conclusion</strong>: The protocol described in this paper constitutes the first method to analyse proteins strongly bound to cell walls in grape skins. However, this method excludes the identification of labile proteins or proteins weakly bound to the cell wall.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This protocol can be used for studying the role that strongly bound cell wall proteins play in development and defense processes in grape skins.</p>

Tissue-Specific and Developmental Modifications of Grape Cell Walls Influence the Adsorption of Proanthocyanidins

Cell wall material from Vitis vinifera L. cv. Cabernet Sauvignon grape skin and flesh was isolated at different stages of grape maturity to determine whether developmental changes in cell wall composition in different tissue types influence the binding of proanthocyanidins (PAs). Trends in cell wall adsorption of, and selectivity for, PAs were determined using two skin PAs that differed in their average molecular masses. Flesh cell walls consistently bound a higher amount of PA than those from skin. Key structural differences that reduced PA adsorption in skin cell walls by comparison with flesh cell walls were endogenously higher concentrations of insoluble PA, Klason lignin, and lower cell wall-bound protein. These differences may confer reduced flexibility and porosity of skin cell walls relative to flesh cell walls. Analysis of skin and flesh cell wall properties revealed that the onset of ripening was associated with a loss of type I arabinogalactan and galacturonic acid, which indicated a degradation of pectin within the cell wall. Flesh cell walls consistently bound PAs of larger molecular mass, and changes in PA adsorption properties after the onset of ripening were minor. For skin cell walls, adsorption of PA was lowest immediately following solubilization of galacturonic acid, and high molecular mass PAs were poorly bound. As ripening progressed, PAs of higher molecular mass were selectively adsorbed by skin cell walls, which indicates that ongoing cell wall remodeling during ripening may confer an increased porosity within the skin cell wall matrix, resulting in a greater adsorption of PA within a permeable structure.

Formation of microcraters on plant cell wall by plasma immersion ion implantation

Ion bombardment of biological cellular material has been used as a tool for the transfer of exogenous DNA macromolecules into the cell interior region. The precise physical mechanisms associated with this transfer of macromolecules through the cell envelope remain unexplained, however it has been observed that the ion bombardment is accompanied by the formation of “microcraters” on the wall of plant cells, and it is possible that these features provide channels for the macromolecule transfer. Thus the nature and origin of the microcraters are of importance to understanding the DNA transfer phenomenon as well as being of fundamental interest. We report here on the formation of microcraters on onion skin cell walls by plasma immersion ion implantation (PIII) using ~20keV Ar+ ions at a dose of about 1×1015ions/cm2. The results indicate that PIII provides a tool for carrying out ion bombardment of living biological materials previously done using beam-line implantation methods.

Closed-Cell Foam Skin Thickness Measurement Using a Scanning Electron Microscope

Closed cell polymer foam skin thickness can be assessed by taking backscatter electron (BSE) images in a scanning electron microscope (SEM) at a series of accelerating voltages. Under a given set of experimental conditions, the electron beam mostly passes through thin polymer skin cell walls. That cell appears dark compared to adjacent thicker-skinned cells. Higher accelerating voltages lead to a thicker skin being penetrated. Monte Carlo modeling of beam-sample interactions indicates that at 5 keV, skin less than ∼0.5 μm in thickness will appear dark, whereas imaging at 30 keV allows skin thicknesses up to ∼4 μm to be identified. The distribution of skin thickness can be assessed over square millimeters of foam surface in this manner. Qualitative comparisons of the skin thicknesses of samples can be made with a simple visual inspection of the images. A semiquantitative comparison is possible by applying image analysis. The proposed method is applied to two example foams. Characterizing foam skin thickness by this method is possible using any SEM that is capable of collecting useful BSE images over a range of accelerating voltages. Imaging in low vacuum, where an electrically conductive metal coating is not required, leads to more sensitivity in skin thickness characterization.

The effect of a commercial pectolytic enzyme on grape skin cell wall degradation and colour evolution during the maceration process

The effect of a macerating enzyme and maceration time on the structure of grape skin cell walls and on wine quality was studied by making wines with different maceration times with and without the addition of enzyme. The results show that the addition of a macerating enzyme accelerates the extraction of phenolic compounds, reducing the maceration time needed for high quality winemaking. The degradation of grape skin cell walls was influenced by the enzyme, which mainly affected the pectin fraction of the cell wall.

