Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X-QUAC, the main anion conductance in xylem-parenchyma cells of barley roots


  • Barbara Köhler,

    Corresponding author
    1. Universität Potsdam, Institut für Biochemie und Biologie, Lehrstuhl für Molekularbiologie, Karl-Liebknecht-Str. 25, Haus 20, 14476 Golm, Germany.
      *For correspondence (fax +49 331 977 2512; e-mail
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  • Lars H. Wegner,

    1. Biozentrum, Am Hubland, 97074 Würzburg, Germany.
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  • Viktor Osipov,

    1. Albrecht-von-Haller-Institut für Pflanzenwissenschaften der Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany.
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  • Klaus Raschke

    1. Albrecht-von-Haller-Institut für Pflanzenwissenschaften der Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany.
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*For correspondence (fax +49 331 977 2512; e-mail bakoehl@rz.uni.potsdam.de)


We report here that NO3 in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around − 40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mm Cl in the bath was replaced by NO3, a shift in the midpoint potential of the depolarisation-activated gate by about − 60 mV from 43 to − 16 mV occurred (Km = 3.4 mm). No such effect was seen when chloride was replaced by malate. Addition of 10 mm NO3 to the pipette solution and reduction of [Cl] from 124 to 4 mm (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO3 effect on gating was considered, an increase of the NO3 concentration in the xylem sap to 5 mm would result in an enhancement of NO3 efflux by about 30%. Although the driving force for NO3 efflux would be reduced simultaneously, NO3 efflux into the xylem through X-QUAC would be maintained with high NO3 concentrations in the xylem sap; a situation which occurs for instance during the night.


The xylem conduits form the main pathway for mineral nutrients from the root to the shoot. It has been shown, that xylem loading is a separate control point for transport of nutrients into the shoot (Herdel et al., 2001; Schurr, 1999; and references therein). Therefore analysis of the underlying mechanisms will identify basic principles in nutrient relations in whole plants and contribute to the understanding of plant growth (Marschner et al., 1996; Schurr, 1999). To date, research into xylem loading has been limited, or as Glass et al. (2001) put it: ‘We are still woefully ignorant concerning the physiology and molecular biology of transport to the vacuole, to the stele and into leaf cells'. Evidence has been obtained that the simultaneous release of anions and cations across the plasma membrane of xylem-parenchyma cells into the xylem occurs through ion channels, which are controlled by membrane voltage (Gaymard et al., 1998; Köhler and Raschke, 2000; Roberts, 1998; Roberts and Tester, 1995; Wegner and DeBoer, 1997a, 1999; Wegner and Raschke, 1994). Channels as specific transporters could provide an important target for control processes that balance nutrient transport into the shoot. While detailed studies have been performed on the electrical properties and the control of K+ channels (Roberts, 1998; Roberts and Tester, 1995; Wegner and De Boer, 1997a, 1999; Wegner and Raschke, 1994), little is known about control of anion channels in the plasma membrane of this cell type. Anion channels have been characterized in a variety of plant cells, including guard cells, where anion efflux during stomatal closure occurs through them (Barbier-Brygoo et al., 2000; Linder and Raschke, 1992; Piñeros and Kochian 2001; Schmidt et al., 1995; Tyerman, 1992; Zhang et al., 2001). Recently we have identified three types of anion conductances in xylem-parenchyma cells from barley roots, which differed vastly in their kinetics, voltage dependence and frequency of appearance in whole-cell recordings (Köhler and Raschke, 2000). X-QUAC was proposed to provide the main pathway for NO3 and other anions into the xylem sap, because (i) X-QUAC is highly permeable to NO3, (ii) X-QUAC, like KORC, which serves K+ loading into the xylem, was active preferably at low cytosolic Ca2+ concentrations (150 nm), and (iii) X-QUAC had by far the highest transport capacity among the anion conductances, matching reported Cl transport rates (Köhler and Raschke, 2000). Therefore we focused on the control of X-QUAC.

In this publication we report first the use of inhibitors to recognise and subtract spurious currents carried by ions other than anions and to describe the voltage dependence of X-QUAC in particular with respect to the hypothesis that X-QUAC possesses two gates. Then the effect of NO3 on X-QUAC was studied against the background of X-QUAC behaviour in the presence of Cl as the major anion and using NO3 in the solutions to get closer to physiological conditions. NO3 is the major anion exported into the xylem (e.g. Engels and Marschner, 1993). In barley plants well supplied with NO3 throughout their growth, the vacuoles of leaf cells contain almost exclusively NO3 as anion (T.A. Cuin and R.A. Leigh, personal communication). This is likely the result of an increased uptake of NO3 into the symplast of the root and a preferred transfer of NO3 to the xylem. We found that NO3 in the xylem changed the voltage dependence of X-QUAC and we discuss a balancing mechanism for NO3 delivery to the xylem.


