Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K⁺-Induced Cytosol Alkalinization

Abstract

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,...

Purification and Identification of the Fusicoccin Binding Protein from Oat Root Plasma Membrane

“…Such a sharp drop in K+-efflux (10-fold) after FC addition has indeed been observed (10). Although it is not immediately clear how the plugging of a K+-leak would lead to hyperpolarization, this hypothesis points out once more the crucial role that K+ plays in the FC response (20). The question whether the FC-receptor acts as a K+-channel will be further pursued with the patch-clamp technique, studying the purified FC-receptor after reconstitution in artificial liposomes.…”

“…8) (20,100), a-lactalbumin (beef milk) (14,200). elution pattern on the hexyl-agarose it can be concluded that the FCBP is one of the more hydrophobic proteins in the plasma membrane (Fig.…”

“…10). The SDS fractions (20 times diluted in the binding-assay) contained 3H-FC binding activity with a peak at 0.05% SDS (Fig. 10).…”

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment

“…The in vivo capacity of the plasma membrane H+-ATPase was evaluated using the phytotoxin FC (Marrè, 1979), which activates this H+ pump (Johansson et al, 1993;Rasi-Caldogno et al, 1993). Although FC stimulated net H+ extrusion in oxygenated HPT and NHPT root tips to similar levels (data not shown), under anoxia, HPT root tips showed much greater stimulation than NHPT tips (Fig.…”

“…The cytoplasmic buffering capacity in plants has been estimated to range from 20 to 100 microequivalents H+ mL-' pH unit-' (Kurkdjian and Guern 1989). The root tips studied here had a cytoplasmic volume approximately 10% of the tissue weight, according to estimates made by Marrè et al (1986). Calculating the impact of the measured net H + fluxes (Figs.…”