JXB Advance Access originally published online on January 17, 2008
Journal of Experimental Botany 2008 59(2):303-313; doi:10.1093/jxb/erm309
© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport
Department of Biological Sciences, University of Toronto, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4
* To whom correspondence should be addressed. E-mail: herbertk{at}utsc.utoronto.ca
Received 8 August 2007; Revised 8 November 2007 Accepted 12 November 2007
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Abstract |
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Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 µmol g–1 h–1 was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5–40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.
Key words: Ammonium, barley, efflux, influx, nitrogen-13, non-selective cation channels, potassium
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Introduction |
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Ammonium (NH4+) is present in many terrestrial ecosystems and over a wide concentration range (Pearson and Stewart, 1993; Miller and Cramer, 2005). At low (micromolar) soil concentrations, NH4+ is adequate as a sole N source for many plant species (Kronzucker et al., 1997, 1999), but most cannot tolerate millimolar concentrations (Britto and Kronzucker, 2002). In this toxic range, NH4+ uptake is mediated by a high-capacity, energetically passive, low-affinity transport system (LATS). However, low-affinity NH4+ influx is accompanied by an efflux of NH4+ nearly equal in magnitude, resulting in the futile cycling of this ion across the plasma membrane (Britto et al., 2001, 2002; Britto and Kronzucker, 2006). The substantial efflux of NH4+ under these conditions has been shown to be energetically costly in NH4+-sensitive plant species (Kronzucker et al., 2001), and it has been postulated that a primary cause of NH4+ toxicity in plants is the energy lost due to the active removal of NH4+ that has entered root cells at an uncontrolled rate (Britto et al., 2001; Kronzucker et al., 2001).
NH4+ nutrition has been shown to influence the mineral composition of plants dramatically, particularly in the reduction of cation content (Kirkby and Mengel, 1967; Vale et al., 1987, 1988a; Gerendas et al., 1997; Santa-María et al., 2000; Szczerba et al., 2006a). The mechanism underlying this has not been unravelled, but it may be through direct competition between NH4+ and other cations for entry through common uptake pathways. In particular, potassium (K+) channels are considered prime candidates for low-affinity NH4+ transport, as NH4+ and K+ are both monovalent cations with similar hydrated atomic radii (Kielland, 1937; Wang et al., 1996; White, 1996). However, a hydrated atomic radius may not be a critical characteristic for use of a common channel, as the hydrated shell has been shown to be removed as ions pass through the selective filter (Doyle, 2004).
A key relationship between K+ and NH4+ nutrition is that an increase in external K+ concentration ([K+]ext) protects sensitive plant species from NH4+ toxicity (Cao et al., 1993; Spalding et al., 1999; Santa-María et al., 2000; Kronzucker et al., 2003b; Szczerba et al., 2006a). This protection is due in part to the restoration of normal K+ status to the plant, a process that ultimately depends on K+ fluxes into roots and its subsequent translocation to the shoot (Kronzucker et al., 2003b; Szczerba et al., 2006a). In studies using intact barley seedlings, Kronzucker et al. (2003b) and Szczerba et al. (2006a) showed that, at low external K+ concentrations, K+ fluxes into the root were much lower in seedlings grown with 10 mM NH4+ than those grown with 10 mM nitrate (NO3–), but at high external K+, these fluxes were independent of N source. Additionally, the K+ flux from root to shoot, which was the flux most suppressed by NH4+ at low external [K+], was nearly identical in plants grown with NO3– or NH4+ at the higher K+ concentration (Kronzucker et al., 2003b). These effects, in particular the suppression of K+ influx at the plasma membrane under low-K+, high-NH4+ conditions, are likely to be due to the inhibitory action of NH4+ upon high-affinity KUP/HAK/KT transporters (Spalding et al., 1999).
While inhibition of K+ uptake and modification of K+ efflux by NH4+ has been demonstrated, the reciprocal effect has only been sparsely investigated (Scherer et al., 1984; Vale et al., 1988b; Wang et al., 1996; Nielsen and Schjoerring, 1998). Nielsen and Schjoerring (1998) found that 100 mM K+ reduced the influx of NH4+ by 50% in leaf apoplasm of Brassica napus L. Other studies have demonstrated moderate suppression of NH4+ isotherms by K+, but never to the same extent as the suppression of K+ influx by NH4+ (Scherer et al., 1984; Vale et al., 1988b; Wang et al., 1996). While many of these studies were conducted at low external concentrations of both NH4+ and K+, none considered growth conditions mediated by LATS for either ion.
