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- Author or Editor: Kenneth C. Gross x
A new assay for nanomole amounts of reducing sugar using 2-cyanoacetamide has been modified and adapted to assay for exo- and endo-polygalacturonase in fruit extracts of tomato (Lycopersicon esculentum Mill. cv. Heinz 1350) with increased sensitivity (140%) and simplicity over currently used methods. Linearity was observed with galacturonic acid as a standard up to 250 nmol; the lower limit of detection was 1 nmol. Polygalacturonase product formation was linear for 3 hours and was proportional to the amount of enzyme in the reaction. Thin-layer chromatography of reaction products revealed a range of uronic acid oligosaccharides as well as galacturonic acid. Thus, both exo- and endo-polygalacturonase were active in the extracts.
Rhamnogalacturonase (RGase) is a new fungal enzyme which degrades the highly branched regions of apple fruit cell wall pectin by cleaving the glycosyl linkage between rhamnosyl and galacturonosyl residues (Schols et al., 1990. Carhohydr. Res. 206:105.). This enzyme, if present in fruit, could play a significant role in fruit softening. Partial purification of RGase was accomplished from a fungal enzyme preparation (Pectinex Ultra SP-L, NOVO Ferment) produced from Aspergillus niger. The crude enzyme hydrolyzed chelator-soluble pectin from red ripe tomato fruit. Methylation linkage analysis of the product suggested that an increase in terminal-rhamnosyl residues accompanied pectin hydrolysis, indicative of RGase activity. Cross-linked alginate, hydroxyapatite, and DEAE-Sephadex chromatography were used to partially purify RGase. Polygalacturonase was efficiently removed using the alginate column. Crude pectin obtained from mature-green tomato fruit cell wall by extracting with 0.5 M imidazole buffer (pH 7) and 50 mM Na-carbonate was incubated with pure polygalacturonase and the residue hydrolyzed with 0.1 N trifluoroacetic acid. This modified pectin was used as a substrate to investigate the presence of RGase in tomato and other fruit.
Experiments were conducted to determine if ethylene influences chilling injury, as measured by percentage of slices exhibiting water-soaked areas in fresh-cut tomato slices of `Mountain Pride' and `Sunbeam' tomato (Lycopersicon esculentum Mill.). Ethylene concentration in containers without ventilation significantly increased during storage at 5 °C, whereas little or no accumulation of ethylene occurred in containers with one or six perforations. Chilling injury was greatest for slices in containers with six perforations, compared to slices in containers with one perforation, and was over 13-fold greater than that of slices in control containers with no perforations. An experiment was also performed to investigate the effectiveness of including an ethylene absorbent pad in containers on subsequent ethylene accumulation and chilling injury. While ethylene in the no-pad controls increased continually during storage of both `Mountain Pride' and `Sunbeam' tomatoes at 5 °C under modified atmosphere conditions, no increase in accumulation of ethylene was observed in containers containing ethylene absorbent pads throughout storage. The ethylene absorbent pad treatment resulted in a significantly higher percentage of chilling injury compared with the no-pad control. In studies aimed at inhibiting ethylene production using AVG during storage of slices, the concentration of ethylene in control containers (no AVG) remained at elevated levels throughout storage, compared to containers with slices treated with AVG. Chilling injury in slices treated with AVG was 5-fold greater than that of controls. Further, we tested the effect of ethylene pretreatment of slices on subsequent slice shelf life and quality. In slices treated with ethylene (0, 0.1, 1, or 10 μL·L-1) immediately after slicing, ethylene production in nontreated controls was greater than that of all other ethylene pretreatments. However, pretreatment of slices 3 days after slicing resulted in a different pattern of ethylene production during storage. The rate of ethylene production by slices treated with 1 μL·L-1 ethylene 3 days after slicing was greater during storage than any of the other ethylene treatments. With slices pretreated with ethylene, both immediately and 3 days after slicing, the rate of ethylene production tended to show a negative correlation with chilling injury. Chemical name used: 1-aminoethoxyvinylglycine (AVG).
