Pectin and hemicellulose were solubilized from cell walls of peach [Prunus persica (L.) Batsch] fruit differing in firmness by extraction with imidazole and sodium carbonate (pectin extracts), followed by a graded series of potassium hydroxide (hemicellulose extracts). The extracts were subjected to size exclusion chromatography. In imidazole extracts, as fruit softened, there was an increase in proportion of a large apparent molecular mass peak, with a galacturonosyl to rhamnosyl residue ratio resembling a rhamnogalacturonan-like polymer. A smaller apparent molecular mass peak was enriched in galacturonic acid and probably represented a broad polydisperse peak derived from more homogalacturonan-like polymers. In sodium carbonate extracts, a homogalacturonan-like polymer appeared to elute primarily as a higher apparent molecular mass constituent, which increased in quantity relative to other constituents as fruit softened. In cold 1 mol·L-1 KOH extracts three peaks predominated. A xyloglucan-like polymer appeared to elute predominantly in the second peak and fucose was strongly associated with it. In 4 mol·L-1 KOH extracts (tightly bound hemicellulose) the higher apparent molecular mass peak was predominantly acidic and presumably of pectic origin. The smaller apparent molecular mass peaks were composed primarily of neutral sugars, the second peak became smaller and the third peak larger as fruit softened. The ability to separate pectin and xyloglucan-like polymer as two separate fractions based on charge suggests that the nature of any pectin-hemicellulose interaction in this fraction is probably one of physical entrapment of pectin fractions by hemicellulose and not principally by covalent crosslinking between the two polysaccharide classes in peach. Flesh firmness serves as an important determinant of quality in peaches. Our results indicate that apparent molecular mass of both pectins and hemicelluloses changed as peaches softened, resulting in alteration of cell wall structure and associated with decreased tissue cohesion.
The hypersensitive response in resistant plants exposed to incompatible pathogens involves structural changes in the plant cell wall and plasma membrane. Cell wall changes may include pectin deesterification resulting in release of methanol. The time course of methanol production was characterized from `Early Calwonder 20R' pepper (Capsicum annuum L.) leaves infiltrated with the incompatible pathogen, Xanthomonas campestris pv. vesicatoria (Doidge) Dye race 1 (XCV). In the first time course experiment, leaves were infiltrated with either 108 colony-forming units/mL of XCV or water control. Leaf panels (1 × 5 cm) were excised after dissipation of water soaking, then incubated in vials at 24 °C. Headspace gas was analyzed at 6-hour intervals up to 24 hours. The rate of methanol production from resistant pepper leaves infiltrated with XCV was greatest during the first 12 hours after excision. In another experiment, leaf panels were harvested at 6-hour intervals up to 24 hours after inoculation and incubated for 12 hours at 24 °C to determine the relationship between the interval from inoculation to leaf excision and methanol production. The highest rate of methanol production was obtained when the interval between bacterial infiltration and leaf excision was 18 hours. The relationship between methanol release and changes in the degree of methylesterification (DOM) of cell wall pectin was determined in near isogenic lines of `Early Calwonder' pepper plants resistant (20R) and susceptible (10R) to XCV race 1. Cell walls were prepared from resistant and susceptible pepper leaves infiltrated with XCV or water. XCV-treated resistant leaves had 18% DOM and 9.7 nmol·g-1·h-1 of headspace methanol, and the susceptible leaves had 48% DOM with 0.2 nmol·g-1·h-1 methanol. Susceptible and resistant control leaves infiltrated with water had 55% and 54% DOM, respectively, with no detectable methanol production. Increased methanol production in resistant pepper leaves inoculated with XCV coincided with an increase in cell wall pH. Intercellular washing fluid of resistant pepper leaves had a significantly higher pH (6.9) compared to susceptible leaves (pH 5.1) and control leaves infiltrated with water (pH 5.1). Both 10R and 20R pepper leaves infiltrated with buffer at increasing pH's of 5.1, 6.9 or 8.7 had increased methanol production. Since deesterified pectin is more susceptible to degradation, demethylation may facilitate formation of pectic oligomers with defensive signalling activity.
To elucidate the role of ethylene in nonclimacteric fruit development and ripening, quantitative (cDNA–amplified fragment length polymorphism) cDNA–AFLP was used to visualize differential gene expression in four stages of ripening of strawberries (Fragaria×ananassa Duch. `Elsanta') treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. The proportion of clones affected by 1-MCP treatment was much higher in green than in white, pink, and red receptacle tissue. Three major cell-wall-related genes were affected by 1-MCP and, thus, are putatively ethylene dependent: a ripening-repressed beta-galactosidase (Faßgal3), up-regulated by 1-MCP; a putative endo-1,3-1,4-beta-D-glucanase (EGase), up-regulated in green and down-regulated in red fruit by 1-MCP; and a pectate lyase B (plB), expressed only in the red stage and significantly down-regulated by 1-MCP. Furthermore, we have identified genes encoding an alcohol dehydrogenase, a protein kinase-related protein, and a putative glutathione S-transferase, all ripening-induced and down-regulated by 1-MCP, suggesting that their regulation is at least partly ethylene dependent.