To investigate how NH4+ fluxes are influenced by external [K+], and how this interaction may underlie potassium's alleviation of ammonium toxicity, NH4+ fluxes were examined in intact barley seedlings using the short-lived positron-emitting radiotracer 13N. It was hypothesized that increasing external [K+] would: (i) decrease unidirectional NH4+ fluxes across the plasma membrane; (ii) reduce the high NH4+ efflux:influx ratio that is symptomatic (and perhaps a causative agent) of NH4+ toxicity; and (iii) lessen the energy burden associated with toxic NH4+ fluxes. All three hypotheses were borne out in the study. It is proposed that low-affinity NH4+ influx is accomplished by two components, the first responding to K+ and the second unaffected by it.
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Materials and methods |
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Plant culture
Seeds of barley (Hordeum vulgare L. cv. ‘Klondike’) were surface-sterilized for 10 min in 1% sodium hypochlorite and germinated under acid-washed sand for 3 d prior to placement in 4.0 l vessels containing aerated, 1/4 strength Johnson's solution, at pH 6–6.5, for an additional 4 d. The solution was modified to provide four concentrations of potassium (as K2SO4), at 0.1, 1.5, 5, and 40 mM, and NH4+ [as (NH4)2SO4], at 10 mM. Solutions were exchanged frequently to ensure that plants remained at a nutritional steady state. Plants were cultured in a walk-in growth chamber under fluorescent lights (Philips Econ-o-watt, F96T12), with an irradiation of 200 µmol photons m–2 s–1 at plant height, for 16 h d–1. Daytime temperature was 20 °C, night-time temperature was 15 °C, and relative humidity was
70%. On day 6 (1 d prior to experimentation), seedlings were transferred to an experimental radiotracer facility that had similar irradiance and temperature to the growth chamber.
Compartmental analysis
Compartmental analysis by tracer efflux was used to estimate subcellular fluxes and compartmental pool sizes (Lee and Clarkson, 1986; Siddiqi et al., 1991; Kronzucker et al., 1995). Each replicate consisted of five plants held together at the shoot base by a plastic collar. Intact roots of these plants were labelled for between 30 min and 55 min in solution identical to growth solution but containing the radiotracer 13N (t1/2=9.97 min; as 13NH4+) provided by the CAMH cyclotron facility (University of Toronto, Ontario, Canada). Labelled seedlings were attached to efflux funnels and eluted of radioactivity with successive 20 ml aliquots of non-radioactive desorption solution, identical to the growth solution. The desorption series was timed as follows: 15 s (four times), 20 s (three times), 30 s (twice), 40 s (once), 50 s (once), 1 min (five times), 1.25 min (once), 1.5 min (once), 1.75 min (once), and 2 min (eight times). All solutions were mixed using a fine stream of air bubbles. Immediately following elution, roots were detached from shoots and spun in a low-speed centrifuge for 30 s prior to weighing. Radioactivity from eluates, roots, shoots, and centrifugates was counted, and corrected for isotopic decay, using a gamma counter (PerkinElmer Wallac 1480 Wizard 3'', Turku, Finland). Linear regression of the function ln 
co(t)*=ln co(i)*–kt [in which co(t)* is tracer efflux at elution time t, co(i)* is initial radioactive tracer efflux, and k is the rate constant describing the exponential decline in radioactive tracer efflux, found from the slope of the tracer release rate; see Fig. 1] was used to resolve the kinetics of the slowest exchanging phase in these experiments, which represents tracer exchange with the cytosolic compartment (Kronzucker et al., 1995; Britto and Kronzucker, 2003). Chemical efflux, co, was determined from co(i)*, divided by the specific activity of the cytosol (SAcyt) at the end of the labelling period; SAcyt was estimated by using external specific activity (SAo), labelling time t, and the rate constant k, which describes tracer exchange with the cytosol, which are related in the exponential rise function SAcyt=SAo(1–e–kt) (Kronzucker et al., 1995). Net flux, net, was found using total plant 13N retention after desorption (Kronzucker et al., 1995). Influx, oc, was calculated from the sum of net and co. Cytosolic [NH4+] ([NH4+]cyt) was determined using the flux turnover equation, [NH4+]cyt=
·oc/k, where is a proportionality constant correcting for the cytosolic volume being 5% of total tissue (Britto and Kronzucker, 2001).
