Plant Physiol, February 2001, Vol. 125, pp. 523-526

SCIENTIFIC CORRESPONDENCE

Cytosolic Concentrations and Transmembrane Fluxes of NH₄⁺/NH₃. An Evaluation of Recent Proposals

Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4 (D.B., A.D.M.G., M.Y.S.); and Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 (H.J.K.)

	INTRODUCTION

TOP INTRODUCTION CYTOSOLIC NH4+ CONCENTRATIONS PASSIVE DIFFUSION OF NH3... CONCLUSIONS LITERATURE CITED

Ammonium (NH₄⁺) is an important, perhaps underestimated, nitrogen source for plant growth (Bloom, 1988; Glass, 1988; Kronzucker et al., 1997). In many agricultural soils it is present in millimolar concentrations in soil solution (Wolt, 1994), whereas in mature forest soils NH₄⁺:NO₃ ratios as high as 56:1 have been reported (Stark and Hart, 1997). In many such soils, NH₄⁺ may constitute the only inorganic nitrogen source available to plants (Van Cleve et al., 1993). Physiological studies have demonstrated that NH₄⁺ fluxes commonly exceed those of NO₃ from equimolar solutions, and when both ions are present, NO₃ influx is reduced within minutes of exposure to NH₄⁺ (Lee and Drew, 1989; Gazzarini et al., 1999; Kronzucker et al., 1999). As a byproduct of photorespiration and the shikimic acid pathway, as well as through the turnover of various nitrogen pools, large quantities of NH₄⁺ are also generated internally (Joy, 1988; Feng et al., 1998). Although much of this NH₄⁺ is re-assimilated through the activity of Gln synthetase (GS), many plants show high values of NH₄⁺ efflux, and some develop symptoms of toxicity when exposed to millimolar concentrations of NH₄⁺ in nutrient media, particularly when NH₄⁺ is the sole source of nitrogen. The actual mechanism of NH₄⁺ toxicity is still unclear, despite extensive studies (Gerendas et al., 1997). For these reasons it is important to characterize subcellular compartmentation and fluxes of NH₄⁺.

Because of the potential toxicity of NH₄⁺/NH₃, it is often assumed that plant cytosolic NH₄⁺ concentrations ([NH₄⁺]_c) are maintained at very low (sub-millimolar) levels, through the high activity and high affinity of GS for NH₄⁺ (Pearson and Stewart, 1993; Gerendas et al., 1997). This is despite the fact that millimolar values for [NH₄⁺]_c have been measured using NMR, efflux analysis, ion-specific microelectrodes, and tissue fractionation (Fentem et al., 1983; Lee and Ratcliffe, 1991; Wang et al., 1993a; Wells and Miller, 2000), and that large plasma membrane fluxes of NH₄⁺ have been reported (Lee and Ayling, 1993; Wang et al., 1993b; Min et al., 1999). Nevertheless, in a recent review of NH₄⁺ transport, Howitt and Udvardi (2000) conclude that some estimates of [NH₄⁺]_c are probably orders of magnitude too high. First, they argue that "it is difficult to reconcile the estimates of cytosolic NH₄⁺ concentrations made by this group (referring to the data of Wang et al., 1993a) with the high affinity of cytosolic GS for NH₄⁺ (K_m = 10-20 µM)." Second, they cite the findings of a single NMR study by Roberts and Pang (1992) who estimated [NH₄⁺]_c to be 3 to 10 µM for external [NH₄⁺] ([NH₄⁺]_o) up to 1 mM. Howitt and Udvardi (2000) go on to suggest that both influx and efflux of NH₃ are mediated by passive diffusion through a reversible low-affinity transport system (LATS), previously considered to be an NH₄⁺ transporter. Finally, they suggest that NH₄⁺ entry to the vacuole is via passive permeation of NH₃ and acid trapping of NH₄⁺. These speculations are at variance with a large body of evidence in the literature and therefore need to be critically examined.

