Molecular cloning and cytochemical analysis of exopolygalacturonase from carrot

Abstract.

Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot (Daucus carota L. cv. Kintoki) cells. The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa (Medicago sativa). However, no significant similarity was found to known PGases. The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family. Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap. A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.