Fluid-phase endocytosis in Citrus juice cells is independent from vacuolar pH and inhibited by chlorpromazine, an inhibitor of PI-3 kinases and clathrin-mediated endocytosis

Abstract

SummaryCompared to their more acidic relatives, sweet lime (Citrus limettioides Tan.) is distinguished by having a high vacuolar pH and a low sugar content. To determine whether variations in vacuolar pH are linked to different sugar uptake systems, resulting in contrasting sugar levels, we investigated developmental changes in V- (tonoplast-bound) and P-(plasmalemma-bound) ATPase activities, and the capacity of energised plasmalemma and tonoplast vesicles to take-up sucrose. Both plasmalemma- and tonoplast-bound ATPase activities, increased during fruit development. Artificially energised plasmalemma vesicles accumulated sucrose against a concentration gradient, whereas tonoplast vesicles did not show a similar uptake capacity, even in the presence of ATP. When juice cells were incubated with sucrose for 18 h with the endocytic marker, dextran-Texas Red (d-TR), an intense red fluorescence was observed in the vacuole. Sucrose-inducible uptake of d-TR was suppressed by the clathrin- and PI-3 kinase-mediated endocytosis inhibitor, chlorpromazine, but not by the caveolin-mediated endocytosis inhibitor, filipin. However, 3-dimensional Z-stack confocal images demonstrated the formation and presence of endocytic vesicles 0.5 – 2 µm in diameter, which were considerably larger than vesicles formed by clathrin-assisted scaffolding. The data indicated that: (i) most sucrose uptake into the vacuole is not mediated by a tonoplast-bound carrier; (ii) uptake of external solutes into the vacuole of C. limettioides juice cells is independent of vacuolar pH; and (iii) sucrose uptake occurs largely by a non-selective endocytic mechanism composed of vesicles, consistent with pinocytosis.