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First published online November 24, 2004; 10.1104/pp.104.046995 Plant Physiology 136:4136-4149 (2004) © 2004 American Society of Plant Biologists Characterization of Anion Channels in the Plasma Membrane of Arabidopsis Epidermal Root Cells and the Identification of a Citrate-Permeable Channel Induced by Phosphate Starvation1Department of Biological Sciences, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, United Kingdom (E.D., M.R., S.K.R.); and Biology Department, University of York, York YO10 5YW, United Kingdom (D.S.)
Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO42– as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of –180 mV exhibiting a maximum peak inward current (Ipeak) at approximately –130 mV. These currents reversed at potentials close to the equilibrium potential for SO42–, indicating that the inward currents represented SO42– efflux. Replacing intracellular SO42– with Cl– or NO3– resulted in inward currents exhibiting similar properties to the SO42– efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO42– was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mM). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.
Anion channels in the plasma membrane of plant cells catalyze anion fluxes both into and out of the cell and serve a variety of functions. They have been implicated in stomatal function, where their activation is thought to be one of the rate-limiting steps in the loss of salts (and thus cell turgor) from guard cells leading to stomatal pore closure (Roelfsema et al., 2004  ). In less specialized cells, anion channel activation is a likely step in the transduction of signals modulating hypocotyl growth, including blue light (Cho and Spalding, 1996) and possibly auxin (Thomine et al., 1997); these signal transduction events arise from depolarizations resulting from anion channel activation. Anion channels are also thought to facilitate the release of organic acids from higher plant roots. Al3+-activated anion channels (ALAACs) in the tips of wheat (Triticum aestivum) and maize (Zea mays) roots have been shown to be permeable to malate and/or citrate (Ryan et al., 1997; Kollmeier et al., 2001; Pineros and Kochian, 2001; Zhang et al., 2001), a function of which is thought to reduce Al3+ stress by chelating this cation. Thus activation of ALAACs by Al3+ and their pharmacological profile, which resembles that for Al3+-induced organic-acid efflux from cereal roots, makes ALAACs likely candidates for mediating Al3+-induced organic-acid secretion from roots.
The biophysical properties of plant anion channels have been best characterized in guard cells. Two types of anion channels have been extensively investigated: rapidly activating (R-type) and slowly activating (S-type) anion channels (e.g. Hedrich et al., 1990
Anion channels in roots have not been well characterized compared to those in guard cells, despite their potential importance in regulating acquisition from soil solution. The ALAACs of wheat and maize roots (see above) resemble S-type channels in that they display slow activation kinetics (Pineros and Kochian, 2001
In this study we address this dearth of knowledge and use the patch clamp technique to investigate anion channel activity in the epidermis of Arabidopsis (Arabidopsis thaliana) roots. We show two types of voltage-dependent channel activity, which resemble the R-type anion channel activity described in guard cells and hypocotyls. One of these channels was ubiquitously expressed in the epidermal cells and was permeable to the inorganic anions, SO42–, NO3–, and Cl– but was impermeable to organic-acid anions, citrate and malate. The second anion channel was less frequently observed, was induced by phosphate starvation, and mediated the efflux of organic-acid anions. It is suggested that the phosphate-regulated anion channel mediates organic-acid anion efflux from Arabidopsis roots, which is thought to be an important strategy for efficient phosphate acquisition by higher plants (Narang et al., 2000
The Arabidopsis line J0841 showed green fluorescent protein (GFP) expression specifically only in root-peripheral cells, namely the epidermal cells (Fig. 1). These included cells of the elongation zone as well as young emerging root hair (trichoblast) and atrichloblast cells. However, GFP expression was not observed in the root tip, or in some of the older root hair or atrichoblast cells. Only cells expressing GFP were used in this study.
