The cloning and characterization of alpha-galactosidase present during andfollowing 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 a n osmoticum, It is also present in the developing and mature dry seed, A cD NA clone for tomato seed alpha -galactosidase (LeaGal) has been isolated an d the characteristics of the protein deduced; the predicted molecular mass of the mature enzyme is 39.8 kDa, with a pl of 4.91, The tomato a-galactosi dase has a high homology (> 62%) at the amino acid level with that of other plant alpha -galactosidases. A hydrophobic signal peptide region is identi fied which is indicative that the enzyme enters the lumen of the endoplasmi c reticulum during its translation, prior to its export to the protein body or cell wall, the presumed sites of its substrates, Using amino acid align ment and phylogenetic analysis, key amino acids have been identified, and r elationships to other alpha -galactosidases inferred. Southern hybridizatio n 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 differ ent isoforms within the seed; post-translational modifications are thus pre sumed to occur. From Northern hybridization studies it is evident that alph a -galactosidase transcripts are present in the lateral and micropylar endo sperm during and following germination, and also to a lesser extent in the embryo.