Retrograde Vesicles From the Vacuole Regulate Tonoplast Surface Area During Sucrose Uptake by Fluid Phase Endocytosis

During transport of external solutes from the apoplast to the vacuole of storage cells by fluid phase endocytosis (FPE), vesicles originating at the plasmalemma merge with the tonoplast to release their contents.  The merging of endocytic vesicles with the vacuole results in a larger surface area/volume ratio than when separated, resulting in dilution of vacuolar contents.  To concentrate solutes in the vacuole, a mechanism(s) to reduce tonoplast surface area must exist.  Three mechanisms have been proposed: 1) Retrograde vesicle formation. 2) Autophagic tubes. 3) Tonoplast autolysis.  The possibility of retrograde vesicles during FPE was investigated using red beet hypocotyls for their red and fluorescent vacuolar pigment, which would identify any structure of vacuolar origin.  Red beet hypocotyl tissue was incubated in sucrose solution for 24 h to induce FPE.  After 24 h, protoplasts were generated and observed under confocal laser scanning microscopy.  Induction of endocytosis by sucrose resulted in the formation of numerous vacuole-derived vesicles as evidenced by betacyanin fluorescence.  Diverse fluorescence intensities of exocytic vesicles reflected their chronological formation as result of betacyanin dilution during FPE. Addition of endocytic inhibitors during incubation with sucrose prevented the formation of exocytic vesicles. The data strongly support the existence of a mechanism by which vesicular structures pinch-off the vacuole reducing tonoplast surface area, and therefore, increasing the vacuolar concentration of FPE transported solutes.