In maize root segments fusicoccin induced a consistent increase in cell sap pH (taken as representative of vacuolar pH). This effect was markedly enhanced by the presence of K' in the medium, whereas in the absence of fusicoccin K' did not significantly influence cell sap pH. Treatment with a weak acid at 2 mm concentration inhibited the uptake of a different ('4C-labeled) weak acid fed at a lower concentration, thus suggesting that acidification of the cytoplasm inhibits weak acid uptake. Fusicoccin and K' increased the rate of uptake of 5,5-dimethyloxazolidine-2,4-dione, butyric acid, or isobutyric acid slightly when fed separately, strongly when fed in combination. The synergism between fusicoccin and K' in stimulating weak acid uptake was parallel to that observed for the stimulation of H' extrusion. Application of the weak acid distribution method to a condition of 'quasi-equilibrium' indicated that fusicoccin induces a cytosolic pH increase of about 0.14 unit. These results are interpreted as providing circumstantial evidence that fusicoccin-and K'-induced stimulation of H' extrusion led to an alkalinization of the cytosol, and that other early metabolic responses, such as an increase in malate level, are a consequence of the increase in cytosolic pH.Recent work emphasizes the importance ofunderstanding how intracellular pH is regulated, and how cytosolic pH changes can influence cell activities. In animal systems the effects of factors or conditions stimulating growth are often associated with changes ofintracellular pH consequent to the activation ofa H / Na+ antiport at the plasma membrane (4). In plants, the main H' translocating system seems to consist of an ATP-driven, electrogenic H+ pump operating at the plasmalemma, apparently influenced by auxin and other natural hormones, and strongly activated by FC2, a toxin closely mimicking various aspects of auxin action (18,19 In the present research we have tried to evaluate qualitatively the FC-induced cytosolic pH changes by measuring in maize root segments the effect of the toxin on the uptake rate and on the accumulation of the permeating weak acids DMO, BA, and IBA. The measurement of the rate of weak acid influx had already been utilized by Marigo et al. (17). This method is based on the assumption that the uptake of an acid passively permeating only in the uncharged form would be proportional to the concentration gradient of this form across the plasmalemma; thus an alkalinization of the cytosol would accelerate the uptake of the acid by increasing its dissociation at the cytoplasmic side ofthe membrane, while an acidification would act in the opposite direction.An objection to this method is that its results are essentially qualitative. But the determination of intracellular pH by measurement of weak acid distribution at equilibrium is difficult in compact tissues because of the time required for equilibration, and other methods (such as 3'P-NMR, intracellular pH indicators, microelectrodes) are either too insensitive, too prone to error,...

ABSTRACFThe rapid uptake of weak acids permnt in the unchaged form is accompanied in maize and wheat root segments by a hyperpolariztion of the transmembrane electrial potential and an increase in K uptake, suggesting a stimulation of the plasmalemma H' pump. The evalation of weak acid-induced H' extrsion must take into account the alkalinization of the medium due to the rapid uptake of the uncagd form of the acid, partially masking the proton pump-mediated extrusion of H+. The data corrected for this interference show that the lipophilic butyric acid and trimethyl acetic acid induce in maize and in wheat root segts a significant increase in 're H+ extrusion, roughly matching the ise in net K+ uptake. The presence of KV significantly incrases the rte of uptake of the weak acid, possibly as a consequence of an alkinitio of the cytosol associated with K absorption. In maize root seents, the effects of fusicoccin and those of butyric acid on both K+ uptake and H+ extrusion are clearly synergistic, thus suggesting distinct modes of action.These results support the view that the activity of the plasmalemma H+ pump is regulated by the value of cytosolic pH.We have previously reported that the treatment with permeant weak acids (such as butyric, isobutyric, trimethylacetic acids, and DMO2) induces in maize root segments a marked hyperpolarization of the PD (12, 14, 15; see also, for similar results in other materials, 1, 3, 6, 16, and 17). The finding that PD hyperpolarization is associated with an increase in the rate of K+ uptake and is correlated with the rate of penetration of the weak acid and with a decrease in cell sap pH suggested that, in these experiments, the electrogenic proton pump at the plasmalemma is stimulated by the acidification of the cytoplasm consequent to the penetration and the dissociation of the weak acid in the cytosol (compare Sanders et al. [16] for the same conclusion from experiments in Neurospora).If this interpretation is correct, the hyperpolarization associated with weak acid uptake should also be associated with an increase in proton secretion, which had not been demonstrated ' in the above-mentioned investigations. On the other hand, the demonstration of weak acid-induced changes in the rate of H+ extrusion presents some experimental difficulties due in part to the strong buffering action of the weak acids, and also, even more seriously, to the fact that the rapid uptake ofthe uncharged form of the weak acid by the tissue results in an alkalinization of the medium, which may mask the acidification due to the secretion of H+. Thus, for a correct evaluation of the 'true' HI movement across the plasmalemma, the titration values must be corrected for the changes in weak acid concentration.Starting from these premises, in the present work we investigated the changes in 'real' H+ extrusion induced in root segments by treatment with permeant weak acids. The proton extrusion values thus obtained were then compared with the results of experiments in which we invesfigated the effects of wea...

Dissociation of active H+ extrusion (−ΔH+) from K+ uptake in pea and maize root segments was attempted by substituting K+ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non‐specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated −ΔH+ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans‐membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC‐promoted −ΔH+. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of −ΔH+. A complete lack of additivity was observed between the effects of lipophilic cations and that of K+ on H+ extrusion. Lipophilic cations severely inhibited K+ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H+ extrusion with K+ influx.

In isolated Elodea densa leaves, the relationships between H+ extrusion (‐ΔH+), K+ fluxes and membrane potential (Em) were investigated for two different conditions of activation of the ATP‐dependent H+ pump. The ‘basal condition’ (darkness, no pump activator present) was characterized by low values of‐ΔH+ and K+ uptake (ΔK+), wide variability of the −ΔH+/ΔK+ ratio, relatively low membrane polarization and Em values more positive than EK for external K+ concentrations (|K+]o of up to 2mol m−3. A net K+ uptake was seen already at [K+]o below 1 mol m−3, suggesting that K+ influx in this condition was a thermodynamically uphill process involving an active mechanism. When the H+ pump was stimulated by fusicoccin (FC), by cytosol acidification, or by light (the ‘high polarization condition’), K+ influx largely dominated K+ and C− efflux, and the −ΔH+/ΔK+ ratio approached unity. In the range 50 mmol m−3−5 mol m−3 [K+]0, Em was consistently more negative than EK. The curve of K+ influx at [K+]0 ranging from 50 to 5000mmol m−3 fitted a monophasic, hyperbolic curve, with an apparent half saturation value = 0–2 mol m−3. Increasing |K+]0 progressively depolarized Em, counteracting the strong hyperpolarizing effect of FC. The effects of K+ in depolarizing Em were well correlated with the effects on both K+ influx and −ΔH+, suggesting a cause‐effect chain: K+0 influx → depolarization → activation of H+ extrusion. Cs+ competitively inhibited K+ influx much more strongly in the ‘high polarization’ than in the ‘basal’ condition (50% inhibition at [Cs+]/[K+]0 ratios of 1:14 and 1:2, respectively) thus confirming the involvement of different K+ uptake systems in the two conditions. These results suggest that in E. densa leaves two distinct modes of interactions rule the relationships between H+ pump, membrane polarization and K+ transport. At low membrane polarization, corresponding to a low state of activation of the PM H+‐ATPase and to Em values more positive than EK, K+ influx would mainly