Characterization of Anion Channels in the Plasma Membrane of ... · plasma membrane of hypocotyl...

Characterization of Anion Channels in the PlasmaMembrane of Arabidopsis Epidermal Root Cells and theIdentification of a Citrate-Permeable Channel Induced byPhosphate Starvation1

Eugene Diatloff, Michael Roberts, Dale Sanders, and Stephen K. Roberts*

Department 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 metaldetoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsisthaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plantswere grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the rootepidermal cells of the elongation zone and young root hairs. Using SO4

22 as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of2180 mVexhibiting a maximum peakinward current (Ipeak) at approximately 2130 mV. These currents reversed at potentials close to the equilibrium potential forSO4

22, indicating that the inward currents represented SO422 efflux. Replacing intracellular SO4

22 with Cl2 or NO32 resulted in

inward currents exhibiting similar properties to the SO422 efflux currents, suggesting that these channels were also permeable

to a range of inorganic anions; however when intracellular SO422 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-cellcurrents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channelactivity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after beinggrown 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, phosphatestarvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channelactivity, which could have a role in phosphate acquisition.

Anion channels in the plasma membrane of plantcells catalyze anion fluxes both into and out of the celland serve a variety of functions. They have beenimplicated in stomatal function, where their activationis thought to be one of the rate-limiting steps in the lossof salts (and thus cell turgor) from guard cells leadingto stomatal pore closure (Roelfsema et al., 2004). In lessspecialized cells, anion channel activation is a likelystep in the transduction of signals modulating hypo-cotyl growth, including blue light (Cho and Spalding,1996) and possibly auxin (Thomine et al., 1997); thesesignal transduction events arise from depolarizationsresulting from anion channel activation. Anion chan-nels are also thought to facilitate the release of organicacids from higher plant roots. Al31-activated anionchannels (ALAACs) in the tips of wheat (Triticumaestivum) 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 toreduce Al31 stress by chelating this cation. Thusactivation of ALAACs by Al31 and their pharmaco-logical profile, which resembles that for Al31-inducedorganic-acid efflux from cereal roots, makes ALAACslikely candidates for mediating Al31-induced organic-acid secretion from roots.

The biophysical properties of plant anion channelshave been best characterized in guard cells. Two typesof anion channels have been extensively investigated:rapidly activating (R-type) and slowly activating(S-type) anion channels (e.g. Hedrich et al., 1990;Schroeder and Keller, 1992; Schmidt and Schroeder,1994). The R-type anion channel exhibits activation/deactivation kinetics in the millisecond range andinactivates in response to prolonged membrane de-polarization. Thus, R-type channels in guard cells arethought to mediate transient anion efflux and mem-brane depolarization. In contrast, S-type channelsactivate and deactivate slowly (with a time constantof seconds), and they do not inactivate. S-type chan-nels may possibly mediate prolonged anion efflux.R- and S-type anion channels also have distinct gating

1 This work was supported by the Biotechnology and BiologicalSciences Research Council (grant no. BRE13629 to S.K.R., D.S., andM.R.).

* Corresponding author; e-mail [email protected]; fax01524–843854.

Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.104.046995.

4136 Plant Physiology, December 2004, Vol. 136, pp. 4136–4149, www.plantphysiol.org � 2004 American Society of Plant Biologists

https://plantphysiol.orgDownloaded on April 12, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

properties. The typical R-type whole-cell current volt-age relationship is exemplified by a pronounced peak-current magnitude at relatively positive voltages andcomplete deactivation at relatively negative (resting)membrane voltages; in contrast, S-type whole-cellcurrent voltage relationships display a less pro-nounced peak current and exhibit inward rectificationin hyperpolarized conditions (Schroeder and Keller,1992; Roelfsema et al., 2004). Similar R- and S-typeanion channels have also been characterized in theplasma membrane of hypocotyl cells (Thomine et al.,1995, 1997; Frachisse et al., 2000).Anion channels in roots have not been well charac-

