α-Galactosidase is synthesized in tomato seeds during development and is localized in the protein storage vacuoles

Abstract

The localization of the enzyme α-galactosidase (EC 3.2.1.22) was investigated during its synthesis in developing tomato (Lycopersicon esculentum Mill.) cv. Trust seeds. This enzyme is also present in germinating seeds, where it is involved in the mobilization of carbohydrate reserves during and following seed germination. Subcellular fractionation of developing tomato seeds revealed that there is a cosedimentation between α-galactosidase activity and protein storage vacuoles in a density gradient, which is dependent upon the presence of membranes. A second approach to localizing this enzyme involved the transient transformation of protoplasts from developing tomato seeds. A reporter construct, coding for tomato α-galactosidase, fused N-terminally to the bacterial enzyme chloramphenicol acetyltransferase was used for transient expression. Immunofluorescence microscopy revealed a colocalization between the α-galactosidase - chloramphenicol acetyltransferase fusion protein and the α-tonoplast intrinsic protein, and a partial colocalization with the dark intrinsic protein (both vacuolar proteins). These data indicate that the protein storage vacuole is the intracellular location for α-galactosidase in developing tomato seeds.Key words: α-galactosidase, protein storage vacuole, seed development, seed protoplasts, tomato, tonoplast intrinsic protein.