Inhibition of X-QUAC by DIDS and IAA-94

In whole-cell patch-clamp recordings, large inward and outward currents were elicited by positive- and negative-going voltage pulses. Despite precautions to eliminate interference by other conductances, reversal potentials of current-voltage relations often deviated from the Nernst potential of Cl: This indicated activity of remaining background conductances like those of electrogenic pumps and Ca2+ channels, particularly in a voltage range in which the membrane exhibited a low overall conductance. To separate X-QUAC from the background conductances we made use of anion channel inhibitors. X-QUAC was inhibited by DIDS (4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid, n = 6) and IAA-94 (Indanyloxyacetic acid 94, n = 4) (Figure 1). The inhibition of X-QUAC was complete after about 3–4 min of perfusion of the bath with one of the blockers. Identical pulse protocols were applied before and after the addition of 100 µm DIDS (Figure 1a,b) or IAA-94 (not shown), respectively. The difference of the currents recorded 10 ms after imposing the voltage step before and after the addition of the inhibitor was assumed to represent the current-voltage curve of X-QUAC (Figure 1c). This approach required that the block was voltage-independent. Indeed, in some experiments with large, rapidly activating anion currents in the absence of either one of the blockers, DIDS or IAA-94 reduced currents to almost zero except at extreme negative or positive voltages (e.g. Figure 1b), indicating complete blockade of X-QUAC in the voltage range required for a quantitative analysis of the data (see below). Similarly, DIDS proved to be an effective blocker of the quickly activating anion channel in guard cells. In these cells, the block was voltage independent (Kd = 0.2 µm, Marten et al., 1993). Inhibition of X-QUAC by DIDS and IAA-94 was irreversible. The difference current-voltage relations obtained with DIDS and IAA-94 were virtually identical (Figure 1c). They were typical for X-QUAC under these conditions with large inward currents negative of − 40 mV, outward currents in the positive voltage range, and a negative slope conductance between 0 and − 40 mV resulting in a concavity (compare Köhler and Raschke, 2000). The direction of currents reversed close to the Nernst potential of Cl.

Figure 1.

Inhibition of X-QUAC by anion channel blockers.

(a) Current responses of X-QUAC without and with 100 µm DIDS added to the bath. From a holding potential of − 53 mV, the voltage was clamped for 100 ms from − 193 to 97 mV in 10 mV increments. Solutions: Bath, 30 mm TEACl, 5 mm Ca(Glc)2, 2 mm MgCl2, 10 mm Mes, pH 5.8, 500 mosmol kg−1; Pipette, 120 mm TEACl, 150 nm free Ca2+, 10 mm EGTA, 5 mm ATP, 2 mm MgCl2, 10 mm Tris, pH 7.2, 530 mosmol kg−1, 1 µm okadaic acid (a phosphatase inhibitor, added to test for a possible requirement for protein phosphorylation of X-QUAC to become active; no effect of okadaic acid on X-QUAC was observed). Filter frequency 500 Hz.

(b) Corresponding current-voltage relationships of X-QUAC in the absence (•) and presence of 100 µm DIDS (○). Inset: Detail to monitor the shift of the reversal potential.

(c) Difference of the curves before and after addition of DIDS (▾, calculated from data shown in (a) and (b)) and IAA-94 (▿).

Two putative gates with opposite voltage dependence

To quantify the voltage dependence of X-QUAC, chord conductances were calculated from the difference current-voltage relations as described in Experimental procedures. Ohmic unitary conductance was tested by plotting the amplitude of tail currents recorded after depolarising or hyperpolarising pulses as a function of membrane voltage (Figure 2a). The resulting curves were linear even though an asymmetric Cl gradient was imposed, indicating that the non-linear features of the current-voltage relation were mainly due to voltage dependent gating of X-QUAC. Instantaneous current-voltage relations following both hyperpolarising and depolarising pre-pulses reversed close to ECl-, indicating that both inward and outward X-QUAC currents were carried by this ion (confirming Köhler and Raschke, 2000).

Figure (a) .

Ohmic unitary conductance of X-QUAC.

The amplitudes of tail currents from − 173 to 87 mV, recorded from double-pulse protocols, were plotted against the voltage. Tail currents (inset) were recorded after activation of X-QUAC by 1-s-pulses to 67 mV (○) or to − 143 mV (□). Linear (ohmic) relationships were obtained in both cases (○, m = 0.86, r2 = 0.99, □, m = 0.13, r2 = 0.98). Due to an interference with the fast activation of the depolarisation-activated gate, data for the hyperpolarisation-activated gate were only collected up to − 40 mV and then extrapolated (dashed line). Standard solutions. Bars, horizontal: 100 ms, vertical: 400 pA.

