Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves' Diurna1 Oscillation of Amylolytic Activity and Starch Content in Chloroplasts

Subcellular localization of the starch-degrading enzymes in Vicia faba leaves was achieved by an electrophoretic transfer method through a starch-containing gel (SCC) and enzyme activity measurements. Total amylolytic and phosphorolytic activities were found predominantly in the extrachloroplastic fraction, whereas the debranching enzymes showed homogenous distribution between stromal and extrachloroplastic fractions. Staining of end products in the SCC revealed two isoforms of a-amylase (EC 3.2.1.1) and very low 8-amylase activity (EC 3.2.1.2) in the chloroplast preparation, whereas aand 8-amylase exhibited higher activities in the crude extract. However, it is unclear whether the low aand 8-amylase activities associated with the chloroplast are contamination or activities that are integrally associated with the chloroplast. Study of the diurna1 fluctuation of the starch content and of the amylase activities under a 9-h/15-h photoperiod showed a 2-fold increase of the total amylolytic activity in the chloroplasts concurrent with the starch degradation in the dark. No fluctuation was detectable for the extrachloroplastic enzymes. l h e possible roles and function of the chloroplastic and extrachloroplastic hydrolytic enzymes are discussed.