The whole-cell configuration of the patch clamp technique and standard bath solution (SBS) was used to record anion currents across the plasma membrane of GFP-expressing protoplasts isolated from the epidermis of Arabidopsis roots. SBS contained 5 mM LaCl3; La3+ is an established broad-spectrum cation channel blocker and, as illustrated in Figure 2, was effective in blocking inward and outward currents at negative and positive potentials, respectively. Although the La3+-sensitive currents illustrated in Figure 2 were not investigated further, they most likely represent cation currents through nonselective and Ca2+-permeable channels, which have been reported previously in Arabidopsis root hairs and epidermal cells (Kiegle et al., 2000 ; Very and Davies, 2000; Demidchik et al., 2002). Pipette solutions were based on the use of caesium salt; although Cs+ permeates nonselective channels (Demidchik et al., 2002), Cs+ does not significantly permeate K+ channels (Tester, 1988), and thus currents in this study were recorded without interference from this channel type.
Anion Currents in Arabidopsis Root Epidermal Cells
Using SBS and standard pipette solution (in which 25 mM SO42– was the main anion), all root epidermal cells exhibited inward currents with strong voltage dependence. SO42– was employed as the main anion because intracellular SO42– has been shown to be both a substrate for and an activator of plasma membrane anion channels in Arabidopsis hypocotyl cells (Frachisse et al., 1999
In experiments using SBS supplemented with 1 mM SO42–, the inward current reversed between the equilibrium potentials for SO42– and Cl– (Fig. 3B), indicating that the channels mediating the inward current are likely to be permeable to other anions. To investigate further the selectivity of the channels that underlie the inward current, intracellular SO42– was substituted by Cl–, NO3–, malate, and citrate (supplied as cesium salts). The substitution of cytosolic rather than extracellular anions was favored because it represented a more physiological condition; that is, the negative potential that usually exists across the plasma membrane of plant cells tends to drive the passive flow of anions from the cells. Furthermore, addition of citrate and malate to the bath solution resulted in an increase in membrane conductance (i.e. leak conductance), consistent with a loss of integrity of the membrane and/or the seal between the membrane and the glass pipette. We suspect that this was the result of cation chelation by citrate and malate. Replacing pipette SO42– with Cl– or NO3– resulted in inward currents, which exhibited similar voltage dependence to that observed for SO42– efflux currents (Fig. 3, C and D). Furthermore, decreasing pipette Cl– or NO3– reduced the mean current density of the inward currents consistent with them representing anion efflux (Table I). However, no inward currents were observed when the pipette SO42– was replaced with citrate or malate, indicating that the anion channels were not significantly permeable to organic-acid anions. It is also significant that the whole-cell currents reversed at ECl when pipette SO42– was replaced with Cl– (Fig. 3C); i.e. Cl– was the principal anion in both the bath and pipette solution. Taken together, these sets of data show that the channels underlying the voltage-gated inward currents in Arabidopsis root epidermal cells are anion selective and mediate at least SO42–, Cl–, and NO3– efflux but not that of malate and citrate.
Regulation of Anion Currents in Arabidopsis Root Epidermis
The following observations indicated that intracellular SO42– was a potent activator of the voltage-dependent anion efflux currents. The magnitude of the Cl– and NO3– efflux currents decreased with time and had usually completely disappeared within 15 min (Fig. 3, C and D); in contrast, SO42– efflux current magnitudes remained stable for at least 2 h. In experiments using standard pipette solution (i.e. with SO42– as the charge-carrying anion), the inward currents exhibited a marked increase in magnitude with time. Specifically, the magnitude of Ipeak increased 733% ± 247% (within a mean time of 8.1 ± 1.8 min; n = 19) after obtaining the whole-cell configuration. Indeed, in four experiments, inward currents were absent immediately after obtaining the whole-cell configuration but developed and increased in magnitude over several minutes—a typical example is shown in Figure 4. It is unlikely that this gradual increase in the inward current reflected a gradual and slow equilibration of the cytosol with SO42–, because the equilibration of small inorganic anions between the pipette media and cytosol would be expected to be relatively immediate and complete within a minute. Rather, these observations are consistent with the activation of the anion efflux currents by intracellular SO42– and are similar to those previously reported by Frachisse et al. (1999)
It is noteworthy that an instantaneously activating, outwardly rectifying current was evident in some root epidermal cells. From Figures 3, C and D, and 4, it is apparent that at least some of this outward current does not run down or increase with time, illustrating that the outward current was mediated, at least in part, by a channel type distinct from the channel responsible for the voltage-dependent inward current. Although the outwardly rectifying current was not investigated further in this study, it is interesting that after the complete rundown of the inward current in Figure 3 and before the runup in Figure 4, the remaining outwardly rectifying current reversed close to ECl, consistent with this current being carried by anion influx. Outwardly rectifying anion-selective channels have also been characterized in the roots of maize and wheat (Skerrett and Tyerman, 1994 ; Pineros and Kochian, 2001) where they are thought to mediate anion influx in high-salt concentration (e.g. during salinity stress).