Postharvest quality of `Climax' rabbiteye blueberries (Vaccinium ashei Read) was evaluated after exposure to dosages of 0, 0.75, 1.5, 2.25, or 3.0 kGy gamma irradiation (0.118 kGy·min-1) and after subsequent storage. Irradiation did not affect weight loss, but irradiated berries were softer than nontreated berries. There was also a trend toward increased decay as dose increased. Irradiation had no effect on powdery bloom or surface color; total soluble solids concentration, acidity, and pH were affected slightly. Flavor preference was highest for nonirradiated berries and generally declined as dosage increased. Irradiation at 2.25 and 3.0 kGy resulted in increased levels of xylosyl residues in cell walls, and xylosyl residues were the most abundant cell-wall neutral sugar detected in blueberries. There was no evidence of cell wall pectin loss in irradiated berries. Irradiation at 21.5 kGy lowered the quality of fresh-market `Climax' blueberries.
Cucumber (Cucumis sativus L.) fruit (‘Heinz 3534’) subjected to mechanical stress followed by storage for 48 hr exhibited visible degeneration of the mesocarp and endocarp, which was accompanied by several-fold increases in the activity of the enzymes pectin methylesterase, peroxidase, polygalacturonase, and xylanase. The activity of all these enzymes increased in the endocarp, whereas only pectin methylesterase and polygalacturonase increased in the mesocarp, and pectin methylesterase, peroxidase, and polygalacturonase increased in the exocarp. Further, the increase in the activity of pectin methylesterase, peroxidase, and polygalacturonase was less when cucumbers were stored at 0° or 10°C vs. 25° or 38° after mechanical stress. Cucumbers stored for only 8 hr after mechanical stress, or not stressed, and stored for 8 or 48 hr showed no consistent significant increases in enzyme activity. Endocarp firmness of fruit stored at 25° or 38° for 8 hr after mechanical stress was lower than that of unstressed fruit, but this decrease was not evident after 48 hr of storage, and mesocarp firmness was not affected by mechanical stress regardless of storage temperature or time. Ethylene production was stimulated significantly by 8-hr storage at 0°, following mechanical stress, and by 48-hr storage at 0°, but was unaffected by all other treatment and storage regimes. These data indicate that mechanical stress induces biochemical and morphological changes in the major tissues of cucumber fruit, but tissue firmness and/or ethylene production will not serve as indicators of these changes. Moreover, the effects of mechanical stress do not appear to be mediated through the action of ethylene.
Galactosidases are thought to play a key role in cell wall metabolism during fruit growth and ripening. In this study we cloned seven β-galactosidase (β-Gal) cDNAs from japanese pear fruit and designated them PpGAL2, PpGAL3, Pp-GAL4, PpGAL5, PpGAL6, PpGAL7, and PpGAL8, in addition to the previously described JP-GAL hereinafter termed PpGAL1. mRNA expression patterns of these clones were characterized throughout fruit growth and on-tree ripening, and in leaves and shoots in three japanese pear cultivars, `Housui', `Kousui', and `Niitaka'. The shared amino acid sequence identity among the eight japanese pear β-Gal (PpGAL) clones ranged from 50% to 60%. They all contained the putative active site containing consensus sequence pattern G-G-P-[LIVM](2)-x(2)-Q-X-E-N-E-[FY] belonging to glycoside hydrolase family 35. Expression of all the clones was both development- and tissue-specific. PpGAL1 and Pp-GAL4 were only expressed in the ripe fruit while PpGAL2 and PpGAL3 were expressed in both expanding and ripening fruit with their abundance being highest in the ripe fruit. The abundance of PpGAL5, PpGAL6, and PpGAL7 mRNAs was highest in expanding fruit but decreased drastically upon the onset of ripening. PpGAL8 was only detected in very young fruit (15 days after full bloom) and not in expanding and ripening fruit. These results indicate that in japanese pear fruit β-Gal is encoded by a multigene family whose members show distinct and overlapping expression during the various phases of fruit development. Some of the members are not only fruit-specific but also ripening-specific and, therefore, may play a crucial role in cell wall disassembly during japanese pear fruit softening.
Studies were conducted to investigate the influence of 50 μl·liter−1 ethylene on the cell wall, polygalacturonase (PG) activity, and electrolyte leakage of harvested watermelon [Citrullus lanatus (thunb) Matsum and Nakai] fruit. Electrolyte leakage was significantly increased in tissues from ethylene-treated fruit. The highest leakage occurred in distilled water, although the net effect of ethylene was less dramatic due to high leakage from control fruit. Leakage was greatly reduced but the ethylene effect more apparent compared to the control when tissues were incubated in an isotonic medium of mannitol or in isotonic medium containing CaCl2. Polygalacturonase activity increased markedly in ethylene-treated fruit, showing a > 10-fold rise during the first 6 days of treatment. Little change in PG activity occurred in melons stored in air, even in fruit stored for as long as 120 days. Cell walls of fruit exposed to ethylene exhibited acute ultrastructural damage. The decline in placental tissue firmness and the development of watersoaking symptoms observed by the third day of 50 μl·liter−1 ethylene treatment were apparently due, in part, to the PG-mediated cell wall breakdown resulting in cell rupture. Additionally, ethylene appeared to enhance membrane permeability.
