Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the a type. Starch is degraded slowly in the first days; during this time, a-and 13-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, ther. is a more rapid depletion of starch which coincides with an increase in aamylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three a-amylases, and one /3-amylase. Based on their heat lability or heat stability, two sets of a-amylase seem to exist in lentil cotyledons.The enzymic degradation of starch in higher plants is due mainly to the phosphorylases and the amylases. They have been studied particularly in seeds, where they are responsible for the breakdown of polysaccharide reserves. Much of our knowledge of starch degradation during seed germination comes from studies with cereals (6,7,15,16,18,19,23) where a number of amylase isozymes have been described. In contrast, available knowledge of starch catabolism in legumes is very limited and almost reduced to the studies with germinating peas (13,(20)(21)(22).The major aim of the work presented here was to determine the mechanism of breakdown of reserve starch in the cotyledons of germinating lentil seeds, in order to improve our knowledge of the carbohydrate catabolism in this group of higher plants. Analyses of starch and activities of starch-degrading enzymes were carried out during the course of germination. Phosphorylases and amylases were separated by polyacrylamide gel electrophoresis to obtain information about the various starch-degrading enzymes of germinating lentil seeds.
MATERIALS AND METHODSLentil seeds (Lens culinaris, Medik = Lens esculenta, Moench) were soaked for 4 hr in a disinfectant solution (0.12% Orthocide-50 wettable, a commercial fungicide) at room temperature. The seeds were then germinated in the dark at 25 C on moist filter paper in Petri dishes for different periods of time, and then separated into cotyledons and embryos. Only the cotyledons were used for all subsequent assays. All operations were performed at 0 to 4 C unless indicated otherwise.Preparation of Crude Enzyme. The cotyledons (1 to 8/ml depending on the germination time) were homogenized with 20 ml of unbuffered distilled H20 in a Sorvall Omni-Mixer. The whole homogenate was strained through 2 layers of cheesecloth, and then centrifuged in a Sorvall RC2-B refrigerated centrifuge at 20,000g for 30 min at 2 C.In order to differentiate between a-and f8-amylase, the super-' Present address: Departamento de Fisiologia Vegetal, Universidad de Salamanca, Facultad de Ciencias, Spain. natant was treated selectively with 10 mm EDTA, 0.1 mM HgCl,, 3 mm CaCl2, and heated at 70 C in the presence of calcium ions.Amylase Assay. Two aliquots of the supernatant were removed...
