Cell Wall Enzyme Activities Research Articles

Physiological changes in boysenberry fruit during growth and ripening

SummaryBoysenberries (Rubus hybrid) were harvested at five developmental stages (green, “turning”, pink, red and purple) to study changes associated with flavour, colour and firmness, with a particular focus on the different enzymes involved in chlorophyll degradation or cell wall degradation. The level of reducing and non-reducing sugars increased 109- and 52-fold, respectively, between the “turning” and the purple stage. Titratable acidity increased by 84% between the green and the pink stage, but dropped by 41% between the red stage and full ripening. Total phenols displayed their highest values in green and “turning” fruit. Chlorophyll a and b degradation occurred mainly during the early stages of development, and was paralleled by a 2.3-fold increase in chlorophyllase activity and a 5-fold increase in Mg-dechelatase activity between the green and the red stages. Chlorophyll peroxidase activity was high at the green stage, but did not change significantly after the “turning” stage. This is the first report on the activity of chlorophyll-degrading enzymes in Rubus spp. fruits. Anthocyanins increased 11-fold between the “turning” and the pink stages, and an additional eight-fold between the pink and the purple stages. Fruit firmness decreased noticeably during ripening, with a concomitant 10.75-fold increase in polygalacturonase activity, beginning at the “turning” stage. Endo- -1,4-glucanase and -galactosidase activities rose 9.2- and 6.7-fold, respectively, after the pink stage and paralleled the major softening process taking place between the pink and purple stages, when fruit firmness decreased from 3.83 N to 0.29 N. Pectin methylesterase activity was high in pink and red fruit, but decreased by 21% as the fruit turned from red to purple. This is the first report on cell wall enzyme activities whose combined action might be required for the breakdown of natural cell wall substrates during ripening in boysenberry, as happens in other soft fruits.

Activity of Cell Wall-associated Enzymes in Cold-stored Tomato Fruit

Tomato fruit (Solanum lycopersicon L.) can develop mealiness and enhanced softening when exposed to chilling temperatures during storage, but the involvement of cell wall-associated enzymes in chilling injury development is not well understood. To study this aspect of injury development, we have exposed breaker stage tomato cv. Trust fruit to a chilling temperature of 3 °C for 0, 7, 14, and 21 days followed by storage at 20 °C for 12 days. Ethylene production was not affected by storage except after 21 days, where production was greater at 20 °C. Exposure of fruit to chilling temperatures delayed the ripening-related color change (chroma and hue) and initially increased compression values, but percentage of extractable juice was not affected consistently. Increased polygalacturonase activity during ripening was reduced by about 50% after 7 days at 3 °C, and further inhibited with increasing storage periods. In contrast, the activities of pectin methylesterase and α-galactosidase were not significantly affected by the cold treatments. β-Galactosidase activity was greater in all chilled fruit compared with fruit ripened at harvest, whereas endo-β-1,4-glucanase activity was lower after 21 days at 3 °C. These results will be compared with equivalent changes in the activities of cell wall enzymes that are associated with wooliness development in chilling-injured peach fruit.

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation ( R 2 = 0.99 ) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaeobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.

Suppression of Avocado (Persea americana Mill.) Fruit Softening and Changes in Cell Wall Matrix Polysaccharides and Enzyme Activities: Differential Responses to 1-MCP and Delayed Ethylene Application

