Enzymically active cell wall isolated from mature-green and ripening tomato (Lycopersicon esculentum Mill cv. `Rutgers') fruit was employed to investigate the mobility of the enzyme polygalacturonase (PG, EC 126.96.36.199). Cell walls from mature-green `Rutgers' fruit or from the ripening mutant rin, which alone exhibits little or no release of pectin, were unaffected by the addition of enzymically active cell wall from ripening `Rutgers' fruit, indicating that PG is either not transferred at all or is not transferred to sites of pectin hydrolysis. The quantity of pectin released by the addition of soluble PG to enzymically active wall depended on the quantity of enzyme added. Similar data were obtained using purified PG2. Pectin solubilization from all wall isolates exhibiting enzymically mediated pectin release diminished with time; however, transfer to fresh buffer initiated a resumption of autolytic activity, indicating that an inhibitor is released during the course of pectin hydrolysis.
Most `Flavortop' nectarines [Prunus persica (L.) Batsch (Nectarine Group)] that were placed directly into 0 °C storage developed chilling injury after removal, while preconditioning fruit for 2 days at 20 °C (delayed storage) reduced chilling injury substantially. Chilling injury was expressed as the development of a dry, woolly flesh texture during ripening. Delayed-storage fruit were as firm as control fruit when placed in storage, but softened more during storage. Analysis of cell wall components showed that in woolly fruit a higher percentage of pectin was retained in the sodium carbonate fraction, although during ripening polymers in this fraction decreased in molecular mass (Mr). In the guanidine thiocyanate hemicellulose fraction of woolly fruit, the associated pectin and hemicellulose remained as large polymers, while in delayed-storage fruit they decreased in Mr during ripening. Endo-polygalacturonase (PG), pectin esterase (PE), and endo-glucanase (EGase) activities of delayed-storage fruit were the same as control fruit at the beginning of storage, although exo-PG was higher. However, differences were observed at the end of storage. Endo-PG activity was lower in control than delayed-storage fruit at the end of storage while PE activity was higher, and exo-PG and EGase activities were similar. These differences in activity were not reflected in the mRNA abundance of the respective enzymes. Endo-PG and PE message was similar in all fruit at the end of storage and increased during ripening, while EGase message was low at all times except in control fruit after storage and development of woolliness. Prevention of chilling injury by delayed storage appears to be due to the ability of the fruit to continue a progressive, slow cell wall degradation in storage which allows normal ripening to proceed when the fruit are rewarmed. Regulation of the softening process did not appear to be by enzyme synthesis, since mRNA levels of the enzymes did not correspond with enzyme activity.
Cell wall changes in `Fantasia' nectarines [Prunus persica (L) Batsch var. nectarina (Ait) maxim] were determined after storage at 0C with or without intermittent warming (at 20C at 2-week intervals) and after ripening. For comparison, fruit were examined at harvest and after ripening without storage. Fruit stored continuously at 0C for 6 weeks became mealy during ripening, whereas fruit subjected to intermittent warming ripened normally. Ripening immediately after harvest was associated with solubilization and subsequent depolymerization of pectic polymers and a net loss of galactosyl residues from the cell wall. No solubilization of pectic polymers from the cell wall occurred during storage of fruit at 0C. Mealy fruit, ripened after continuous storage at 0C, showed only limited solubilization of pectins and depolymerization, high relative molecular weight (M) polymers being predominant. During ripening after storage, pectic polymer solubilization was not as extensive in intermittently warmed fruit as in fruit undergoing normal ripening but solubilized polymers were depolymerized, low M uronic acid-rich polymers becoming predominant. Intermittent warming of fruit resulted in significant softening during storage, alleviating the development of mealiness by promotion of cell wall changes associated with normal ripening.