Figure 5 shows typical currents resulting from stepping the voltage from values either more negative (–216 mV) or more positive (+64 mV) than the Ipeak voltages. Upon stepping the potential from –216 mV, inward currents at potentials negative of –96 mV were characterized by a fast time-dependent activation, which could be roughly fitted by a single exponential, whereas inward currents at potentials positive of –96 mV exhibited instantaneous activation. In contrast, stepping from holding potentials positive of the Ipeak voltage, whole-cell inward currents instantaneously increased with the driving force for SO42– efflux before decreasing to reach a new steady-state value. The time-dependent decrease of the whole currents reflected a fast deactivation, which could be roughly fitted by a single exponential (note that deactivation of the inward currents was apparent only at voltages negative of the Ipeak voltage). The time constants for exponential activation and deactivation were in the millisecond range and were voltage dependent (Fig. 5B).
A slight decay (or inactivation) of the anion efflux current was evident at activating voltages of more than 2 s in duration (see currents at –136 mV in Fig. 5A). To determine the extent of inactivation in response to prolonged stimulation, voltage protocols were applied for up to 7 min. During prolonged depolarizations to –136 mV, inward-current magnitude decayed to approximately 50% of the initial peak-current amplitude (Fig. 5C) within 1 min, after which current magnitude remained stable or decayed slightly over 6 min. Note the fast (millisecond) time-dependent activation of the inward current when the time scale is expanded to a higher resolution (Fig. 5C inset).
To characterize further the channel activity underlying the whole-cell inward-anion currents, we recorded single-channel activity in the outside-out patch clamp configuration. Figure 6 shows standard single-channel activity. These channels did not exhibit rundown in their activity; however, many patches contained up to 50 channels (e.g. Fig. 6A) making single-channel analysis difficult. As a consequence, data analysis of single-anion channel activity was restricted to three patches from which single-channel currents could be resolved. Figure 6B illustrates the single-channel activity of the anion channel using SBS containing 1 mM SO42–. Plotting single-channel current amplitudes as a function of voltage revealed a single-channel conductance of 12.4 ± 0.1 picoSiemens (pS; n = 3; Fig. 6C). The following observations suggest that the single-channel activity shown in Figure 6B underlay the whole-cell inward currents. First, averaging single-channel recordings resulted in current traces that displayed similar deactivation kinetics to that observed for whole-cell currents (compare traces in Figs. 6D with whole-cell currents). Second, increasing extracellular SO42– from 1 to 25 mM significantly increased channel activity at potentials negative of the Ipeak voltage (Fig. 6E). This increase in channel activity is consistent with the SO42–-dependent shift of the activation potential to more negative voltages observed for the whole-cell recordings (Fig. 3A).
