Cell Wall Polyuronides Research Articles

Compositional changes in cell wall polyuronides and enzyme activities associated with melting/mealy textural property during ripening following long-term storage of ‘Comice’ and ‘d’Anjou’ pears

Irregular ripening is a major deterrent for the purchase of European pears. Cell wall polyuronides and related- modifying enzymes were investigated in two European winter pears, ‘Comice’ and ‘d’Anjou’, during ripening at 20°C following 5 and 8 months of storage at −1.1°C. Both pear cultivars developed a melting (buttery-juicy) texture and higher water-soluble polyuronides (WSP) in flesh tissue after 5 months, but the pears’ texture quality decreased and a mealy (coarse-dry) texture presented in ‘Comice’ pear after long-term storage. Activities of pectin methylesterase (PME) and α-arabinofuranosidase (α-ARF) in ‘Comice’ pears was positively correlated with texture development, whereas in ‘d’Anjou’ pears polygalacturonase (PG), PME, cellulase (CEL), and β-galactosidase (β-GAL) were more highly correlated with texture. 1-MCP reduced pear ripening by inhibiting ethylene production and the activities of cell wall-modifying enzymes, maintaining higher antioxidants, and alleviating membrane lipid peroxidation. These results indicated melting texture developed in European pears as a result of the synthesis of WSP and increase of PME activity during pear fruit softening.

Cell wall modification in 1-methylcyclopropene-treated post-climacteric fresh-cut and intact papaya fruit

Papaya is a climacteric fruit in which ripening is greatly regulated by ethylene often associated with stress responses such as wounding. The changes in cell wall compositions in papaya fruit at an advanced stage of ripening under stress conditions including chilling temperature of 5°C and wounding employed as fresh-cut and how these changes were affected by an ethylene action inhibitor of 1-methylcyclopropopene (1-MCP) were examined in the study. The recovery of ethanol-insoluble solids, total soluble sugars, water-soluble polyuronides, neutral hemicelluloses, and neutral sugars of rhamnose, arabinose, mannose and glucose were not affected by 1-MCP or fresh-cut processing. The fresh-cut processing, however, caused a higher loss of total polyuronides and the neutral sugar galactose while increasing the recovery of chelator-soluble polyuronides. Few significant differences due to 1-MCP application were recorded in the recoveries of alkali-soluble polyuronides, hemicellulosic polyuronides extracted with 4% KOH, and the neutral sugar xylose. Modifications of cell wall polyuronides and hemicelluloses in ripe fresh-cut papaya fruit exhibited mostly similar patterns to those in intact ripe papaya fruit under the chilling temperature of 5°C while minimally affected by 1-MCP.

Compositional Changes in Cell Wall Polysaccharides from Japanese Plum (Prunus salicina Lindl.) during Growth and On-Tree Ripening

Climacteric Japanese plums were harvested at six developmental stages with no intermediate storage period, and cell wall compositional changes were analyzed. Arabinose proved to be the principal neutral monosaccharide constituent in cell walls during growth and the most dynamic neutral sugar in pectic fractions. Arabinose loss from tightly bound pectins was found to be a relatively early feature in the sequence of cell wall biochemical modifications, thus suggesting a softening-related role during Japanese plum on-tree ripening. Depolymerization of matrix glycans started at the end of the cell expansion phase and increased throughout ripening. Pectin solubilization was first detected during early ripening. Firmness loss did not correlate with polyuronide depolymerization early in ripening, but the last softening phase was associated with a strong depolymerization of cell wall polyuronides as well as a decrease in the arabinose/galactose ratio in loosely bound pectins. This is the first work that characterizes the temporal sequence of cell wall polysaccharide changes in Japanese plum.