terized compared to those in guard cells, despite theirpotential importance in regulating acquisition fromsoil solution. The ALAACs of wheat and maize roots(see above) resemble S-type channels in that theydisplay slow activation kinetics (Pineros and Kochian,2001; Zhang et al., 2001) and exhibit inwardly rectify-ing current voltage relationships. Other reports ofanion channel activity in roots are limited to an out-wardly rectifying anion-selective channel in wheatand maize, which allows anion influx from the soilsolution (Skerrett and Tyerman, 1994; Pineros andKochian, 2001) and three different anion conductancesin the xylem parenchyma cells of barley (Hordeumvulgare) roots, which are thought to mediate anionefflux to the xylem vessels during salt delivery to theshoot (Kohler and Raschke, 2000; Kohler et al., 2002).In particular, there has been no systematic study ofanion channels in roots with respect to root soilinteraction and nutrient acquisition.In this study we address this dearth of knowledge

and use the patch clamp technique to investigate anionchannel activity in the epidermis of Arabidopsis

(Arabidopsis thaliana) roots. We show two types ofvoltage-dependent channel activity, which resemblethe R-type anion channel activity described in guardcells and hypocotyls. One of these channels wasubiquitously expressed in the epidermal cells andwas permeable to the inorganic anions, SO4

22, NO32,

and Cl2 but was impermeable to organic-acid anions,citrate and malate. The second anion channel was lessfrequently observed, was induced by phosphate star-vation, and mediated the efflux of organic-acid anions.It is suggested that the phosphate-regulated anionchannel mediates organic-acid anion efflux from Ara-bidopsis roots, which is thought to be an importantstrategy for efficient phosphate acquisition by higherplants (Narang et al., 2000).

RESULTS

The Arabidopsis line J0841 showed green fluores-cent protein (GFP) expression specifically only in root-peripheral cells, namely the epidermal cells (Fig. 1).These included cells of the elongation zone as well asyoung emerging root hair (trichoblast) and atrichlo-blast cells. However, GFP expression was not observedin the root tip, or in some of the older root hair oratrichoblast cells. Only cells expressing GFP were usedin this study.

The whole-cell configuration of the patch clamptechnique and standard bath solution (SBS) was usedto record anion currents across the plasma membraneof GFP-expressing protoplasts isolated from the epi-dermis of Arabidopsis roots. SBS contained 5 mM

LaCl3; La31 is an established broad-spectrum cation

channel blocker and, as illustrated in Figure 2, was

Figure 1. Cell-specific expression of GFP in Arabidopsis roots (line N9093). A, Bright-field image of root showing tip, elongationzone, and emerging root hair zone. Note that the image results from two independent but overlapping scans that have beenjoined at the overlap to provide an image representing a continuous length of root. B, Flourescent image of A showing GFPexpression in epidermal cells. C, Enlarged bright-field image of section highlighted in A. D, Fluorescent image of C showing GFPexpression in both trichoblast and atrichoblast epidermal cells.

Anion Channels in Arabidopsis Roots

Plant Physiol. Vol. 136, 2004 4137



effective in blocking inward and outward currents atnegative and positive potentials, respectively. Al-though the La31-sensitive currents illustrated in Figure2 were not investigated further, they most likelyrepresent cation currents through nonselective andCa21-permeable channels, which have been reportedpreviously in Arabidopsis root hairs and epidermalcells (Kiegle et al., 2000; Very and Davies, 2000;Demidchik et al., 2002). Pipette solutions were basedon the use of caesium salt; although Cs1 permeatesnonselective channels (Demidchik et al., 2002), Cs1

does not significantly permeate K1 channels (Tester,1988), and thus currents in this study were recordedwithout interference from this channel type.