Ripening-Induced Changes in Grape Skin Proanthocyanidins Modify Their Interaction with Cell Walls

Proanthocyanidins were isolated from the skins of Cabernet Sauvignon grapes at different stages of grape development in order to study the effect of proanthocyanidin modification on the interaction with grape cell wall material. After veraison, the degree of proanthocyanidin polymerization increased, and thereafter was variable between 24 and 33 subunits as ripening progressed. Affinity of skin cell wall material for proanthocyanidin decreased with proanthocyanidin ripeness following veraison. A significant negative relationship (R2=0.93) was found for average proanthocyanidin molecular mass and the proportion of high molecular mass proanthocyanidin adsorbed by skin cell wall material. This indicated that as proanthocyanidin polymerization increased, the affinity of a component of high molecular mass proanthocyanidins for skin cell wall material declined. This phenomenon was only associated with skin proanthocyanidins from colored grapes, as high molecular mass proanthocyanidins of equivalent subunit composition from colorless mutant Cabernet Sauvignon grapes had a higher affinity for skin cell wall material.

EXPRESSION, ACTIVITY AND CELLULAR LOCALIZATION OF PECTIN METHYLESTERASE IN GRAPE BERRY SKIN DURING RIPENING

Kipening ot grape berry involves a series ot complex physiologic changes in which senescing tissues undergo programmed changes in firmness, texture, coloration, flavour and susceptibility to microbial infection. Softening is accompanied by modifications in cell wall polymers that involve the co-ordinated and independent action of a range of cell wall-modifying enzymes such as pectin methylesterase (PME). In the present work, expression of VVPME, activity and cellular localization of the enzyme were studied in berry skin during ripening. Our results show that expression and activity were well correlated. VVPME and PME activity decrease during the colour change period then increase at the end of ripening in relation with the loosening of skin cell-wall and the increase in susceptibility to pathogens. The subcellular localization of PME in developing skin berry was performed via immunogold electron microscopy technique and shows that few gold particles representing PME are found on the cell-wall. This work will provide new insight for the regulation of structural changes in pectin chains and hemicellulose and its early may constituted a key event in the induction of grape softening.

Grape berry skin features related to ontogenic resistance to Botrytis cinerea

This work investigated the structural and biochemical changes during grape berry development which account potentially for the onset and increase in susceptibility to Botrytis cinerea. Using the cv. Sauvignon blanc, we quantified at seven developmental growth stages from herbaceous to over-mature berries: (1) fruit ontogenic resistance using three strains (II-transposa), (2) the morphological and maturity fruit characteristics and (3) preformed biochemical compounds located in the berry skin. From the mid-colour change stage onwards, susceptibility of unwounded fruit increased sigmoidally in both rot and sporulation severities at the berry surface. A principal component analysis identified a very close connection between fruit susceptibility and the level of fruit maturity. Berry susceptibility was significantly and positively correlated with the phenolic compounds in the skin cell walls and negatively correlated with the total tannin content in the skin and with water activity (Aw) at the fruit surface. On the berry, Aw decreased from 0.94 at bunch closure to 0.89 at berry maturity, with a relatively low value (0.90) at the stage of mid-colour change. Using artificial media, different Aw levels led to significant differences in mycelial growth (Aw ≤0.95 resulted in the lowest growth rate ≤0.34 mm day−1). Thus, besides the level of fruit maturity, both water activity on the fruit and the total tannin content in the skin may affect fungal growth and berry colonisation. The potential of these variables for use as indicators of grape berry susceptibility as well as associated mechanisms for the development of disease are discussed.

Experimental evidence for eukaryotic fossil preservation: Onion skin cells in silica solution

Research on the origin of eukaryotes often focuses on the exceptional preservation found in silicified Precambrian fossils. Nuclei like subcellular structures in well-preserved fossil becomes confusion and arguments, which focus on whether the partial degradation of prokaryotes produces artifacts that resemble a ‘nucleus’, or fossilized nuclei of eukaryotes. In order to understand the mechanisms of silicification and identify the fossilized subcellular and microstructures in rocks, a series of laboratory controlled experiments were performed for simulating the silicification process. The effects of different silica solutions in eukaryote fossilization were studied in our experiments by exposing onion skin cells (epidermis) to silica solutions. Onion skin provides a good experimental model because of its well characterized cellular structures which are easily observed. The designed experiments revealed that the possibility of onion cell preserved as a “fossil” with nuclear structures, the first week fossilization, or mineralization as rapid as 1 week is important. And the experiment also revealed interactions between silica and the onion skin cell wall surface functional groups were weak. The preservation of nuclei in the onion skin model was due to precipitation in highly supersaturated silica solutions rather than simply the high silica concentration. When the silica gel precipitates slowly at low supersaturation states, the nuclei were not well preserved, but the rapid precipitation at high supersaturated silica conditions preserved nuclear structures. A better understanding of the processes involved in onion skin fossilization will further contribute to issues concerning the silicification of other eukaryotic materials.