Pre-ripe `Booth 7' avocado (Persea americana Mill.) fruit, a cross of West Indian and Guatemalan strains, were treated with 0.9 μL·L-1 1-methylcyclopropene (1-MCP) for 12 hours at 20 °C. After storage for 18 days in air at 13 °C, at which time whole fruit firmness values averaged about 83 N, half of the 1-MCP-treated fruit were treated with 100 μL·L-1 ethylene for 12 hours and then transferred to 20 °C. 1-MCP delayed softening, and fruit treated with 1-MCP retained more green color than air-treated fruit when full ripe (firmness 10 to 15 N). 1-MCP affected the activities of pectinmethylesterase (EC 3.2.1.11), α-(EC 3.2.1.22) and β-galactosidases (EC 3.2.1.23), and endo-β-1,4-glucanase (EC 3.2.1.4). The appearance of polygalacturonase (EC 3.2.1.15) activity was completely suppressed in 1-MCP-treated fruit for up to 24 days, at which time the firmness of 1-MCP-treated fruit had declined nearly 80% compared with initial values. The effect of exogenous ethylene applied to partially ripened 1-MCP-treated fruit differed for different ripening parameters. Ethylene applied to mid-ripe avocado exerted no effect on the on-going rate or final extent of softening of 1-MCP-treated fruit, even though polygalacturonase and endo-1,4-β-glucanase activities increased in response to ethylene. β-galactosidase decreased in 1-MCP-treated fruit in response to ethylene treatment. 1-MCP delayed the increase in solubility and depolymerization of water- and CDTA (1,2-cyclohexylenedinitrilotetraacetic acid)-soluble polyuronides, likely due to reduced polygalacturonase activity. At the full-ripe stage, the levels of arabinose, galactose, glucose, mannose, rhamnose, and xylose associated with the CDTA-soluble polyuronide fraction were similar among all treatments. In contrast, the galactose levels of water-soluble polyuronides declined 40% and 17% in control and 1-MCP treated fruit, respectively. Hemicellulose neutral sugar composition was unaffected by 1-MCP or ethylene treatment. The data indicate that the capacity of avocado fruit to recover from 1-MCP-mediated suppression of ripening can be only partially amended through short-term ethylene application and differs significantly for different ripening parameters.

Mannan transglycosylase: a novel enzyme activity in cell walls of higher plants.

Mannan transglycosylase is a novel cell wall enzyme activity acting on mannan-based plant polysaccharides in primary cell walls of monocotyledons and dicotyledons. The enzyme activity was detected by its ability to transfer galactoglucomannan (GGM) polysaccharides to tritium-labelled GGM-derived oligosaccharides generating tritium-labelled GGM polysaccharides. Mannan transglycosylase was found in a range of plant species and tissues. High levels of the enzyme activity were present in flowers of some kiwifruit (Actinidia) species and in ripe tomato (Solanum lycopersicum L.) fruit. Low levels were detected in mature green tomato fruit and activity increased during tomato fruit ripening up to the red ripe stage. Essentially all activity was found in the tomato skin and outermost 2 mm of tissue. Mannan transglycosylase activity in tomato skin and outer pericarp is specific for mannan-based plant polysaccharides, including GGM, galactomannan, glucomannan and mannan. The exact structural requirements for valid acceptors remain to be defined. Nevertheless, a mannose residue at the second position of the sugar chain and the absence of a galactose substituent on the fourth residue (counting from the non-reducing end) appear to be minimal requirements. Mannan-based polysaccharides in the plant cell wall may have a role analogous to that of xyloglucans, introducing flexibility and forming growth-restraining networks with cellulose. Thus mannan transglycosylase and xyloglucan endotransglycosylase, the only other known transglycosylase activity in plant cell walls, may both be involved in remodelling and refining the cellulose framework in developmental processes throughout the life of a plant.

Skin adhesion in sweetpotato and its lack of relationship to polygalacturonase and pectinmethylesterase during storage

Sweetpotatoes have a tendency to experience epidermal loss during harvest and postharvest handling. Epidermal detachment from underlying periderm (skin loss) causes weight loss, shriveling of the root surface, increased susceptibility to pathogen attack and inferior appearance. ‘Beauregard’ is very prone to skin loss, while ‘Jewel’ is thought to have stronger skin. Our objective was to determine the variation in skin strength in relation to cell wall enzyme activity and to determine possible correlations among these variables that could explain skin loss on the basis of enzyme activity during storage of the roots. Skin adhesion, polygalacturonase (PG) and pectinmethylesterase (PME) activity were measured during storage of ‘Beauregard’ and ‘Jewel’ roots in 1999 and again in 2000. Skin adhesion varied among years and cultivars. Overall, roots of ‘Beauregard’ were more susceptible than ‘Jewel’ to skin loss after several weeks of storage. Roots of ‘Jewel’ had lower skin adhesion at harvest, which increased after several weeks of storage and decreased again at the end of the storage period. Enzyme activity exhibited a random pattern during the storage period, and skin adhesion did not correlate with PG or PME activity.

Ripening-related changes in ethylene production, respiration rate and cell-wall enzyme activity in goldenberry (Physalis peruviana L.), a solanaceous species

Abstract The ripening of goldenberry (Physalis peruviana) is associated with a conspicuous climacteric rise in carbon dioxide and ethylene production. Its respiration rate and ethylene biosynthesis can be classified as extremely high. Ethylene yields between 7 and 24 nmol h−1 per g in the ripe/overripe stages thus compare favorably with production rates previously reported for tomato fruit. As the fruit color turns from green (chlorophyll) to yellowish orange (carotenoids) and a progressive softening occurs, several cell-wall enzyme changes arise. Pectinmethylesterase and α- and β-galactosidase reach activity levels similar to those in tomato fruit. Pectinmethylesterase and α-galactosidase increase toward the ripe stage. α-Arabinofuranosidase and β-glucosidase show lower activities but with an increasing pattern during ripening. On the other hand, polygalacturonase and α-glucosidase activities are hardly noticeable.

Firmness, pectin components and cell wall hydrolases of mango fruit following low-temperature stress

SummaryMango (Mangifera indica L. cv. Nam Dokmai) fruits chilled for 21 d at 48C were firmer than non-chilled fruits during subsequent ripening at 258C. The cell wall contents of chilled fruit contained less water-soluble pectin, more ammonium oxalate-soluble pectin and less alkali-soluble pectin than non-chilled fruits. The increase in ammonium oxalate-soluble pectin was at the expense of the alkali-soluble pectin and correlated with high pectinesterase activity in the fruit from low-temperature storage. Polygalacturonase and b-galactosidase activities were inhibited in chilled fruits. The relationships between delay in loss of firmness, and differences between activity of cell wall enzymes and pectin content of chilled and non-chilled fruits are discussed.

Apoplastic pH and Mineral Composition during Ripening of Tomato Pericarp

Ionic conditions and pH affect the catalytic activity of polygalacturonase and other cell wall enzymes, as well as non-enzymic pectin solubilization. Despite its potential role in regulating fruit ripening, apoplastic pH and mineral composition in ripening fruit are poorly documented. A pressure-extracted fluid was obtained from tomato pericarp, using a constant pressure of≈0.6–0.7 MPa for immature and mature-green fruit and 0.3–0.5 MPa for pink and red fruit. The osmolality of the pressure-extracted fluid ranged from 66 to 138 mmol·kg–1 compared with 268 to 293 mmol·kg–1 for the bulk pericarp homogenate, which attests to the putative apoplastic origin of the fluid. The pH of the apoplastic fluid was 6.7 at the immature and mature-green stages and dropped to 4.4 by the ripe stage, whereas bulk pericarp pH dropped from 4.6 to 4.3. [K+] in the apoplast increased from 13.2 mM at the mature-green stage to 37.3 mM at the ripe stage, whereas the bulk tissue [K+] ranged from 44 to 49 mM without relation with ripening stage. [Mg2+] declined from 11.9 mM in immature fruit to 8.7 mM in mature-green fruit and then increased to 12.9 mM in ripe fruit. [P] increased from 1.5 to 4.8 mM during ripening, and [Cl–] remained around 3.8 to 4.0 mM until the pink stage, increasing to 6.5 mM at the ripe stage. [Na+] remained constant at 6.2 to 6.5 mM, whereas [Ca2+] ranged from 3.5 to 4.9 with no consistent relationship with ripening stage. The changes in pH and ionic composition of the pericarp apoplast may affect the binding and catalysis of cell wall hydrolases and physical interactions between structural polysaccharides.

