Electrogenic Proton Translocation by the ATPase of Sugarcane Vacuoles

Abstract

Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation.In recent years, availability ofbetter methods ofisolating intact functional vacuoles from higher plants has permitted better insight into the physiology and biochemistry of this organelle (5,20,25). A tonoplast-bound ATPase which functions in energizing the membrane exists in higher plants (4,24,26). The evidence for this phenomenon comes predominantly from small fungal vacuoles (10), lysosome-like membrane vesicles (13), and microsomal membrane fractions (3,6,19

Temporal and Spatial Regulation of Sucrose Accumulation in the Sugarcane Stem

“…However, the vacuole preparations were apparently defective; sucrose was taken up by the isolated vacuoles at a rate 10 times less than the in situ uptake rate and 100 times less than that of 3-OMG (Thom et al 1982b). Nevertheless, tonoplast vesicles isolated from the vacuoles confirmed the functioning of a H+-pumping ATPase capable of maintaining a protonmotive potential difference of 125 mV (Thom and Komor 1985). This is comparable with the protonmotive values for other plants (see review by Bush 1993).…”

“…These results indicated that sugarcane has a tonoplast-bound ATPase that translocates protons into the vacuole thereby generating the ;+ H+ H+ proton gradient (Thom and Komor 1985) needed for operating a hexose H+-antiport system ([3] of Fig. 4) (Thom and Komor 198417).…”

Analysis of the Reaction Products from Incubation of Sugarcane Vacuoles with Uridine-Diphosphate-Glucose: No Evidence for the Group Translocator

“…On one side, addition of sucrose in presence of MgATP to vacuoles from sugarcane stalk gave barely perceptible uptake of sucrose (14), while on the other hand MgATP even inhibited the UDP-glucose group translocator (26). The delicate coexistence of tonoplast energization by the ATPase (22) and sucrose storage by the UDP-glucose group translocator was puzzling, especially in the case of sugar beet vacuoles, where the coexistence of the proton-driven sucrose-proton antiport system and the UDP-glucose group translocator was visualized (23). Therefore, as a first step to obtain ideas about the mutual interaction of energization and sucrose storage, a careful analysis of the reaction products, which are produced by incubation of sugarcane vacuoles with UDP-glucose, was performed.…”

Ion Transport at the Vacuole during Stomatal Movements

“…Thus, measurements were performed on cell types amenable to microelectrodes, like root cells, mesophyll cells, or unicellular green algae, and using fluorescent dyes on isolated vacuoles. The reported transtonoplast potential values varied between 0 and 266 mV (Thom and Komor, 1985;Bethmann et al, 1995;Walker et al, 1996;Miller et al, 2001;Wang et al, 2015). In Arabidopsis, measurements of tonoplast potentials in mesophyll and root hair cells yielded a value of about 230 mV (Miller et al, 2001;Wang et al, 2015).…”