Anion Currents in Arabidopsis Root Epidermal Cells

Using SBS and standard pipette solution (in which25 mM SO4

22 was the main anion), all root epidermalcells exhibited inward currents with strong voltagedependence. SO4

22 was employed as the main anionbecause intracellular SO4

22 has been shown to be bothasubstrate for andanactivatorofplasmamembranean-ion channels in Arabidopsis hypocotyl cells (Frachisseet al., 1999). Polarization of the membrane to potentialsmore positive than 2180 mV activated an inwardcurrent, which peaked at approximately 2130 mV(Fig. 2). At potentials more positive than the peakinward current (Ipeak), the current decreasedwith a pro-nounced rectification at voltages close to the reversalpotential (Erev). Using SBS containing 1 mM Cs2SO4,whole-cell currents reversed close to the equilibriumpotential of SO4

22 (141 mV), which is distinct from

the equilibrium potentials for Cl2 (262 mV) and Cs1

(282 mV) and indicates that the inward currents werelikely to be mainly carried by SO4

22 efflux (Fig. 3). Toinvestigate further the nature of the ion responsible forthe inward current, reversal potentials were recordedin varying extracellularCs2SO4 concentrations. Increas-ing extracellular SO4

22 to 10 and 25 mM shifted Erev ofthe whole-cell currents to 13.2 6 2.6 and 2.4 6 0.9 mV,respectively (Fig. 3, A and B); thus, Erev followedpredicted changes in the equilibrium potential forSO4

22. Furthermore, lowering pipette SO422 concentra-

tion from 25 to 1 mM reduced the mean current densityfrom 32.7 6 5.99 to 1.27 6 0.34 pA/pF (n 5 13). Thesedata are consistent with the inward currents beingcarried by SO4

22 efflux. It is also apparent that in-creasing extracellular SO4

22 induced a shift in the Ipeakpotential to more negative voltages illustrating thatextracellular SO4

22 was able to modulate the gating ofthe inward current such that the activation potentialwas shifted to more negative potentials. Similar gatingby extracellular anions has been reported for anionchannels in a variety of plant cells including guard cells(Dietrich and Hedrich, 1998) and hypocotyl cells inArabidopsis (Colcombet et al., 2001).

In experiments using SBS supplemented with 1 mM

SO422, the inward current reversed between the equi-

librium potentials for SO422 and Cl2 (Fig. 3B), in-

dicating that the channels mediating the inwardcurrent are likely to be permeable to other anions. Toinvestigate further the selectivity of the channelsthat underlie the inward current, intracellular SO4

22

was substituted by Cl2, NO32, malate, and citrate

(supplied as cesium salts). The substitution of cyto-

Figure 2. Isolation of whole-cell voltage-dependent inward currents from epidermal root cells using extracellular La31. A,Whole-cell currents measured across the plasma membrane of a GFP-expressing protoplast. Currents resulted from voltagepulses ranging from 2216 mV to 164 mV in 10-mV steps (for clarity only currents from 40-mV intervals are shown). Holdingvoltage was 216 mV. Standard pipette solution was used. Extracellular solution was as for SBS but with LaCl3 omitted andsupplemented with 1 mM Cs2SO4. B, As A except extracellular solution was SBS supplemented with 1 mM Cs2SO4. C, Currentvoltage relationship of steady-state whole-cell currents shown in A (d) and B (:).

Diatloff et al.

4138 Plant Physiol. Vol. 136, 2004



Figure 3. Anion efflux underlies the whole-cell voltage-dependent inward currents from the root epidermis. A, Current voltagerelationships of steady-state voltage-dependent currents using standard pipette solution and SBS supplemented with 1 mM (d),10 mM (:), and 25 mM (n) 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 SO4