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K+ concentration shift experiments followed a protocol identical to that above except that, upon 12.25 min of elution, subsequent aliquots were no longer identical to the growth and labelling solutions, but contained a new K+ concentration (0.1 mM or 5 mM).
Direct influx
Influx of NH4+ was also determined directly, by short-term labelling with 13N. Seedlings were placed for 5 min in growth solution for equilibration, followed by immersion in labelling solution (containing 13NH4+), for either 1 min or 5 min (the two labelling times were used to investigate the rapidity of NH4+ influx response to changing external [K+]). The labelling solution was either identical to the growth solution, for steady-state experiments, or contained a new [K+]ext, for K+ concentration shift experiments. After labelling, plants were transferred to non-radioactive growth solution for 5 s, to reduce tracer carryover to the desorption solution, which was also identical to growth solution, and in which roots were then desorbed for 5 min. Radioactivity remaining in roots and shoots was quantified by gamma counting. Influx values obtained in this way were very close to those determined using compartmental analysis, indicating that the effect of efflux on the measurement of influx was negligible. K+ influx was determined as described for NH4+, using 42K (provided by the McMaster University Nuclear Reactor, Hamilton, Ontario, Canada), and a single labelling time of 5 min.
Pharmacological agents
Similar to the procedure described above, direct influx measurements by short-term labelling with 13N (or 42K) were conducted, in the presence of the channel inhibitors caesium (Cs+), lanthanum (La3+), and tetraethylammonium (TEA). Seedlings were placed for 10 min in growth solution for equilibration, containing 10 mM Cs+, La3+, or TEA. Labelling with 13N (or 42K), and subsequent solution exchanges, were identical to the above procedure except that all solutions contained the appropriate channel inhibitor.
Tissue ammonium determination
To measure tissue NH4+ content, barley seedlings were harvested and desorbed for 5 min in 10 mM CaSO4 to remove extracellular NH4+. Roots and shoots were then separated and weighed, then transferred to polyethylene plastic vials and frozen in liquid N2 for storage at –80 °C. Approximately 0.5 g of root or shoot tissue was homogenized under liquid N2 using a mortar and pestle, followed by the addition of 6 ml of formic acid (10 mM) for the purpose of extracting NH4+ (Husted et al., 2000). Subsamples (1 ml) of the homogenate were centrifuged at 2.5x104 g at 2 °C for 10 min. The supernatant was transferred to 2 ml polypropylene tubes with 0.45 µm nylon filters (Costar, Corning Inc., USA) and centrifuged at 5x103 g (2 °C) for 5 min. The resulting supernatant was analysed by either the indophenol colorimetric (Berthelot) method or the o-phthalaldehyde (OPA) method to determine total tissue NH4+ content.
Indophenol method
This method has been described in detail elsewhere (Solorzano, 1969; Husted et al., 2000). Briefly, three solutions were combined with 1.6 ml of tissue extract: (i) 200 µl of 11 mM phenol in 95% (v/v) ethanol; (ii) 200 µl of 1.7 mM sodium nitroprusside (prepared weekly); and (iii) 500 µl of solution containing 100 ml of 0.68 M trisodium citrate in 0.25 M NaOH with 25 ml of commercial strength (11%) sodium hypochlorite. The colour was allowed to develop for 60 min at room temperature (25 °C) in the dark, and sample absorbance was measured at 640 nm.
OPA method
This method has been described in detail elsewhere for use with spectrophotometry (Goyal et al., 1988). Briefly, 100 ml of OPA reagent was prepared by combining 200 mM potassium phosphate buffer (composed of equimolar amounts of potassium dihydrogen phosphate and potassium monohydrogen phosphate), 3.75 mM OPA, and 2 mM 2-mercaptoethanol 1 d before use. Prior to the addition of 2-mercaptoethanol, the solution pH was adjusted to 7 with 1 M NaOH, and filtered through a grade 2 Whatman filter paper. A 10 µl aliquot of tissue extract was combined with 3 ml of OPA reagent, the colour was allowed to develop in the dark for 30 min at room temperature (25 °C), and sample absorbance was measured at 410 nm.