Regulation of cell wall glucanase activities by non-enzymic proteins in maize coleoptiles

Auxin stimulates cell wall glucanase activities in isolated segments of maize coleoptiles with no detectable change in the amounts of the enzymic peptides. To disclose the regulatory mechanism for this enhanced growth, we examined the possibility that protein interactions account for stimulating cell wall enzyme activities. One putative regulatory peptide, an acidic wall protein (AW), was separated from the wall protein fractionation profile, and this non enzymic protein was able to stimulate both exo- and endoglucanase activities in vitro. The results are consistent with the hypothesis that a pertinent glucanase/glucan interaction might be facilitated by AW mobilization within the wall in response to auxin-treatment.

Irradiation-induced Changes in Tomato Fruit and Pericarp Firmness, Electrolyte Efflux, and Cell Wall Enzyme Activity as Influenced by Ripening Stage

Mature green and pink tomato (Lycopersicon esculentum Mill.) fruit were subjected to ionizing irradiation in the range of 0.7 to 2.2 kGy from gamma-or X-ray sources. Firmness of whole fruit and pericarp tissue, pericarp electrolyte leakage, and pericarp cell wall hydrolase activities were measured following irradiation and during postirradiation ripening at 20 °C. Irradiation-induced softening was evident in mature-green and pink fruit within hours following irradiation, and differences between irradiated and control fruit persisted throughout postirradiation storage. Trends of firmness loss were much more consistent and showed much greater dose dependency in pericarp tissue than whole fruit. Irradiation enhanced electrolyte efflux in fruit of both maturity classes. Fruit irradiated at the mature-green stage softened during postirradiation storage but exhibited an apparently irreversible suppression in polygalacturonase activity, with levels remaining <10% of those of nonirradiated fruit. Polygalacturonase activity was less strongly affected in irradiated pink fruit than in mature-green fruit, but activity remained reduced relative to the controls. Pectinmethylesterase and β-galactosidase activities were significantly enhanced in irradiated fruit of both ripening stages in the early period following irradiation, but reductions were noted after prolonged storage.

To PG or Not to PG?

The absence of endo-α-1,4-d-galacturonanase (PG, EC 3.2.1.15) in some fruits and the molecular suppression of PG in tomato fruit have collectively provided evidence that this protein is neither required nor sufficient to achieve normal softening in fleshy fruits. On the other hand, initial claims that down-regulation of PG was without effect on tomato softening were overstated. The influence of PG on softening does appear to be minimal during the initial stages of ripening, during which time changes in the locule tissues can significantly alter texture as monitored in whole fruit. Enzymes, including pectinmethylesterases, cellulases, rhamnogalacturonanase, and glycosidases may also play pivotal roles in softening. β-galactosidases have attracted much attention as potential determinants of fruit texture; however, conclusive evidence for this role is lacking, and increased levels of β-galatosidase (and net cell wall galactosyl residue loss) have been noted in senescing vegetative and floral organs as well as in fruit. Apoplastic pH, ionic activity, and composition are likely to contribute to tissue and wholeorgan texture through weakening of polymer aggregates and/or through modulation of cell wall enzyme activity. During the latter stages of ripening and overripening, the role of PG is apparent from the persistent structural integrity of fruit transformed with PG antisense constructs. Patterns of softening and deterioration in normal tomato fruit suggest that the catalytic activity of PG in vivo is initially queued and does not parallel the accumulation of PG protein. Developmental changes in membrane permeability, physical injury, and other stress conditions can alter the apoplastic environment, releasing constraints on PG action.

Wirkung verschiedener W�rmevorbehandlungen auf das Gewebe eingefrorener Kirschen (Prunus avium L.) und dazu in Beziehung stehende Enzym-Aktivit�ten

The effect produced on the texture of cherries by heating at 50, 60 and 70° C for 3, 6, 9, and 12 mins before freezing followed by 3 and 6 months of frozen storage was studied. Variations in cell-wall enzyme activity were also analysed. To this end, objective measurements were carried out on the firmness of the cherries, by means of mechanical penetration, shear and Kramer Shear Cell tests. Pectinesterase and polygalacturonase activity were also measured. Correlations were established between the mechanical test results and pectinesterase activity in cherries subjected to different treatments. Significant correlations were found between this activity and the final firmness of the frozen fruit. Low-temperature heating (70° C) prior of freezing significantly improves the final texture of the product.

Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls.

At 'low' ionic strength, acid phosphatase bound to plant cell walls exhibits an apparent negative co-operativity, whereas it displays classic Michaelis-Menten kinetics in free solution. Conversely, at 'high' ionic strength, the bound enzyme and the soluble enzyme behave identically. This apparent negative co-operativity is explained by the existence of an electrostatic partition of the charged substrate by the fixed negative charges of the cell wall. Raising the ionic strength suppresses these electrostatic repulsion effects. Calcium may be removed from the cell walls by acid treatment and the acid phosphatase is apparently strongly inhibited. This inhibition occurs together with an increased apparent negative co-operativity of the enzyme. Incubating cell wall fragments previously depleted of calcium with CaCl2 restores the initial behaviour of the enzyme. Calcium, which tightly binds to cell wall pectic compounds, has by itself no effect on the enzyme in free solution. It affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects. Non-linear least-square fitting methods make it possible to estimate the density of fixed negative charges as well as the electrostatic partition coefficient, for both the 'native' and 'calcium-deprived' cell wall fragments. It may be shown directly that calcium loading and unloading in the cell wall controls the electrostatic effects, by monitoring proton extrusion from cell wall fragments upon raising the ionic strength. Proton outflux in the bulk phase is considerably enhanced upon removal of calcium from the cell walls. The main conclusion is that loading and unloading of calcium during cell elongation and division may regulate the activity of cell wall enzymes.

Localization and Properties of Cell Wall Enzyme Activities Related to the Final Stages of Lignin Biosynthesis

Cell wall activities which are related to the final stages of lignin biosynthesis, that is the generation of hydrogen peroxide and peroxidase activity, were investigated using biochemical and histochemical methods. Peroxidases involved in both reactions appeared to be restricted to lignifying tissues. Isolated cell walls exhibited a very high affinity for syringaldazine. Cell walls were able, in vitro as well as in vivo, to oxidize guaiacol in the absence of H2O2 when NADH was present in the incubation medium together with p-coumarate and MnCl2 as cofactors. The origin of the NADH used to form H2O2 is discussed.

Effects of auxin on wall polysaccharide composition and enzyme activity during extension‐growth of Pellia (Bryophyta)

Auxin‐induced changes in cell wall polysaccharide composition and enzyme activity of seta segments from the liverwort Pellia epiphylla (L.) Corda were studied using colorimetric, gas chromatographic, radioactive tracer, and viscometric techniques. Extension‐growth of segments doubled in the presence of aqueous 10 μM indole‐3‐acctic acid (IAA) ± 50 mM glucose. IAA‐enhanced growth was accompanied by (1) enhanced synthesis of all wall polysaccharides but cellulose, (2) increase in the relative glucose content of neutral wall sugars, and (3) change in the activity of wall‐bound glycosidase relative to controls, but no change in the activity of cellulase. Galactose and mannose (50 mM) suppressed auxin enhancement of both growth and wall synthesis. These findings suggest that auxin‐mediated extension‐growth of Pellia setae is dependent upon the maintenance of non‐cellulosic cell‐wall synthesis.

Role of calcium in ripening and senescence

Abstract In view of the possibility that senescence may be a consequence of the deterioration of cell walls and membrane compartments, calcium was studied as a possible agent which might defer senescence. Calcium treatment delayed leaf senescence in corn leaf discs. The effect of calcium on senescencse was additive to the cytokinin deferral of senescence. An increase in hydraulic permeability to [3H] water during senescence was demonstrataed and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Analysis of inorganic ions in the pericarp of nonripening rin mutant tomajç fruits and isogenic Rutgers revealed higher levels of bound Ca in rin at advanced stages of maturity. In the normal fruits, bound Ca decreased about 3‐fold during maturity. In rin fruits, no change in polygalacturonase (PG) activity was detected, whereas in Rutgers, an increase in PG activity was observed at advanced stages of maturity. Each of the measured changes associated with senescence (pigment changes, protein decrease, hydraulic permeability increase, and an increase in cell wall enzyme activity) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cell wall structure and membrane integrity.