	CYTOSOLIC NH4+ CONCENTRATIONS

TOP INTRODUCTION CYTOSOLIC NH4+ CONCENTRATIONS PASSIVE DIFFUSION OF NH3... CONCLUSIONS LITERATURE CITED

Howitt and Udvardi (2000) suggest that [NH₄⁺]_c must be considerably lower than the 3.6 mM estimated by Wang et al. (1993a) for plants grown at low external [NH₄⁺] because of the reported low K_m values for NH₄⁺ (10-20 µM) of GS. However, it must first be emphasized that K_m values of enzymes determined in vitro might be very different from those operating in vivo because of regulation by allosteric effectors. Furthermore, the extensively studied glycolytic enzymes have K_m/[substrate]_c ratios that vary from 0.02 to 333 (Fersht, 1985), and similar deviations of K_m/[substrate]_c from unity can be found for the enzymes nitrate reductase and pyruvate kinase. In the first case, K_m/[NO₃^-]_c can be as low as 0.01 (Lee and Clarkson, 1986; Siddiqi et al., 1991; Miller and Smith, 1992; Kanayama et al., 1999), whereas in the second, the ratio can be as low as 0.02 (Memon et al., 1985a, 1985b; Walker et al., 1996).

Furthermore, catalytic rates of enzymes that are subject to allosteric regulation are typically determined by the concentration of regulatory molecules rather than by substrate concentration. For example, the hexokinase of erythrocytes has a K_m value of 0.1 mM for Glc, despite a cellular Glc concentration around 5 mM. Hexokinase activity is not regulated by the available Glc but as a result of allosteric inhibition from its product Glc-6-phosphate, with the result that large changes of Glc concentration hardly alter the rate of glycolysis (Fersht, 1985). GS is also known to be a highly regulated enzyme, located at a pivotal biochemical position to regulate plant nitrogen assimilation (Eisenberg et al., 2000), and available data indicate that [NH₄⁺]_c can vary considerably even in the same system under different conditions of [NH₄⁺]_o supply (Lee and Ratcliffe, 1991; Roberts and Pang, 1992; Wang et al., 1993a; Kronzucker et al., 1995). The rationale that an enzyme's K_m should necessarily track ambient substrate concentration to optimize reaction rate, therefore, fails to apply to such enzymes.

Roberts and Pang (1992), cited by Howitt and Udvardi (2000) in support of micromolar values of [NH₄⁺]_c, incubated excised maize root tips in solutions containing from 0 to 10 mM NH₄⁺, and used NMR signals from ³¹P and ¹³C to estimate cytosolic and vacuolar pH. [NH₄⁺]_c was then calculated from total root NH₄⁺ to be from 2 to 438 µM (the upper limit being already 20 times higher than the reported GS K_m for NH₄⁺), on the presumption that NH₃ rapidly equilibrated across the tonoplast and was trapped as NH₄⁺ according to cytosolic and vacuolar pH values. In contrast, Lee and Ratcliffe (1991) used a more direct ¹⁴N-NMR method to obtain [NH₄⁺]_c of 3 to 8 mM, almost 3 orders of magnitude higher than the Roberts and Pang results, when comparing values obtained at the same [NH₄⁺]_o. All other methods (tissue fractionation, efflux analysis, and NH₄⁺-specific microelectrodes) have yielded [NH₄⁺]_c values in the millimolar range (Kronzucker et al., 1995, and references therein; Wells and Miller, 2000).

	PASSIVE DIFFUSION OF NH3 THROUGH AN LATS?

TOP INTRODUCTION CYTOSOLIC NH4+ CONCENTRATIONS PASSIVE DIFFUSION OF NH3... CONCLUSIONS LITERATURE CITED

Transport of NH₄⁺ across the plasma membrane is biphasic (Ullrich et al., 1984; Wang et al., 1993b), corresponding to an active, saturable, high-affinity transport system and a passive, non-saturable LATS. Howitt and Udvardi (2000), however, propose that LATS transport occurs via a reversible NH₃ transporter. In support of this proposal, the authors cite a study of ¹⁴C-methylamine (CH₃NH₂/CH₃NH₃⁺) uptake by Phaseolus vulgaris leaves (Raven and Farquhar, 1981), but in fact this study showed that the relationship of influx to external pH and the equilibrium concentration of methylamine were "far higher than could be explained by the transport of CH₃NH₂ alone." Raven and Farquhar concluded that methylamine uptake, at least at pH values below 7, was predominantly as CH₃NH₃⁺, and driven by the membrane electrical potential difference ().