(b) Chord conductance of X-QUAC calculated from difference current-voltage relations obtained with 100 µm DIDS (solid symbols, 6 experiments) or IAA-94 (open symbols, 4 experiments). Each symbol represents an independent experiment. Data were normalized with respect to the value measured at 67 mV to facilitate comparison of data obtained with different protoplasts. Equation 3 was fitted to the data (continuous line; fit parameters: Gd = 1.61 ± 0.03 (rel. units), Ud0.5 = 58 ± 1 mV, δd = 1.3 ± 0.06, Gh = 0.12 ± 0.05 (rel. units), Uh0.5 =− 84 ± 34 mV, δh = 0.82 ± 0.52, for abbreviations, see Experimental procedures). Data were only plotted down to − 140 mV since inhibition of X-QUAC may have been incomplete negative of that value (Figure 1b).

Chord conductances, calculated from difference current-voltage relations obtained with DIDS (n = 6) and IAA-94 (n = 4) (as in Figure 1c), increased strongly from a minimum of almost zero at about − 40 mV by a shift towards more positive, and also slightly towards more negative potentials (Figure 2b). We interpreted this kind of voltage dependence in terms of two gates with opposite voltage dependence existing in X-QUAC, and therefore described the behaviour of X-QUAC by a sum of two Boltzmann equations (Equation 3, Figure 2b). Voltage dependence of the gates is indicated by the midpoint potentials (U0.5) that denote the voltage at which 50% of the maximum conductance (G) is established. The midpoint potential was around 50 mV for the depolarisation-activated gate, and around − 85 mV for the gate opened by membrane hyperpolarisation (Figure 2b). Generally, analysis of X-QUAC gating at membrane hyperpolarisation was less precise than at depolarisation because currents were relatively small. A comparison of the G-values indicates that depolarisation makes the membrane about 10 times more permeable to Cl than hyperpolarisation. Absolute G-values for the depolarisation-activated gate ranged between 0.3 and 0.98 mS cm−2, and for the hyperpolarisation-activated gate it ranged between 0.04 and 0.07 mS cm−2. For a process of simple electrodiffusion (as described by the Goldman-Hodgkin-Katz equation), the opposite would have been predicted from the Cl gradient imposed on the membrane, indicating that other processes are involved in Cl permeation through X-QUAC besides simple electrodiffusion.

In the majority of the experiments in which X-QUAC dominated the membrane conductance and the reversal potential was close to the Nernst potential of Cl, analysis of X-QUAC gating properties did not require correction for background conductances. This approach rendered results similar to those obtained with the difference current-voltage technique (see superimposed curve in Figure 3). Midpoint potentials were 43 ± 1 mV for the depolarisation-activated gate and − 84 ± 12 mV for the hyperpolarisation-activated gate. The slightly less positive Ud0.5 value can be explained by the presence of a linear leak current. The absolute G-values ranged from 0.3 to 2.5 mS cm−2 at membrane depolarisation and from 0.04 to 0.46 mS cm−2 at membrane hyperpolarisation.

Figure 3.

Effect of external NO3 and malate on the voltage dependence of X-QUAC. Chord conductances per membrane surface area were measured on a protoplast after 30 mm TEACl in the bath was replaced by TEANO3 (•) or (TEA)2malate (○). Equation 3 was fitted to the data (continuous lines; fit parameters: •, Gd = 1.83 ± 0.04 mS cm−2, =− 3 ± 1 mV, δd = 1.48 ± 0.08, Gh = 0.17 ± 0.04 mS cm−2, = − 90 ± 12 mV, δh = 1.75 ± 1.1, and ○, Gd = 0.69 ± 0.02 mS cm−2, = 41 ± 2 mV, δd = 0.93 ± 0.03, Gh = 0.24 ± 0.01 mS cm−2, = − 85 ± 2 mV, δh = 1.23 ± 0.11). For comparison, the fitted curve, which had been obtained with 30 mm TEACl in the bath, was superimposed (fit parameters: Gd = 1.33 ± 0.03 (rel. units), = 43 ± 1 mV, δd = 1.26 ± 0.05, Gh = 0.17 ± 0.03 (rel. units), = − 84 ± 12 mV, δh = 1.03 ± 0.31). Midpoint potentials are indicated by perpendicular dotted lines. The horizontal arrow indicates the displacement of the midpoint potential by NO3. Bath, 30 mm TEANO3 or (TEA)2malate, 5 mm Ca(Glc)2, 2 mm MgCl2, 10 mm Mes, pH 5.8, 500 mosmol kg−1; Pipette, 120 mm TEACl, 10 mm EGTA, 150 nm free Ca2+, 2 mm ATP, 2 mm MgCl2, 10 mm Tris, pH 7.2, 530 mosmol kg−1.