Antisense technology has shown that neither polygalacturonase nor pectin methylesterase alone are responsible for tomato fruit softening, leading to the likelihood that other enzymes or factors are important. Our laboratory recently found that α and β-galactosidase from avocado fruit solubilized tomato fruit pectin in vitro. Previously, Pressey (Plant Physiol. 1983,71:132) found that the activity of one of three α-galactosidase isozymes from tomato fruit increased during ripening and was capable of degrading cell wall galactan, suggesting a role for the enzyme in fruit softening. Increased β-galactosidase activity was observed in a number of other fruit during ripening. In the present study, NaCl extraction of tomato pericarp yielded relatively high levels of cc- and β-galactosidase activity. At least two isozymes of each were resolved during Mono-Q HPLC α-Galactosidase was further purified by additional Mono Q and Superose 12 gel filtration HPLC. Gel filtration and SDS-PAGE yielded an apparent molecular weight of 44 kD. The partially pure α-galactosidase had a specific activity of 294 μmol product/min per mg protein, a Km of 317 μm, a pl of 5.0, and a pH optimum of 5.5. Activity was inhibited 67% by α-d-galactose. Preliminary results show that β-galactosidase can also be purified by the same techniques. Following further purification, the isozymes will be sequenced and cloned. A second approach being used in an attempt to identify cDNA clones for the α- and β-galactosidase genes from tomato fruit involves using heterologous cDNA clones from guar (Overbeeke et al., 1989; Plant Molecular Biology 13:541-550) and carnation (Raghothama et al., 1991; Plant Molecular Biology 17:61-71), respectively, to screen a ripening tomato fruit cDNA library. Basic molecularbiological techniques will be used to elucidate the role of these enzymes in tomato fruit ripening.
Respiration, ethylene production, firmness, polygalacturonase activity, cell wall composition, and soluble uronide content were measured during ripening of two tomato (Lycopersicon esculentum Mill.) genotypes, ‘Manapal’ and dark green (dg). Respiration rates and cell wall uronide contents of the two genotypes were similar. Climacteric ethylene production rates of dg fruit were about half that of ‘Manapal’ fruit. Firmness and polygalacturonase activity of dg tomatoes were similar to that of ‘Manapal’ fruit until 55 days postpollination, when dg fruit were twice as firm as ‘Manapal’ fruit and exhibited greater polygalacturonase activity. Soluble uronide content did not differ between the two genotypes, except at 50 days postpollination, when that of dg fruit was 60% that of ‘Manapal’ fruit. Cell wall uronide content of dg fruit was 1.5 times greater than ‘Manapal’ fruit at 55 days postpollination. Although dg fruit contained larger, absolute amounts of cell wall noncellulosic neutral sugars than ‘Manapal’ fruit, net changes in sugar composition were similar throughout ripening. Also, ratios of cell wall arabinosyl or galactosyl residues to cell wall galacturonic acid were similar in both genotypes. These data suggest that firmness differences between dg and ‘Manapal’ fruit are not due to differing activities of polygalacturonase or changes in cell wall composition during ripening, but to other factors that may affect solubilization of cell wall uronides.
Free galactose was detected in outer pericarp tissue from fruit of ‘Heinz 1350’ and nor tomato (Lycopersicon esculentum Mill.). The amount of free galactose increased almost 8-fold in ‘Heinz 1350’ fruit during ripening. In contrast, it did not change significantly in 32- to 52-day-old (post-pollination) nor fruit. A 25 μl drop of water ± 100 μg galactose was applied to the locular surface of 1.5 cm diameter pericarp disks. While disks from 40- and 60-day-old nor tomatoes, and from mature green and pink ‘Heinz 1350’ tomatoes, were able to reduce the level of free galactose to levels found in control tissue within 48 hr, disks of red ripe tissue were only able to metabolize 45% of the added galactose within this time. Pink ‘Heinz 1350’ tomatoe disks lost galactose more slowly than disks of mature green tomatoes. The results suggest that the increased amount of free galactose in pericarp tissue of ripening tomatoes may be the result of their progressive inability to metabolize galactose as they ripen.