Howitt and Udvardi go on to claim that both NH₃ influx and efflux in roots, as well as leaves, probably occurs by diffusion through a reversible LATS, citing studies by Wang et al. (1993a) and Kronzucker at al. (1995). In fact, neither of these studies claimed that the efflux of ¹³N was in the form of ¹³NH₃ because it is not possible to distinguish between the efflux of NH₃ and NH₄⁺. Any NH₃ effluxing from cells into the external solution would immediately be protonated in the relatively acidic cell wall compartment (see Fig. 1, A and B), and thus all NH₄⁺/NH₃ leaving the cytosol will "appear" as NH₄⁺.

View larger version (30K): [in this window] [in a new window]   Figure 1.   A, Directions of passive NH4+ (dotted arrows) and NH3 (solid arrows) fluxes as functions of external [NH4+], (all concentrations are in µmol L1), calculated from the Nernst equation, with cytosolic [NH4+] set at 10 µM and vacuolar [NH4+] at 20,000 µM. External [NH4+] was set at values from 10 to 10,000 µM. The pKa for dissociation of NH4+ was set at 9.32; external, cytosolic, and vacuolar pH values were set at 5, 7, and 5, respectively; plasma membrane and tonoplast were set at 100 and +10 mV, respectively. B, As in Figure 1A, except that cytosolic [NH4+] is set at 10,000 µM.

A further prediction arising from the suggestion that NH₃ efflux occurs passively via LATS is that NH₃ efflux should diminish as [NH₄⁺]_o (and thus [NH₃]_o) increases. However, measurements of ¹³N tracer efflux have demonstrated that this flux increased from approximately 10% of influx to as high as 86% of influx as [NH₄⁺]_o is increased from 10 or 100 µM to 1 or 1.5 mM (Wang et al., 1993a; Kronzucker et al., 1995; Min et al., 1999). Such findings are inconsistent with passive efflux of NH₃.

PLASMA-MEMBRANE FLUXES OF NH₄⁺/NH₃: A THERMODYNAMIC EVALUATION

To evaluate the hypotheses of Howitt and Udvardi, we have constructed a model for the directions of passive fluxes of NH₃ and NH₄⁺ (Fig. 1, A and B), assuming [NH₄⁺]_c of 10 µM (Howitt and Udvardi, 2000) or 10,000 µM (Wang et al., 1993a). In this model, [NH₄⁺]_o was varied from 10 to 10,000 µM, pH values were set at 5, 7, and 5, respectively, for external solution, cytosol, and vacuole, values of 100 and +10 mV were selected for plasma membrane and tonoplast, respectively, (Wang et al., 1994), and [NH₄⁺]_v was set at 20,000 µM. This value was selected to represent a midpoint of a quite wide range of literature values (Lee and Ratcliffe, 1991; Roberts and Pang, 1992; Wang et al., 1993a; Wells and Miller, 2000). The quantitative analyses of the distribution of NH₄⁺/NH₃, and directions of passive fluxes of these molecules that follow, are therefore based upon realistic values of pH, [NH₄⁺]_o, , and [NH₄⁺]_v. These values may vary somewhat under natural conditions but differences corresponding with orders of magnitude are unlikely.

The model predicts that when [NH₄⁺]_c is 10 µM (Fig. 1A), influx of NH₄⁺ is passive until [NH₄⁺]_o falls below approximately 100 nM, a rare situation in most soils. Active influx of NH₄⁺ would therefore be virtually unnecessary. This is a surprising conclusion given the abundant physiological and molecular evidence suggesting that the high-affinity transport system actively transports NH₄⁺ at [NH₄⁺]_o of up to 655 µM (Ullrich et al., 1984; Wang et al., 1994; Gazzarini et al., 1999). In contrast, when [NH₄⁺]_c is 10,000 µM (Fig. 1B), active influx of NH₄⁺ is required in the range of [NH₄⁺]_o  500 µM. This corresponds with the [NH₄⁺]_o at which a break between high- and low-affinity influx systems (Ullrich et al., 1984; Wang et al., 1993b; Kronzucker et al., 1996) and changes in membrane depolarization are observed (Wang et al., 1994). Figure 1A also indicates that if [NH₄⁺]_c is 10 µM, passive NH₃ influx is not energetically feasible unless [NH₄⁺]_o is 1 mM. It is entirely impossible if [NH₄⁺]_c is 10 mM (Fig. 1B), because the gradient for NH₃ is always from cytosol to cell wall. In this regard, it is instructive to examine the effects of pH on ¹³NH₃/¹³NH₄⁺ influx. Published data reveal that low-affinity putative ¹³NH₄⁺ fluxes declined from 18.63 to 11.44 µmol g¹ h¹ as external pH increased from 4.5 to 7.5 in rice roots (Wang et al., 1993b). Thus, despite a 1,000-fold increase of [¹³NH₃]_o, tracer influx actually declined by 40%; a similar decline of ¹³NH₄⁺ fluxes between pH 5 and 7, was observed by Kosegarten et al. (1997) using rice roots. The results argue against NH₃ influx at typical values of soil pH. This is not to suggest that NH₃ influx may not occur as the pK_a for NH₃/NH₄⁺ (9.32) is approached or exceeded. However, this situation is rare in most soils.

