The cloning and characterization of alpha-galactosidase present during and following germination of tomato (Lycopersicon esculentum Mill.) seed.

alpha-Galactosidase (EC 3.2.1.22) is present in the embryo, micropylar and lateral endosperm of seeds of tomato during and following germination. Its activity is unchanged even when germination of the seeds is prevented by an osmoticum. It is also present in the developing and mature dry seed. A cDNA clone for tomato seed alpha-galactosidase (LeaGal) has been isolated and the characteristics of the protein deduced; the predicted molecular mass of the mature enzyme is 39.8 kDa, with a pI of 4.91. The tomato alpha-galactosidase has a high homology (>62%) at the amino acid level with that of other plant alpha-galactosidases. A hydrophobic signal peptide region is identified which is indicative that the enzyme enters the lumen of the endoplasmic reticulum during its translation, prior to its export to the protein body or cell wall, the presumed sites of its substrates. Using amino acid alignment and phylogenetic analysis, key amino acids have been identified, and relationships to other alpha-galactosidases inferred. Southern hybridization analyses show that the enzyme is derived from a single gene (for which a partial sequence has been obtained) and yet there are at least three different isoforms within the seed; post-translational modifications are thus presumed to occur. From Northern hybridization studies it is evident that alpha-galactosidase transcripts are present in the lateral and micropylar endosperm during and following germination, and also to a lesser extent in the embryo.