Root respiration and energy cost of transport
Root respiration was determined in intact seedlings using a Hansatech oxygen electrode and Oxygraph control system (Hansatech Instruments, Norfolk, UK). Seedlings were placed in a cuvette with 2.5 ml of air-saturated growth solution. The decline in O2 concentration was monitored for 15 min, but only the initial linear decline was used to calculate O2 depletion rates. The energy costs of ion transport were calculated based upon the following equation:
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5 ATP:O2. Therefore, in the application of this model to the primary unidirectional influx of NH4+ or K+, across the root plasma membrane, H/Ij=1, H/P=1, and P/O2=5, resulting in 1/Utheor=0.2 (mol O2 per mol ion transported). This value of 1/Utheor was multiplied by the influx of NH4+ or K+ to determine the theoretical O2 consumed to sustain the flux. For further details on the application of this model to passive cation influx operating concomitantly with active cation efflux and proton pumping, see Britto and Kronzucker (2006).
Statistical analysis
Statistical analyses were conducted using one-way analysis of variance (ANOVA) with the statistical package SPSS (version 12).
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Results |
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Steady-state NH4+ fluxes are strongly affected by K+
Figure 1 shows the time-dependent efflux of 13NH4+ from roots of 7-d-old intact barley seedlings. The semi-logarithmic plots displayed a compoundly exponential character, and could be precisely resolved into three kinetically distinct linear phases, each representing tracer released from a separate subcellular compartment (Siddiqi et al., 1991; Kronzucker et al., 1995; Britto and Kronzucker, 2003). Slopes of each linear phase yielded half-times of exchange (t1/2) for each compartment. The more rapidly exchanging phases, representing the extracellular surface film and Donnan free space, had t1/2 values of 7 s and 59 s, respectively, while the slowest exchanging compartment, identified as the cytosol (Kronzucker et al., 1995; Britto and Kronzucker, 2003), had a t1/2 of 14 min for the high external K+ conditions, and 21 min for the lowest [K+]ext (Fig. 1). Compartment identification was rigorously ascertained in previous studies (Kronzucker et al., 1995; Britto and Kronzucker, 2003), and the magnitude of NH4+ influx as determined using compartmental analysis (Fig. 2) was confirmed by direct influx measurements (Fig 2, inset; see Szczerba et al., 2006b).
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Root and shoot NH4+ tissue content was determined using two independent methods, indophenol and OPA (Table 1). The values obtained by both methods were in excess of what is necessary to account for the [NH4+]cyt estimates calculated by compartmental analysis. Following the pattern of changing NH4+ activity in the cytosol (which dropped from 240–580 mM to 90–150 mM, depending on activity coefficients used; see Fig. 6), raising the external [K+] from 0.1 mM to 40 mM dramatically reduced the root tissue content of NH4+, from 70 to 14 µmol g–1 (root FW).
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, from Szczerba et al., 2006a). Also included are measured cytosolic NH4+ concentrations and ranges of NH4+ activities calculated using two estimates for the ionic strength (I) of the cytosol: (i) I based on K+, NH4+, and a monovalent anion—upper value; or (ii) I based on a simple solution of (NH4)2SO4—lower value.Net fluxes found with compartmental analysis were similar across treatments, while efflux and influx varied dramatically with external K+ supply (Fig. 2). At the lowest [K+]ext condition of 0.1 mM, under which K+ influx is mediated by a high-affinity transport system (HATS), NH4+ influx was significantly greater than under all other conditions, with a rate of 92 µmol g–1 h–1. However, when [K+]ext was elevated into the low-affinity K+ transport range (
1.5 mM), NH4+ influx declined by as much as 63%, to 34 µmol g–1 h–1. Even more dramatic was the effect of elevated K+ on NH4+ efflux, which was reduced by as much as 75%, from 69 µmol g–1 h–1 to 17 µmol g–1 h–1. Because of this differential effect on unidirectional NH4+ fluxes, the ratio of efflux to influx declined substantially when [K+]ext was raised, from 0.75 to as little as 0.42. Increasing external potassium beyond the LATS threshold value of 1.5 mM did not further reduce NH4+ influx or the efflux:influx ratio. Thus, the constant residual flux observed throughout the LATS range is identified as the K+-insensitive component of NH4+ influx.
