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First published online October 29, 2004; 10.1104/pp.104.046201 Plant Physiology 136:3771-3783 (2004) © 2004 American Society of Plant Biologists
Citrate-Permeable Channels in the Plasma Membrane of Cluster Roots from White Lupin1Wine and Horticulture, School of Agriculture and Wine, The University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia (W.-H.Z., S.D.T.); and Commonwealth Scientific and Industrial Research Organization Plant Industry, Canberra, Australian Capital Territories 2601, Australia (P.R.R.)
White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (–P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3– than Cl– (PCit/PCl = 26.3), while ORAC was selective for Cl– over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of –P plants, as well as from normal (noncluster) roots of plants grown with 25 µM phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of –P plants. However, the IRAC current from +P plants occurred less frequently than in the –P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.
The development of cluster roots is an important strategy used by some plant species to adapt to infertile soils (Dinkelaker et al., 1995  ; Neumann and Martinoia, 2002). These bottlebrush-like roots were initially called proteoid roots because they were first characterized in members of the Proteaceae (Purnell, 1960). Cluster roots are groups of short, secondary lateral rootlets that are covered with a dense mat of root hairs (Lamont et al., 1984). In white lupin (Lupinus albus), the development of cluster roots is greatly enhanced under phosphorus (P) deficiency, and this species has been widely used as a model system for studying the morphology and physiology of these specialized root structures (Dinkelaker et al., 1995; Keerthisinghe et al., 1998; Neumann et al., 1999; Watt and Evans, 1999a, 1999b).
Cluster roots are characterized by specific metabolic and physiological changes that help plants overcome the nutrient deficiency that induces their development (Neumann and Martinoia, 2002
Exudation of organic acids by cluster roots could result from altered organic acid metabolism, as indicated by the enhanced in vitro activities of phosphoenolpyruvate carboxylase and malate dehydrogenase (Johnson et al., 1994
Since citrate exists predominantly as a trivalent anion in the cytoplasm, its movement out of the root cells is an energetically passive process due to the large negative potential difference across the plasma membrane. Therefore, citrate efflux could feasibly occur through a citrate-permeable anion channel in the plasma membrane. The observation that citrate efflux from the cluster roots of white lupin is sensitive to anion channel blockers, such as anthracene-9-carboxylic acid (A-9-C) and ethacrynic acid, is consistent with this proposition (Neumann and Römheld, 1999 To investigate the mechanism underlying the exudation of citrate anions from cluster roots of white lupin, we used the patch-clamp technique to measure the whole-cell currents in protoplasts isolated from mature cluster roots (Fig. 1) of P-deficient (–P) plants. We compared these currents with those in noncluster roots of –P plants, as well as those in noncluster roots from P-replete (+P) plants.
Whole-Cell Currents in Protoplasts Prepared from Mature Cluster Roots According to convention, the efflux of anions across the plasma membrane is defined as an inward or negative current. To detect inward currents associated with citrate efflux from protoplasts of white lupin roots, we included citrate3– as the main anion in the patch-pipette solution and tetraethylammonium as the main cation, due to its low permeability across biological membranes. When the holding potential was near 0 mV, two main types of current were detected in the protoplasts: (1) an inwardly rectifying current that activated rapidly and inactivated slowly (Fig. 2A and inset), and (2) an outwardly rectifying current that only showed deactivating currents at hyperpolarized potentials (Fig. 2C and inset). The reversal potentials for the two types of current were 16.7 ± 6.0 mV (n = 12) and –7.9 ± 3.7 mV (n = 26), respectively (compare Fig. 2, B with D). The outwardly rectifying current also displayed slow current relaxation (tail currents) following depolarizing pulses (Fig. 2C). The inwardly rectifying current was observed in 34% of protoplasts (n = 65) and the outwardly rectifying current was observed in 45% of protoplasts (Table I). The remaining protoplasts contained small inward and outward currents (Fig. 2E), which often displayed reversal potentials less than –40 mV and more negative than the equilibrium potentials for the ions present in the system (Fig. 2F). This implies that the H+ pump may be contributing to these small conductances, but this was not investigated further. On a few occasions, the inwardly rectifying current and the outwardly rectifying current were observed in the same protoplast, but not at the same time. The current appeared to switch from one type to the other along with the expected shift in the reversal potential. This switch occurred in a matter of minutes, even though the experimental conditions remained unchanged (Fig. 3).
