Monica Fischer

In vivo and in vitro swelling of cell walls during fruit ripening

Abstract. Swelling properties of the cell walls of nine temperate fruit species, selected for their different ripening and textural characteristics, were studied during ripening. Cell wall swelling was examined in intact fruit using microscopy techniques and in vitro, using cell wall material isolated from fruit tissue. In fruit which ripened to a soft melting texture (persimmon, avocado, blackberry, strawberry, plum), wall swelling was pronounced, particularly in vitro. In-vivo swelling was marked only in avocado and blackberry. Fruit which ripened to a crisp, fracturable texture [apple (two cultivars), nashi pear, watermelon] did not show either in-vivo or in-vitro swelling of the cell wall. There was a correlation between swelling and the degree of pectin solubilisation, suggesting that wall swelling occurred as a result of changes to the viscoelastic properties of the cell wall during pectin solubilisation. Chemical and enzymatic removal of pectin from kiwifruit cell wall material supported the idea that swelling is associated with movement of water into voids left in the cellulose-hemicellulose network by the solubilised pectin. However, the results also suggested that swelling in vivo was more complex than this, and that the physicochemical changes which led to swelling included other elements of cell wall modification involving the site and mechanism of pectin solubilisation and-or the cellulose-xyloglucan complex.

Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls

Abstract. Cell wall material (CWM) was prepared from nine fruit species at two ripening stages (unripe and ripe) and extracted sequentially with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na2CO3 and 4 M KOH. Each solubilised fraction and the CWM-residue remaining after 4 M KOH extraction was analysed for non-cellulosic sugar composition. A common pattern of distribution for polyuronide and pectin-associated neutral sugar was observed for all unripe fruit. Most polyuronide was extracted in the CDTA/Na2CO3 fractions while 70–93% of the neutral sugar was located on pectic polysaccharides in the 4 M KOH-soluble and CWM-residue fractions. During ripening, most of the galactose was lost from pectic polysaccharides in the CWM-residue. Partial solubilisation of these polysaccharides was achieved by treating the CWM-residue with endopolygalacturonase. The solubilised polysaccharides were separated into two fractions by ion-exchange chromatography. One of these contained polysaccharides with average molecular weights of 400 kDa or larger and consisted of between 70 and 90% arabinogalactan. The galactosyl residues were 80–90% β-1→4 linked, indicating largely unbranched side-chains. The arabinosyl residues were distributed among terminal, 3-, 5-, 2,5-, and 2,3,5-linked residues, indicating a highly ramified structure. The results are discussed with regard to the relationship between pectin solubilisation and galactose loss and their respective contribution to fruit softening.

Polysaccharides of green Arabica and Robusta coffee beans

Two independent procedures for the quantitative determination of the polysaccharide content of Arabica Caturra (Coffea arabica var. Caturra) and Robusta ROM (Coffea canephora var. ROM) green coffee beans showed that they both contained identical amounts of polysaccharide. Cell wall material (CWM) was prepared from the beans and partial solubilisation of component polysaccharides was effected by sequential extraction with water, 1 M KOH, 0.3% NaClO2, 4 M KOH and 8 M KOH. The monosaccharide compositions of the CWMs were similar, although Arabica beans contained slightly more mannose than Robusta. In the latter, more arabinogalactan was solubilised during preparation of the CWM and the water-soluble fraction of the CWM contained higher amounts of galactomannan than in Arabica. Linkage analysis indicated that the galactomannans possessed unbranched to branched mannose ratios between 14:1 and 30:1 which is higher than previously reported. No major difference in the structural features of the galactomannans between species was found. The arabinogalactans were heterogeneous both with regard to the degree of branching and the degree of polymerisation of their arabinan side-chains. Compared to Arabica, Robusta appeared to contain greater amounts of arabinogalactans with longer side chains. It is concluded that there was no detectable difference between the Arabica and Robusta varieties of this study in their absolute polysaccharide content or in the gross structural features of their galactomannans. Differences were apparent both in the structural features and ease of solubility of the arabinogalactans but a more detailed study of several varieties of Arabica and Robusta will be required to determine whether these differences occur consistently between species.

Effect of roasting on degradation and structural features of polysaccharides in Arabica coffee beans

The degree and nature of polysaccharide degradation at different roasting levels was determined for three Arabica (Coffea arabica) bean varieties. Between 12 and 40% of the bean polysaccharides were degraded depending on the roasting conditions. The thermal stability of the arabinogalactans, (galacto)mannans and cellulose was markedly different. The arabinogalactans and mannans were degraded up to 60 and 36%, respectively, after a dark roast, while cellulose showed negligible evidence of degradation. Roasting led to increased solubility of both the arabinogalactans and (galacto)mannans from the bean but the structural modifications, which accompanied this change in solubility, were different for each polysaccharide. Despite the moderate degradation of the (galacto)mannans, those remaining in the bean after roasting showed no evidence of change to their molecular weight even after a dark roast. In contrast, arabinogalactans were depolymerised after a light roast both by fission of the galactan backbone and loss of arabinose from the sidechains. The recently discovered covalent link between the coffee bean arabinogalactans and protein survived roasting. The glucuronic acid component of the AG was degraded markedly after a dark roast, but approximately 30% of the original content remained as part of the AG polymer. The results show that polysaccharide degradation during roasting is more marked than previously documented, and points to roasting induced changes to the polysaccharides as major factors in the changing physicochemical profile of the coffee bean during processing.