Elevated K+ rapidly decreases unidirectional NH4+ influx
Figure 3 shows the influx of NH4+ into intact barley seedlings, as determined by short-term (5 min) accumulation of 13NH4+. In agreement with compartmental analysis, NH4+ fluxes were maximal at 0.1 mM [K+]ext (reaching a peak value of 84 µmol g–1 h–1). However, when low-potassium seedlings were exposed to elevated (5 mM) [K+]ext, NH4+ influx was drastically and immediately reduced, by 26% after the first minute of exposure, and by nearly 50% within 5 min (Fig. 3).
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A sudden increase in [K+]ext decreased not only the influx of NH4+, but also its efflux (Fig. 4). After introducing an elevated (5 mM) concentration of K+ midway through an elution protocol, NH4+ efflux declined notably within a few minutes. Within 15 min following the shift in [K+]ext, the half-time of cytosolic NH4+ exchange appeared to have been re-established to the value seen prior to the shift (Fig. 4). The reverse change in [K+]ext, from high to low, however, did not immediately elevate NH4+ efflux (Fig. 4, inset).
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La3+ application mimics K+ inhibition of NH4+ influx
The channel inhibitors TEA, Cs+, and La3+, which have been shown to reduce channel-mediated fluxes of K+ and NH4+ (Wegner et al., 1994; Nielsen and Schjoerring, 1998), were used to help identify the mechanisms underlying the K+-sensitive and -insensitive components of NH4+ transport (Fig. 5). Interestingly, Cs+ and TEA both stimulated NH4+ influx, under high (40 mM) and low (0.1 mM) [K+]ext, with TEA in particular increasing NH4+ influx by nearly 40%. In contrast, La3+ application reduced the influx of NH4+ by 60% under low [K+]ext (0.1 mM). At elevated [K+]ext, no reduction in NH4+ influx was observed. The effects of La3+ upon K+ influx were also tested, using 42K as a tracer (Fig. 5, inset). Surprisingly, K+ influx was reduced by 70% at the HATS concentration of 0.1 mM, but only by 45% at the LATS concentration of 1.5 mM.
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Active NH4+ and K+ fluxes can be energetically costly
The energetics of NH4+ transport were analysed using previously reported plasma membrane electrical potentials from the same plant system (Szczerba et al., 2006a) and cytosolic concentrations of NH4+ measured in the present study using compartmental analysis (Fig. 6). The range of activities presented in Fig. 6 were determined using activity coefficients corresponding either to a cytosol dominated by K+, NH4+, and a univalent anion (upper estimate), or to a simple solution of (NH4)2SO4 (lower estimate) (Nobel, 1991; Lide, 2007). Although the cytosolic activities of NH4+ were significantly different at low and high [K+]ext, the electrochemical potential gradient for NH4+ was inwardly directed in both cases. Thus, NH4+ influx was determined to occur via facilitated diffusion into the plant cell, and its efflux, in turn, would be energy demanding.
To test the model and relate it to growth and NH4+ toxicity, root respiration experiments were conducted (Fig. 7). Specific respiratory costs of the active components of K+ and NH4+ fluxes were determined based on current models of energy usage (Kurimoto et al., 2004) and by use of K+ fluxes determined previously (Szczerba et al., 2006a). In all conditions tested, the respiratory costs not associated with NH4+ and K+ fluxes were similar. The two conditions with the lowest energy requirement for NH4+ and K+ transport (1.5 mM and 5 mM K+) had the lowest overall root respiration, but the largest root and shoot masses (Fig. 7).
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Discussion |
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Rapid, futile NH4+ cycling
Efflux of ions from plant cells into the external environment can be monitored using several techniques, but none is as comprehensive as compartmental analysis by tracer efflux, which facilitates the simultaneous measurement of unidirectional fluxes, subcellular concentrations, and compartmental exchange rates of labelled ions (Lee and Clarkson, 1986; Siddiqi et al., 1991; Britto et al., 2001; Kronzucker et al., 2003b; Szczerba et al., 2006a). This technique initially led to the discovery of rapid, futile cellular NH4+ cycling in a number of plant systems (Britto et al., 2001, 2002; Kronzucker et al., 2003a; Britto and Kronzucker, 2006), and to the close association of this phenomenon with NH4+ toxicity (Kronzucker et al., 2001). In the present study, the high unidirectional fluxes of NH4+ in both directions across the plasma membrane of barley root cells were confirmed, as well as the high ratio of efflux to influx, both of which are key characteristics of the futile cycling condition (Fig. 2).
