Figure 4.

Dependence of V‐PPase‐mediated proton pump currents on pyrophosphate concentration and luminal pH. (a) Vacuolar pH of Nicotiana benthamiana mesophyll cells, transformed with or without NbVHP1 in the absence or presence of 200 mM NaCl, was visualized using BCECF‐AM (n = 10). Images in the upper panel were taken from salt‐untreated leaves. The fluorescence ratio is shown in pseudocolor images and the emission at the corresponding excitation wavelength is presented in grayscale. Quantification of the vacuolar pH ratio value with (dark gray) or without (light gray) 200 mM NaCl infiltration is displayed (cf. Supporting Information Fig. S8 and Methods S6 for pH calibration with BCECF). Data are means ± SE (*, P < 0.05; **, P < 0.01, Student's t‐test). (b) Representative current responses of one N. benthamiana mesophyll vacuole to the application of a range of pyrophosphate concentrations as indicated above the current traces. Using symmetrical solute conditions with pH 7.5 at both sides of the vacuolar membrane, the experiment was conducted with a vacuole released from an NbVHP1/GFP‐overexpressing mesophyll cell. Duration of pyrophosphate treatment is indicated by the superimposed gray bars. (c) Maximal pyrophosphate‐induced changes in current density of vacuoles released from NbVHP1/GFP‐overexpressing N. benthamiana mesophyll cells, plotted against the pyrophosphate concentration. Current responses were normalized to those recorded from the same vacuole during the application of 150 μM pyrophosphate. Experiments were performed at a luminal pH of 7.5 (triangles, n = 4–10) or pH 5.5 (circles, n = 3–5). Data points (means ± SE) were globally fitted with a Michaelis–Menten equation (solid line).