22.Extracellular sulfate was varied using Cs2SO4 added to SBS. Data represent the mean (6SEM) for three separate experiments.Standard pipette solution was used. The dashed lines represent values where Erev 5 Esulfate, ECs, or ECl. C, Current voltagerelationship 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 (d) and 7 (:) min afterobtaining the whole-cell configuration. Inset, Whole-cell currents (used for the construction of the current voltage relationship)result from voltage pulses ranging from 2204 to 176 mV in 10-mV steps (for clarity only currents every 40-mV interval areshown). Holding voltage was136 mV. D, Current voltage relationship of steady-state whole-cell currents using standard pipettesolution but with 25mM Cs2SO4 replacedwith 50mM CsNO3. Currents shown are representative of 21 independent experiments.Currents were recorded 1 (d) and 11 (:) min after obtaining the whole-cell configuration. Inset, Whole-cell currents (used forthe construction of the current voltage relationship) result from voltage pulses ranging from2209 to171 mV in 10-mV steps (forclarity only currents every 40-mV interval are shown). Holding voltage was 131 mV.





solic rather than extracellular anions was favoredbecause it represented a more physiological condition;that is, the negative potential that usually exists acrossthe plasma membrane of plant cells tends to drive thepassive flow of anions from the cells. Furthermore,addition of citrate and malate to the bath solutionresulted in an increase in membrane conductance (i.e.leak conductance), consistent with a loss of integrity ofthe membrane and/or the seal between the membraneand the glass pipette. We suspect that this was theresult of cation chelation by citrate and malate.

Replacing pipette SO422 with Cl2 or NO3

2 resultedin inward currents, which exhibited similar voltagedependence to that observed for SO4

22 efflux currents(Fig. 3, C and D). Furthermore, decreasing pipette Cl2

or NO32 reduced the mean current density of the

inward currents consistent with them representinganion efflux (Table I). However, no inward currentswere observed when the pipette SO4

22 was replacedwith citrate or malate, indicating that the anion chan-nels were not significantly permeable to organic-acidanions. It is also significant that the whole-cell currentsreversed at ECl when pipette SO4

22 was replaced withCl2 (Fig. 3C); i.e. Cl2 was the principal anion in boththe bath and pipette solution. Taken together, these setsof data show that the channels underlying the voltage-gated inward currents in Arabidopsis root epidermalcells are anion selective andmediate at least SO4

22, Cl2,and NO3

2 efflux but not that of malate and citrate.

Regulation of Anion Currents in ArabidopsisRoot Epidermis

The following observations indicated that intracel-lular SO4

22 was a potent activator of the voltage-dependent anion efflux currents. The magnitude of theCl2 and NO3

2 efflux currents decreased with time andhad usually completely disappeared within 15 min

(Fig. 3, C and D); in contrast, SO422 efflux current

magnitudes remained stable for at least 2 h. In experi-ments using standard pipette solution (i.e. with SO4

22

as the charge-carrying anion), the inward currentsexhibited a marked increase in magnitude with time.Specifically, the magnitude of Ipeak increased 733% 6247% (within a mean time of 8.1 6 1.8 min; n 5 19)after obtaining the whole-cell configuration. Indeed, infour experiments, inward currents were absent imme-diately after obtaining the whole-cell configuration butdeveloped and increased in magnitude over severalminutes—a typical example is shown in Figure 4. It isunlikely that this gradual increase in the inwardcurrent reflected a gradual and slow equilibration ofthe cytosol with SO4

22, because the equilibration ofsmall inorganic anions between the pipette media andcytosol would be expected to be relatively immediateand complete within a minute. Rather, these observa-tions are consistent with the activation of the anionefflux currents by intracellular SO4

22 and are similar tothose previously reported by Frachisse et al. (1999) inArabidopsis hypocotyl epidermal cells.

It is noteworthy that an instantaneously activating,outwardly rectifying current was evident in some rootepidermal cells. From Figures 3, C and D, and 4, it isapparent that at least some of this outward currentdoes not run down or increase with time, illustratingthat the outward current was mediated, at least in part,by a channel type distinct from the channel responsiblefor the voltage-dependent inward current. Althoughthe outwardly rectifying current was not investigatedfurther in this study, it is interesting that after thecomplete rundown of the inward current in Figure 3and before the runup in Figure 4, the remainingoutwardly rectifying current reversed close to ECl,consistent with this current being carried by anioninflux. Outwardly rectifying anion-selective channelshave also been characterized in the roots of maize andwheat (Skerrett and Tyerman, 1994; Pineros andKochian, 2001) where they are thought to mediateanion influx in high-salt concentration (e.g. duringsalinity stress).

