Apple Cell Walls Research Articles

Degradation of apple fruit xyloglucan by endoglucanase

A purified, alkali-extractable apple fruit xyloglucan (APfxg) was treated by endoglucanase (endoIV) from Trichoderma viride. The degradation products were fractionated by size-exclusion chromatography on BioGel P-2; the pentamer to dodecamer fractions were further fractionated by semi-preparative high-performance anion-exchange chromatography. The purified oligosaccharides were characterized by monosaccharide analysis, mass spectrometry and degradation with Driselase. Based on these data, tentative structures were proposed for most products. Apple xyloglucan is composed of a diversity of repeating units. Next to the major oligosaccharides XXXG, an octamer, XXFG and XLFG (Renard, C.M.G.C, Lomax, J.A. & Boon, J.J. (1992). Carbohydr. Res., 232, 303–320), XG, XXG, FG and two new fucose-containing oligosaccharide building blocks (hexasaccharide and dodecasaccharide) were found. Larger xyloglucan fragments were obtained by partial degradation of APfxg by endoIV, and treatment of apple cell wall material by endoI. These fragments were predominantly composed of fucose-containing oligosaccharides. Our results show that both endoIV and endoI are hindered by fucosylated side chains.

A xylogalacturonan subunit present in the modified hairy regions of apple pectin

The high molecular weight fraction (fraction A) of the modified hairy regions (MHR) from apple cell walls was treated by rhamnogalacturonase (RGase) after saponification and after deacetylation by rhamnogalacturonan acetylesterase (RGAEase). Three fractions could be recognized by size-exclusion chromatography: rhamnogalacturonan oligomers, residual stubs of the rhamnogalacturonan backbone rich in arabinan side-chains, and a fraction rich in xylose and galacturonic acid. The “xylogalacturonans” obtained after saponification and after enzymic deacetylation had rather similar sugar compositions (xylose:galacturonic acid ratios of 0.4–0.9) and molecular weights. After saponification, the xylogalacturonan was eluted as a single peak on anion-exchange chromatography whereas three peaks were obtained when the MHR was deacetylated by RGAEase, indicating variations in the degrees of methylation. One of the methyl-esterified xylogalacturonan fractions was characterized by NMR spectroscopy: the xylose residues were β-(1 → 3)-linked to some of the galacturonic acid residues within a rather high molecular weight xylo-(1 → 4)-α-galacturonan and a degree of methylation of 39 was calculated. The methyl esters were found to be equally divided between the substituted and unsubstituted galacturonosyl residues.

Different populations of pectic hairy regions occur in apple cell walls

Alcohol insoluble solids from apple were extracted in sequence by buffer at 20°C and at 70°C, EDTA/oxalate, and mild alkali, yielding four populations of pectins. These pectins and the insoluble residue were characterized by their sugar composition, degree of esterification (methyl ester and O-acetyl groups), molecular weight distribution, and degradability by the combination of endopolygalacturonase (PG) and pectin esterase (PE) and by rhamnogalacturonase (RGase) after chemical saponification. After PG/PE treatment, the remaining high molecular weight material representing the pectic hairy regions was isolated and characterized. Clear differences were found in the sugar composition of the fractions obtained, while only small variations were observed in the sugar linkage composition. The pectic hairy regions were further degraded by RGase and the digests separated into high molecular weight and oligomeric degradation products. These “RGase oligomers” consisted of between 4 and 9 sugar units with a backbone of alternating rhamnose and galacturonic acid residues, partly substituted with galactose linked to C-4 of the rhamnose moiety. Both the absolute amount of RGase oligosaccharides released as well as the degree of galactose-substitution of the oligomers increased when severer extraction conditions were used. Relatively more RGase oligomers were released from the low molecular weight hairy regions as compared to the high molecular weight fraction. Typical high molecular weight fragments isolated from the RGase digests of various hairy regions included residual segments of the rhamnogalacturonan backbone rich in arabinose and a polymer presumably enriched in xylogalacturonan segments.

Pectin esterase activity and pectin methyl esterification in heated golden delicious apples

Pectin methylesterase activity and the degree of methyl esterification of cell wall pectins were measured in Golden Delicious apples after a 4 day heat treatment at 38° and after subsequent storage at 0° of heated and unheated apples. Enzyme activity increased similarly during storage in both heated and unheated apples, although heated fruit softened much less than unheated. During storage, the degree of esterification decreased in both heated and unheated apples in water- and CDTA-soluble pectin, but not in insoluble pectin. In a comparison of three methods of determining methyl groups, similar values were obtained for degree of bulk esterification of apple cell walls, regardless of treatment. Results presented do not confirm a role for pectin methylesterase or for de-esterification in apple fruit softening.