Eight-one percent of the harvest firmness in kiwifruit (Actinidia chinensis Planch.) was lost during the first 8 weeks of storage in air at 0°C. As softening proceeded, a solubilization of uronic acids and the neutral sugar residues usually associated with pectic polymers (galactose, arabinose, and rhamnose) was detected. No consistent changes were noted in cellulose or the neutral sugars usually associated with hemicelluloses. Starch degradation also occurred coincident with softening. The amount of cell wall components soluble in water following fruit homogenization and the proportion of ethanol-precipitable pectic neutral sugars in this fraction increased during the first 8 weeks of storage. Once the rate of softening slowed (8 to 20 weeks), an equilibrium situation apparently was established between the amounts of the sugars formed in the ethanol-precipitable (i.e., polymeric) and ethanol-soluble fractions, suggesting that digestion of wall components continues after their excision from the insoluble wall matrix. Controlled atmosphere (2% O2+ 5% CO2; CA) storage retarded flesh softening relative to that measured in fruit held at 0° in air. A comparison of the changes in the cell wall components of air-stored and CA-stored kiwifruit suggests that, in addition to cell wall degrading processes contributing to fruit softening, starch degradation (possibly causing cell turgor changes) also may be involved in low-temperature softening of kiwifruit. The losses in water-insoluble cell wall pectic neutral sugars and uronic acids in air and CA storage were similar during the first 8 weeks of storage. Once softening slowed in CA, small but consistent reductions in the amount of cell wall turnover were observed as compared to air storage.
`Gemini II' cucumber (Cucumis sativus L.) fruits were stored for 2, 4, or 6 days at 5 and 6C in 1989 and for 5 days at SC or 10 days at 3C in 1990. Chilling injury (CI) symptoms were rated after 2 to 4 days at 25C. Cell wall polysaccharide concentrations in the peels and in injured and noninjured portions of the peels were determined only in 1990. High CO2 and low O2 delayed the onset of CI symptoms, but did not prevent symptom development. Chilling injury symptoms increased with longer exposure to chilling temperatures. Solubilization of cell wall polysaccharides was associated with development of CI symptoms. Variations in low methoxyl pectinates accounted for 70% of the variation in CI.
`Keitt' and `Tommy Atkins' mango (Mangifera indica L.) fruit were evaluated for selected ripening criteria at six ripening stages, from mature green to overripe. `Tommy Atkins' mangos developed more red and yellow pigmentation (CIE a* and b*) in peel and mesocarp tissues than `Keitt'. The outer mesocarp of `Keitt' remained firm longer than `Tommy Atkins', and the inner mesocarp was softer than the outer at each stage in both cultivars. Cell wall neutral sugars, particularly arabinosyl, rhamnosyl, and galactosyl residues, decreased with ripening in both cultivars. `Keitt' had more loosely associated, chelator-soluble pectin, accumulated more soluble polyuronides, and retained more total pectin at the ripe stage than `Tommy Atkins'. Both cultivars had similar polygalacturonase (EC 188.8.131.52) activity which increased with ripening. The amount and molecular weight of cell wall hemicellulose decreased with ripening in both cultivars. These data indicate that enzymatic and/or nonenzymatic processes, in addition to polygalacturonase activity, are involved in the extensive softening of mango fruit.
Easter lily flower buds at five stages of development (stage 1, 3–4 cm in length; stage 2, 6–7 cm; stage 3, 9–10 cm; stage 4, unopened buds, 13–14 cm; and stage 5, open flower 1 day after anthesis) were harvested, and flower organs were dissected for invertase assay. On a fresh weight (FW) basis, anthers had the highest soluble invertase activity (about 10-fold greater) than all other organs reaching to 15 units/g FW by the stage 2. The activity dropped to about 3 units/g FW at stage 3 and 4, and then increased up to 10 units/g FW in open flowers. Specific activity (units per mg of protein) also showed the same trend. On a specific activity basis, sepal invertase activity steadily increased during bud development, but was relatively constant on a fresh weight basis. stigma, style, and ovary, soluble invertase activity expressed on a FW and specific activity basis steadily increased as bud development. Filament soluble invertase activity on FW basis dropped at the stage 2 and 3, while specific activity steadily increased during bud development. Cell wall-bound invertase activity (released with 1 m NaCl) was present in all flower organs. However, soluble activity accounted for the most of total activity in sepal, ovary and filament (about 90%). About 75% of total activity was soluble in anther and style, whereas nearly equal amounts of soluble and cell wall activities were present in the stigma. The cell wall bound invertase activity increased throughout the bud development in sepal, stigma, style, and ovary parallel to soluble activity. Anther cell wall-bound activity fluctuated in a similar pattern as the soluble activity.
