Ripening-Related Polygalacturonase cDNA from Avocado

Abstract

During the ripening of avocado (Persea americana) fruit, extensive cell wall degradation leads to a dramatic softening of the mesocarp tissue. Two enzymes have been suggested to play a critica1 role in this ripening-associated cell wall degradation in many fruits, including avocado, viz. cellulase (endo-~-1,4-glucanase) and endo-PG (Awad and Young, 1979; Fischer and Bennett, 1991). Although the avocado cellulase cDNA (Tucker et al., 1987) and gene (Cass et al., 1990) have been cloned and sequenced, the avocado PG has not previously been characterized at the molecular level. The ripe avocado fruit is known to contain numerous electrophoretic variants of the PG protein (Kanellis et al., 1991), but whether these represent different gene products or posttranslational modifications of one or more primary polypeptides is unknown. In tomato fruit, the various PG isoforms that accumulate during ripening (PG1, PG2A, and PG2B) are derived from a single gene (Fischer and Bennett, 1991). Using heterologous hybridization with a tomato cDNA probe, we have identified a putative avocado PG cDNA (pAVOpg) from a library generated with RNA from ripe avocado fruit (Table I). The cDNA contains an insert of 1725 bp that detects a 1900-nucleotide mRNA on hybridization to ripe fruit RNA and no detectable signal in mRNA from unripe fruit. This pattem of expression during ripening in avocado parallels that previously reported for PG enzyme activity (Awad and Young, 1979). Sequence analysis of the pAVOpg cDNA revealed an open reading frame of 453 amino acids, which is similar in size to that reported for avocado PG protein (Kanellis et al., 1991). This putative avocado PG sequence shows close similarity to PG sequences derived from both tomato and com, 52 and 36% identity, respectively. In addition, it contains an octapeptide motif that is conserved in PG sequences derived from plant, fungal, and bacterial species (Bairoch, 1992). This octapeptide is contained within a span of 14 residues (TCGPGHGISIGSLG) that is identical among the known plant PG sequences and thus should allow characterization of other plant PG genes using degenerate oligonucleotide primers in polymerase chain reaction experiments.