リンゴ果実の軟化時におけるβ-D-ガラクトシダーゼとα-L-アラビノフラノシダーゼ活性

Four β-D-galactosidases (GA-ase I, II, III, and IV) and one α-L-arabinofuranosidase (AF-ase) activities were detected in cell wall extracts from apples (Mains dornestica Borkh. cv. Starking Delicious) stored at 0°C for five months.The GA-ase fractions separated by hydroxylapatite column chromatography degraded a polyuronide prepared from apple cell walls, releasing galactose. GA-ase II, III, and IV fractions contained arabinogalactan degrading and galactose releasing activities, but GA- ase I failed to degrade arabinogalactan. AF-ase fraction degraded polyuronide and arabinogalactan releasing arabinose and other sugars.The activities of GA-ase II, III and IV decreased gradually as the apples softened in storage. The activities of GA-ase I and AF-ase which were not detectable at harvest, appeared and increased during storage.These galactose and arabinose releasing activities, may be involved in the degradation and solubilization of polyuronide, araban, and galactan in the cell walls of apples during softening.

Enzymatic degradation of cell walls of apples and characterization of solubilized products

Partially depectinated apple cell walls were digested by pectin lyase or endoglucanase or a combination. By combining these commercial enzymes, a higher yield of 22.2% of carbohydrate material was obtained compared with only 13.9% and 5.7%, respectively, when using them singly. Only small amounts of carbohydrates were extracted by buffer (0.8%). The solubilized extracts were fractionated using a combination of ion-exchange chromatography and gel filtration. The indidividual subfractions were analysed for neutral sugar and uronic acid content. The results indicated the existence of a synergistic effect between pectin lyase and endoglucanase based on the percentage of material extracted.

Binding of calcium by cell walls and estimation of calcium in apple fruit tissue with an ion selective electrode

Calcium is a critical nutrient for postharvest handling and quality of apples. Difficulties of analysis by atomic absorption spectrophotometry (AAS) prompted an examination of the use of a “standard addition” procedure with an ion selective electrode (ISE) to estimate calcium in apple tissue after homogenization in water. Calcium could be reliably estimated in synthetic mixtures, containing potassium malate at pH values representative of apple tissue homogenates. However, ISE estimates of calcium in apple homogenates were consistently lower than AAS estimates. The error in ISE estimates was attributed to cell wall binding of calcium. Titration of isolated apple cell walls with calcium showed that the proportion of calcium bound was dependent on the free calcium concentration. An empirical relationship between the reciprocal of the square of bound calcium and the reciprocal of free calcium concentration was derived. Incorporating this relationship into the calculation of calcium in apple homogenates led to closer agreement between ISE and AAS analyses than with the uncorrected calculation.

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.

Solid-State 13C NMR Investigation of Molecular Ordering in the Cellulose of Apple Cell Walls

A resolution-enhanced 13 C nuclear magnetic resonance (NMR) spectrum of cell wall material isolated from Braeburn apples has revealed a pair of signals assigned to cellulose chains exposed on crystal surfaces, providing evidence for a high degree of molecular ordering on those surfaces. Proton spin relaxation editing revealed no detectable amorphous cellulose and provided an estimate of about 23 chains in a typical crystallite. Cellulose in apple cell walls was found to be distinctive, with triclinic Iα and monoclinic Iβ crystalline forms present in similar proportions. Hence, its composition is intermediate between the family of bacterial and Valonia celluloses, rich in the triclinic crystalline form, and the family of cotton and ramie celluloses, in which the monoclinic form is dominant

Spectroscopic method for the authentication of vegetable matter

Infrared spectroscopy, in combination with classical discriminant analysis, was used to investigate the composition of the cell walls extracted from a number of plant species. It was shown that apple cell walls gave rise to a characteristic spectrum that allowed the discrimination of apple from a range of other plant species. The conclusion drawn is that infrared spectroscopy is potentially of value for the identification of plant cell walls, opening up the possibility that the technique may be useful for the authentication of fruit-containing products.

Studies on apple protopectin VI: extraction of pectins from apple cell walls with rhamnogalacturonase

Apple cell walls were extracted by treatment with rhamnogalacturonase, an enzyme that cleaves the rhamnogalacturonic backbone in the rhamnose-rich regions. It solubilised 13·6% of the cell walls after 24h. The extracted material was composed of galacturonic acid (42%) and neutral sugars (46%), especially arabinose, and had a high rhamnose: galacturonic acid ratio; it was also rich in methyl and acetyl groups (58 and 24 mg/g, respectively). The material was separated into two fractions by anion-exchange chromatography on DEAE Sepharose CL-6B. The neutral, non-retained fraction was composed mostly of arabinose and had a low hydrodynamic volume as shown by its elution pattern on Sephacryl S500. The retained fraction was enriched in galacturonic acid and had a higher hydrodynamic volume. Methylation analysis of the side-chains showed mostly arabinans and type I arabinogalactans. Some terminal rhamnose could be detected, and was relatively more abundant in the non-retained fraction. Admixture with pectinlyase or polygalacturonase plus pectinmethylesterase during treatment of the apple cell walls resulted in degradation of the high-molecular-weight fractions, and with arabinanases and galactanase in degradation of the low-molecular-weight fraction of the rhamnogalacturonase extract.