Activation/Deactivation Kinetics

Figure 5 shows typical currents resulting from step-ping the voltage from values either more negative(2216 mV) or more positive (164 mV) than the Ipeakvoltages. Upon stepping the potential from 2216 mV,inward currents at potentials negative of296mVwerecharacterized by a fast time-dependent activation,which could be roughly fitted by a single exponential,whereas inward currents at potentials positive of296mVexhibited instantaneous activation. In contrast,stepping from holding potentials positive of the Ipeakvoltage, whole-cell inward currents instantaneouslyincreased with the driving force for SO4

22 efflux beforedecreasing to reach a new steady-state value. The time-dependent decrease of the whole currents reflected

Table I. Frequency of occurrence and mean (6SEM) peak-currentdensity of whole-cell inward currents with varying intracellularanions and anion concentrations

All measurements were in SBS. Pipette solution was based onstandard pipette solution and modified by replacing Cs2SO4 withappropriate anion (as a cesium salt) and adjusted to 700 mosmol kg21

using sorbitol when necessary.

Internal Anion

Percentage of Cells with Current

(Total Number of Whole-Cell

Measurements)

Current Density

pA/pF

25 mM SO4 100 (20) 32.7 6 6.025 mM Cl 42 (19) 1.8 6 0.750 mM Cl 56 (26) 6.7 6 2.025 mM NO3 49 (37) 4.0 6 1.250 mM NO3 38 (21) 13.8 6 4.960 mM Citratea 0 (35) –60 mM Malate 0 (12) –

aThirteen of these experiments used pipette solution that was thesame as that detailed in Figure 7A.

Diatloff et al.




a fast deactivation, which could be roughly fitted bya single exponential (note that deactivation of theinward currentswas apparent only at voltages negativeof the Ipeak voltage). The time constants for exponentialactivation and deactivation were in the millisecondrange and were voltage dependent (Fig. 5B).A slight decay (or inactivation) of the anion efflux

current was evident at activating voltages of more than2 s in duration (see currents at2136 mV in Fig. 5A). Todetermine the extent of inactivation in response toprolonged stimulation, voltage protocols were appliedfor up to 7 min. During prolonged depolarizations to2136 mV, inward-current magnitude decayed to ap-proximately 50% of the initial peak-current amplitude(Fig. 5C) within 1 min, after which current magnituderemained stable or decayed slightly over 6 min. Notethe fast (millisecond) time-dependent activation of theinward current when the time scale is expanded toa higher resolution (Fig. 5C inset).

Single Channels

To characterize further the channel activity under-lying the whole-cell inward-anion currents, we re-corded single-channel activity in the outside-out patchclamp configuration. Figure 6 shows standard single-channel activity. These channels did not exhibitrundown in their activity; however, many patchescontained up to 50 channels (e.g. Fig. 6A) makingsingle-channel analysis difficult. As a consequence,data analysis of single-anion channel activity wasrestricted to three patches from which single-channelcurrents could be resolved. Figure 6B illustrates thesingle-channel activity of the anion channel using SBScontaining 1mM SO4

22. Plotting single-channel current

amplitudes as a function of voltage revealed a single-channel conductance of 12.4 6 0.1 picoSiemens (pS;n 5 3; Fig. 6C). The following observations suggestthat the single-channel activity shown in Figure 6Bunderlay the whole-cell inward currents. First, aver-aging single-channel recordings resulted in currenttraces that displayed similar deactivation kinetics tothat observed for whole-cell currents (compare tracesin Figs. 6D with whole-cell currents). Second, increas-ing extracellular SO4

22 from 1 to 25 mM significantlyincreased channel activity at potentials negative of theIpeak voltage (Fig. 6E). This increase in channel activityis consistent with the SO4

22-dependent shift of theactivation potential to more negative voltages ob-served for the whole-cell recordings (Fig. 3A).

