β-Glucosidase in the indigo plant: Intracellular localization and tissue specific expression in leaves

Abstract

β-Glucosidase of indigo plant (Polygonum tinctorium) has a high substrate specificity for indican (indoxyl β-D-glucoside). To examine the localization of this β-glucosidase, we fractionated the cells of the leaves and analysed them immunocytochemically. Immunoelectron micrographs with specific antibodies against the β-glucosidase clearly showed that the β-glucosidase was localized in the stroma of the chloroplasts in mesophyll cells, but not in the thylakoid membrane. Chloroplasts were isolated from the crude homogenate of the fresh leaves by Percoll density gradient centrifugation and then subjected to suborganellar fractionation. β-Glucosidase activity was specifically detected in the stromal fraction, but not in the thylakoid membrane. This was also supported by the result of an immunoblot of the fractions with anti-β-glucosidase antibodies. The β-glucosidase was immunocytochemically localized in the chloroplasts of mesophyll cells, but not in any chloroplasts in marginal cells of the vascular bundle or epidermal cells; ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromal protein, was observed in all chloroplasts in these cells. These results suggest that β-glucosidase is tissue specific in its expression in the leaves of the indigo plant.