Discs from outer pericarp of mature green (MG) and light red (LR) tomatoes were incubated with 13C6-glucose as precursor to cell wall constituents, to determine biosynthetic capacity of the outer 2mm (including cuticle) and adjacent inner 2mm of tissue. Cell wall material was fractionated into pectic and hemicellulosic classes by sequential extraction, and alditol acetates and partially-methylated alditol acetates were prepared. Neutral sugars (NS), glycosidic linkage compositions and incorporation of label were determined by GC-FID and GC-MS. Rhamnose, arabinose and galactose accounted for ca. 90% of both labeled and total NS in the pectic fractions (sugar ratios within ripeness stage were the same for labeled and total NS). Xylose and glucose accounted for ca. 70% of both labeled and total NS in the hemicellulosic fraction (sugar ratios within ripeness stage were different between labeled and total NS). In the crude cell wall, galactose and glucose contents were significantly higher in the inner than in outer tissues for both MG and LR tomatoes. Loss of galactose during ripening was higher from outer tissues. These results show compositional differences between inner and outer tissues, and suggest that ripening-related wall synthesis may give rise to pectic polymers similar in NS composition to existing polysaccharides, and hemicellulosic polymers which may differ in composition.
Fruits of mid- (`O'Henry'), late (`Airtime'), and extra-late-season (`Autumn Gem') peach [Prunus persica (L.) Batsch] cultivars were examined for changes in cell wall structure and cytochemistry that accompany the onset of mealiness and leatheriness of the mesocarp due to chilling injury. The peaches were stored at 10C for up to 18 days or at SC for up to 29 days. Plastic-embedded sections were stained by the Schiff's-periodic acid reaction, Calcofluor white MR2, and Coriphosphine to demonstrate total insoluble carbohydrates, ß-1,4 glucans, and pectins, respectively. Mealiness was characterized by separation of mesocarp parenchyma cells leading to increased intercellular spaces and accumulation of pectic substances in the intercellular matrix. Little structural change was apparent in the cellulosic component of the cell walls of these fruits. In leathery peaches, the mesocarp parenchyma cells collapsed, intercellular space continued to increase, and pectin-positive staining in the intercellular matrix increased greatly. In addition, the component of the cell walls that stained positively for ß-1,4 glucans became thickened relative to freshly harvested or mealy fruit. At the ultrastructural level, dissolution of the middle lamella, cell separation, irregular thickening of the primary wall, and plasmolysis of the mesocarp parenchyma cells were seen as internal breakdown progressed.
Tomato fruit (Solanum lycopersicum 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 `Trust' tomato 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 percent extractable juice was not affected consistently. Increased polygalacturonase (PG) 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 (PME) 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. In chilled fruits, transcript accumulations for PG, PME (PME1.9), and expansin (Expt.1) were lower during storage at 20 °C compared with those of nonchilled fruits. Transcript accumulation for β-galactosidase (TBG4) was affected only at 14 days of cold storage, when transcript accumulation decreased. Cold treatment increased transcript accumulation of endo-β-1,4-glucanase (Cel1) after 12 days at 20 °C and decreased transcript accumulation after 7 days and 21 days at 21 °C. Cell wall analyses to investigate relationships among enzyme activities and cell wall disassembly are ongoing.