PATTERNS OF TEMPORAL MRNA EXPRESSION AND ENZYME ACTIVITY OF PECTOLYTIC AND NON-PECTOLYTIC CELL-WALL DEGRADING ENZYMES IN APPLE FRUIT GROWTH AND RIPENING

Abstract

Textural changes that lead to softening of apples are accompanied by enzymatic degradation and loss of neutral sugars of the pectic polysaccharides of the cell-wall. Recently, it has been suggested that the process is the result of the combined action of several cell-wall degrading enzymes. This work aims to establish a temporal sequence of the action of cell-wall degrading enzymes during apple growth and ripening. A cDNA enriched with mRNA isolated from over-ripe fruits was constructed and screened to clone: an endo-1,4-β-glucanase (EGase), two xyloglucan endotransglycosylase/hydrolases (XTH), an expansin, an α-L-arabinofuranosidase (AFase), a pectin methylesterase (PME), and a pectate lyase (PL) full-length cDNAs encoding late isoforms. These clones were characterized and their accumulation during fruit growth and ripening was examined by semi-quantitative RT-PCR analyses. Expression of β-galactosidase (βGal) and polygalacturonase (PG) mRNAs previously reported was also included in the analyses. Transcripts of all enzymes, except PME, could be detected in fruits at harvest. However, transcripts of EGase were more abundant at fruit set and both expansin and pectate lyase had high expression before ripening. The strongest expression in over-ripe fruits was observed for βGal and PG clones. RT-PCR analyses were also conducted in flowers, peduncles, young and expanded leaves and senescent petioles, and the results showed that the cloned cDNAs are not fruit-specific. The activity for each enzyme was also monitored in fruits at the same stages as for mRNA accumulation. Comparison of the patterns of enzyme activity with mRNA expression suggests the presence of isoforms acting in fruit growth and ripening-specific isoforms or post-transcriptional regulation. Furthermore, the results indicate that the pattern of softening of apple fruits different from the model plant tomato.