Complexity of potassium acquisition

Abstract

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 However, when external NH₄⁺ was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K⁺ influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K⁺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 potassium (K⁺)-selective Shaker-type channel, AKT1, has been shown to mediate both high- and low-affinity K⁺ acquisition in roots of higher plants,^1-3 and a sophisticated model involving its molecular regulation and relative contribution to K⁺ uptake has emerged, based largely on the work in the model system Arabidopsis (Arabidopsis thaliana L.).^4-7 According to this view, approximately 80% of high- and low-affinity K⁺ uptake in Arabidopsis can be attributed to the sum of functions of AtHAK5 (a member of the HAK/KUP/KT family of transporters)⁸ and AtAKT1, while the remaining ~20% is mediated by as yet unidentified components.⁵ Of special interest and the source of some controversy, has been the involvement of AtAKT1 in mediating K⁺ uptake from low (micromolar) external K⁺ concentrations ([K⁺]_ext) in the presence of high (millimolar) external ammonium (NH₄⁺).^1,9 Under such conditions, high-affinity K⁺ uptake is severely suppressed at the functional level, which, in Arabidopsis, has been directly linked to AtHAK5 inhibition.¹⁰ It has been shown that under such conditions, AtAKT1 can conduct the majority of K⁺ uptake from [K⁺]_ext as low as 10 µM.¹ However, such findings have been difficult to reconcile with thermodynamic considerations that suggest channel-mediated K⁺ acquisition at such low [K⁺]_ext is generally not feasible.^11,12 Our recent study¹³ showed that the Arabidopsis model of K⁺ acquisition is not a universally applicable one, and that, in particular, it may not apply to cereals. Our work demonstrated that in roots of intact barley (Hordeum vulgare L.) seedlings grown under high (10 mM) NH₄⁺, K⁺ channels could not conduct K⁺ acquisition when [K⁺]_ext was low (22.5 µM), but at intermediate (112.5 µM) [K⁺]_ext, channels operated jointly with high-affinity transporters. At high (5 mM) [K⁺]_ext, K⁺ channels dominated and stimulations of K⁺ influx by anions chloride (Cl⁻) and nitrate (NO₃⁻) were observed. K⁺ efflux was found to be channel-mediated at low and intermediate [K⁺]_ext, albeit with differing pharmacological profiles and no K⁺ efflux was found at high [K⁺]_ext. When external NH₄⁺ was withdrawn, significant stimulations in K⁺ influx were observed (176% increase compared with control), coincident with a thermodynamic shift from active to passive conditions that permitted channel-mediated K⁺ influx even at low [K⁺]_ext. This ammonium-withdrawal effect, termed AWE, was also observed in wild-type and mutant lines of Arabidopsis (athak5, atakt1 and athak5 atakt1). AWE was suppressed by high (1–5 mM) levels of external calcium (Ca²⁺) at low and intermediate, but not at high, [K⁺]_ext, which was attributed to Ca²⁺-sensitivity of AKT1. The effect was additionally enhanced by the presence of Cl⁻ and NO₃⁻ at high [K⁺]_ext, conditions under which we observed some of the highest trans-plasma-membrane K⁺ fluxes ever reported [~36 µmol g (root fresh weight)⁻¹ h⁻¹]. Furthermore, AWE was sustainable (and at times indeed increasing) over a 24 h period, at low and intermediate [K⁺]_ext and persisted for up to ~8 h at high [K⁺]_ext. This resulted in significant total-tissue K⁺ accrual at all [K⁺]_ext over 24 h. These results raise a suite of interesting questions about the nature of channel-mediated K⁺ influx in intact plants. Although we showed that K⁺ channels cannot “do it all,” as suggested by early thermodynamic considerations,¹¹ our work sheds light on the wide-ranging and complex operation of K⁺ channels in planta.

