Apple fruit undergoes three stages of postharvest fruit softening: an initial slow softening phase, followed by a rapid softening phase, and a final slow softening phase, thus adversely affecting fruit quality (Johnston et al., 2001; Knee, 1975). At the cell-wall level, fruit softening is a result of changes in interactions among cell-wall polysaccharides and their breakdown (Knee, 1975). These changes in cell walls result from the activities of cell-wall–modifying enzymes. Ripening-specific or postharvest softening-specific genes for various cell-wall–modifying enzymes have been characterized or cloned from apple, but data on how transcript levels of these genes change during storage is lacking.
β-Galactosidase increases wall porosity and enables other cell-wall–modifying enzymes to access cell-wall polysaccharides (Brummell and Harpster, 2001). It is known that the initial slow softening phase of apple fruit softening is marked by the loss of galactose (Knee, 1975; Peña and Carpita, 2004) and an increased transcript accumulation of the fruit ripening-specific β-galactosidase gene, pABG1 (Ross et al., 1994).
Xyloglucan endo-transglycosylase/hydrolase (XET/H) can break down hemicelluloses of the cell wall (Rose and Bennett, 1999). XET/H activity increased in ripe apple fruit and appeared to be positively correlated with the initial slow softening phase of postharvest apple fruit softening (Johnston et al., 2002; Percy et al., 1996). Data on transcript accumulation of the XET/H gene during postharvest storage are lacking.
Arabinose loss precedes the rapid softening phase during storage (Peña and Carpita, 2004; Tong et al., 1999). Arabinose is removed from pectic polysaccharides by arabinofuranosidase. A gene for arabinofuranosidase, MdAFase1, has been cloned from overripe ‘Royal Gala’ apple fruit (National Center for Biotechnology Information accession number AY309436).
Expansins are enzymes that allow loosening of the cell wall by breaking hydrogen bonds between cellulose and hemicellulose, making cell-wall polysaccharides accessible to the other cell-wall–degrading enzymes (Brummell et al., 1999; McQueen-Mason and Cosgrove, 1994). mRNA for MdEXPA2, an apple expansin gene, is detected primarily after harvest and for up to 12 d of storage in ‘Golden Delicious’ fruit (Wakasa et al., 2003).
Cell-wall polysaccharides exposed by expansin are further acted upon by endo-polygalacturonase, an enzyme that breaks down pectic polysaccharides of the cell wall and the middle lamella (Brummell et al., 1999). The loss of polygalacturonic acid, a pectic polysaccharide, is correlated with the rapid softening phase in apple fruit (Knee, 1975). MdPG is an apple fruit ripening specific gene for endo-polygalacturonase (Atkinson, 1994; Wu et al., 1993).
The extent of fruit softening varies with genotype (Tong et al., 1999). A comparative study of four different apple cultivars by Tong et al. (1999) established that ‘Honeycrisp’ fruit maintains its texture during storage for 6 months at low temperature, compared with ‘Macoun’ fruit, which is significantly less crisp and less firm after 6 months of storage. Transmission electron micrographs showed little or no cell-wall degradation in ‘Honeycrisp’ after 6 months of storage, whereas the cell walls of ‘Macoun’ fruit had completely deteriorated in that time period. In addition, ‘Honeycrisp’ fruit did not lose uronide or arabinose when compared with ‘Macoun’ fruit after 6 months of storage. An exploration of the molecular bases of the above-mentioned phenotypic differences between fruit from ‘Honeycrisp’ and ‘Macoun’ could provide us with candidate genes that might regulate or cause postharvest fruit softening in apple.
The pattern of changes in transcript levels of a set of known cell-wall–modifying genes was studied for fresh and 3-month-stored fruit from ‘Honeycrisp’ and ‘Macoun’ apples. In an attempt to identify additional genes that might have a role in regulation of postharvest fruit softening in apple, a suppression-subtractive hybridization and cDNA array technique was used. Messinger RNA transcripts with differential accumulation in fresh or 3-month-stored ‘Macoun’ apple fruit tissue were identified. Differentially accumulated transcripts were further quantified with qRT-PCR from fresh and 3-month-stored fruit from both ‘Macoun’ and ‘Honeycrisp’. Results and discussions of these experiments are presented below.
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