Cell and tissue localization of β-glucosidase during the ripening of olive fruit (Olea europaea) by in situ activity assay

Olea europaea is an endemic tree in the Mediterrranean basin of large economic interest for both oil and fruits production. One of the main problems to solve for improving the fruit quality is olive debittering. Thus we aimed to study, at cytological level, the beta-glucosidases involved in the enzymatic degradation of oleuropein, a bitter defence phenol glucoside. This work represents the first insight on the in situ localization of beta-glucosidase activity in the mesocarp tissue of olive during fruit ripening. The cytological localization is performed by the hydrolysis of X-Glc, a chromogenic synthetic substrate developing an insoluble blue precipitate in the cells. A strong reaction is detected within nucleus, chloroplasts and cytoplasm oil droplets. The enzymes kept in chloroplasts and oil droplets showed high specificity for the exogenous oleuropein respect to those toward X-Glc in the in situ competitive assay, thus indicating that two different oleuropein-degradative-beta-glucosidases are present in these cell compartments. Following the fruit ripening, significant variations in the number and distribution of reactive cells in mesocarp tissues are observed. In fact in immature fruits the reactive cells are very few and located in the inner mesocarp zone; thereafter, in green mature fruits a great number of reactive cells are found distributed in the whole mesocarp. Finally in black fruits numerous reactive cells are located only in the inner mesocarp close by the woody endocarp, whereas the outer mesocarp cells are devoted to polymeric anthocyanins accumulation. These results clearly showed that the variations of oleuropein-degradative-beta-glucosidases activity during ripening are due to changes in the competence of single mesocarp cells to synthesize the enzyme isoforms. (c) 2006 Elsevier Ireland Ltd. All rights reserved.