Phosphate Starvation Induces an AnionChannel-Mediated Citrate Efflux

It is well established that Arabidopsis roots secretecitrate in soils depleted in phosphate. To investigatefurther citrate efflux from Arabidopsis roots, we ap-plied the whole-cell patch clamp technique to epider-mal protoplasts isolated from Arabidopsis roots thathad been exposed to phosphate-free media (see ‘‘Ma-terials and Methods’’ for growth conditions). Experi-ments were conducted using 60 mM citrate in thepipette as the dominant anion. Voltage-dependentinward currents (Fig. 7A) were observed in 11 out of78 cells (14%) with a mean current density of 2.66 0.56pA/pF; notably, these currents were not observed inprotoplasts derived from Arabidopsis roots culturedin phosphate-replete media (Table I). Unfortunately,the inward currents displayed rapid rundown andthey were completely abolished within 10 min after

Figure 4. Runup of whole-cell SO422 efflux currents. A,Whole-cell currents resulting from voltage pulses from2216mV to164

mV in 10-mV steps (for clarity only currents every 40-mV interval are shown). Holding voltage was 124 mV. Currents wererecorded 10 s after obtainingwhole-cell configuration. Standard pipette solution and SBSwere used. B, As A except currentswererecorded 8 min after obtaining whole-cell configuration. C, Current voltage relationships of steady-state currents recorded fromthe same cell as used for A and B. Currents were recorded at 10 s (d), 1 (:), 3 (;), 6 (¤), and 8 min (n) after obtaining whole-cellconfiguration. Arrow denotes reversal potential for Cl2. The currents shown are representative of four independent experiments.





obtaining the whole-cell configuration (Fig. 7, A andB). As a consequence of this rapid rundown, analysis ofthe single channels that underlie the currents shownin Figure 7 was not possible. However, we generateda difference current voltage (DI-V) relationship bysubtracting the current measured before rundownfrom that remaining after complete rundown (SeeFig. 7A inset). TheDI represented citrate efflux currents(assuming no cation permeation), which reversed at120 mV (Fig. 7A inset), predicting a permeability ratio(Pcit/PCl) of 26 (see Kohler and Raschke, 2000, equation4). The identity of the inward currents was furtherconfirmed in separate experiments using citrate-con-taining pipette media supplemented with 4 mM Cl2

and a bath solution containing 10 mM HEPES. In theseexperiments ECl and EHEPES were set at 262 mV and276mV, respectively. Figure 7C shows a representativeexample from three independent recordings in whichIpeak voltage was close to ECl and EHEPES, demonstratingthat the inward currents were not the result of Cl2 orHEPES efflux but rather represented citrate efflux.Similar experiments, in which intracellular citrate

was replaced with equimolar malate, showed 5 out of27 epidermal root protoplasts isolated from phos-phate-starved roots also exhibited small anion effluxcurrents. However, due to their small current magni-tude (1.06 6 0.7 pA/pF), high variability, and lowfrequency of occurrence, these currents were notanalyzed further (data not shown).

Finally,we investigated the possibility that the citrateefflux currents were mediated by the anion channelactivity responsible for SO4

2, Cl2, and NO32 efflux. To

investigate this possibility, we compared the magni-tudes ofwhole-cell currents (i.e. Ipeak) for SO4

22 andCl2

effluxes from epidermal protoplasts isolated from theroots of Arabidopsis grown in phosphate-replete andphosphate-free media. In experiments using standardpipette solution (containing 25 mM SO4

22 or with theSO4

22 replaced with 50 mM Cl2), phosphate starvationdid not significantly affect either the frequency ofoccurrence or current density of SO4

22 or Cl2 effluxcurrents (Fig. 7D). This suggests that organic-acidefflux is mediated by a distinct and novel channelactivity, which is induced by phosphate starvation.