Activities of several membrane and cell-wall hydrolases, ethylene biosynthetic enzymes, and cell wall polyuronide degradation during low-temperature storage of intact and fresh-cut papaya (Carica papaya) fruit

Abstract Fresh-cut fruit tissue deteriorates more rapidly than its intact counterpart. A study was conducted to determine changes in firmness, cell-wall polyuronides, and the activities of cell-wall and membrane hydrolases and ethylene biosynthetic enzymes in intact and fresh-cut papaya fruit during storage at 5 °C. Processing of papaya fruit was performed under sanitized conditions at 5 °C, and fruit pieces were stored for 8 days at 5 °C. Levels of total and CDTA-soluble polyuronides in intact fruit did not change during storage, but water-soluble polyuronides increased significantly by day 8 (15%). In contrast, total polyuronide content of fresh-cut papaya decreased (9.5%), whereas levels of CDTA- and water-soluble polyuronides increased 45 and 30%, respectively. Firmness and mol mass of polyuronides decreased more rapidly in fresh-cut fruit than in intact fruit. The activities of polygalacturonase (EC 3.2.1.15), alpha- (EC 3.2.1.22) and beta-galactosidases (EC 3.2.1.23), lipoxygenase (EC 1.13.11.12), phospholipase D (EC 3.1.4.4), and ACC synthase (EC 4.4.1.14) and ACC oxidase increased within 24 h in fresh-cut fruit, and remained significantly higher compared with levels in intact fruit throughout the storage period. Pectin methyl esterase (EC 3.2.1.11), and phospholipase C (EC 3.1.4.3) activities showed no consistent differences between fresh-cut and intact fruit. The data suggest that a wound-induced increase in enzymes targeting cell walls and membranes contributes to the rapid deterioration of fresh-cut fruit. The significantly less pronounced changes observed for intact fruit stored under identical conditions indicate that the enhanced deterioration of fresh-cut fruit does not reflect low-temperature injury.

Cell wall metabolism during development of rambutan fruit

SummaryCell wall metabolism during fruit development in ‘Rongrien’ rambutans (Nephelium lappaceum L.) was investigated. Around 80% of the cell wall composition in the skin and around 60% in the pulp was cellulose. The firmness of skin increased towards harvest. In the skin, water-soluble pectic substances (WSP), hemicellulose and cellulose decreased with days after full bloom (DAFB), but hexametaphosphate-soluble pectic substances (HMP) increased. Cellulose concentrations were high throughout fruit development, that is, they never fell below 27 g kg–1 FW. The inconsistency of the skin firmness with cellulose and hemicellulose suggests that if they remained at a certain minimum level, their concentrations might not be reflected in the firmness. In the skin, HMP concentrations may influence firmness. Pectin esterase (PE) activity in the skin decreased towards harvest, but endo-polygalacturonase (PG) activity increased. Thus their activities may not be associated with the cell wall polyuronide degradation in the skin. The firmness of pulp decreased towards harvest, and coincided with the changes of HMP, hemicellulose and cellulose, but WSP increased. From 56 DAFB to 98 DAFB (harvest), cellulose decreased 70% from 10 g kg–1 FW and hemicellulose decreased 60% from 5 g kg–1 FW. Although PE activity in the pulp did not change during fruit development, endo-PG increased towards harvest. This result suggests that endo-PG may relate to cell wall polyuronide degradation in the pulp. However, the WSP concentration was very low compared with other components. In contrast to the skin, the decreases of cellulose and hemicellulose may be related to the softening of pulp.

INTERNAL BREAKDOWN IN MANGO FRUIT: CHANGES IN CELL WALL COMPOUNDS

Tommy Atkins' mangoes (Mangifera indica, L.) were harvested at mature green stage (Texture = 126 N; total soluble solids = 9,8%; tritable acidity = 0,86%) and submitted to X-ray analysis to identify the incidence of internal breakdown (IB) disorder. Both the healthy and damaged fruits were stored at 12±2°C and 88±3% RH for 28 days. Analyses of firmness, cell wall cellulose and polyuronides were made at 7-day intervals. As ripening progressed, all those fruit parameters decreased to low values in both groups of the fruit, but fruit with IB had higher cell wall polyuronides content than the healthy ones. indicating depolymerization. For both the groups, significant levels of arabinose. galactose, rhamnose, xylose, manose and glucose, with predominance of arabinose galactose and xyiose in damaged fruit were observed.

