Endopolygalacturonase in Apples (Malus domestica) and Its Expression during Fruit Ripening

Abstract

The activity of polygalacturonase (PC) has been detected in ripe Mclntosh apples (Malus domestica Borkh. cv Mclntosh) both by enzyme activity measurement and immunoblotting using an antitomato-PC antibody preparation. PC activity increased during fruit ripening and remained steady, or decreased slightly, after 5 months of controlled atmospheric storage. l h e enzyme had a relative molecular weight of 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 56,000 to 61,000 when determined by gel filtration. Viscosity and reducing end group measurements with a commercial pectin preparation showed that the enzyme is endo acting. In RNA and DNA blot hybridization experiments, a full-length tomato PC cDNA hybridized with the apple RNA and DNA, showing the identity of genes encoding the activity of the enzyme in tomato and apple.Structural changes in the cell walls of climacteric fruits that lead to softening of the tissues are initiated by the action of ethylene, the fruit-ripening hormone (Abeles, 1985). The softening of the fruit tissues is the consequence of cell walldegrading enzymes , of which endo-PG (EC 3.2.1.15) was reported to cause the most prominent changes (Crookes and Grierson, 1983). Endo-PG was implicated in tissue softening because its appearance during fruit ripening corresponds to the increase in fruit softening. In a number of cultivars, a correlation between PG activity and fruit softening has been observed (Crookes and Grierson, 1983). Also, application of isolated endo-PG preparations to fruit tissue discs resulted in ultrastructural changes that were similar to those occurring during the normal ripening process (Ben-Arie et al., 1979). Ripening mutants with delayed or decreased softening that were deficient in PG activity Koch and Nevins, 1990) gave further indications that the enzyme plays a major role in the softening of fruits.Results of recent experiments with transgenic tomato fruits, however, are not in agreement with the above assessment. Fruit softening was not affected significantly in tomatoes with down-regulated PG activity (Giovannoni et al., 1989; ' This work was supported in part by a grant from the Cornell showed that when the expression of the PG gene has been up-regulated using the promoter region of another ripeningassociated gene (Schuch et al., 1989), PG production and polyuronide degradation are increased without any detectable effect on fruit softening. These data suggest that although endo-PG activity increases during the ripening process of tomatoes, and the increased activity of this enzyme results in structural changes of the middle lamella, the softening of fruits is a more complex process than anticipated. The ripening process of fruits has been best investigated in tomatoes; however, other fruits such as peaches (Lee et al., 1990), papayas (Lazan et al., 1989), and pears (Pressey and Avants, 1976) also showed elevated activity of an endo-PG during ripening. From biological considerations it is expected that the basis of the biochemi...

A cDNA Clone for Endopolygalacturonase from Apple

“…This pattern of expression parallels that reported for PG enzyme activity by Wu et al (1993). The coding region of pGDPG-1 contains 460 amino acids and shows 52, 55, and 62% identity to endo-PG genes from tomato, avocado, and kiwifruit, respectively.…”

“…Recently, however, the activity of endo-PG has been detected in apple (Wu et al, 1993). These authors also reported cross-reactivity between an anti-tomato-PG antibody and a protein in apples of M, 45,000.…”

Polygalacturonases: Many Genes in Search of a Function1

“…Alternatively, PG activity may be present but at as-yet-undetectable levels. For example, apple pectins are disassembled at the late stages of ripening (Knee, 1973), and recently, under rigorous examination, endo-PG activity and PG mRNA accumulation have been detected but the levels are much lower than that observed in tomato (Wu et al, 1993).…”

Postharvest softening of apple (Malus domestica) fruit: A review

“…The enzyme originally considered responsible for pectin solubilisation and therefore softening in most fruits was polygalacturonase (PG), with activities of both exo-PG and endo-PG detected in ripening apples (Bartley 1978;Wu et al 1993;Atkinson et al 1998). Electron microscopy revealed that treatment of unripe apple discs with PG caused similar disruption in the middle lamella to that observed in ripe apple tissue (BenArie et al 1979).…”