Potato (Solanum tuberosum L.) periderm forms a barrier at the surface of the tuber that protects it from infection and dehydration. Immature periderm is susceptible to excoriation (skinning injury), which results in costly storage loses and market quality defects. The periderm consists of three different cell types: phellem (skin), phellogen (cork cambium), and phelloderm (parenchyma-like cells). The phellogen serves as a lateral meristem for the periderm and is characterized by thin radial walls that are labile to fracture while the periderm is immature and the phellogen is actively dividing, thus rendering the tuber susceptible to excoriation. As the periderm matures the phellogen becomes inactive, its cell walls thicken and become resistant to fracture, and thus the tuber becomes resistant to excoriation. Little is known about the changes in cell wall polymers that are associated with tuber periderm maturation and the concurrent development of resistance to excoriation. Various changes in pectins (galacturonans and rhamnogalacturonans) and extensin may be involved in this maturational process. The objectives of this research were to compare immunolabeling of homogalacturonan (HG) epitopes to labeling of rhamnogalacturonan I (RG-I) and extensin epitopes to better understand the depositional patterns of these polymers in periderm cell walls and their involvement in tuber periderm maturation. Immunolabeling with the monoclonal antibodies JIM5 and JIM7 (recognizing a broad range of esterified HG) confirmed that HG epitopes are lacking in phellogen walls of immature periderm, but increased greatly upon maturation of the periderm. Labeling of a (1,4)-β-galactan epitope found in RG-I and recognized by the monoclonal antibody LM5 was abundant in phelloderm cell walls, but sparse in most phellem cell walls. LM5 labeling was very sparse in the walls of meristematically active phellogen cells of immature periderm, but increased dramatically upon periderm maturation. Deposition of a (1,5)-α-l-arabinan epitope found in RG-I and recognized by LM6 was abundant in phelloderm and phellogen cell walls, but was sparse in phellem cell walls. LM6 labeling of phellogen walls did not change upon periderm maturation, indicating that different RG-1 epitopes are regulated independently during maturation of the periderm. Labeling with the monoclonal antibody LM1 for an extensin epitope implied that extensin is lacking in phellem cell walls, but is abundant in phelloderm cell walls. Phellogen cell walls did not label with LM1 in immature periderm, but were abundantly labeled with LM1 in mature periderm. These immunolabeling studies identify pectin and extensin depositions as likely biochemical processes involved in the thickening and related strengthening of phellogen walls upon inactivation of the phellogen layer as a lateral meristem and maturation of the periderm in potato tuber. These results provide unique and new insight into the identities of some of the biological processes that may be targeted in the development of new technologies to enhance resistance to tuber skinning injury for improved harvest, handling and storage properties.
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 22.214.171.124), α-(EC 126.96.36.199) and β-galactosidases (EC 188.8.131.52), and endo-β-1,4-glucanase (EC 184.108.40.206). The appearance of polygalacturonase (EC 220.127.116.11) 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.
Oxygen uptake and glycosidase activities were examined in normal and granulated juice vesicles of several citrus fruit. Oxygen uptake was low in normal juice vesicles isolated from freshly harvested `Lee' tangelos [Citrus reticulate Blanco cv. Clementine × (Citrus paradisi Macf. cv. Duncan × Citrus reticulate Blanco cv. Dancy)] and stored `Dancy' tangerine (C. reticulate Blanco) and `Marsh' grapefruit (Citrus paradisi Macf.) (35.7, 17.9, and 11.6 μl O2/hr per g fresh weight, respectively), but was 2- to 3-fold higher in granulated juice vesicles. As severity of granulation increased in grape. fruit, O2 uptake increased. Oxygen uptake in normal and disordered juice vesicles of all citrus fruit examined was reduced to nondetectable levels with 0.1 mM KCN and was insensitive to salicylhydroxamic acid. α - and β -galactosidase and α- and β -glucosidase activities were present in extracts of normal grapefruit juice vesicles (123, 214, 51, and 25 nmol·hr-1·g-1 fresh weight, respectively) and was 2- to 3-fold higher in extracts of granulated tissue. α- and β -mannosidase activities, nondetectable in normal juice vesicle extracts, were present in extracts from granulated tissue. The results suggest that increased metabolic activity occurs in granulated juice vesicles and the energy produced may be used to support cell wall synthesis and modification. Increases in O2 uptake and glycosidase activities correlate well with observed symptoms of section-drying in citrus.