Endo-PGase in peach fruits with different flesh firmness : study of gene expression and of enzyme levels and activity

In peach, the Melting Flesh (M) locus encodes the enzyme endo-polygalacturonase (endo-PG) being thus involved in the control of ripe fruit firmness. The Non Melting Flesh (NM) phenotype appears to arise from different alterations of the M locus, ranging from massive deletion to slighter alterations depending on the genotype (Lester et al., 1996, J. Am. Soc. Hort. Sci. 121: 231; Callahan et al., 2004, Funct. Plant Biol. 37:159). We have previously shown (Negrini et al. 2003, 6th CNB Congress) that polyclonal antibodies raised against a synthetic polypeptide constructed on the basis of a conserved sequence (NCBI CAA54150) of ripe peach fruits recognized (SDS-PAGE/Western analysis), in a cell-wall protein-enriched fraction from the flesh of peach fruits, an endo-PG like polypeptide which was expressed relevantly in M (Bolero) fruits and also, though at very low extent, in NM (Oro A) fruits. The present results show that the different levels of the endo-PG-like polypeptide were accompanied by consistent differences in the expression (Northern analysis) of the endo-PG gene, indicating presence of a complete form of the gene in both genotypes and suggesting that the different levels of the endo-PG protein in the cell walls may be due to differences in the gene transcription activity, even if possible differences at the post-translational and/or secretive pathway level can not be excluded. The identity between the endo-PG-like polypeptide and endo-PG protein was confirmed by nondenaturing PAGE followed by activity staining and, in duplicate gels, immunodetection with the same antibodies. In fact, endo-PGase activity co-localized with antibody cross-reaction, and both were completely removed when the cell wall protein extracts were previously immunoprecipitated. Further work is now being conducted to extend these results to the other peach genotypes at our disposal characterised by different flesh softening patterns.