The Inwardly Rectifying Current Is Carried by Citrate Efflux Inward currents can be caused by the influx of cations (i.e. Ca2+) or the efflux of anions (i.e. citrate3–, Cl–, or even HEPES). To determine whether Ca2+ influx was responsible for the inwardly rectifying current, we tested the effect of the Ca2+ channel blocker Gd3+. Figure 4A shows that the inward current was not inhibited by the addition of 100 µM GdCl3 to the bath. Furthermore, both the current magnitude and reversal potential of the inwardly rectifying current were independent of CaCl2 concentrations in the bath (Fig. 4B). These results suggest that the inwardly rectifying current is likely to result from anion efflux rather than Ca2+ influx and was accordingly termed inwardly rectifying anion conductance (IRAC). The contributions of HEPES efflux to the observed inward current appeared to be small, as the substitution of external solution from 10 mM CaCl2, 5 mM MES, pH 6.0, to 10 mM CaCl2, 5 mM HEPES, pH 7.0, did not shift the reversal potential or the current magnitude (data not shown). Therefore, the inward current could result from the efflux of citrate3– and/or Cl–, since a small amount of Cl– was required in the pipette solution to reduce the junction potentials associated with the Ag/AgCl half-cell. The relative permeability of citrate3– and Cl– through the underlying channels can usually be calculated from the reversal potential under biionic conditions (same solution either side of the membrane) because the reversal potential will track closer to the equilibrium potential of the more permeable ions. Attempts to perform these experiments were unsuccessful because the inclusion of citrate in the bath solution caused the protoplast membranes to become unstable and collapse. Therefore, the following two experiments were conducted to examine whether the observed inward current was mainly caused by Cl– or citrate3– efflux.
The first method monitored the shift in reversal potential when the Cl– concentration in the bath solution was changed 10-fold. Reducing CaCl2 from 10 to 1 mM CaCl2 shifted the theoretical equilibrium potential for Cl– (ECl) from –40 mV to 17 mV (Fig. 4B). The reversal potential of the IRAC current was 21.6 ± 8.9 mV in 10 mM CaCl2 and 24.6 ± 6.8 mV (n = 3) in 1 mM CaCl2, respectively. Since the reversal potential was unaffected by Cl– concentration in the bath, we can conclude that Cl– efflux does not contribute significantly to the IRAC.
In the second method, Cl– was removed from the pipette solution altogether and the Ag/AgCl half-cell was replaced with a platinum electrode. A rapidly activating, slowly inactivating inward current, typical of IRAC, was still observed under these experimental conditions (Fig. 5A). The reversal potential of this current was 15.4 ± 4.4 mV (n = 3; Fig. 5B), which is comparable to the reversal potential measured with 4 mM Cl– in the pipette (Erev = 16.7 ± 6.0; n = 12), confirming that Cl– efflux contributes little to the observed inward current. Since Ca2+ influx and Cl– efflux are not responsible for IRAC, we deduce that this inward current is carried predominantly by citrate3– efflux. The relative permeability of the IRAC channels to citrate and Cl– is determined later. Identical currents were also observed when
To determine whether the IRAC was permeable to malate2–, the 20 mM citrate salt usually included in the pipette solution was substituted with an osmotically equivalent concentration of malate salt (30 mM). An IRAC-like inward current was observed using malate2– as the main permeant anion in the pipette (data not shown). Current densities at –150 mV were –189.7 ± 35.4 mA m–2 (n = 4) with malate in the pipette, and –216.5 ± 32.2 (n = 8) with citrate3– in the pipette. The reversal potential of the current with malate2– in the pipette was 14.4 ± 4.6 mV (n = 4), which is comparable to the reversal potential of 16.7 ± 6.0 (n = 12) measured with citrate3– in the pipette using the same bath solution. These findings suggest that the IRAC is permeable to both citrate3– and malate2– anions.