Figure 5. Kinetic properties of whole-cell anion efflux currents using standard pipette and bath solutions. A, Activation anddeactivation of whole-cell currents in response to the voltage protocol as detailed in the bottom of the figure. Inset, Expandedtime scale for current trace at 2136 mV illustrating time dependent (exponential) activation. B, Time constants for deactivation(:) and activation (d) of whole-cell currents plotted as a function of voltage. Current deactivation was recorded by pulsing to testvoltages from a holding voltage of126mV, and current activation was recorded by pulsing to test voltages from a holding voltageof 2216 mV. Values for activation time constants are the mean (6SEM) from 5 independent experiments, and values fordeactivation time constant are the mean (6SEM) from 10 independent experiments. C, Inward currents in response todepolarization for 7 min to 2136 mV from a holding voltage of 2216 mV. The currents are representative of three independentexperiments. Note that the expansion of the time scale immediately following depolarization reveals time-dependent activation(and slight decay) as shown for equivalent depolarization in A.

Diatloff et al.




DISCUSSION

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 expres-sion exclusively in the root epidermal cells (Fig. 1; seealso Kiegle et al., 2000; Diatloff et al., 2004), ensuringthat protoplasts isolated from the root epidermis wereused in this study.

Figure 6. SO422 permeation through single voltage-dependent anion efflux channels. A, Current voltage relationship of an

outside-out patch of membrane in which single channel activity could not be resolved. Currents are in response to voltage rampfrom 2216 to 164 mV of 2-s duration. Currents are representative of four independent experiments. Peak current magnitude at2120 mV corresponds to approximately 50 simultaneous single-channel openings (calculated using corresponding values fromC). B, Representative single-channel sulfate currents recorded from an outside-out patch of membrane (n 5 3). Voltages areindicated to the right of the current traces, and downward deflections represent channel openings. c and o, Closed and openstate, respectively. Standard pipette solution and SBS supplemented with 1 mM Cs2SO4 were used. C, Single-channel currentvoltage relationship for SO4

22 currents (n 5 3). Linear regression fit revealed a single-channel conductance of 12.4 6 0.13 pS.Standard pipette and bath solutions were used. D, Current recordings from an outside-out patch for test voltages 2176 mV,2156 mV, and 2116 mV. Holding voltage was 216 mV. Each trace represents the average current trace from 30 consecutiverecordings at each test voltage. Solutions used are as for B. E, Activation of single-channel activity by extracellular SO4

22. Single-channel currents were recorded in an outside-out patch of membrane at a voltage of2176 mV. The top trace was obtained usingSBS supplemented with 1 mM Cs2SO4. The bottom trace is from the same patch of membrane following perfusion with SBSsupplemented with 25 mM Cs2SO4. Standard pipette solution was used. Currents shown are representative of two independentexperiments.





Figure 7. Voltage-dependent citrate efflux currents from epidermal root protoplasts isolated from phosphate-starved roots. A,Current voltage relationships of whole-cell steady-state currents from the cell shown in B. Currents are representative of 11independent experiments. Currents were recorded 3 (d), 4 (:), 5 (;), 6 (¤), and 10 (n) min after obtaining the whole-cellconfiguration. Extracellular solution was SBS. Pipette solution was as standard pipette solution except 25 mM Cs2SO4 wasreplaced with 60 mM Cs3citrate and 2 mM MgCl2 was omitted. Inset, Difference current voltage relationship obtained bysubtracting current values at 3 min from current values at 10 min after obtaining whole-cell configuration. Reversal potential ofthe difference current was120mV. B, Example of whole-cell currents used to construct the current voltage relationship shown inA. Currents were recorded 3 and 10 min after obtaining whole-cell configuration and result from voltage pulses ranging from2226 to154 mV in 10-mV steps (for clarity only currents every 40-mV interval are shown) from a holding voltage of124 mV. C,Current voltage relationship of steady-state whole-cell currents using pipette solution containing 60 mM Cs3citrate, 0.5 mM