Changes in skin cell wall composition during the maturation of four premium wine grape varieties

AbstractBACKGROUND: Grape skin cell walls form a barrier against the diffusion of colour. The colour of red wines is mainly due to anthocyanins, although their concentration in wine is not always correlated with the anthocyanin content of grape skins. The cell wall composition changes during fruit ripening, so it was thought that a study of the ripening behaviour of four premium varieties might provide information on how the composition changes during ripening and explain the technological differences.RESULTS: The largest quantities of skin cell wall material (CWM) were isolated from Monastrell grapes, probably owing to the greater number of cell layers they contain and the thicker cell walls. During ripening, a loss of skin CWM (e.g. 120–60 and 65–45 mg CWM g−1 fresh skin for Monastrell J and Cabernet Sauvignon respectively) and galactose (e.g. 36–20 and 55–30 mg g−1 CWM for Cabernet Sauvignon and Monastrell J respectively) was observed, together with a decrease in the degree of pectin methylation and acetylation, except in Syrah, in which pectin methylation remained unchanged. These changes were accompanied by the accumulation of glucose, while other neutral sugars showed no significant variations. A correlation was found between the degree of ripening and those constituents changing in the grape skin cell wall.CONCLUSION: The cell wall composition differs sufficiently between grape varieties to allow discrimination between them. Ripening was associated with a decrease in the quantity of CWM and galactose in the cell walls. Monastrell showed the largest quantities of both throughout ripening. The differences observed between Monastrell and the other varieties would explain the technological differences between them. Copyright © 2007 Society of Chemical Industry

Differences in morphology and composition of skin and pulp cell walls from grapes (Vitis vinifera L.): technological implications

The morphology and composition of skin and pulp cell walls of four premium red wine grape (Vitis vinifera L.) varieties were studied to determine whether the technological differences observed in previous studies between the four different varieties during the winemaking process (especially, the anthocyanin extractability) could be partly explained by their respective cell wall compositions. Monastrell grapes showed the highest amount of cell wall material in their skins (55–57 mg cell wall/g skin), while Syrah showed the largest amount of cell wall material in the pulp (5.1 mg cell wall/g pulp). The highest percentage of galactose and cellulosic glucose in skin and pulp cell walls was also found in Monastrell grapes. The cell wall composition of Monastrell grapes, together with their morphology, suggests that the firmer pulp and skin are responsible for the difficulties this variety shows for anthocyanin extraction (extractability index varying from 37 to 60). As cell wall composition may be modulated by the action of enzymes, the activity of pectinmethylesterase, polygalacturonase, cellulase and α- and β-galactosidase was studied. No polygalacturonase and cellulase activity was found. The two galactosidases showed higher activity than pectinmethylesterase (25–290 units/g tissue versus 0.3–0.4 units/g tissue), especially in Cabernet Sauvignon grapes, although in Monastrell grapes α- and β-galactosidase activity was low, which may explain the large amount of galactose in Monastrell grapes cell wall.

Electronic microscopy examination of the influence of purified enzymatic activities on grape skin cell wall

<p style="text-align: justify;">Pectolytic enzymes act differently on the degradation of the cell wall of grape skin and on the libération of tannins. PG and PL degrade the pectin from the middle lamella and the primary wall which favours the liberation of granulate tannins present inside the vacuole only ones. Cellulase degrade the cellulose fibbers and allows the liberation of tannins bound to the cellular wall. These last ones being bound to cellulosic molecules.</p>

Polysaccharides from grape berry cell walls. Part I: tissue distribution and structural characterization of the pectic polysaccharides

Buffer-soluble arabinogalactan-proteins (AGPs) and pectins from grape berry skin and pulp tissues have been isolated and their structure has been partly determined. Pectic polysaccharides from the cell wall material were solubilized by treating pulp and skin cell walls with homogeneous glycosyl hydrolases. Homogalacturonans, rhamnogalacturonans I (RG-I), and rhamnogalacturonan II (RG-II) of each tissue have been fractionated by high resolution size exclusion chromatography and their relative distribution and major structural features have been determined. It has been shown that pulp tissue contains two-fold more buffer-soluble AGPs and pectins than skin tissue and we have determined that 75% of the grape berry walls originates from the skin tissue. There is three-fold more RG-I and RG-II in skin tissue than in pulp tissue and three-fold more RG-I than RG-II in the grape berry cell walls. The results of this study have shown that the grape polysaccharide content of a wine is related to the type of tissue used for wine making and to the solubility of the grape polysaccharides and their resistance to fragmentation by grape and yeast glycanases.

Peroxidase isozyme patterns in the skin of maturing tomato fruit

ABSTRACTThe cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent Mr 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.

Cell wall composition of grape berry skins

Cell wall materials (CWM) were prepared from the skins of Chardonnay and Carignan mature grape berries by the sodium deoxycholate-(phenol-acetic acid-water) extraction procedure. Dried skin contained ca 15% of CWM with similar features for the two cultivars. Skin cell walls were made up of ca 30–35% of neutral polysaccharides [cellulose, xyloglucan, arabinan, types I and II (arabino)-galactans, xylan, and mannan] and ca 20% of acidic pectic substances moderately methyl-esterified (62%). CWM were also contaminated by insoluble proanthocyanidins (>15%).