Phosphate Starvation Induces an Anion Channel-Mediated Citrate Efflux
It is well established that Arabidopsis roots secrete citrate in soils depleted in phosphate. To investigate further citrate efflux from Arabidopsis roots, we applied the whole-cell patch clamp technique to epidermal protoplasts isolated from Arabidopsis roots that had been exposed to phosphate-free media (see "Materials and Methods" for growth conditions). Experiments were conducted using 60 mM citrate in the pipette as the dominant anion. Voltage-dependent inward currents (Fig. 7A) were observed in 11 out of 78 cells (14%) with a mean current density of 2.6 ± 0.56 pA/pF; notably, these currents were not observed in protoplasts derived from Arabidopsis roots cultured in phosphate-replete media (Table I). Unfortunately, the inward currents displayed rapid rundown and they were completely abolished within 10 min after obtaining the whole-cell configuration (Fig. 7, A and B). As a consequence of this rapid rundown, analysis of the single channels that underlie the currents shown in Figure 7 was not possible. However, we generated a difference current voltage ( Finally, we investigated the possibility that the citrate efflux currents were mediated by the anion channel activity responsible for SO4–, Cl–, and NO3– efflux. To investigate this possibility, we compared the magnitudes of whole-cell currents (i.e. Ipeak) for SO42– and Cl– effluxes from epidermal protoplasts isolated from the roots of Arabidopsis grown in phosphate-replete and phosphate-free media. In experiments using standard pipette solution (containing 25 mM SO42– or with the SO42– replaced with 50 mM Cl–), phosphate starvation did not significantly affect either the frequency of occurrence or current density of SO42– or Cl– efflux currents (Fig. 7D). This suggests that organic-acid efflux is mediated by a distinct and novel channel activity, which is induced by phosphate starvation.
Epidermal cells are in direct contact with the soil solution and are most likely to be involved in nutrient absorption and modification of the rhizosphere (see below). Arabidopsis line N9093 showed GFP expression exclusively in the root epidermal cells (Fig. 1; see also Kiegle et al., 2000 ; Diatloff et al., 2004), ensuring that protoplasts isolated from the root epidermis were used in this study.
Using the whole-cell configuration of the patch clamp technique we have identified a voltage-dependent inward-rectifying anion channel activity in the epidermal cells of Arabidopsis roots, which we will refer to hereafter as Arabidopsis root anion channel (ARAC). SO42– was chosen as the permeable intracellular anion to investigate ARAC activity because it has been shown to prevent anion channel rundown (Frachisse et al., 1999
ARAC possessed properties characteristic for R-type anion channels, which have been previously reported in guard cells (Keller et al., 1989
There have been only a few studies of anion channel activity in higher plant roots. Kiegle et al. (2000)
Anion efflux channels have also been characterized in the xylem parenchyma cells of barley roots (Kohler and Raschke, 2000
ARAC is regulated by voltage (see above) and cytosolic factors. This study indicates that SO42– is a potent activator of ARAC and, in the absence of intracellular SO42–, ARAC exhibited rundown. Thus, the loss of cytosolic factors necessary for ARAC activity could be compensated for by intracellular SO42–. The basis of this channel regulation is unknown, but it is proposed to reflect the binding of SO42– to an intracellular regulatory site (Frachisse et al., 1999
We also observed that ARAC whole-cell current magnitude increased by approximately 7-fold within 10 min after obtaining the whole-cell configuration, indicating that intracellular SO42– activated or recruited inactive plasma membrane anion channels in Arabidopsis epidermal root cells. Interestingly, Table I shows that all cells exhibited SO42– efflux currents, but only approximately 50% of cells exhibited NO3– or Cl– efflux. This result was initially surprising because it was expected that the same channel type was responsible for SO42–, NO3–, and Cl– efflux (which is likely; see review by White and Broadly, 2001