VACUOLAR FLUXES OF NH₃/NH₄⁺

With respect to vacuolar accumulation of NH₄⁺, Howitt and Udvardi (2000) propose that the flux of NH₄⁺ from cytosol to vacuole must be active. This is confirmed in our model for conditions where [NH₄⁺]_c is 10 µM, or when [NH₄⁺]_c is 10 mM and vacuolar [NH₄⁺] ([NH₄⁺]_v) exceeds 5 mM. However, the authors consider it more likely that passive transport of NH₃ across the tonoplast, and acid trapping in the vacuole, provide the mechanisms for vacuolar NH₄⁺ accumulation. This situation might apply when [NH₄⁺]_v is 1 mM, corresponding to an [NH₃]_v  0.048 µM. Above this value, however, as shown in Figure 1A, the gradient for NH₃ permeation across the tonoplast is in the opposite direction (from vacuole to cytosol), requiring active transport of NH₃ to the vacuole. The data reported by Lee and Ratcliffe (1991), Roberts and Pang (1992), and Wang et al. (1993a) provide [NH₄⁺]_v values that are >1 mM. This leads to the unlikely scenario whereby transport of NH₃ to the vacuole requires an active NH₃ flux, not a passive flux as they propose. Together with a passive leak of NH₃ in the opposite direction, this scenario would result in a futile cycling of NH₃ between cytosol and vacuole. In contrast, when [NH₄⁺]_c = 10,000 µM (Fig. 1B), the flux of NH₃ to the vacuole will always be passive, and remobilization of NH₄⁺ to the cytosol is also passive.

	CONCLUSIONS

TOP INTRODUCTION CYTOSOLIC NH4+ CONCENTRATIONS PASSIVE DIFFUSION OF NH3... CONCLUSIONS LITERATURE CITED

[NH₄⁺]_c values in the millimolar range are confirmed by four different methodologies.

The single literature value for [NH₄⁺]_c that conforms to a micromolar value (Roberts and Pang, 1992) is predicted from measurements of cellular pH and total-tissue [NH₄⁺] in roots, rather than being based upon direct measurements of [NH₄⁺]_c.

Conjectures regarding cytosolic [ion] based upon in vitro measurements of enzyme K_m values are untenable.

If [NH₄⁺]_c were 10 µM, then (a) Active transport of NH₄⁺ would be unnecessary, unless [NH₄⁺]_o falls below 100 nM; (b) Passive influx of NH₃ is feasible at [NH₄⁺]_o  1000 µM, but the pH profile for ¹³N tracer influx appears to contradict this possibility; (c) Passive efflux of NH₃ is thermodynamically feasible, but appears to be contradicted by the observation that ¹³N efflux increases as [NH₄⁺]_o (and hence [NH₃]_o) is increased; and (d) If [NH₄⁺]_c is 10 µM, NH₃ can only traverse the tonoplast passively as long as [NH₄⁺]_v  1 mM, an unlikely situation when NH₄⁺ is available in external media.

	FOOTNOTES

Received September 27, 2000; accepted November 8, 2000.

^* Corresponding author; e-mail aglass{at}interchange.ubc.ca; fax 604-822-6089.

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TOP INTRODUCTION CYTOSOLIC NH4+ CONCENTRATIONS PASSIVE DIFFUSION OF NH3... CONCLUSIONS LITERATURE CITED

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