Robert J. Redgwell

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.

Structural features of cell-wall polysaccharides of onion Allium cepa

Abstract Cell-wall material has been isolated from immature onion tissues and extracted in sequence with cyclohexane- trans -1,2-diaminetetra-acetate (CDTA) at 20°, 0.05 m Na 2 CO 3 at 1°, 0.05 m Na 2 CO 3 at 20°, and 0.5, 1, and 4 m KOH at 20° to leave the α-cellulose residue, which contained a significant amount of pectic material. The polymers isolated from the extracts were fractionated by anion-exchange chromatography and subjected to methylation analysis. This study helped to distinguish between the pectic polysaccharides of the middle lamellae (solubilised by CDTA) and those of primary cell walls (solubilised by dilute alkali); the latter contained more highly branched rhammogalacturonan backbones. All the rhamnogalacturonans were substituted to various degrees with side chains comprising galactans or arabinoglactans which contained mainly (1»4)-linked galactose, lesser amounts of (1»4,1»)- and (1»2,1»6)-linked galactose, and (1»5)-linked arabinose, and small proportions of (1»2)-linked galactose. Most of the branched residues were terminated by galactopyranosyl and arabinofuranosyl groups. The major hemicellulose was a xyloglucans which showed structural features in common with the xyloglucans of dicotyledonous plants. Small amounts of hemicellulosepectic complexes were also isolated.

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.

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A β-galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl-β-d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the β-configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na2CO3-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the β-galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A [beta]-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-[beta]-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing [beta]-(1–>4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61–71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple [beta]-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.

Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit

Abstract.Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The Km was 0.6 mg · mL−1 for kiwifruit xyloglucan and 100 μM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA. The six cDNA clones share 93–99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.

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.

Cell-wall polysaccharides of kiwifruit (Actinidia deliciosa): Chemical features in different tissue zones of the fruit at harvest

Abstract Cell-wall material (CWM) was isolated from cryo-milled (− 196°) powders prepared from 4 different tissue zones of kiwifruit ( Actinidia deliciosa ). Polysaccharides were solubilised by stepwise extraction with cyclohexane- trans -1,2-diaminetetra-acetate (CDTA), 0.05 m Na 2 CO 3 , 6 m guanidinium thiocyanate (GTC), and 4 m KOH. A heterogeneous mixture of pectic galactans accounted for 40–50% of the CWMs, while hemicelluloses, the bulk of which were xyloglucans, accounted for 15–25%. Each tissue zone contained similar types of polysaccharide. Variability in their amount and sugar composition are thought to reflect different stages in the physiological development of the fruit at harvest, in the 4 zones. Polymers from the outer pericarp tissue were fractionated by anion-exchange chromatography and subjected to methylation analysis. The CDTA- and Na 2 CO 3 -soluble polymers were rhamnogalacturonans substituted to varying degrees with galactan and arabinogalactan side-chains containing 4-, 2,4-, 3,4- and 4,6-linked galactose and 5- and 3,5-linked arabinose. Side chains were terminated by galactose and arabinose and lesser amounts of rhamnose, fucose, xylose, and galacturonic acid. The pectic polysaccharides of the GTC- and KOH-soluble fractions had more highly branched rhamnogalacturonan backbones than the CDTA- and Na 2 CO 3 -soluble polymers and contained hemicellulosic elements. The major hemicellulose was a xyloglucan, but lesser amounts of a 4- O -methylglucuronoxylan and a branched mannan were partially characterised. Several polymers were associated with proteins low in hydroxyproline. Evidence is presented that a polysaccharide of the rhamnogalacturonan II type is associated with the pectic polymers of kiwifruit.

Re-interpreting the role of endo-β-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall

BACKGROUND Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose-cellulose network with a similar function to xyloglucans. Endo-beta-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-beta-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-beta-mannanases to be reinterpreted. AIMS In this review, the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported. FUTURE OUTLOOK There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.

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.