Because NH4+ toxicity in plants can be relieved by increasing potassium availability (Cao et al., 1993; Spalding et al., 1999; Santa-María et al., 2000; Kronzucker et al., 2003b; Szczerba et al., 2006a), it was hypothesized that changes in external [K+] would alter the magnitude of NH4+ fluxes and the degree of futile cycling in NH4+-susceptible barley plants. As shown in Fig. 2, this hypothesis was borne out on both counts. Changing the steady-state K+ supply from 0.1 mM to 1.5 mM significantly reduced both influx and efflux of NH4+, and substantially decreased the ratio of efflux to influx. Increasing [K+]ext to higher values (5 mM and 40 mM) had no further effect on these flux parameters. Importantly, this shift is also associated with relief from NH4+ toxicity (Fig. 7). Increasing [K+]ext from 0.1 mM to 1.5 mM or above caused a reduction in both root tissue and cytosolic NH4+ of 70%, showing that the amelioration of NH4+ toxicity by increasing [K+]ext was paralleled by a reduction in NH4+ tissue content (Fig. 6, Table 1). The [NH4+]cyt values for the toxic condition, while high, are in agreement with values reported in a separate study on NH4+ toxicity in barley seedlings (Britto et al., 2001). Moreover, the tissue NH4+ values are well in excess of what is required to account for the [NH4+]cyt estimates, demonstrating that a substantial NH4+ pool is also present in the vacuole. While vacuolar pools were not directly measured here, since the focus of the study was the futile cycling of NH4+ at the plasma membrane, subtraction of cytosolic content from whole-root content of NH4+ yields estimates of vacuolar pools that range between 2.4 and 25 µmol g–1 (FW) (depending on N and K status; not shown), in good agreement with prior studies (Lee and Ratcliffe, 1991; Wang et al., 1993).
While the dramatic differences in key flux parameters shown in Fig. 2 were determined under steady-state nutritional conditions, it was of further interest to examine the time scale over which K+-induced alterations in NH4+ transport occur. Figures 3 and 4 show that these potent effects become manifest within 1–5 min of increased K+ supply, for both influx and efflux of NH4+. The rapidity of this response suggests that K+ regulates NH4+ fluxes possibly by acting allosterically on an ammonium transporter, or by competing directly with NH4+ for a common transport mechanism. Such short-term changes are likely to precede longer-term changes that involve alterations in gene expression, that would bring about the further lowering of NH4+ influx seen in the rightmost column in Fig. 3. The lack of an immediate stimulatory effect on NH4+ influx by a reduction in external [K+] may indicate that the shift from the high-K+ condition to the low-K+ condition entails the up-regulation of NH4+-sensitive transporters, which would occur over a longer time scale. Alternatively or in addition, it may indicate that the release of K+ from inhibitory binding sites on NH4+ transporters also occurs over a longer time scale.
K+-sensitive NH4+ influx pathway
It is not yet fully resolved how NH4+ enters the plant cell, particularly in the low-affinity range. The present study provides new insight into possible candidates and characteristics of low-affinity NH4+ transport. The dramatic reduction of NH4+ fluxes and cycling brought about by elevating [K+]ext from 0.1 mM to 1.5 mM or higher (Fig. 2) is strong evidence that at least two pathways of NH4+ influx operate simultaneously, one sensitive and the other insensitive to [K+]ext. Because NH4+ influx under LATS conditions is high capacity and energetically passive (Fig. 6; also see section on energetics below), it is very likely that ion channels are responsible for catalysing both components of the flux.