The depolarisation-activated gate of X-QUAC is affected by external nitrate, but not by malate or potassium ions.

External NO3 strongly affected the voltage dependence of X-QUAC. Substituting NO3 for Cl in the bath resulted in a shift of the voltage dependence of the depolarisation-activated gate (Figure 3, •). The midpoint potential went negative by about − 60 mV (from 43 ± 1 mV (n = 8) to − 16 ± 5 mV (n = 4)). The gating charge was not affected, nor was there a clear effect on the hyperpolarisation-activated gate. The corresponding fit parameters were δd = 1.46 ± 0.1, Uh0.5 = − 85 ± 5 mV, and δh = 1.46 ± 0.3 (n = 4). Tail currents under these conditions displayed an ohmic voltage dependence (not shown). Remarkably, the maximum conductances of the two gates differed by about the same factor under standard conditions and after replacing external chloride by NO3. G-values ranged between 0.3 and 1.83 mS cm−2 (Gd) and 0.03 and 0.17 mS cm−2 (Gh).

The effect on gating could mostly be reversed when NO3 was replaced by malate (Figure 3, ○). Fit parameters were Gd = 1.29 ± 0.14 (rel. units), Ud0.5 = 30 ± 8 mV, δd = 0.84 ± 0.24, Gh = 0.39 ± 0.05 (rel. units), Uh0.5 =− 59 ± 15 mV, and δh = 1.25 ± 0.76 (n = 6). Absolute G-values ranged between 0.09 and 0.69 mS cm−2 (Gd) and between 0.04 and 0.27 mS cm−2 (Gh). The values for Ud0.5 were clearly more positive than those obtained with NO3 solution in the bath (Figure 3). Like the Ud0.5 values obtained with Cl or NO3 in the bath, they were significantly different, whereas the midpoint potentials obtained with Cl or malate in the bath were not (t-test, P = 0.05). The deviation of the midpoint potentials for the hyperpolarisation-activated gate was statistically not significant. In addition to a positive shift in U0.5 for the depolarisation-activated gate, replacement of NO3 by malate also caused a decrease in Gd from 1.83 to 0.66 mS cm−2 (Figure 3). Taking into account the fact that malate was in its divalent form, a relative permeability for malate versus NO3 of 0.18 could be calculated from the Gd values from this experiment, compared with a value of about 0.08 determined from reversal potentials (Köhler and Raschke, 2000).

The importance of the NO3 effect on X-QUAC for the physiology of xylem loading was tested with pipette solutions containing 4 mm Cl and 10 mm NO3, which resemble physiological anion concentrations (Miller and Smith, 1996). With NO3 on both sides of the membrane, the current-voltage curve intersected the voltage axis at − 27 ± 3 mV (n = 5) close to the Nernst potential of NO3 (ENO3- = − 28 mV). The deviation of the two curves in the positive voltage range was again due to a shift in the voltage dependence of gating induced by external NO3 (Figure 4a, inset). The composition of the pipette medium had no effect on the voltage dependence of the depolarisation-activated gate (compare data recorded with identical 30 mm NO3 medium in the bath, but different pipette solutions in Figure 3 and Figure 4a, respectively). The midpoint potential was − 16 ± 5 mV (n = 4) with internal 124 mm Cl and − 17 ± 7 mV (n = 4) for the more ‘physiological’ pipette solution. The other parameters were δd = 1.37 ± 0.1, Uh0.5 = − 69 ± 4 mV, δh = 1.9 ± 0.2. G values ranged between 0.02 and 0.22 mS cm−2 (Gd) and between 0.008 and 0.04 mS cm−2 (Gh). No attempt was made to fit the data with 6 mm chloride in the pipette and standard bath medium (34 mm Cl) in Figure 4a, because the membrane had not been depolarized sufficiently to observe maximum conductance. For the hyperpolarisation-activated gate, the midpoint potential was slightly more positive with the ‘physiological’ pipette solution than with standard pipette medium. However, this difference was statistically not significant.

Figure 4.

Voltage dependence of X-QUAC recorded with ‘physiological’ pipette media.