Changes to Physical Properties of the Cell Wall and Polyuronides in Response to Heat Treatment of `Fuyu' Persimmon that Alleviate Chilling Injury

Modifications to solubilized cell wall polyuronides of sweet persimmon (Diospyros kaki L. `Fuyu') were examined during development of chilling injury (CI) during storage and in response to heat treatments that alleviated CI. Storage at 0 °C caused the solubilization of a polyuronide fraction that possessed a higher average molecular mass than polyuronide solubilized during normal ripening. The viscosity of this fraction was 30-times that of normally ripened fruit. Fruit heat-treated before or following storage contained a soluble polyuronide fraction with a markedly lower average molecular mass and decreased viscosity than in chilling injured fruit. Heat treatment also impeded an increase in viscosity of the cell wall material if applied before storage. CI (gelling) was related to the release of polyuronide from the cell wall during storage and its lack of subsequent degradation. Heat treatments retarded polyuronide release but promoted degradation of solubilized polyuronides.

Increase in low molecular size uronic acid in ripening banana fruit

AbstractUronic acid soluble in ethanol/water (4:1 v/v) increased from 9 ± 1 mg kg−1 fresh weight in pulp of unripe banana fruit. Musa (AAA Group, Cavendish subgroup) ‘Williams’, to 53 ± 13 mg kg−1 fresh weight in fruit ripened for 13 days. This increase began within the first 2 days of ripening. Uronic acid soluble in phenol/acetic acid/water (2:1:1 w/v/v) increased from 15 ± 5 to 86 ± 17 mg kg−1 fresh weight during the first 8 days of ripening, accompanied by a decrease in cell wall uronic acid from 10·2 ± 0·8 to 4·4 ± 0·4 g kg−1 fresh weight. Most or all of the uronic acid in extracts of ripe pulp cochromatographed with monogalacturonic acid. The results were consistent with hydrolysis of cell wall polyuronides by exopolygalacturonase (EC 3.2.1.67).

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.

Cell Wall Dissolution in Ripening Kiwifruit (Actinidia deliciosa)

Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa ;Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na(2)CO(3), 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na(2)CO(3)-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na(2)CO(3)-soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA-soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation. Pectolytic enzymes such as endopolygalacturonase and beta-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo.

Relationship between softening and the polyuronides in ripening banana fruit

AbstractBanana fruits, Musa (AAA Group, Cavendish subgroup) ‘Williams’ were ripened in air at 20°C with ethylene. Pulp began softening between days 1 and 2 of ripening and reached a maximum by the fourth day. Total pulp cell wall uronic acid and uronic acid soluble in 40 mM EDTA, 50 mM acetate, pH 4.5, also began to decrease and increase respectively between days 1 and 2. By day 8, total uronic acid had decreased from 10.2 to 4.4 mg g−1 fresh weight, and had become entirely soluble in EDTA‐buffer, while EDTA‐buffer‐soluble uronic acid had increased from 2.3 to 4.5 mg g−1. The molecular size distribution of the EDTA‐buffer‐soluble uronic acid was unchanged up to day 4, when there was a slight loss in the proportion of smaller species. The average molecular size of this uronic acid did not change significantly during 8 days of ripening (relative to dextrans, Mn 36 kDa; Mw 173 kDa). The large change in total content and extractability of cell wall polyuronides that correlated with softening was inconsistent with depolymerisation by endopolygalacturonase (EC 3.2.1.15), reduced cross‐linking of polyuronides by calcium, or extraction artefacts.

Homopolygalacturonan nitroxyl amides: matrix deformation-induced motional perturbations of cell wall polyuronides

Marquage de polymeres contenant de l'acide galacturonique, sur la position C 6 par une liaison amide pour etudier la structure microscopique du reseau complexe de paroi cellulaire a l'etat sec et solide hydrate

Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

Tomato fruit ripening is accompanied by extensive degradation of pectic cell wall components. This is thought to be due to the action of a single enzyme, polygalacturonase, whose activity is controlled, at least in part, at the level of gene expression. At the onset of tomato fruit ripening, polygalacturonase enzyme activity, mRNA levels, and relative rate of gene transcription all increase dramatically. To elucidate the role of polygalacturonase during tomato fruit ripening, we utilized a pleiotropic genetic mutation, rin, that blocks many aspects of ripening, including the activation of polygalacturonase gene transcription. The polygalacturonase structural gene was ligated to a promoter that is inducible in mature rin fruit and inserted into the fruit genome, and plants were regenerated. This allowed expression of the polygalacturonase gene in transgenic rin fruit at a time corresponding to ripening in wild-type fruit. Expression of this gene resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in transgenic rin fruit. However, no significant effect on fruit softening, ethylene evolution, or color development was detected. These results indicate that polygalacturonase is the primary determinant of cell wall polyuronide degradation, but suggest that this degradation is not sufficient for the induction of softening, elevated rates of ethylene biosynthesis, or lycopene accumulation in rin fruit.

Analysis of the molecular size of tomato (Lycopersicon esculentum Mill) fruit polyuronides by gel filtration and low-speed sedimentation equilibrium.

The cell-wall structures of tomato (Lycopersicon esculentum Mill) and other fruit are intimately linked with the nature of their polyuronides. Cell-wall polyuronides from unripe and ripe tomato fruit were isolated and purified and their molecular size and molecular-size distributions were compared. It was demonstrated that there is a considerable decrease in the weight-average Mr upon ripening (from 160,000 +/- 10,000 to 96,000 +/- 4000) and a corresponding increase in polydispersity, particularly at the low-Mr end of the distribution. The estimates of polyuronide molecular size and molecular-size distribution were obtained without the need for polyuronide standards of known Mr by using gel-filtration chromatography combined with the absolute method of low-speed sedimentation equilibrium.

ESR spectroscopic evidence for hydration- and temperature-dependent spatial perturbations of a higher plant cell wall paramagnetic ion lattice

To study the influence of cell wall polyuronide structure on bound paramagnetic ion interactions, spin-spin coupling measurements were made on intact cell walls exchanged with a wide range fo Mn 2+ and Cu 2+ concentrations. These experiments were performed so that dimer-only intercationic nearest neighbor distances ( d) and lattice constants (κ) could be calculated from the linewidth-concentration dependency. d values were estimated to be 12 and 14 Å for Cu 2+ and Mn 2+, respectively. At the maximal bound ion concentration, κ was 2.3–2.6, indicating that about 5–7 paramagnetic ion nearest neighbor spin-spin interactions occur per dipole in the nearly filled lattice. This latter observation strongly argues for the egg-box model of the cell wall-polyuronide lattice structure. Mn 2+ linewidths of hydrated cell wall-bound paramagnetic ions displayed an unusual temperature dependency, whereby linewidths increased between 20°C and the temperature at which maximal linewidths were observed ( T max). T max was inversely proportional to the degree of lattice hydration, indicating that the temperature dependency was not associated with the freezing of bound water. The relative change in Mn 2+ linewidths, between 20°C and T max, was affected by binding site-associated 1H spin-lattice relaxation times, indicating that the temperature dependency is at least partially controlled by cell wall polyuronide structure.

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn 2+ and Ca 2+. These experiments were performed to find the Mn 2+ nearest-neighbor distance and thereby determine whether carboxylate-Mn 2+ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn 2+ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca 2+ was found to reduce the Mn 2+ spin-spin line broadening at similar total bound Mn 2+ concentrations. This is expected only if Ca 2+ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.

Role of membranes of mycelial Mucor rouxii in synthesis and secretion of cell wall matrix polymers.

Membrane fractions of the mycelial form of Mucor rouxii contained endogenous polyuronides and glycoproteins with sugar compositions similar to the cell wall polyuronide (mucoran) and extracellular (wall and filtrate) glycoproteins, respectively. The polyuronide pool was rapidly labeled with D-[U-14C]glucose and on pulse-chase experiments appeared to turn over, suggesting that these polymers were precursors of the wall mucoran. In contrast, the glycoproteins appeared to accumulate. Although the membrane-associated glycoproteins had molecular weights similar to those of the extracellular glycoproteins, the bulk of the membrane-associated polyuronides were of a molecular weight lower than that of the wall polyuronide.