Citrate efflux from the cluster roots of white lupin is reportedly correlated with rhizosphere acidification via enhancement of H+-ATPase activity (Yan et al., 2002
Since citrate efflux from intact cluster roots of white lupin is inhibited by the anion channel blockers A-9-C and ethacrynic acid (Neumann et al., 1999
We used the reversal potential of this A-9-C-sensitive current in a modified Goldman-Hodgkin-Katz (GHK) equation (Eq. 2; see "Materials and Methods") to estimate the relative permeability of the IRAC channels to citrate3– and Cl– (PCit/PCl) by assuming A-9-C inhibits the anion conductance only. The A-9-C-sensitive current-voltage curve is generated by subtracting the currents measured after A-9-C addition from the currents measured before A-9-C addition. The reversal potential of the resulting curve was 20 mV (Fig. 6C), which predicts a permeability ratio, PCit/PCl, of 26.3. Note that the calculation has accounted for the effect of ionic strength on activities of the ions. If the effect of ionic strength on activities of the ions is not taken into account, a much lower value for PCit/PCl (1.7) can be obtained.
The second type of current observed in about 45% of protoplasts was an outwardly rectifying current that exhibited deactivating inward currents during hyperpolarizing pulses (Fig. 2C). When the holding voltage was shifted from near 0 mV to –171 mV, the character of these currents changed. The deactivating inward currents disappeared, but the magnitude of the tail currents increased (Fig. 7A). The amplitude of the tail current began to saturate as the membrane voltage was returned from increasingly more positive potentials. The relative conductance of the tail currents was plotted against voltage (Fig. 7B) and the resulting curve was fitted with a Boltzmann function (Eq. 1):
The reversal potential of protoplasts showing the outwardly rectifying conductance in 10 mM CaCl2 was more positive than the equilibrium potential for Cl– (ECl), but more negative than the equilibrium potentials for citrate and Ca2+ (which are very positive; Fig. 8C). The magnitude of the outward currents and tail currents was reduced when external CaCl2 was reduced from 10 to 1 mM (Fig. 8, A–C) and the reversal potential shifted from –20.7 ± 7.4 mV to –5.3 ± 1.2 mV (n = 3), which follows ECl (Fig. 8C). An identical shift in the reversal potential with changes in external CaCl2 was observed in the presence of the Ca2+ channel blocker, Gd3+ (data not shown). These results suggest that Cl– carries this outward current and that the deactivating inward currents observed at negative potentials (see Fig. 2C) are caused by the partial closure of the channels. This conductance was designated outwardly rectifying anion conductance (ORAC).
The effect of external  (with removal of Cl–) on both IRAC and ORAC was tested by substituting the 10 mM KCl in the bath solution with 10 mM KH2PO4 in a background of 1 mM Ca-(gluconate)2. As shown in Figure 8, D to F, the outward current through ORAC was markedly reduced and the reversal potential shifted from –15.3 ± 2.8 mV to 32.6 ± 3.7 mV (n = 3) with this substitution. If we assume the contribution of cations to this current is negligible, then the relative permeability of the channel to and Cl–  can be estimated to be 0.15 using the GHK equation (Goldman, 1943). More interesting was the inhibition of inward current (Cl– and citrate3– efflux) through ORAC despite there being a much larger electrochemical gradient for Cl– efflux. This effect could be observed both in the initial and transient inward currents, the steady-state inward currents, and the tail currents (n = 3). No effect of external on IRAC was observed (data not shown).