(a) Current density versus voltage plot of X-QUAC in the presence of TEACl (•) or TEANO3 (○) in the bath. The arrows indicate the Nernst potentials of Cl and NO3, respectively. Bath, 30 mm TEANO3 or TEACl, 5 mm Ca(Glc)2, 2 mm MgCl2, 10 mm Mes, pH 5.8, 500 mosmol kg−1; Pipette, 10 mm TEANO3 (○) or 1 mm NMGCl (•), respectively, 150 nm free Ca2+, 2 mm ATP, 2 mm MgCl2, 10 mm EGTA, 10 mm TRIS, pH 7.2, 530 mosmol kg−1. Inset, chord conductances per membrane surface area calculated from the current-voltage curves shown in (a). Data with NO3 in the bath (○) were fitted with Equation 3 (fit parameters: Gd = 0.13 ± 0.005 mS cm−2, Ud0.5 = − 24 ± 8 mV, δd = 1.49 ± 0.47, Gh = 0.03 ± 0.004 mS cm−2, Uh0.5 = − 63 ± 24 mV, δh = 1.93 ± 1.9).

(b) Current density versus voltage curve of a protoplast with 30 mm NO3 in the bath (▵ solutions as in a) and after lowering the external NO3 concentration to 5 mm (○). Ionic strength was maintained by adding 25 mm TEAgluconate. In the inset, chord conductances normalized to 40 mV at 5 mm NO3 in the bath calculated from this (○) and two other independent experiments is shown. Equation 3 was fitted to the data (continuous line, fit parameters: Gd = 1.4 ± 0.24 (rel. units), Ud0.5 = 5 ± 11 mV, δd = 0.8 ± 0.34, Gh = 0.38 ± 0.11 (rel. units), Uh0.5 =− 75 ± 22 mV, δh = 1.2 ± (1). Absolute G-values ranged between 0.016 and 0.16 mS cm−2 (Gd) and between 0.001 and 0.04 mS cm−2 (Gh).

(c) Concentration dependence of the NO3 induced shift of Ud0.5. The midpoint potential was plotted against the external NO3 concentration. Since the pipette solution did not affect the NO3 induced shift of Ud0.5, data obtained with 120 mm TEACl and 10 mm TEANO3 in the pipette were pooled. A Hill equation was fitted to the data (continous line, parameters: Km = 3.4, Hill coefficient = 1.25, and maximum shift of =− 63 mV).

In order to match physiological conditions both in the pipette and in the bath, external NO3 was reduced to 5 mm (Schurr and Schulze, 1995). The outward current decreased by more than 50%, and the reversal potential shifted in the positive direction, although less than the Nernst potential of NO3 (Figure 4b). The Ud0.5 value was already shifted by this lower NO3 concentration (Figure 4b, inset). The midpoint potential at depolarisation (5 ± 11 mV, n = 3) was between the voltages measured with 30 mm NO3 in the bath and in the absence of NO3. The determination of the concentration dependence of the shift resulted in a Km value of 3.4 mm (Figure 4c). The apparent Hill coefficient was close to 1 indicating no co-operative binding of NO3 ions.

Experiments with 120 mm KCl in the pipette and 30 mm KCl in the bath were undertaken to explore possible effects of internal and external K+ concentrations on X-QUAC gating. Protoplasts without measurable KIRC currents (Wegner and DeBoer, 1997b; Wegner and Raschke, 1994; Wegner et al., 1994) were selected. Outward K+ currents could be separated from X-QUAC on the basis of their different kinetic properties (Köhler and Raschke 2000; Wegner and DeBoer, 1999). Replacing 30 mm KCl in the bath by TEACl under these conditions did not affect the current-voltage curve of the protoplasts significantly (not shown). Boltzmann parameters of X-QUAC obtained with KCl in pipette and bath were Gd = 1.35 ± 0.02 (rel. units), Ud0.5 = 42 ± 1 mV, δd = 1.13 ± 0.03, Gh = 0.17 ± 0.03 (rel. units), Uh0.5 = − 89 ± 8 mV, and δh = 1.47 ± 0.47 (n = 4). Absolute G-values ranged between 0.63 and 0.98 mS cm−2 (Gd) and between 0.12 and 0.18 mS cm−2 (Gh). These parameters did not deviate from those determined with NMG or TEA-salts, indicating that K+ did not modify gating of X-QUAC.