The above observations indicate that ARAC is under strong posttranslational regulation in planta. The physiological significance of this is unknown, but it probably reflects the fact that the epidermal root cells used in this study represent a variety of cell types (e.g. atrichloblast and trichloblast cells and cells from the root elongation zone) that have different physiological roles and demands as influenced by the range of different extracellular environments individual root cell types will encounter (see below). The basis of this posttranslational regulation was not investigated in this study but could reflect the absence of a cytosolic factor on which channel activity is dependent (see above); candidates could include cytosolic calcium, kinases or phosphatases, pH or nucleotides—all of which have been shown to regulate anion channel activity in plant cells (for review, see White and Broadly, 2001
Plasma membrane anion efflux channels in plants play a number of fundamental roles in plant cell biology and root cell physiology. Osmoregulation has been best studied in guard cells, in which stomatal pore closure is initiated by salt loss effected principally by the opening of anion channels. Epidermal root cells are likely to experience large variations in the osmotic potential of the soil solution requiring cells to osmoregulate. Using Arabidopsis suspension cell cultures, Teodoro et al. (1998)
Organic-acid efflux has been well documented from higher plant root cells, and in Arabidopsis, malate and citrate efflux from the roots has been shown to be necessary for efficient phosphate acquisition (Narang et al., 2000
Phosphate is a major mineral nutrient required by plants, but it is one of the most immobile and inaccessible nutrients present in soils (Holford, 1997 Root epidermal cells from Arabidopsis plants grown in phosphate-free conditions possessed citrate (and malate) efflux currents, which exhibited channel-like activation/deactivation kinetics and voltage dependence (i.e. similar to that exhibited by ARAC-mediated anion efflux currents). This raised the possibility that either (1) phosphate starvation induced a citrate- and malate-permeable channel activity distinct from ARAC, or (2) citrate and malate conductance through ARAC is small and beyond the resolution of the patch clamp technique but that phosphate starvation up-regulated the ARAC activity to levels that allow citrate and malate efflux currents to be resolved. However, the absence of any significant regulation of ARAC activity by phosphate supply (Fig. 7D) suggests that phosphate starvation induces a novel channel activity distinct from ARAC activity and permeable to organic-acid anions. Thus, we refer to the phosphate-regulated ARAC as PR-ARAC. In some experiments, Cl– was omitted from the pipette media to avoid the contamination of citrate efflux currents by Cl– efflux. From a technical viewpoint, Ag/AgCl electrodes (which were used in this study) ideally require the presence of high millimolar amounts of Cl– in the pipette solution. However, in this study, junction potentials could be offset by the amplifier, and no drift in the junction potential during the experiment was evident, suggesting that the Ag/AgCl electrodes were stable in this study. Possible reasons for this are that only small currents (<50 pA) were recorded using citrate-containing pipette solutions and that there was sufficient contaminant Cl– (calculated to be at least 0.3 µM from the reported impurities in the citric acid) for reliable stable operation of the Ag/AgCl electrodes. Taking intracellular Cl– to be 0.3 µM, ECl was set at –305 mV; thus, the inward currents shown in Figure 7A could not be the result of Cl– efflux.
PR-ARAC differs significantly from other channels from higher plant roots that show a degree of permeability to organic-acid anions. First, PR-ARAC has significantly greater selectivity for organic-acid anions over Cl– compared to that reported for ALAACs from maize root tips (Pcit/PCl = 0.18 and Pmal/PCl = 0.25; Kollmeier et al., 2001
The low frequency of occurrence for PR-ARAC currents indicates that relatively few epidermal root cells were active in organic-acid anion efflux in response to phosphate starvation. It is expected that the sites of organic-acid efflux in the root system will probably reflect sites of active phosphate uptake. Indeed, organic-acid efflux has been shown to be a key factor influencing the phosphate acquisition efficiency of Arabidopsis (Narang et al., 2000 In summary, we have characterized a novel anion conductance in the plasma membrane of epidermal cells from Arabidopsis roots, which is permeable to citrate and malate and regulated by extracellular phosphate supply. It is likely that the channels underlying this conductance are involved in phosphate acquisition and represent the pathway for organic-acid anion efflux associated with phosphate nutrition in Arabidopsis roots.
