High V-PPase activity is beneficial under high salt loads, but detrimental without salinity

Abstract

The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H⁺ -ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PP_i hydrolysis and proton-pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging. NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants. The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na⁺ sequestration.

Keywords: cell death; plasma membrane voltage; proton pump currents; salt; vacuolar pH; vacuolar proton-ATPase (V-ATPase); vacuolar proton-pyrophosphatase (V-PPase).

Figure 1

Expression and function of NbVHPs in Nicotiana benthamiana mesophyll cells. (a) Transcript abundances of NbVHP1 and NbVHP2 in mesophyll protoplasts isolated from noninfiltrated leaves. n = 4 independent experiments, means ± SE; **, P < 0.01, Student's t‐test. (b) Confocal fluorescence images of a protoplast (left) and vacuole (right) released from different mesophyll cells overexpressing NbVHP1 together with free green fluorescent protein (GFP). Red fluorescence is a result of chloroplast autofluorescence. Bars, 20 μm. (c) Representative pyrophosphate‐induced current responses of vacuoles released from mesophyll cells overexpressing free GFP together with either NbVHP1 or NbVHP2 after agroinfiltration. As a control, GFP was overexpressed alone. Duration of pyrophosphate treatment (150 μM) is indicated by the superimposed gray bars. Total number of experiments with individual vacuoles is given in (d). (d) Maximal pyrophosphate‐induced changes in current density of vacuoles released from mesophyll cells overexpressing free GFP alone (control, n = 10) or free GFP together with either NbVHP1 (n = 14) or NbVHP2 (n = 4) after agroinfiltration. Pyrophosphate was applied at a concentration of 150 μM. Data points represent means ± SE. Asterisks indicate significant differences compared with the control (Student's t‐test): ***, P < 0.001.

Figure 2

Salt stress rescues V‐PPase‐overloaded Nicotiana benthamiana leaves from cell death. (a) Images from leaves overexpressing free green fluorescent protein (GFP) together with either NbVHP1 or NbVHP2 at 3 d after agroinfiltration with or without 200 mM NaCl. (b) Maximum photochemical quantum yields of photosystem II (F _v/F _m) determined from detached leaves overexpressing free GFP alone (control) or free GFP together with the indicated pyrophosphatases at 3 d after agroinfiltration with or without 200 mM NaCl. Number of independent experiments is n = 6 (means ± SE). Significant differences between values at P < 0.05 (Duncan test), determined by statistical analysis with the software Sas 9.0 (http://www.sas.com/), are indicated by different letters.

Figure 3

Effect of salt treatment on pump activity of V‐PPase and V‐ATPase. (a) Maximal V‐PPase‐mediated proton pump current responses of vacuoles from Nicotiana benthamiana mesophyll protoplasts overexpressing free green fluorescent protein (GFP) alone or together with NbVHP1 for 2 d. Agroinfiltration was carried out with or without 200 mM NaCl. Pump activity of V‐PPase (n = 4–10; **, P < 0.01; ***, P < 0.001, Student's t‐test) was triggered by cytosolic application of 150 μM pyrophosphate. (b) Maximal V‐ATPase‐mediated proton pump current responses of vacuoles from N. benthamiana mesophyll protoplasts (n = 7; P = 0.059, Student's t‐test), overexpressing free GFP at 2 d after agroinfiltration in the presence or absence of 200 mM NaCl. For studies of the V‐ATPase pump activity, the Ca²⁺ concentration in the bath and pipette medium was increased to 1 mM and Kgluconate was replaced by KCl. The pump activity was triggered by cytosolic application of 5 mM ATP. Data points represent means ± SE.

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).

Figure 5

Effect of V‐PPase overexpression on membrane voltage control. Membrane voltages of Nicotiana benthamiana epidermal leaf cells expressing free green fluorescent protein (GFP) without (control) or together with NbVHP1 (***, P < 0.001, Student's t‐test). Measurements (n = 6–10) were performed 2–3 d after infiltration of transformed agrobacteria ± 200 mM NaCl. The horizontal line in the box represents the median while the upper and lower whiskers give the maximum and minimum values. Additionally, the mean value is indicated by a square.