More specifically, the present evidence suggests that the K+-sensitive pathway, which catalyses the greater amount of NH4+ influx, involves the operation of either non-selective cation channels (NSCCs) or inward rectifying K+ channels, such as AKT1. NSCCs comprise a large group of relatively uncharacterized transporters that have been shown to transport a variety of ions, including Na+, Ca2+, K+, and NH4+ (Demidchik et al., 2002b). NSCCs are inhibited by lanthanides, but tend to be insensitive to traditional K+ channel blockers, particularly Cs+ and TEA (Tyerman and Skerrett, 1999). In the present study, NH4+ influx at 0.1 mM [K+]ext was strongly inhibited by the relatively broad-spectrum channel blocker La3+, but no La3+ effect was observed on the K+-suppressed flux at 40 mM [K+]ext (Fig. 5). This is consistent with the idea that, at this supply level, K+ much more effectively competes for a La3+-sensitive NSCC pathway, making it unavailable for NH4+ transport. Evidence that K+ is indeed also transported through the La3+-sensitive pathway is seen in the inhibitory effect of La3+ on K+ influx (Fig. 5, inset; the inhibitory effect on the low-K+ control is most likely to be due to the suppression of high-affinity KUP/HAK/KT transporters by NH4+; see Spalding et al., 1999, and Introduction). The lack of inhibition of NH4+ influx by Cs+ and TEA is also consistent with permeation through NSCCs. Indeed, it is tempting to attribute K+-sensitive, La3+-sensitive NH4+ transport to the activity of the weakly voltage-dependent NSCC described by White and Lemtiri-Chlieh (1995), White (1996), and Davenport and Tester (2002), because this channel displays several physiological attributes strongly reminiscent of the characteristics defined here: transport of both NH4+ and K+ as competing substrates; relative insensitivity to TEA and Cs+; and strong inhibition by the lanthanide Gd3+ as well as by La3+ itself. The observation that, in the present study, TEA and Cs+ actually stimulated NH4+ influx at both K+ conditions tested (Fig. 5) is perhaps surprising, but supports the finding that both of these agents can increase Na+ influx (Wang et al., 2006), and TEA has also been shown to increase the influx of both Ca2+ (Demidchik et al., 2002a) and Cs+ (Hampton et al., 2004) in roots of Arabidopsis. In addition, both of the latter studies postulated that the transporters involved were NSCCs, and showed that Gd3+ was effective in inhibiting the TEA-stimulated fluxes, a result very similar to the La3+ suppression of TEA-stimulated NH4+ influx observed in the present study.
Many studies, in a variety of organisms including bacteria, yeast, animals, and plants, have suggested that NH4+ enters the cell through K+-specific channels (Wang et al., 1996; Nielsen and Schjoerring, 1998; Hess et al., 2006), and the K+ suppression of NH4+ influx (Fig. 2) supports the idea that K+ channels are responsible for the K+-sensitive component of low-affinity NH4+ uptake, instead of, or in addition to, NSCCs. At higher [K+]ext, these channels would be occupied by K+, limiting NH4+ influx to NH4+-specific pathways. Consistent with this observation was the finding that La3+ (known to block K+ channels as well as NSCCs; Wegner et al., 1994) blocked NH4+ influx at low [K+]ext, in addition to reducing K+ influx at both high and low [K+]ext (Fig. 5). However, the stimulation of NH4+ influx by the K+-channel blockers Cs+ and TEA does not support NH4+ permeation through K+ channels. Thus, the proposal that NSCCs are responsible for the K+-sensitive component of root NH4+ influx in the LATS range is more congruent with the present data. Low-affinity fluxes in shoots may be mediated by a different mechanism, as was shown by Nielsen and Schjoerring (1998), who observed in leaves of B. napus a 30% and 47% reduction in NH4+ influx with La3+ and Cs+ treatments, respectively.
K+-insensitive NH4+ influx pathway
In addition to K+-sensitive NH4+ conductance, a substantial portion of low-affinity NH4+ entry into barley root cells is mediated by a K+-insensitive mechanism (Fig. 2). This mechanism is resistant to increases in [K+]ext from 1.5 mM to 40 mM, suggesting that, because of the lack of a competitive effect, the transporter involved is neither a K+-specific channel nor an NSCC. Several other possibilities arise as to its molecular identity. One is that it is a high-affinity NH4+ transporter, such as AMT1 (Ninnemann et al., 1994; Rawat et al., 1999), which may have some dual-affinity character, such as has been seen for nitrate and potassium transporters (Fu and Luan, 1998; Liu et al., 1999). However, AMT1-mediated NH4+ transport is down-regulated by high NH4+, both genetically and functionally (Rawat et al., 1999), which eliminates its likelihood as a candidate for NH4+ influx under the NH4+ supply (10 mM) used in the present study. Another possibility is that NH4+ permeates via aquaporins. Several recent studies have shown that in addition to water, Xenopus oocytes expressing Arabidopsis TIP genes (encoding aquaporins) could mediate the transport of small molecules such as CO2, glycerol, urea, NH4+, and NH3 (Uehlein et al., 2007). However, Detmers et al. (2006) found that TEA effectively inhibited aquaporin-mediated water transport, while in the present study TEA failed to inhibit NH4+ influx, casting doubt on the role of aquaporins in low-affinity NH4+ transport. The elimination of these two candidates suggests that NH4+ enters root cells under high-K+, high-NH4+ conditions via NH4+-specific channels, the molecular identity of which remains to be determined.