The current through ORAC was almost completely inhibited when 100 µM A-9-C were added to the bath solution (Fig. 9). This inhibition was largely reversible if the blocker was washed out of the bath (data not shown). The inhibition demonstrated that ORAC could carry significant inward current (anion efflux). By subtracting the currents before and after A-9-C addition, we constructed a current-voltage curve for the A-9-C-sensitive current (see above). The reversal potential of this curve was applied to the modified GHK equation (Eq. 2) and the relative permeability of citrate3– to Cl– (PCit/PCl) was estimated to be 0.27. Like the IRAC, ORAC was relatively insensitive to 100 µM ethacrynic acid (n = 3; data not shown). To identify the single channels responsible for the observed whole-cell IRAC and ORAC currents, inside-out and outside-out patches were obtained. However, we were unable to observe any single-channel activity that was sensitive to A-9-C, whether the channel blocker was added to the cytoplasmic or the extracellular side of the plasma membranes (data not shown). The lack of single-channel activity implies that critical components essential for activation of the IRAC and ORAC are lost upon excising the patches from the whole cell.
Mature cluster roots of white lupin release more citrate than noncluster roots from the same P-deficient plants (Keerthisinghe et al., 1998
We investigated whether K+ currents could be detected in the cluster roots of white lupin. The KCl in the bath was replaced with either K2-malate, K-Glu, or K2SO4 to avoid confusion with the ORAC-mediated Cl– currents. With 100 mM K+ in the pipette solution, a small, rapidly activating outward current and a noisy inward current were elicited by depolarizing and hyperpolarizing pulses, respectively (Fig. 10A). Identical currents were found with all three bath solutions (data not shown), suggesting that the outward currents were due to K+ efflux and not anion influx. Support for this conclusion was obtained by increasing the external K+ concentration from 5 to 50 mM, which increased the magnitude of inward current and shifted the reversal potential in a positive direction, toward EK (Fig. 10B). It is noteworthy that the time-dependent K+ inwardly rectifying channels, often reported in root cells (Maathuis et al., 1997
Cluster roots form on the lateral roots of white lupin during the onset of phosphorus deficiency. The special physiology exhibited by these roots assists lupin plants to extract phosphorus from sparingly soluble pools in the soil that are not accessible to most other species. To investigate the mechanism underlying citrate efflux from the roots of white lupin, protoplasts were isolated from the cluster and noncluster roots of –P plants and from the normal (noncluster) roots of +P plants. Whole-cell currents in these protoplasts were then measured using the patch-clamp technique. Unlike most studies of this kind, in which protoplasts were isolated by digesting finely chopped root tissue for several hours (Zhang et al., 2001 ), we prepared protoplasts by treating intact root tissue with digestive enzymes for relatively short periods, which preferentially digested epidermal cells (see "Materials and Methods"). With citrate3– as the main permeant anion in the cytoplasm, 10 mM CaCl2 in the bath solution, and a holding potential near 0 mV, we found two main anion conductances: an IRAC that displayed a fast activation and slow inactivation in response to membrane hyperpolarization (Fig. 2A), and an ORAC with deactivating inward currents (Fig. 2C). However, ORAC could carry significant inward current in the steady state. We also detected proton pump-like conductances in some protoplasts, but these were not investigated in detail (Fig. 2, E and F). On occasion, more than one of these conductances occurred in the same protoplasts, but not at the same time. Instead, it appeared that one type of conductance was replaced by another within several minutes (Fig. 3). Similar changes in conductance have been reported in protoplasts derived from wheat root cells (Tyerman et al., 2001), as were the fast and slow anion conductances in guard cells, which were suggested to be different modes of action for the same channel (Dietrich and Hedrich, 1994). We did not investigate this further, but it remains possible that IRAC and ORAC represent different conductance states of the same transporter. Activation kinetics for the IRAC are reminiscent of hyperpolarization-activated anion channels in carrot suspension cells (Barbara et al., 1994), as well as the aluminum-activated malate channel in wheat roots (Zhang et al., 2001), whereas ORAC shared some features with the slow anion channel in guard cells (Schroeder and Keller, 1992).
