Transport of sucrose into the vacuole of Vitis vinifera cell suspensions

Abstract

The accumulation of sugar in plant cells seems to be mediated by the combined action of plasma membrane and tonoplast sugar transporters, but much work remains to be done to clarify the route(s) of sugar import and how this transport and storage is regulated. Tonoplast vesicles prepared from suspension cultured cells of Vitis vinifera were used as a model system to study the transport of sucrose into the vacuole. The purity of the tonoplast preparations was checked by the marker enzymes vanadate-sensitive H+-ATPase and H+-PPase. ATP- and PPi-dependent fluorescence quenching of ACMA indicated that these vesicles were capable of building up a pH gradient (∆pH, inside acid). Ca2+-induced dissipation of ∆pH was observed on energized vesicles, as a result of Ca2+/H+ antiport activity. However, H+-dependent sucrose transport did not seem to be involved on sucrose uptake since the disaccharide did not induce ∆pH dissipation. Initial velocities of [U-14C]sucrose measured in the absence of a transmembrane H+ gradient showed a simple Michaelis-Menten kinetics, suggesting the involvement of mediated transport with the following kinetic parameters: Km, 57 mM sucrose; Vmax, 49 nmol sucrose s-1 mg-1 protein. Influx and efflux experiments suggested that the affinity of the transport system for sucrose is similar on both sides of the membrane, and the pair sucrose/[U-14C]sucrose exhibited countertransport. It is proposed that sucrose concentrated in the cytoplasm by the activity of energy-dependent plasma membrane sugar carriers is imported into the vacuole by a facilitated diffusion mechanism.