Here we focus on X-QUAC as a potential control point for NO3 release into the xylem and its control by NO3. Anion channels with fast activation kinetics have been reported for several cell types in higher plants, e.g. in guard cells (Dietrich and Hedrich, 1998; Hedrich et al., 1990), tobacco culture cells (Zimmermann et al., 1994) and epidermal cells of Arabidopsis hypocotyls (Frachisse et al., 1999; Thomine et al., 1995). Similar to these channels, X-QUAC is activated by membrane depolarisation, but unlike X-QUAC, none of these channels is activated by membrane hyperpolarisation as well. Both branches of activation could be inhibited by the anion channel blockers DIDS and IAA-94 (Figure 1). We interpret the two branches of activation in terms of two gates with opposite voltage dependence (Figure 2), in accordance with the one-conductance hypothesis of Köhler and Raschke (2000) for X-QUAC: Anion currents with rapid kinetics activated by membrane depolarisation and hyperpolarisation are considered to pass a single conductance with complex voltage dependence rather than two separate conductances. Different maximum conductances associated with the two gates (different by a factor of approximately 10) are not necessarily at variance with the one-conductance hypothesis, since the unitary conductance or the fraction of active channels, or both, may be different at hyper- and depolarisation, as shown previously for the fast vacuolar channel in barley mesophyll (Tikhonova et al., 1997). Unfortunately, we failed to record single-channel events of X-QUAC. Similarly, single anion channel events could not be resolved in some animal cells (Kunzelmann et al., 1994; Pusch et al., 1994), and for anion channels in the root cortex of wheat, single channel events were rare and could only be recognized after the chloride concentration had been raised to 300 mm (Skerrett and Tyerman, 1994). Possibly, the single channel conductances associated with the two gates of X-QUAC are very small. Generally, patch-clamp data are inadequate to decide whether both gates are located on the same protein.

Although X-QUAC and the quickly activating anion channel from guard cells share inhibition by DIDS and IAA-94 (Figure 1; Marten et al., 1993), X-QUAC differs from the fast activating channels of epidermal cells from Arabidopsis hypocotyl and the root cortex from wheat, which are much less or even insensitive to these inhibitors (Skerrett and Tyerman, 1994; Thomine et al., 1997). On the other hand, we have not obtained any evidence for a role of apoplastic malate in X-QUAC gating, as was demonstrated for the quickly activating anion channel in guard cells (Figure 3; Hedrich and Marten, 1993).

Extracellular (but not cytoplasmic) NO3 caused a shift of the midpoint potential of the depolarisation-activated gate towards more negative voltages (Figure 3 and Figure 4), increasing the transport capacity of the membrane in the voltage range between − 60 and + 100 mV not only for NO3, but for all permeating anions. We can rule out that the observed shift in the voltage dependence has been due to the simultaneous reduction of the Cl concentration in the bath from 34 to 4 mm, because the effect on gating could be reversed when malate was substituted for NO3 (Figure 3), leaving external Cl at a concentration of 4 mm. X-QUAC may possess a binding site for NO3 that is exposed to the apoplast. Alternatively, or in addition, a mechanism could be envisaged in which gating is intrinsically linked to ion permeation, as suggested for the gating of ClC-0 (Chen and Miller, 1996). Half-maximum shift occurred at a NO3 concentration of 3.4 mm (Figure 4c). This lies within the physiological concentration range (Herdel et al., 2001; Mattsson et al., 1988). In Ricinus, the NO3 concentration in the xylem sap reached values up to 10 mm during the night (Herdel et al., 2001), we measured 20 mm in barley root exudates (not shown).

We showed here, that it is possible to record currents in ionic conditions, which mimic physiological conditions, although currents were smaller and more difficult to analyse than with high anion concentrations. That gives a direction to future experiments. It would be worthwhile to also explore under physiological conditions whether other substrates (e.g. plant hormones or amino acids) modify the current-voltage relationship of X-QUAC and other ion channels in a way that they function as an effector mechanism during the regulation of nutrient transport within the plant according to requirement and availability.

To assess the physiological impact of the feature of X-QUAC described here on NO3 transport into the xylem, a simple analogue was used to model the membrane potential of xylem-parenchyma cells and NO3 flux into the xylem (Figure 5a, inset; for details of the model, see Experimental procedures). Since cation and anion fluxes are coupled to each other by the criterion of electroneutrality — and indeed K+ and NO3 fluxes are closely coupled (Herdel et al., 2001) — the voltage dependences of KORC (for K+ release, Wegner and Raschke, 1994) and X-QUAC were taken into account. Calculations were based on patch-clamp data obtained at physiological ion gradients (Figure 4b, inset). For simulation the effect of NO3 on X-QUAC gating was either included (bold lines) or omitted (thin lines) (Figure 5). In our example, equal maximum permeability for K+ and NO3 (GdX-QUAC/GKORC = 1) results in a membrane potential of − 25 mV under physiological conditions with 5 mm NO3 in the bath (bold arrow) versus (hypothetical) − 33 mV in the absence of a NO3 effect on X-QUAC gating (thin arrow) (Figure 5a). We do not know if these values correspond to membrane potentials of xylem-parenchyma cells in vivo. Roberts and Snowman (2000) showed a hyperpolarisation of stelar cells caused by ABA. However, they had to disrupt the root symplast in order to get access to the stele. Obviously this did not disturb the measurement of the hormone effect, but it is likely that the values of the membrane potential in this disrupted system do not figure the absolute values of the membrane potentials in vivo.