We conclude that IRAC is caused by citrate3– efflux rather than Cl– efflux or Ca2+ influx, and several results support this position. First, IRAC was insensitive to the Ca channel blocker Gd3+ (Fig. 4A), and the reversal potential and magnitude of IRAC current were relatively unaffected by changes in Ca2+ concentrations in the bath (Fig. 4B). These results indicate that Ca2+ fluxes are unlikely to make substantial contributions to the current mediated by IRAC. IRAC was similarly independent of Cl– concentrations in the bath (Fig. 4B), and identical currents were detected in experiments when Cl– was removed from the pipette solution (Fig. 5). This is strong evidence that Cl– fluxes do not contribute significantly to IRAC. Last, the disappearance of IRAC when citrate was removed from the pipette solution (Fig. 9) also supports the conclusion that citrate3– anions are the main carriers of charge for the IRAC currents.
Attempts to determine the relative permeability of IRAC to citrate–and Cl– in bi-ionic conditions were hampered by the tendency for protoplasts to collapse after citrate was added to the external solution. This injury was probably related to the formation of Ca-citrate complexes that would reduce the free Ca2+ activity and destabilize the membranes (Bertl and Slayman, 1990
Several types of anion channels have been identified in the plasma membrane of root cells, including an outwardly rectifying anion channel in wheat (Skerrett and Tyerman, 1994
Citrate efflux from cluster roots of white lupin is inhibited by A-9-C and, to a lesser extent, ethacrynic acid (Neumann et al., 1999
ORAC was also frequently observed in protoplasts derived from cluster and noncluster roots of –P as well as from noncluster roots of +P plants (compare with Fig. 2C and Table I). We estimated that the channel responsible for the outward current had lower, but significant, citrate3– permeability relative to Cl– (PCit/PCl = 0.27), which is similar to the majority of anion channels that have been ascribed also to function in organic anion release (Schmidt and Schroeder, 1994
Previous studies have shown that there are considerable variations in citrate efflux from different types of roots and from different stages of cluster root development. For example, citrate efflux from the mature segments of the cluster roots is approximately 5 times greater than that from noncluster root tissue on a fresh-weight basis (Keerthisinghe et al., 1998
The IRAC and ORAC current types were also observed in protoplasts derived from noncluster roots of plants grown in the presence of 25 µM KH2PO4 (Table I). However, the currents tended to be smaller (P0.062) and occurred less frequently in the noncluster roots of +P plants compared to the roots of –P plants (Table I). These differences, while small, combined with the effect of
It is likely that H+-ATPase activity would balance citrate efflux from cluster roots (Neumann and Martinoia, 2002 In summary, we have identified two anion conductances in the plasma membranes of cells from the cluster and noncluster roots of white lupin that are permeable to citrate3–. These conductances differ with respect to rectification, activation, and selectivity. The hyperpolarization-activated IRAC was highly selective for citrate over Cl–, while the depolarization-activated ORAC was selective for Cl– over citrate3–. Several results suggest that IRAC is a likely pathway for citrate efflux at hyperpolarized membrane potentials, but the ORAC may become significant at more depolarized membrane potentials.
