The effects of external salt and inorganic phosphate (Pi) on the concentrations of vacuolar Pi, and cytoplasmic Pi, ATP, glucoe-6-phosphate and UDP-glucose in maize root tips were examined using 31P nuclear magnetic resonance spectroscopy. We observed a more than twofold stimulation of Pi uptake from 10 millimoar KH2PO4 solutions when root tips were exposed to 100 millimolar NaC + CaCI2. This stimulation of Pi uptake was associated with an increase in the concentration of cytoplasmic Pi in root tip cells. Thus, the molar ratio of cytoplasmic Pi to Pi + ATP + glucose-aphosphate + UDP-glncose increased greatly in root tips exposed to salt and Pi. We speculate that it is this disturbance in relative concentrations of cytoplasmic phosphates (which we show are normally tightly regulated) that is responsible for both the greater rte of uptake of Pi by vacuoles of excised maize root tips, and the previously documented stimulation of Pi translocation from root to shoot in whole maize plants exposed to salt and Pi.A significant interactive effect of salinity and nutrient Pi on yield in maize has been described by Bemstein et al. (1). High concentrations of Pi (2 mM) caused lower yields in salt-treated plants than low concentrations of Pi (0.05 mM). This lower yield appears to result from excessive uptake of Pi, with translocation to the leaves, leading to symptoms of phosphorus toxicity (1, 9). Thus, some aspect of uptake and/or translocation is disturbed by the combination of salt and high Pi. An inability to regulate Pi transport has also been observed in phosphorus-deficient barley plants when they were supplied with Pi (4, 6).The study of Pi transport and metabolism in plants is complicated by the existence of metabolically distinct pools of Pi, in the vacuolar and cytoplasmic intracellular compartments (2), and by the rapid exchange of P between Pi and organic phosphates in the cytoplasm (8). 3"P-NMR2 spectroscopy permits observation of both these aspects of Pi metabolism. The method permits simultaneous monitoring of the cytoplasmic and vacuolar Pi pools in plant tissues (11,15) and also can be used to estimate rates of exchanges between cytoplasmic Pi and ATP in maize root tips (14). Recently, 3'P-NMR has been used to 'Supported by grants from the-National Institutes of Health (RROO7 11), the National Science Foundation (PCM 82-04877 and GP 23633), and a United States Department of Agriculture cooperative agreement (AGRIC 58 9AZ-2-665).2Abbreviation: NMR, nuclear magnetic resonance. measure cytoplasmic and vacuolar Pi pool sizes in cell suspension cultures (10) and pea root tips (7) exposed to Pi. In both studies uptake of Pi resulted in large increases in the concentration of vacuolar Pi, while the concentration of cytoplasmic Pi was essentially unchanged; this result was observed over external concentrations of Pi ranging from 0.2 to 45 mm. In this paper we describe the time course for changes in cytoplasmic and vacuolar Pi pool sizes in maize root tips exposed to solutions of salt and Pi. We show that s...

A proton motive force (AjAH+) of 70 to 130 mV was measured across the membrane of Mycoplasma gallisepticum celLs. The membrane potential was measured by utilizing the lipid-soluble cation tetraphenylphosphonium. The method was validated by showing that in the presence of valinomycin the ratio of the concentrations (in/out) of tetraphenylphosphonium agreed well with those for K+ and Rb+. The pH gradient was calculated from the measured distribution ratio of benzoic acid. The proton motive force was approximately the same in cells harvested at early exponential, midexponential, and stationary phases of growth. The proportion of pH gradient to membrane potential varied with external pH. In the absence of glucose, cells incubated in an isosmotic NaCl solution showed low adenosine triphosphate and AIH+ levels and a tendency to swell and lyse compared with cells incubated with added glucose. It is concluded that energy is required for normal cell volume regulation.

When washed cells of Mycoplasma gallisepticum were incubated at 37°C in 250 mM 22NaCl, the intracellular Na+ increased, and the K+ decreased. The addition of glucose to these Na+-loaded cells caused Na+ efflux and K+ uptake (both ions moving against concentration gradients). This effect of glucose was blocked by the ATPase inhibitor dicyclohexylcarbodiimide, which prevents the generation of a proton motive force in these cells. In additional experiments, Na+ extrusion was studied by diluting the 22Na+-loaded cells into Na+-free media and following the loss of 2Na' from the cells. Glucose stimulated 22Na' extrusion in such cells by a dicyclohexylcarbodiimide-sensitive mechanism. Proton movement was studied by measuring the pH gradient across the cell membrane with the 9-atninoacridine fluorescence technique. Glucose addition to cells preincubated with cations other than Na+ resulted in cell alkalinization (which was prevented by dicyclohexylcarbodiimide). This observation is consistent with the operation of a proton-extruding ATPase. When glucose was added to Na+-loaded cells and diluted into Na+-free media, intracellular acidification was observed, followed several minutes later by a dicyclohexylcarbodiimide-sensitive alkalinization process. The initial acidification was probably due to the operation of an Na+-H+ antiport, since Na+ exit was occurring simultaneously with H+ entry. When Na+-loaded cells were diluted into Na+-containing media, the subsequent addition of glucose resulted in a weak acidification, presumably due to H+ entry in exchange for Na+ (driven by the ATPase) plus a continuous passive influx of Na+. All of the data presented are consistent with the combined operation of an ATP-driven proton pump and an Na+-H+ exchange reaction.

Mycoplasma gallisepticum cells incubated in 250 mM NaCl solutions in the absence of glucose showed a progressive fall in intracellular ATP concentration over a period of 2 to 3 h. When the ATP level fell below 40 ,uM the cell began to swell and become progressively permeable to [14C]inulin and leak intracellular protein and nucleotides. The addition of nondiffusable substances such as MgSO4 or disaccharides prevented swelling, suggesting that NaCl (and water) entry was due to Gibbs-Donnan forces. The addition of glucose after the initiation of cell swelling increased intracellular ATP, induced cell shrinkage, and prevented the release of intracellular components. The ATPase inhibitor dicyclohexylcarbodiimide, which collapsed the chemical and electrical components of the proton motive force, caused rapid cell swelling in the presence of glucose (and high intracellular ATP levels). Extracellular impermeable solutes such as MgSO4 and disaccharides prevented swelling of dicyclohexylcarbodiimide-treated cells incubated in NaCl. It was postulated that Na+ that diffused into the cell was extruded by an electrogenic Na+-H+ exchange (antiport) energized by the proton motive force established by the dicyclohexylcarbodiimide-sensitive H+-ATPase.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37°C, with 50% inhibition occurring at 22 ,IM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 ,ug/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 ,uM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.