Energetics of NH4+ and K+ unidirectional fluxes
It is instructive to examine, from an energetics perspective, the unidirectional NH4+ fluxes observed here. The thermodynamic analysis shows that, under all experimental conditions, NH4+ exchange across the plasma membrane takes the form of a ‘leak–pump’ scenario, i.e. with passive NH4+ influx coupled to active NH4+ efflux (Fig. 6). In this respect, bidirectional NH4+ transport follows a pattern that has been observed in the low-affinity exchange of other major cations, such as K+ (Szczerba et al., 2006a) and Na+ (Wang et al., 2006; Kronzucker et al., 2006). Based upon current models of ion transport (Kurimoto et al., 2004; Britto and Kronzucker, 2006), which consider the coupling and stoichiometry of ion fluxes in relation to proton fluxes and ATP hydrolysis, the respiratory costs of NH4+ and K+ transport weres estimated, and they were compared with measured respiration rates and growth (Fig. 7). In agreement with estimates indicating that, under certain conditions, as much as 70% of total root respiration can be invested in the transport of the NO3– anion (Scheurwater et al., 1999), it was found that as much as 64% of the measured respiration rates could be accounted for by the combined plasma membrane fluxes of the two cations NH4+ and K+. The plants that displayed rapid, futile cycling of NH4+ or K+ not only showed the highest respiration, but had significant reductions in total plant biomass (Fig. 7). This effect is attributed to the differential allocation of carbohydrate supply in the various treatments, with a greater proportion directed towards the wasteful process of futile ion cycling, in the case of the growth-compromised plants.
Concluding remarks
This study provides the first demonstration of the parallel operation of K+-sensitive and -insensitive root NH4+ fluxes in the low-affinity transport range, and offers insight into the mechanism by which K+ is able to alleviate NH4+ toxicity. Elevated K+ eliminates a major fraction of low-affinity NH4+ influx, and substantially reduces the amount of futile cycling of this toxic ion. Intriguingly, this effect contrasts sharply with the effect of NH4+ on K+ transport, where high-affinity influx is diminished by NH4+, but low-affinity influx remains unaffected (Spalding et al., 1999; Kronzucker et al., 2003b; Szczerba et al., 2006a). It is proposed that low-affinity NH4+ transport may be mediated by the dual operation of non-selective, K+-sensitive cation channels on the one hand, and K+-insensitive, NH4+-specific channels on the other. However, it should be pointed out that the present study does not rule out the existence of other mechanisms of low-affinity NH4+ transport, in addition to the two proposed here. The shift between low and high external [K+] steady states may entail the expression of a genetically and mechanistically distinct complement of transporters, a possibility that only extensive new genetic analyses can unravel. Nevertheless, the physiological observations presented here, that: (i) the NH4+ fluxes under both high- and low-K+ conditions show virtually the same degree of La3+ resistance (Fig. 5); and (ii) NH4+ influx is rapidly suppressed, when the low-K+ condition is suddenly altered to a high-K+ condition, almost to the same extent as observed at a high-K+ steady-state, strongly suggest that the La3+- and K+-insensitive component of low-affinity NH4+ influx is operative under all K+ conditions. This study demonstrates how pivotal a role K+ plays in the regulation of NH4+ toxicity, reducing the energy burden of toxic NH4+ fluxes, and substantially improving growth under a high-NH4+ nutritional regime.
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We thank Dr A Wilson and the staff at the Centre for Addiction and Mental Health (CAMH) in Toronto, Ontario, Canada, for supplying the 13NH4+, and M Butler and staff at McMaster University in Hamilton, Ontario, Canada, for supplying the 42K required to conduct these experiments. We also would like to thank S Ebrahimi and AB Vesterberg for assistance with experiments. The work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Research Chair (CRC) program, Canada's National Green Crop Network (NSERC), and the International Plant Nutrition Institute [formerly the Potash & Phosphate Institute (PPI)].
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