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Potassium (K(+)) acquisition in roots is generally described by a two-mechanism model, consisting of a saturable, high-affinity transport system (HATS) operating via H(+)/K(+) symport at low (<1mM) external [K(+)] ([K(+)]ext), and a linear, low-affinity system (LATS) operating via ion channels at high (>1mM) [K(+)]ext. Radiotracer measurements in the LATS range indicate that the linear rise in influx continues well beyond nutritionally relevant concentrations (>10mM), suggesting K(+) transport may be pushed to extraordinary, and seemingly limitless, capacity. Here, we assess this rise, asking whether LATS measurements faithfully report transmembrane fluxes. Using (42)K(+)-isotope and electrophysiological methods in barley, we show that this flux is part of a K(+)-transport cycle through the apoplast, and masks a genuine plasma-membrane influx that displays Michaelis-Menten kinetics. Rapid apoplastic cycling of K(+) is corroborated by an absence of transmembrane (42)K(+) efflux above 1mM, and by the efflux kinetics of PTS, an apoplastic tracer. A linear apoplastic influx, masking a saturating transmembrane influx, was also found in Arabidopsis mutants lacking the K(+) transporters AtHAK5 and AtAKT1. Our work significantly revises the model of K(+) transport by demonstrating a surprisingly modest upper limit for plasma-membrane influx, and offers insight into sodium transport under salt stress., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Futile transmembrane NH3/NH4(+) cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4(+) toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4(+)) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope (13)N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4(+) ion. Influx of (13)NH3/(13)NH4(+), which exceeded 200 µmol g(-1) h(-1), was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4(+)), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g(-1) h(-1)). Efflux of (13)NH3/(13)NH4(+) responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.

The involvement of potassium (K(+))-selective, Shaker-type channels, particularly AKT1, in primary K(+) acquisition in roots of higher plants has long been of interest, particularly in the context of low-affinity K(+) uptake, at high K(+) concentrations, as well as uptake from low-K(+) media under ammonium (NH₄(+)) stress. We recently demonstrated that K(+) channels cannot mediate K(+) acquisition in roots of intact barley (Hordeum vulgare L.) seedlings at low (22.5 µM) external K(+) concentrations ([K(+)](ext)) and in the presence of high (10 mM) external NH₄(+), while the model species Arabidopsis thaliana L. utilizes channels under comparable conditions. However, when external NH₄(+) was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K(+) influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K(+) influx, illustrating a hitherto unexplored magnitude and rapidity of K(+)-uptake capacity and plasticity. Here, we expand on our previous work by offering further characterization of channel-mediated K(+) fluxes in intact barley, with particular focus on anion effects, root respiration and pharmacological sensitivity and highlight key additions to the current model of K(+) acquisition.

The role of potassium (K(+)) transporters in high- and low-affinity K(+) uptake was examined in roots of intact barley (Hordeum vulgare) and Arabidopsis (Arabidopsis thaliana) plants by use of (42)K radiotracing, electrophysiology, pharmacology, and mutant analysis. Comparisons were made between results from barley and five genotypes of Arabidopsis, including single and double knockout mutants for the high-affinity transporter, AtHAK5, and the Shaker-type channel, AtAKT1. In Arabidopsis, steady-state K(+) influx at low external K(+) concentration ([K(+)]ext = 22.5 µm) was predominantly mediated by AtAKT1 when high-affinity transport was inhibited by ammonium, whereas in barley, by contrast, K(+) channels could not operate below 100 µm. Withdrawal of ammonium resulted in an immediate and dramatic stimulation of K(+) influx in barley, indicating a shift from active to passive K(+) uptake at low [K(+)]ext and yielding fluxes as high as 36 µmol g (root fresh weight)(-1) h(-1) at 5 mm [K(+)]ext, among the highest transporter-mediated K(+) fluxes hitherto reported. This ammonium-withdrawal effect was also established in all Arabidopsis lines (the wild types, atakt1, athak5, and athak5 atakt1) at low [K(+)]ext, revealing the concerted involvement of several transport systems. The ammonium-withdrawal effect coincided with a suppression of K(+) efflux and a significant hyperpolarization of the plasma membrane in all genotypes except athak5 atakt1, could be sustained over 24 h, and resulted in increased tissue K(+) accumulation. We discuss key differences and similarities in K(+) acquisition between two important model systems and reveal novel aspects of K(+) transport in planta.

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.

Potassium (K(+) ) flux into plant cells is a well-characterized ion transport phenomenon. By contrast, little is known about the mechanisms and regulation of K(+) flux from the cell. Here, we present a radioisotopic analysis of K(+) fluxes from roots of intact barley (Hordeum vulgare), in the context of recent discoveries in the molecular biology and electrophysiology of this process. Plants were labelled with (42)K(+), and kinetics of its release from roots were monitored at low (0.1 mM) or high (1.0 mM) external K concentration, [K(+)](ext), and with the application of channel modulators and nutrient shifts. At 0.1 (but not 1.0) mM [K(+)], where K(+) efflux is thought to be mediated by K(+)-outward-rectifying channels, (42)K(+) efflux was inhibited by the channel blockers barium (Ba(2+)), caesium (Cs(+)), tetraethylammonium (TEA(+)), and lanthanum (La(3+)). Ammonium and nitrate (10 mM) stimulated and inhibited (42)K(+) efflux, respectively, while 10 mM [K(+)](ext) or [Rb(+) ](ext) decreased it. No evidence for the involvement of ATP-binding cassettes, nonselective cation channels, or active K(+)-efflux pumps was found. Our study provides new evidence for the thermodynamic transition between high- and low-affinity transport, from the efflux perspective, identifying the operation of channels at low [K(+)], and the cessation of transmembrane efflux at high [K(+)]., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

*Stimulation of potassium (K(+)) efflux by sodium (Na(+)) has been the subject of much recent attention, and its mechanism has been attributed to the activities of specific classes of ion channels. *The short-lived radiotracer (42)K(+) was used to test this attribution, via unidirectional K(+)-flux analysis at the root plasma membrane of intact barley (Hordeum vulgare), in response to NaCl, KCl, NH(4)Cl and mannitol, and to channel inhibitors. *Unidirectional K(+) efflux was strongly stimulated by NaCl, and K(+) influx strongly suppressed. Both effects were ameliorated by elevated calcium (Ca(2+)). As well, K(+) efflux was strongly stimulated by KCl, NH(4)Cl and mannitol , and NaCl also stimulated (13)NH(4)(+) efflux. The Na(+)-stimulated K(+) efflux was insensitive to cesium (Cs(+)) and pH 4.2, weakly sensitive to the K(+)-channel blocker tetraethylammonium (TEA(+)) and quinine, and moderately sensitive to zinc (Zn(2+)) and lanthanum (La(3+)). *We conclude that the stimulated efflux is: specific neither to Na(+) as effector nor K(+) as target; composed of fluxes from both cytosol and vacuole; mediated neither by outwardly-rectifying K(+) channels nor nonselective cation channels; attributable, alternatively, to membrane disintegration brought about by ionic and osmotic components; of limited long-term significance, unlike the suppression of K(+) influx by Na(+), which is a greater threat to K(+) homeostasis under salt stress.