Here, we expand on our previous work by further characterizing channel-mediated K⁺ uptake in roots of barley. Figure 1 illustrates the varying sensitivity of AWE to Ca(NO₃)₂, which is clearly a function of [K⁺]_ext: Ca²⁺-induced suppressions are seen at low and intermediate [K⁺]_ext (Fig. 1A and B), while a NO₃⁻-induced stimulation is seen at high [K⁺]_ext (Fig. 1C). Interestingly, under all AWE + Ca(NO₃)₂ combinations, we observed effective suppression by the K⁺-channel inhibitors¹⁴ tetraethyl ammonium (TEA⁺), barium (Ba²⁺) and cesium (Cs⁺) (Fig. 1A–C). Thus, although Ca²⁺ can block K⁺ channels (specifically AtAKT1)¹³ under some AWE conditions, it appears that K⁺ channels continue to operate under such suppressed conditions, resulting in a higher flux compared with control (NH₄⁺-background) conditions (Fig. 1A and B). This is consistent with previous thermodynamic analyses¹³ that showed AWE + Ca(NO₃)₂ hyperpolarizes root plasma-membrane potentials away from the equilibrium potential for K⁺ (E_K⁺) at low and intermediate [K⁺]_ext. At high [K⁺]_ext, it is evident that NO₃^--induced stimulations in K⁺ influx are also linked to K⁺ channels (Fig. 1C), illustrating their complexity of function and regulation (also see below). Interestingly, these elevations in K⁺ influx compared with control (albeit minor at low and intermediate [K⁺]_ext) are sufficient to noticeably increase tissue-K⁺ content over 24 h at all three [K⁺]_ext (data not shown), similar to what AWE entails on its own.¹³

Figure 1. K⁺ influx into intact 7-d old barley (Hordeum vulgare L.) seedlings. (A–C) Steady-state (control) flux in plants grown in a full nutrient medium (0.25-strength Johnson’s solution)¹³ with 10 mM NH₄⁺ and either 0.0225 (A), 0.1125 (B) or 5 mM (C) K⁺ (as sulfate salts) and the effect of ammonium-withdrawal (AWE) ± Ca(NO₃)₂ (5 mM) and AWE + Ca(NO₃)₂ ± TEA⁺ (10 mM), Ba²⁺ (5 mM), or Cs⁺ (10 mM) (as chloride salts). All treatments involved 5 min incubation prior to ⁴²K⁺ uptake (5 min protocol).¹³ (D) Influx into plants grown in a full nutrient medium (as above) with 5 mM K⁺ and 10 mM NH₄⁺ (both as either SO₄^2- or Cl⁻ salts) and the effect of switching (K⁺ and NH₄⁺) counter-ions (10 min incubation prior to uptake, as above). Influx measured on a per g (root fresh weight) basis. Error bars indicate ± SEM of minimum four replicates. Letters in each panel denote significantly different means (p < 0.05, one-way ANOVA with Tukey post-hoc test).