Figure 5.

Modelling (a) membrane potential of xylem-parenchyma cells and (b) NO3 flux into the xylem using X-QUAC fit parameters at 5 mm NO3 in the bath (Ud0.5 = 5 mV, bold lines), and in the absence of NO3 (Ud0.5 = 43 mV, thin lines).

(a) The membrane potential of xylem-parenchyma cells is a function of GdX-QUAC/GKORC, the ratio of the maximum conductances of X-QUAC and KORC (Equation 4). Values at equal maximum conductances (GdX-QUAC/GKORC = 1) are indicated by the bold arrow for Ud0.5 = 5 mV and by the thin arrow for = 43 mV. The ionic milieus are assumed to be as follows (concentrations in mM, activities in parenthesis): cytosol 120 (91) K+, 10 (8) NO3, 4 (3) Cl, 2 Mg2+, 1.5*10−4 Ca2+xylem sap 5 (4) K+, 5 (4) NO3, 4 (3) Cl, 2 Mg2+, 5 Ca2+. The reversal potential of X-QUAC was equated with the Nernst potential of NO3 calculated from ion activities. In the case of KORC, the experimentally determined value taken from Wegner and DeBoer (1997a) was taken that deviates from EK+. Data for midpoint potentials and gating charges were taken from Figure 4b (inset) for X-QUAC and from Wegner and DeBoer (1999) for KORC. The inset shows the analogue.

(b) NO3 flux into the xylem, Φcx, is computed as a function of the membrane potential at cytosolic and extracellular NO3 concentrations of 10 and 5 mm, respectively (Equation 5). The curves reflect the range of maximum conductances found experimentally, as indicated in the figure. Arrows indicate the NO3 efflux rate at equal maximum conductances taken from (a) for midpoint potentials of 5 mV (bold arrows) and 43 mV (thin arrows).

Changes in the conductances of KORC and X-QUAC alter membrane potential and magnitude of fluxes. Interestingly, KORC was likewise gated by the apoplastic concentration of its own substrate, K+ (Wegner and DeBoer, 1999). An increase in the external K+ concentration led to a decrease of K+ efflux. The NO3 effect on X-QUAC would counteract this effect. Apoplastic K+ did not directly affect X-QUAC, indicating that coupling of K+ and NO3 fluxes is rather indirect via the membrane potential or specific substances.

In the model, NO3 efflux is enhanced by about 30% due to the NO3-induced shift in voltage dependence (Figure 5b). To demonstrate the variation in NO3 efflux it was calculated for two different Gd values for X-QUAC (obtained on different protoplasts). The effect of NO3 on X-QUAC gating can therefore be described in terms of positive feedback during NO3 loading into the xylem. However, the magnitude of the resulting flux depends not only on the conductance of X-QUAC but also on the driving force for NO3 efflux. An increase in NO3 concentration in the xylem sap reduces the driving force, which produces negative feedback. To maintain NO3 efflux the conductance for NO3 has to be increased to the same extent as the driving force is reduced. In our example, the driving force for NO3 was reduced roughly 2-fold. The increase of the conductance was larger than that. Therefore the opposing effects of NO3 in the xylem on driving force and anion conductance can produce stimulation of NO3 efflux, despite increased NO3 concentration in the xylem. In the measurements of Herdel et al. (2001) both NO3 concentration in the xylem sap and NO3 efflux increased during the afternoon and partly during the night. This could not be explained entirely by changes in transpiration rate (Herdel et al., 2001). The experiments were done with Ricinus, but diurnal variations of these NO3 concentrations and NO3 fluxes might be a general phenomenon. As already mentioned in the introduction, the observation of T.A. Cuin and R.A. Leigh indicates that a high transfer of NO3 to the xylem is likely in barley supplied with NO3 throughout growth. X-QUAC with its NO3 sensitive depolarisation-activated gate provides a mechanism to compensate for a shallow NO3 gradient and to ensure further NO3 efflux into the xylem with high NO3 concentrations in the xylem sap.

Experimental procedures

Electrical recording and solutions

Anion currents were recorded applying the patch-clamp technique in the whole-cell configuration (Hamill et al., 1981) using the same setup as in previous studies (for details see Köhler and Raschke, 2000). Capacitive currents, access resistance, and liquid junction potentials were corrected for. To get access to the plasma membrane, xylem-parenchyma protoplasts were isolated enzymatically from roots of barley (Hordeum vulgare L. cv Apex; Lochow-Petkus GmbH, Bergen, Germany). Plant cultivation and protoplast isolation are described fully in Köhler and Raschke (2000) and Wegner and Raschke (1994).

