measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

University of Toronto

*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.

42K analysis of sodium-induced potassium efflux in barley: mechanism and relevance to salt tolerance.

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(+)].

Regulation and mechanism of potassium release from barley roots: an in planta 42K+ analysis.

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.

Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots.

Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K(+)) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world's most important and salt-sensitive crop species. Using efflux analysis with (42)K, coupled with growth and tissue K(+) analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K(+) set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K(+) release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K(+) status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na(+) accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K(+) efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K(+) status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na(+) accumulation is the key factor in performance decline on NaCl stress.

K+ efflux and retention in response to NaCl stress do not predict salt tolerance in contrasting genotypes of rice (Oryza sativa L.).

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.

Capacity and plasticity of potassium channels and high-affinity transporters in roots of barley and Arabidopsis.

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.

Complexity of potassium acquisition: how much flows through channels?

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.

Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.

K(+) channels are among the best-characterized classes of membrane protein in plants. Nevertheless, in-planta demonstrations of traits emerging from molecular characterizations have often been insufficient or lacking altogether. Such linkages are, however, critical to our basic understanding of plant nutrition and to addressing 'real-world' issues that are faced in environmental and agricultural settings. Here, we cover some of the recent advances in K(+) acquisition with particular focus on voltage-gated K(+) channel functioning and regulation in roots, and highlight where linkages to in-planta behavior have been successfully made and, conversely, where such linkages are yet to be made.

Devrim Coskun

Department of Biological Sciences

University of Toronto

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers