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Jongkee Kim and Kenneth C. Gross

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.

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David A. Starrett and Kenneth C. Gross

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.

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Ji Heun Hong and Kenneth C. Gross

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).

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Elizabeth J. Mitcham, Kenneth C. Gross, and Timothy J Ng

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.

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Megumi Ishimaru, David L. Smith, and Kenneth C. Gross

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.

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Ji Gang Kim*, Yaguang Luo, Yang Tao, and Kenneth C. Gross

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.

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Chae Shin Lim, Seong Mo Kang, Jeoung Lai Cho, and Kenneth C. Gross

To study ripening-related chilling injury in pepper (Capsicum annuum L.), chilling-tolerant ‘Buchon’ and chilling-sensitive ‘Nockgwang’ fruit were harvested at mature green (MG), breaker (BR), and red-ripe (RR) stages and stored at 1, 5, and 10 °C for 21 d. ‘Buchon’ did not show surface pitting (SP) regardless of ripeness stage and storage temperature, whereas ‘Nockgwang’ at MG and BR exhibited SP at 1 and 5 °C. After 14 days of storage at 1 °C, chilling-sensitive ‘Nockgwang’ did not show SP when fruit were at the RR stage. Compared with ‘Buchon’, ‘Nockgwang’ at MG and BR had more electrolyte leakage increase during storage at 1 and 5 °C. ‘Buchon’ at all ripeness stages showed significantly higher ethylene production during storage regardless of storage temperatures. Contents of β-carotene and lycopene increased in both cultivars as ripening progressed. The contents of β-carotene and lycopene were similar between the two cultivars regardless of storage temperatures and ripeness stages. Susceptibility of pepper fruit to chilling appeared to be related to superoxide dismutase (SOD) and catalase (CAT). Activities of SOD and CAT were much higher in ‘Buchon’ than ‘Nockgwang’, more apparently at MG and BR. The results suggest that chilling-tolerant ‘Buchon’ and fruit at RR could have been equipped with a more efficient antioxidizing system, even if it was not clear whether oxidative stress is a cause or an effect of the CI in pepper.

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Chae Shin Lim, Seong Mo Kang, Jeoung Lai Cho, Kenneth C. Gross, and Allan B. Woolf

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.

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Ji Gang Kim, Yaguang Luo, Robert A. Saftner, and Kenneth C. Gross

Fresh-cut tissues are subjected to severe injury during preparation that leads to increased respiratory activity and quality deterioration. Modified atmosphere packaging (MAP) has been used to maintain quality of fresh-cut produce, but O2 depletion and excessive CO2 accumulation can be injurious. This study was conducted to evaluate the effect of delayed packaging and MAP using two different oxygen transmission rate (OTR) films on quality maintenance and shelf stability of fresh-cut romaine lettuce (Lactuca sativa L.). Romaine lettuce leaves were cut, washed, dried, and placed for 0, 4, 8, and 12 hours at 5 °C in ambient air before packaging. Fresh-cut samples were placed into packages prepared from films having OTRs of 8.0 and 16.6 pmol·s-1·m-2·Pa-1, flushed with N2 to reach an initial headspace O2 level of 1.5 kPa O2, and stored at 5 °C for up to 14 days. Delayed packaging affected gas composition, fermentative volatile production, off-odor development, color, CO2 injury, and tissue electrolyte leakage. With increasing delay before packaging, fermentative volatile production, off-odor development, and CO2 injury progressively decreased and discoloration increased. The modified atmospheres obtained with 16.6 OTR film increased discoloration when present, and generally had less off-odor development and CO2 injury compared to MAP with 8.0 OTR film. Delayed packaging affected overall quality of fresh-cut romaine lettuce packaged with both films. A 12-hour delayed packaging into packages prepared from 8.0 OTR film maintained quality by inhibiting CO2 injury, off-odor development, and tissue electrolyte leakage. However, an 8-hour delayed packaging into packages prepared from 16.6 OTR film was better at maintaining the quality of fresh-cut romaine lettuce at 5 °C for 14 days. The results indicated that delayed packaging could be an alternative method to optimize or balance package O2 during suboptimal OTR film packaging conditions.

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Raymond Fung*, Chien Wang, David Smith, Kenneth Gross, Yang Tao, and Meisheng Tian

Methyl salicylate (MeSA) and Methyl jasmonate (MeJA) treatments increased chilling resistance of light red tomato (Lycopersicon esculentum cv. Beefsteak) and extended shelf life and fresh-cut quality. We previously showed induction of AOX expression by low temperature and that induction of AOX transcript by MeSA and MeJA is correlated with resistance against chilling injury in peppers. Here, we investigate tomato, which is genetically closely related to peppers and belongs to the same Solanaceae family. In particular, we used four EST tomato clones of AOX from the public database that belong to two distinctly related families, 1 and 2 defined in plants. Three clones designated as LeAOX1a, 1b and 1c and the fourth clone as LeAOX2. Probes for these four genes were designed and Southern blotting done to confirm that they do not cross-hybridize. We will present data from Southern, Northern hybridization and RT-PCR to show: (1) gene copy number of each of these AOX members in the tomato genome; (2) gene-specific expression profiles in response to MeSA and MeJA in cold stored tomato; and (3) the relative transcript abundance of these four AOX genes.