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Jong-Pil Chun and Donald J. Huber

The catalytic capacity of tomato polygalacturonase (PG) toward soluble pectic polymers is in excess of activity expressed in vivo; however, in vitro assays of PG have traditionally been performed under conditions (pH 4.0 to 4.5, 150 mM NaCl) that likely do not reflect the apoplastic environment of ripening tomato fruit. In this study, hydrolysis of pectin by purified tomato PG (isozyme 2) was examined in response to K+ (the predominate apoplastic cation) and over the pH range from 3.0 to 6.0. In the presence of K+, PG activity toward polygalacturonic acid measured reductometrically increased nearly 3.5-fold from pH 4.0 to pH 5.5. In the presence of Na+, activity decreased 90% over the same pH range.

PG-mediated degradation of cell wall from mature-green fruit showed divergent hydrolytic patterns in response to pH and K+. At pH 4.5 in the presence of K+ (as KCl), catalysis resulted in both solubilization and extensive depolymerization of cell wall pectin, with oligomers accounting for a significant portion of the hydrolysis products. At pH 5.5, the total quantity of wall pectin released in response to PG2 was similar to that at pH 4.5; however, oligomer production was strongly suppressed at the higher pH. At pH values favoring extensive depolymerization, low mol mass products were produced at 5 mM K+ and increased to a maximum at 100 mM K+. At higher pH, hydrolysis patterns were not affected by [K+]. pH and ionic effects may contribute to the distinctive patterns of pectin hydrolysis observed for different fruits.

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Jong-Pil Chun and Donald J. Huber

The high catalytic potential of PG evident in reactions with soluble pectic polymers is typically not expressed in vivo. In this study, the binding and catalytic properties of PG isozyme 2, and the influence of the B-subunit protein, were investigated using cell walls prepared from tomato fruit expressing the B-subunit antisense gene. Cell walls were prepared from mature-green fruit and treated to remove/inactivate endogenous enzymes. Walls were then preloaded with rate-limiting quantities of purified PG 2, and incubated under catalysis-promoting conditions over the range of pH from 4.5 to 6.0. Cell walls of both B-subunit antisense and wild-type fruit retained comparable quantities of loaded PG 2. The enzymic release of pectin from PG-loaded walls was proportional to the quantity of wall-bound PG 2. In walls lacking the B-subunit protein, the quantity of pectin released by a given dose of wall-associated PG was as much as 2-fold higher than from wild-type walls. The B-subunit protein also influenced the extent of pectin depolymerization during ripening. The release of pectin from cell walls during periods of catalysis was not the sole indicator of the range of pectins hydrolyzed. Treating postcatalytic loaded cell walls to inactivate PG, and subsequent extraction of residual wall pectins using 50 mm CDTA solutions solubilized polymers of significantly lower mol mass compared with pectins solubilized directly from nonloaded cell walls.

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Yong-Soo Hwang, Jong-Pil Chun and Jae-Chang Lee

Physiological disorder occurred in a recently developed oriental melon cultivar, `Gumssaragi-Bunchun' (Cucumis melo var. makuwa), is involved with the appearance of water soaking area in placenta and can be extended to the pulp when severely affected. Physiological changes between normal and disordered fruits were compared. Ethanol soluble sugars were significantly decreased in both pulp and placenta tissue of disordered fruits whereas acidity was increased. Ethanol and acetaldehyde accumulation were confirmed in juice from disordered fruits, which were net detectable in normal ones. The contents of boron and calcium, especially water and HCl soluble calcium, were fairly low in disordered pulp. Also, there was a great difference in pectin content between both fruit tissue and severe hydrolysis of water soluble pectins isolated from disordered placenta was found by gel chromatography. However, the hydrolysis of pectins seemed not to be associated with the increase of wall hydrolase activities such as polygalacturonase and β-galactosidase.

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Jong-Pil Chun, Jae-Chang Lee and Yong-Soo Hwang

Pectins isolated from three cultivars with different maturity were compared to find a potential role of pectin modification on the fruit softening during fruit development and ripening. There was an increase of total pectins in developing fruit and no significant decrease of pectins was confirmed even after storage in `Tusgaru' (30 days) and `Fuji' (120 days), whereas soluble pectins, except NaOH-soluble ones, gradually increased in all cultivars. Gel-filtration profile and ion exchange chromatographic evidence of soluble pectins revealed that pectin degradation in apple fruit may not be associated with softening. However, a degree of esterification probably has an important role on softening of fruits. Further results will be discussed in the presentation.