Cell wall synthesis during development and ripening of `Rutgers', rin and nor tomato (Lycopersicon esculentum Mill.) fruit was quantified by monitoring incorporation of 14C into outer pericarp cell walls after pedicel injection of (U-14C) - sucrose. Fruit color (Hunter “a” and “b” values) and firmness (Instron) were also monitored. 14C-Incorporation continued throughout development and ripening in `Rutgers' cell walls and exhibited a transient increase from late maturegreen to the turning stage. Incorporation of 14C into cell walls of rin pericarp tissue was similar to `Rutgers' at 20 days pest-anthesls (DPA) (immature-green) but decreased to a level similar to red `Rutgers' fruit by 35 DPA. Incorporation of 14C into nor pericarp cell walls was low throughout the experimental period (20 to 75 DPA). In contrast to previous reports, rin and nor pericarp tissue exhibitad a decrease in firmness of the outer pericarp. However, the rate of softening was slower than in `Rutgers'. Pericarp tissue from rin and nor fruit at 70 and 75 DPA, respectively, resisted compression as much as pink `Rutgers' pericarp tissue.
Fruit softening occurs by several mechanisms, including modifications of cell wall structure by wall degrading enzymes. The most prominent change in tomato fruit pericarp wall composition is the loss of galactosyl residues throughout development and especially during ripening. In order to understand the role of galactosyl turnover in fruit softening, we successfully produced three recombinant tomato β-galactosidase/exo-galactanase (TBG) fusion proteins in yeast. TBG1, 4 and 5 enzyme properties and substrate specificities were assessed. Optimum pH of TBG1, 4 and 5 was 5.0, 4.0, and 4.5 and optimum temperature was 40∼50, 40, and 40 °C, respectively. The K ms for TBG1, 4 and 5 were 7.99, 0.09, and 2.42 mm, respectively, using p-nitrophenyl-β-D-galactopyranoside as substrate. Using synthetic and plant-derived substrates, TBG1 and 5 released galactosyl residues from 1 → 4 linkages. TBG4 released galactosyl residues from a wide range of plant-derived oligosaccharides and polysaccharides. Using tomato fruit cell wall material, TBG1, TBG4 and TBG5 released galactosyl residues from a variety of fruit stages and cell wall fractions. TBG4 released the most galactosyl residues from the ASP fraction and especially the ASP fraction from fruit at the turning stage. Interestingly, even though walls from Turning fruit stage contain less total galactosyl residues than at the Mature Green stage, TBG4 released 3–4 fold more galactose from the CSP and ASP fractions from Turning fruit. These results suggest that changes in structure of wall pectic polysaccharides leading up to the Turning stage may cause the wall to become more susceptible to hydrolysis by the TBG4 product.
The effects of 1-methylcyclopropene (MCP), sanitizer and their combination on ethylene action, microbial growth and storage life of fresh-cut cilantro were studied. Fresh cilantro was treated with 1.5 μL·L-1 MCP for 18 hours at 10 °C. The treated and nontreated cilantro leaves were cut and washed in water, chlorine, and mixed solution of sodium chlorite and citric acid (SANOVA). Samples were dried, packaged with 29.2μmol·kg-1 Pa s oxygen transmission rate films, and stored for 14 days at 5 °C. Results indicated that MCP affected respiration rate of fresh-cut cilantro and the headspace gas composition (O2 and CO2) of sample packages. The combined treatment had lower tissue electrolyte leakage and ethanol concentration, and delayed color changes during storage. SANOVA and the combination of MCP and SANOVA were effective in reducing aerobic microbial population and coliform population. Samples treated with MCP and SANOVA had good quality with high overall quality score at the end of storage.
To study ripening-related chilling injury (CI) of bell pepper (Capsicum annuum L.), fruit at mature green, breaker, and red-ripe stages were stored at 1, 5, 7, and 10 °C for 4 weeks. Surface pitting was evaluated after storage at 1 °C for 2 weeks followed by a 2-day exposure to room temperature (20 °C). Exposing fruit to 1 °C enhanced water loss, respiration, ethylene production, and electrolyte leakage, but slowed color change. Weight loss, respiration, ethylene production, electrolyte leakage, and color change increased more in breaker than in mature green and red-ripe fruit. No pitting symptom was observed at temperatures of 5 to 10 °C. After storing peppers at 1 °C for 2 weeks, breaker stage fruit exhibited chilling symptoms of severe surface pitting with more sheet pitting and deeper peel depression. Mature green fruit showed only moderate pitting. However, red-ripe peppers showed no injury and cells showed a normal appearance after low-temperature storage (1 °C). These results show that bell peppers tended to be more susceptible to chilling temperature while at the breaker stage and that the increase in visible CI is correlated with increased water loss, respiration, ethylene production, electrolyte leakage, and color change during storage.