Coffee bean arabinogalactans: acidic polymers covalently linked to protein.

The arabinogalactan content of green coffee beans (Coffea arabica var. Yellow Caturra) was released by a combination of chemical extraction and enzymatic hydrolysis of the mannan-cellulose component of the wall. Several arabinogalactan fractions were isolated, purified by gel-permeation and ion-exchange chromatography and characterised by compositional and linkage analysis. The AG fractions contained between 6 and 8% glucuronic acid, and gave a positive test for the beta-glucosyl-Yariv reagent, a stain specific for arabinogalactan-proteins. The protein component accounted for between 0.5 and 2.0% of the AGPs and contained between 7 and 12% hydroxyproline. The AG moieties displayed considerable heterogeneity with regard to their degree of arabinosylation and the extent and composition of their side-chains. They possessed a MW average of 650 kDa which ranged between 150 and 2000 kDa. An investigation of the structural features of the major AG fraction, released following enzymatic hydrolysis of the mannan-cellulose polymers, allowed a partial structure of coffee arabinogalactan to be proposed.

Purification and characterisation of a galactoglucomannan from kiwifruit (Actinidia deliciosa)

A galactoglucomannan (GGM) has been purified from the primary cell walls of ripe kiwifruit. A combination of barium hydroxide precipitation, anion exchange- and gel-permeation chromatography gave a chemically homogeneous polymer with a 1:2:2 galactose-glucose-mannose ratio and a molecular weight range of 16-42 kDa. Complete hydrolysis of the polymer with endo-1,4-beta-mannanase (EC 3.2.1.78) from Aspergillus niger gave a mixture of oligosaccharides, three of which (II, III, IV) accounted for more than 80% of the GGM. Structural characterisation of these oligosaccharides and the original polysaccharide was achieved by linkage analysis, 1D and 2D NMR spectrometry and enzymatic hydrolysis. Oligosaccharide II beta-D-Glcp-(1-->4)-beta-D-Manp-(1-->, III beta-D-Glcp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Manp-(1-->, and IV beta-D-Glcp-(1-->4)-[beta-D-Galp-(1-->2)-alpha-D-Galp-(1-->6)]-beta-D-Manp-(1-->4)-beta-D-Glcp-(1-->4)-beta-D-Manp-(1-->, appeared in the molar ratio of 2:1:1. A trace amount of mannobiose (I) was detected, indicating that some of the mannosyl residues were contiguous. It is concluded that the predominant structural feature of kiwifruit GGM is a backbone of alternating beta-(1-->4)-linked D-glucopyranosyl and D-mannopyranosyl residues, with approximately one third of the latter carrying side-chains at 0-6 of single alpha-D-Galp-(1--> residues (50% of the branches) or the disaccharide beta-D-Galp-(1-->2)-alpha-D-Galp-(1--> (50% of the branches), the substituted residues being separated by three or five unsubstituted monosaccharide units.

Solid-state NMR characterization of hydration effects on polymer mobility in onion cell-wall material

The effect of hydration on the mobility of polysaccharides in onion cell-wall material (CWM) was studied by solid-state NMR. The application of proton T-1 rho, carbon T-1 relaxation measurements, and 2D-WISE (wideline separation) experiments led to a coherent overall picture of the mobility inside the cell wall, combining domain-averaged as well as site-specific motional information. Whereas the mobility of all individual chemical sites increased upon hydration, only the average chain mobility of the pectin was strongly affected, indicating the predominant interaction of water molecules with the pectin. The 2D-WISE experiment revealed a spatial heterogeneity of the polysaccharide dynamics across the sample, showing at least two different motional regimes for pectin and cellulose domains

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans

Summary.The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (1→5)-α-arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.

CHEMICAL AND STRUCTURAL FEATURES OF KIWIFRUIT CELL WALLS : COMPARISON OF FRUIT AND SUSPENSION-CULTURED CELLS

Abstract Chemical and structural features were compared in cell-wall polysaccharides isolated from intact kiwifruit (Actinidia deliciosa) and suspension-cultured cells initiated from the same fruit. Morphological features of the cells and whole fruit tissue were also examined by electron microscopy. Cell-wall material (CWM) from cultured cells contained less cellulose, more protein, and had a higher degree of acetylation than cell-wall material from intact fruit. Whereas galactose was the major neutral sugar in the pectic polysaccharides of intact fruit, arabinose was predominant in cultured cells. The arabinose occurred as terminal and 5-substituted residues, presumably as neutral side-chains attached to the galacturonan backbone which was more highly branched in the cultured cells. The most significant structural difference in the hemicelluloses occurred in a galactoglucomannan purified from the 4 M KOH-soluble fraction. The backbone of the galactoglucomannan of the cultured cells was more branched than that in the intact fruit, with 84% of the 4-linked mannosyl residues substituted at O-6 with either single galactosyl or galactosyl-(1 → 2)-galactosyl groups. In intact fruit ∼ 30% of the mannosyl residues were substituted at O-6. Rhamnogalacturonan II (RG-II) was partially purified from CWM and shown to possess a very similar composition of glycosyl residues in both intact fruit and cells. However, the primary cell walls of the cultured cells contained twice the amount of RG-II found in the cell walls of intact fruit.