Plant Material and Growth Conditions Seeds of a GFP-expressing Arabidopsis (Arabidopsis thaliana) line, Haseloff donor number J0841 (Nottingham Stock no. N9093), were surface sterilized first for 10 min with 80% (v/v) ethanol, then for 10 min with 1.2% (v/v) active chlorine bleach (NaOCl), and finally thoroughly rinsed with sterile water. These seeds were planted onto the surface of sterile agar plates (90-mm diameter) containing 0.8% phytagel (Sigma-Aldrich, St. Louis), full-strength Murashige and Skoog basal medium (Sigma-Aldrich), and 2% Suc, pH 5.5. The plates were placed vertically in a Sanyo (Sanyo Electric Biomedical, Sakata, Japan) MLR-350 environmental chamber with a light intensity of 100 µmol–1 m–2 s–1 for 16 h at a constant 22°C. Roots were harvested after 7- to 20-d growth. For phosphate starvation, 7- to 10-d-old plants were transferred to phosphate-free agar plates containing full-strength Murashige and Skoog basal medium without phosphate, 0.8% purified agarose (MBI Fermentas, Vilnius, Lithuania), and 2% Suc, pH 5.5.
Roots were imaged live in situ on the agar plates using a laser-scanning confocal microscope (TCS SP2, Leica Microsystems, Wetzlar, Germany). Imaging was performed using an excitation wavelength of 488 nm and emission window of 510 to 525 nm. Images are presented without manipulation.
Roots were removed from the agar plants and finely chopped in a solution (10 mM CaCl2, 10 mM KCl, 2 mM MgCl2, 2 mM MES/KOH, pH 6.0) containing (w/v) 1.5% cellulase (Onozuka RS, Yakult Honsha, Tokyo), 0.1% pectolyase Y-23 (Kikkoman, Japan), and 1% cellulase (Calbiochem, UK), 0.1% bovine serum albumin, and adjusted to 500 mosmol kg–1 with sorbitol. The chopped tissue was agitated at 30°C for 90 min. The digest was filtered using 50-µm nylon mesh and centrifuged at 60g for 5 min. The pellet was resuspended in 5 mL of ice-cold Solution A (500 mM sorbitol, 1 mM CaCl2, 5 mM MES/KOH, pH 6.0) and protoplasts were isolated using a Suc gradient as previously described (Roberts and Tester, 1995
GFP fluorescence in root protoplasts was detected using a fluorescence microscope equipped with an excitation filter of 460 to 500 nm and an emission filter at 510 to 560 nm. Whole-cell currents from GFP-fluorescent protoplasts were recorded at approximately 20°C with an Axopatch 200A amplifier (Axon Instruments, CA) using conventional patch clamp techniques (Hamill et al., 1981
Giga- Received May 25, 2004; returned for revision July 16, 2004; accepted July 16, 2004.
1 This work was supported by the Biotechnology and Biological Sciences Research Council (grant no. BRE13629to S.K.R., D.S., and M.R.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046995. * Corresponding author; e-mail s.k.roberts{at}lancaster.ac.uk; fax 01524–843854.
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ABSTRACT
RESULTS
) and B (
).
) Cs2SO4. Inset, An expanded view of the region corresponding to the current reversal potentials (Erevs). Arrows indicate calculated values for Esulfate. B, Erev of whole-cell currents plotted as a function of extracellular SO42–. Extracellular sulfate was varied using Cs2SO4 added to SBS. Data represent the mean (±SEM) for three separate experiments. Standard pipette solution was used. The dashed lines represent values where Erev = Esulfate, ECs, or ECl. C, Current voltage relationship of steady-state whole-cell currents using standard pipette solution but with 25 mM Cs2SO4 replaced with 50 mM CsCl. Currents shown are representative of 26 independent experiments. Currents were recorded 1 (
), 6 (
), and 10 (
I-V) relationship by subtracting the current measured before rundown from that remaining after complete rundown (See 
in sealing solution (see below). Using sealing solution, seals >10 G