Apple-fruit xyloglucans: a comparative study of enzyme digests of whole cell walls and of alkali-extracted xyloglucans

Apple cell walls or alkali-extracted xyloglucans were digested with an endo-glucanase from Trichoderma viride and the resulting oligosaccharides were isolated by chromatography on Bio-Gel P-4. Three main oligosaccharides were present in similar proportions, and their structures were shown to be [Xyl(Glc)] 3-Glc, [Xyl(Glc)] 2-(FucGalXyl)Glc-Glc, and XylGlc-(GalXyP)Glc-(FucGalXyl)Glc-Glc. Each non-reducing-end Glc was 6-linked, each reducing-end Glc was 4-subsituted, and each other Glc was 4,6-disubstituted. The Xyl was either terminal or 2-substituted, the Fuc was terminal, and the Gal was either terminal or 2-substituted. The 1H-NMR spectra of the oligosaccharides extracted directly from the cell wall showed that they are not acetylated. Other oligosaccharides, notably GalXyl 3Glc 4, Xyl 2Glc 4, and Xyl 2Glc 3, were present in smaller proportions in the digest of the cell walls.

Molecular Weight and Degree of Methoxylation in Cell Wall Polyuronide during Softening in Pear and Apple Fruit

The concentrations of water-soluble polyuronides in apples [Malus domestica Borkh.) and pears (Pyrus communis L.) increased, but those of EDTA- and HCl-soluble polyuronides decreased during softening. Total polyuronide content decreased slightly during softening in both fruits. Depolymerization of polyuronides was observed only in the water-soluble fraction in pear fruit during softening, concomitant with an increase in polygalacturonase (PG) activity. No detectable depolymerization was observed in any of the polyuronide fractions during softening of apple fruit nor was any PG activity detected. The polyuronide fractions extracted from pear and apple cell walls contained various amounts of methoxyl groups. Polyuronides with a high degree of methoxylation were preferentially lost from EDTA- and HCl-soluble polyuronides during softening of both fruit. The water-soluble polyuronide had a lower degree of methoxylation than those lost in the EDTA- and HCl-soluble fractions. These results suggest de-esterification of polyuronides with a high degree of methoxylation rather than the depolymerization of polyuronides in the solubilization of polyuronides during ripening of apples and pears.

Cellulase activity in developing apple fruits

Changes in cellulase, polygalacturonase and total protein were measured during the growth and ripening of three apple cultivars ( Malus sylvestris Mill ‘Stark Lodi’, ‘Paulared’ and ‘Golden Delicious’) with the view of establishing the role of these cell wall-degrading enzymes in fruit development. In agreement with earlier work, no endo-polygalacturonase activity was evident at any stage of apple development. Cellulase was present in young expanding fruit and decreased in activity as the fruit reached full size and ripened. Separation of cellulase on MonoQ columns indicated the presence of three isoforms, two of which had molecular weights of 67 kDa. Electrophoresis of proteins from developing fruit indicated that proteins with molecular weights of 24, 26 and 67 kDa increased during ripening. Unlike other climacteric fruit, ripening in apples was not associated with an increase in extractable cellulase or polygalacturonase. The sodium dodecylsulfate-polyacrylamide gel electrophoresis studies suggest that a 67 kDa protein increased during ripening. The decrease in enzyme activity may be due to the inability of available extraction techniques to release this enzyme from apple cell walls.

Comparison between enzymatically and chemically extracted pectins from apple cell walls

The pectic material that was not extractable by cyclohexane-diamino-tetracetic acid was extracted from apple cell walls (i) successively by hot buffer, hot acid and cold dilute sodium hydroxide, and (ii) in parallel by buffer, arabinanases and galactanase and pectin-lyase. The pectin extracts were analysed for uronic acid and neutral sugar contents. Their molecular weight distribution was obtained by gel-permeation chromatography. Subfractions from gel-permeation chromatography were also analysed for uronic acid and individual neutral sugars. The pectin-lyase extracted the highest amount of uronic acid (55% of the strongly bound uronides). The dilute sodium hydroxide, solubilizing 34% of the uronic acid, was the most efficient chemical treatment. Hot acid and arabinanases and galactanase led to extraction of limited amounts of pectins. The sodium hydroxide and pectin-lyase extracts showed two subfractions on gel-permeation chromatography, one of them rich in neutral sugars and of high molecular weight.