Expanding on the anion effects observed at high [K⁺]_ext, we also found that switching the counterions for K⁺ and NH₄⁺ from sulfate (SO₄²⁻) to Cl⁻ (a 10 min treatment was used in SO₄²⁻grown plants) approximately doubles K⁺ influx (from ~10 to 20 µmol g⁻¹ h⁻¹) and the reverse scenario (switching from Cl⁻ to SO₄²⁻, in Cl⁻-grown plants), reduces influx by half (from ~20 to 10 µmol g⁻¹ h⁻¹, Fig. 1D). This suggests that anion effects related to K⁺-channel functioning are highly effective in both the short (10 min) and long (steady-state) term. These findings are consistent with early reports^15,16 demonstrating higher low-affinity K⁺ influx in the presence of Cl⁻ over SO₄²⁻, possibly as a result of a coupling of the more rapidly absorbed anion (Cl⁻) with K⁺ influx. Surprisingly, there has been little, if any, advance in understanding this phenomenon. It does, however, raise interesting questions about the mechanism and regulation of channel-mediated K⁺ influx. For one, when one considers that the fluxes of all anions tested (Cl⁻, SO₄²⁻and NO₃⁻) are coupled to H⁺ influx and are electrogenic (net positive),^17-19 anion fluxes (particularly those of NO₃⁻ and Cl⁻, which show high rates of uptake)²⁰ should theoretically decrease the gradient for K⁺ uptake, i.e. working against what is observed for rates of influx (Fig. 1C and D; refs. 15 and 16). Moreover, to our knowledge, there are no demonstrations of possible allosteric modulations of Shaker-type K⁺ channels by anions in the plant literature;^7,21 by contrast, some evidence exists in the animal literature²² that such allosteric modulation may occur. This warrants further investigation. Lastly, the reasons why Cl⁻/NO₃⁻-induced stimulations are only observed at high (low-affinity-range) [K⁺]_ext remain largely unknown. Reports on the allosteric modulation of Shaker-type K⁺ channels by [K⁺]_ext appear exclusive to outward rectifiers,²³ and studies on the post-translational modification (e.g., phosphorylation/dephosphorylation networks) of inward rectifiers are restricted to low-[K⁺]_ext sensing.^24-26 Thus, it is clear that as yet insufficiently understood mechanisms exist regarding the regulation of K⁺ channels by anions, a phenomenon that can have profound effects on rates of K⁺ acquisition.

We also observed that NH₄⁺ withdrawal (for 5 min) results in significant reductions in root respiration at low [K⁺]_ext (from ~30 to 20 µmol g⁻¹ h⁻¹, as measured by oxygen [O₂] consumption; Fig. 2). Based on earlier suggestions, this may be attributable to a reduction in futile NH₄⁺ cycling at root-cell plasma membranes, an energetically demanding scenario linked to NH₄⁺ toxicity in barley,²⁷ or other, more generic respiratory stress responses when NH₄⁺ levels are high. Since futile cycling of NH₄⁺ can be reduced by high [K⁺]_ext,²⁸ as evident in the significantly lower steady-state root respiration at high [K⁺]_ext (Fig. 2), this may explain the lack of any further respiratory drop upon NH₄⁺ withdrawal. Given the substantial increases in K⁺ influx (from ~10 to 25 µmol g⁻¹ h⁻¹) (Fig. 1C), it is interesting that no change in root respiration was observed, in keeping with the contention that the energy cost per K⁺ transported is significantly lower for channels than for high-affinity transporters,²⁹ i.e., plants already engaging channels may show no further significant energetic requirement upon NH₄⁺ withdrawal even when K⁺ influx is greatly enhanced. Thus, AWE carries no major cost to the plant, while a significant benefit in terms of net K⁺ accumulation in tissue is seen (see above). Indeed, at low [K⁺]_ext, barley expends significantly less energy than prior to NH₄⁺ withdrawal, while accruing large quantities of K⁺. This highlights an important feature of engaging channels episodically in the acquisition of potassium that may be of importance under fluctuating nutrient conditions,³⁰ especially in NH₄⁺-dominated systems that normally suppress K⁺ uptake,^31-33 which, integrated over an extended time frame, may result in significant growth benefits.³⁴

Figure 2. Steady-state (control) root O₂ consumption, as measured using a Hansatech oxygen electrode and Oxygraph control system (Hansatech Intruments), in intact barley (Hordeum vulgare L.) seedlings grown in a full nutrient medium ( Fig. 1) at 0.0225 or 5 mM K⁺ and 10 mM NH₄⁺ (both as sulfate salts) and the effect of sudden (5 min treatment) NH₄⁺ withdrawal (AWE). O₂ flux measured on a per g (root fresh weight) basis. Error bars indicate ± SEM of 4 replicates. Letters denote significantly different means (p < 0.05, one-way ANOVA with Tukey post-hoc test).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.