A Mutant of Arabidopsis thaliana with a Reduced Response to Fusicoccin. I.

Because fusicoccin (FC) has the the capacity to promote solute uptake, a selective procedure for isolating mutants of Arabidopsis thaliana with a reduced response to the toxin has been developed. The procedure is based on the incubation of A. thaliana seedlings in a solution containing the cation Paraquat (Pq) at a concentration that per se does not produce bleaching of the leaves upon illumination but does in the presence of FC because of the increased uptake of the toxic cation. Using this procedure, we identified, among the progenies of 2010 M1 ethyl methanesulfonate-mutagenized plants, two mutants that stay green after exposure to FC and Pq. Some properties and inheritance of one of the two mutants (5-2) are described. Morphology of mutant plants is almost indistinguishable from that of the wild type. However, 5-2 seeds germinate and produce viable seedlings in the presence of FC plus the aminoglycoside antibiotic hygromycin B: plants of the mutant do not wilt when exposed to FC and stomata do not open or open only partially. In the presence of FC, the mutant appears less responsive than the wild type as far as the increment in fresh weight, the enlargement of leaf disc area, or the stimulation of H+ extrusion is concerned. Inheritance of the trait is monogenic dominant or semidominant, depending on the test used.

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves.

In Elodea densa leaves, ethanol up to 0.17 m stimulates H(+) extrusion activity. This effect is strictly dependent on the presence of K(+) in the medium and is suppressed by the presence of the plasmalemma H(+)-ATPase inhibitor vanadate. Stimulation of H(+) extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K(+) influx. These results suggest that ethanol-induced H(+) extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H(+) extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H(+)-extruding mechanism.

Plasmalemma redox activity and h extrusion: I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm acidification.

Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K(+) to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H(+) extrusion no longer requires the presence of K(+) in the medium. The (ferricyanide reduced)/(H(+) extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H(+) extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K(+) release to the medium. The ratio (ferricyanide reduced)/(H(+) extruded + K(+) released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump, stimulated by both intracellular acidification and depolarization. K(+) efflux would depend on potential depolarization.

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

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 ((14)C-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.

H extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments treated with permeant weak acids.

The rapid uptake of weak acids permeant in the uncharged form is accompanied in maize and wheat root segments by a hyperpolarization of the transmembrane electrical potential and an increase in K(+) uptake, suggesting a stimulation of the plasmalemma H(+) pump. The evaluation of weak acid-induced H(+) extrusion must take into account the alkalinization of the medium due to the rapid uptake of the uncharged 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 segments a significant increase in ;real' H(+) extrusion, roughly matching the increase in net K(+) uptake. The presence of K(+) significantly increases the rate of uptake of the weak acid, possibly as a consequence of an alkalinization of the cytosol associated with K(+) absorption. In maize root segments, 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.

Transmembrane electrical potentials in growing maize roots : Anti-auxin effects.

The anti-auxin 4-chlorophenoxyisobutyric acid (PCIB) applied at a concentration of 10(-2) mol m(-3) to maize root segments was found to induce a transmembrane electrical potential of up to-130 mV (Δpd of 30 mV). The kinetics of this response were comparable to the time scale for PCIB-stimulated H(+)-extrusion. Both effects are eliminated by the addition of p-fluoromethoxycarbonyl cyanide phenylhydrazone (FCCP). Treatment with fusicoccin (FC) and PCIB together does not result in a hyperpolarization greater than with FC alone. Benzoic acid (10(-2) mol m(-3)) had no effect on the transmembrane electrical potentials. These results are discussed in relation to a possible electrogenic proton pump which may be regulated by perturbations in the cellular auxin content or activity.