K+-currents were suppressed by using tetraethylammonium (TEA+) or N-Methylglucamine (NMG+) salts in the solutions. The standard extracellular solution (bath) contained (mM) 30 TEACl (or NMGCl), 5 Calcium-gluconate (Ca(Glc)2), 2 MgCl2, 10 Mes, pH 5.8 (adjusted with Tris), 500 mosmol kg−1. The standard intracellular solution (pipette) contained (mM): 120 TEACl (or NMGCl), 150 nm free Ca2+ (added as Ca(Glc)2), 10 EGTA, 2 ATP, 2 MgCl2, 10 Tris, pH 7.2 (adjusted with Mes), 530 mosmol kg−1 (adjusted with mannitol). Total and free concentrations of divalent cations were computed with the program ‘Calcium’ (Führ et al., 1993). A cytosolic free Ca2+ concentration of 150 nm suppressed the activities of NORC (= non-specific outward rectifying conductance, Wegner and De Boer, 1997a) and X-SLAC (= slowly activating anion conductance from xylem parenchyma, Köhler and Raschke, 2000). The inhibitor DIDS was dissolved in distilled water; IAA-94 was added as an ethanolic solution; the ethanol content of the bath was 0.3% v/v. Ethanol on its own had no effect. For deviations from the standard solutions, see figure legends.

Data analysis

Data were analysed with the software packages Review (Instrutech, Elmont, NY, USA) and SigmaPlot (SPSS Science, Chicago, USA). Membrane voltages were defined as voltages on the cytoplasmic side of the membrane relative to the physiological outside. A negative current corresponds to an anion efflux from the protoplast. Means are given with standard error.

Current-voltage curves were constructed from series of voltage pulses as indicated in the figures. Currents recorded 10 ms after imposing the voltage steps were plotted. Chord conductances were calculated from current-voltage curves according to g = I/(U-Erev), with Erev being the reversal potential of the anion current, and plotted against voltage to analyse the voltage dependence of gating. To compare measurements on different cells, currents and conductances were related to membrane area. The specific capacity of xylem-parenchyma protoplasts was 0.9 µF cm−2 (Wegner and Raschke, 1994). The capacities of the protoplasts were between 8 and 23 pF. Note that each parenchyma cell disintegrated into an average of six protoplasts during preparation (Wegner and Raschke, 1994).

Quantitative analysis of data

The following Boltzmann function describes the voltage dependence of the open probability of a channel:


with δ = gating charge, U0.5 = voltage at which the open probability is half-maximum (the 'midpoint potential′), U = voltage, F = Faraday's constant, R = gas constant, and T = absolute temperature.

Provided that the open-channel conductance is voltage independent (Figure 2a), the relation of the conductance (g (U)) to the maximum conductance (G) is a measure for the open probability (Po (U)):


In the case of X-QUAC, the dependence of the chord conductance on membrane potential (g(U)) can be described by the sum of two Boltzmann functions, representing two independent gates with opposite voltage dependence:


with G = maximum conductance, for δ, U0.5, U, F, R, T, see above. Indices d and h mark two different gating processes; d stands for depolarisation-activated and h for hyperpolarisation-activated. Parameters are given with standard error.

Modelling membrane potential and NO3 flux

X-QUAC and KORC are considered to be the main pathways for salt transport into the xylem. Ion currents are considered to result from two current generators operating in parallel (and maintaining electroneutrality). They are driven by the electrochemical potential differences of anions and cations (represented by the two batteries in the inset in Figure 5a) and determined in magnitude by the ion conductances X-QUAC and KORC (compare Buschmann et al., 1996; Spanswick, 1981). If the contribution of other conductances is negligible, the membrane potential UMP of xylem-parenchyma cells results from the activity of KORC and X-QUAC according to:


with ENO3- = Nernst potential of nitrate, EKORC = reversal potential of KORC, and g(U) = conductance of X-QUAC and KORC, respectively.

Voltage dependence of the NO3 conductance is given by Equation 3. For KORC, the corresponding relation was published (Equation 9 in Wegner and DeBoer, 1999). By insertion into Equation 4, the membrane potential can be calculated as a function of GdX-quac/GKORC, the ratio of maximum anion to cation conductance. The partial inactivation of X-QUAC was negative of the Nernst potential of K+ and could therefore be neglected here.

The flux of NO3 (or any other permeable anion) from the stelar symplast into the xylem, φcx, can be calculated according to the following equation:


with gNO3-(U) = chord conductance for NO3 per surface area of the membrane, j = current density, F = Faraday's constant, A = effective membrane area involved in salt release per cm length of the root, and w = FW, likewise normalized to the root length.


This work was supported by grants from Deutsche Forschungsgemeinschaft (SPP 717, ‘Apoplast’) to K.R.