Studies on apple protopectin. III: Characterization of the material extracted by pure polysaccharidases from apple cell walls

Plant cell walls contain polymers with complex and badly understood interactions that can be studied by enzymic extraction. Apple cell wall material, partially depectinated by a chelating agent, was treated with pure polysaccharidases (pectin-lyase, polygalacturonase, pectinesterase, endo-β-(1,4)-glucanases, exo-β-(1,4)-glucanase, endo-α-(1,5)-arabinanase, arabinofuranosidase, endo-β-(1,4)-galactanase and endo-β-(1,4)-xylanase) and combinations of these enzymes. Glucanases, arabinanases and galactanase extracted neutral oligomers. Enzyme formulations active on highly methylated pectins freed galacturonate oligomers and high-molecular-weight material. Addition of endo-β-(1,4)-glucanase to these enzyme formulations increased the amounts of high-molecular-weight material in the extracts. One of the endo-β-(1,4)-glucanases had a more marked action. The high-molecular-weight material was rich in neutral sugars, notably arabinose and galactose, and was degraded to a large extent by arabinanases and galactanase. The results were analysed by principal component analysis.

Studies on apple protopectin. IV: Apple xyloglucans and influence of pectin extraction treatments on their solubility

Hemicelluloses were extracted from apple cell walls with 1 m and 4 m sodium hydroxide and 8 m urea after depectinisation by a chelating agent, by a chelating agent and dilute sodium hydroxide or by a chelating agent and a pectin-lyase. The extracts were fractionated on Sephacryl S 500 and DEAE Sepharose CL-6B. The bulk of the hemicelluloses were solubilised by 4 m sodium hydroxide. The main hemicellulose was a fucogalactoxyloglucan. Some low-molecular-weight mannans were also present. Part of the xyloglucans could be extracted by urea after pectin extraction by a chelating agent or by pectin-lyase but not after pectin extraction by dilute sodium hydroxide. Dilute sodium hydroxide probably insolubilised some of the pectins and hemicelluloses.

Studies on apple protopectin. II: Apple cell wall degradation by pure polysaccharidases and their combinations

Plant cell walls are complex associations of polymers, the interconnections of which can be studied by sequential enzyme degradation. Partially depectinated apple cell wall material was degraded by pure polysaccharidases (pectin-lyase, polygalacturonase, pectinesterase, endo-β-(1,4)-glucanases, exo-β-(1,4)-glucanase, endo-α-(1,5)-arabinanase, arabinofuranosidase, endo-β-(1,4)-galactanase and endo-β-(1,4)-xylanase) and combinations of these enzymes. Amounts of extracted material and composition of the residues were determined. Principal component analysis permitted an objective assessment of these results. Extraction of significant amounts of uronides was only possible with enzyme formulations active on highly methylated pectins. Addition of endo-β-(1,4)-glucanase enhanced cell wall degradation by enzyme formulations active on highly esterified pectins, but arabinanases and galactanase did not increase the extraction of uronides. Principal component analysis indicated that the effects of the endo-β-(1,4)-glucanases were related to degradation of a fucogalactoxyloglucan, and emphasized the specificity of the action of one of the endo-β-(1,4)-glucanases.

Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins

A rhamnogalacturonase (RGase), that could degrade the modified hairy regions (MHR) prepared from apple cell walls, was isolated and purified from a technical preparation of Aspergillus aculeatus. The RGase cleaved galactopyranosyluronic-rhamnopyranosyl linkages. No activity was observed towards other cell-wall polysaccharides or p-nitrophenyl glycosides. The optimal conditions for RGase were pH 3–4 and 40–50°. MHR was degraded by RGase, and methylation analysis and 13C-n.m.r. spectroscopy indicated the products to have a tetrasaccharide backbone of alternating rhamnose and galacturonic acid residues. Some oligomers had a galactose residue 4-linked to rhamnose. The potential value of this new enzyme is considered.

Static and dynamic light-scattering studies of pectic polysaccharides from the middle lamellae and primary cell walls of cider apples

Mild, selective, and sequential methods have been used to extract pectic polysaccharides from cider apple pomace. The extracts have been characterised by static and dynamic light-scattering. CDTA (cyclohexane- trans-1,2-diaminetetra-acetate)-soluble pectic polysaccharides from the middle lamellae were characterised as having a broad-molecular-weight distribution ( M w = 4.2 × 10 6)_of non-free-draining stiff coils. CDTA-insoluble and Na 2CO 3-soluble pectic polysaccharides ( M w = 1.1 × 10 6) from the primary cell walls showed properties consistent with branched, cross-linked, microgel structures.