Plant Materials
Seeds of white lupin (Lupinus albus L. cv Kive mutant) were germinated in damp sand for 6 d and transferred to black 10-L hydroponic tanks. The composition of the nutrient solution used is described by Neumann and Römheld (1999)
Mature cluster roots and noncluster lateral roots (Fig. 1) were excised from –P plants (grown without KH2PO4) and lateral roots (noncluster) were excised from +P plants (grown with 25 µM KH2PO4). The noncluster root tissues included the apical 2 to 4 cm of lateral roots that were devoid of any emerging laterals that could be incipient cluster roots (Fig. 1). Tissues from the –P plants were incubated in the following enzyme solution for 1 h at 30°C: 1.6% cellulase (Onozuka RS; Yakult Honsha, Tokyo), 0.12% pectolyase (Sigma, St. Louis), 0.5% (w/v) polyvinylpyrrolidone, 0.5% (w/v) bovine serum albumin, 1 mM CaCl2, 500 mM sorbitol, 2 mM ascorbic acid, and 10 mM MES/Tris, pH 6.0. Lateral roots from +P plants were incubated in a slightly different enzymatic solution that consisted of 0.8% cellulase (Onozuka RS; Yakult Honsha), 0.08% pectolyase (Sigma-Aldrich), 0.5% (w/v) polyvinylpyrrolidone, 0.5% bovine serum albumin, 1 mM CaCl2, 500 mM sorbitol, 2 mM ascorbic acid, and 10 mM MES/Tris, pH 6.0. The digest was filtered through fine muslin, centrifuged at 60g for 5 min, and the pellet resuspended in 2 to 3 mL of standard bath solution. The protoplasts were kept on ice until experiments were started. The mean diameter of protoplasts derived from the mature cluster roots, noncluster roots of –P plants, and lateral roots of +P plants was 15.5 ± 0.3 µm (n = 31), 16.4 ± 0.7 µm (n = 15), and 15.2 ± 0.4 µm (n = 15), respectively, which suggests they were derived from the epidermal cells of the root tissue (see Watt and Evans, 1999a
The patching pipettes were pulled from borosilicate glass blanks (Clark Electromedical, Reading, UK) and coated with Sylgard (184 silicone elastomer kit; Dow Corning, Midland, MI). The resistance of pipettes containing solution (see below) ranged from 20 to 40 M
Three types of pipette solutions were used. Type I pipette solution (mM) was composed of 20 citric acid, 2 CaCl2, 2 MgSO4, 2 Na2ATP, 2 EGTA, and 10 HEPES. Type II pipette solution (mM) was composed of 20 citric acid, 2 CaCl2, 2 MgATP, 2 EGTA, and 10 HEPES. Type III solution was the same as that of type II, but without CaCl2. All pipette solutions were adjusted to pH 7.2 and osmolality of 720 mosmol kg–1 with tetraethylammonium hydroxyl and Suc, respectively. Free calcium concentrations of the above pipette solutions were approximately 50 nM, calculated using the chemical speciation program GEOCHEM (Parker et al., 1987
We thank Wendy Sullivan for expert technical assistance. Received May 11, 2004; returned for revision June 12, 2004; accepted June 22, 2004.
1 This work was supported by the Australian Research Council. 
2 Present address: Institute of Botany, Chinese Academy of Sciences, Beijing 100093, People's Republic of China. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046201. * Corresponding author; e-mail steve.tyerman{at}adelaide.edu.au; fax 81–8–8303–7116.
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ABSTRACT
RESULTS
was removed from the pipette solution (type II pipette solution; see 
concentrations on the outwardly rectifying current. Data in A and B show the superimposed current traces from a single cluster root protoplast elicited by voltage pulses between –171 to 59 mV in 20-mV intervals from a holding potential of –1 mV. External solutions contained 10 mM CaCl2 (A) and 1 mM CaCl2 (B). Steady current-voltage curves from A and B are presented in C. Data in D and E show the superimposed current traces from a single –P cluster root protoplast in response to voltage pulses ranging from –173 to 57 mV in 20-mV intervals from a holding potential of –3 mV. External solutions contained 1 mM Ca-(gluconate)2 together with either 10 mM KCl (D) or 10 mM KH2PO4 (E). Steady current-voltage curves from D and E are presented in F. Pipette solution was type I.
than physiologically important organic anions, such as malate and citrate (Barbier-Brygoo et al., 2000
in 10 mM CaCl2. The voltage across the plasma membrane was controlled and monitored with the Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Pulse protocols with sampling frequencies of 2 kHz and corresponding filtering frequencies of 0.5 kHz were used to elicit the current from a holding potential. Unless stated otherwise, holding potentials were near 0 mV. Sufficient time between voltage pulses was given to allow currents to settle to the steady clamp current for the particular holding potential before a new pulse was applied. Records were stored and analyzed using pClamp 6.0 (Axon Instruments). Junction potentials were corrected for according to Amtmann and Sanders (1997)
