Molecular cloning and characterization of the beta-subunit of tomato fruit polygalacturonase isoenzyme 1.

Abstract

The β-subunit of PG1 was purified and cloned from tomato fruit. It is immunologically distinct from the catalytic PG2 subunit and heterogeneous in size (37 to 39 kD). DNA sequencing analysis of a full length β -subunit cDNA indicates that the β-subunit is encoded as a 69-kD precursor consisting of four distinct domains. The mature protein domain contains six N-linked core glycosylation sites and a novel 14-amino acid motif, FTNYGxxGNGGxxx, in which many of the phenylalanine residues are post-translationally modified. We propose that the β-subunit represents a class of bifunctional plant proteins that interact both with structural components of cell walls and catalytic proteins to localize and/or regulate metabolic activities within the cell walls. β-subunit and PG2 expression patterns were investigated in both wild-type and ripening inhibitor mutant (rin) fruit. The results show that (1) β-subunit expression is ethylene-independent and unrelated to catalytic PG2 expression, and (2) β-subunit expression is regulated primarily by developmental cues. In addition, β-subunit antigen and mRNA were detected in root, leaf and flower tissues. Ethylene treatment of immature wild-type and rin fruit resulted in accumulation of PG2 transcript but not PG2 protein. These results contrast with previous reports suggesting PG2 mRNA accumulation is ethylene independent and indicate that PG2 expression is a complex process involving integration of developmental and hormonal signal cues at the transcriptional and post-transcriptional levels. Several β-subunit antisense plants were successfully obtained from transformation of wild-type plants with a β-subunit antisense construction. Western analysis showed that β-subunit expression was inhibited in transgenic fruit by up to 96% relative to wild-type controls. Preliminary analysis indicates that the transgenic antisense fruit developed and ripened normally. These plants will allow investigation of the in vivo functions of the β-subunit in the future.