HEPES, 2 mMMgCl2, 1 mMMgATP, 5 mM EGTA, p 7.2, and adjusted to 700mosmol kg21 using sorbitol. Extracellular solution wasSBS supplemented with 10 mM HEPES. Arrows denote values for ECl and EHEPES. Inset, Whole-cell currents used for theconstruction of current voltage relationship resulting from voltage pulses ranging from 2226 to 154 mV in 10-mV steps (forclarity only currents every 40-mV interval are shown). Holding voltage was 1 24 mV. Currents are representative of threeindependent experiments. D, Peak inward-current densities from protoplasts isolated from roots growing in phosphate replete(1P) and phosphate-free (2P) media (see ‘‘Materials and Methods’’ for details). SO4

22 efflux currents (black bars) were recordedusing standard pipette solution, chloride efflux currents (white bars) were recorded using standard pipette solution with 25 mM

Cs2SO4 replaced with 50 mM CsCl, and citrate efflux currents (hatched bars) were recorded using pipette media as in A. Currentdensities were calculated only from cells exhibiting a voltage dependent inward current with a defined current peak. Numbers inparentheses represent the number of cells exhibiting voltage-dependent efflux current/total number of cells tested.

Diatloff et al.




Whole-Cell Currents

Using the whole-cell configuration of the patchclamp technique we have identified a voltage-dependent inward-rectifying anion channel activityin the epidermal cells of Arabidopsis roots, which wewill refer to hereafter as Arabidopsis root anionchannel (ARAC). SO4

22 was chosen as the permeableintracellular anion to investigate ARAC activity be-cause it has been shown to prevent anion channelrundown (Frachisse et al., 1999); consequently, the useof intracellular SO4

22 permitted prolonged investiga-tion of ARAC activity for up to 3 h. ARAC activity wasalso recorded free from the interference from cation-permeable channels as a result of using the establishedcation channel blockers, La31 and Cs1. Surprisingly,extracellular La31 has been reported to block the blue-light activated anion channel activity in the hypocotylsof Arabidopsis (Lewis and Spalding, 1998); however,increasing extracellular La31 from 0.5 to 5 mM had noappreciable effect on ARAC activity (data not shown).ARAC possessed properties characteristic for R-type

anion channels, which have been previously reportedin guard cells (Keller et al., 1989; Hedrich et al., 1990),Arabidopsis hypocotyls (Thomine et al., 1995, 1997),and suspension cells of tobacco (Nicotiana tabacum;Zimmerman et al., 1994), and carrot (Daucus carota;Barbara et al., 1994). Specifically, activation of ARAC istightly controlled by transmembrane voltage, beingdeactivated at negative (resting) membrane potentialsand activated by subsequent depolarization. Theactivation/deactivation kinetics are rapid, being inthe millisecond range, and ARAC is permeable toother inorganic anions, including NO3

2 and Cl2 (forreview of R-type anion channel properties, see Whiteand Broadly, 2001). However, ARAC differed fromR-type channels in guard cells in that ARAC was onlypartially inactivated (i.e. by 50%) during prolongedapplication of activating voltages compared to 90%inactivation of R-type channels in guard cells. Thus,whereas guard cell R-type anion channels are thoughttomediate a transient anion efflux, ARACs are likely tomediate sustained anion efflux. It is also noteworthythat ARAC was not appreciably permeable to theorganic-acid anions, malate and citrate. This is incontrast to that reported for guard cells and hypocotylsin which malate is reported to permeate R-type chan-nels (e.g. Hedrich and Marten, 1993; Frachisse et al.,1999).

ARAC Activity: Comparisons with Other RootAnion Channels

There have been only a few studies of anion channelactivity in higher plant roots. Kiegle et al. (2000), ina preliminary study, identified inward currents, whichwere probably carried by SO4

22 efflux and exhibitingsimilar voltage dependence to ARAC; however, norigorous investigation of their gating, selectivity, orpossible physiological function was performed. More

rigorous characterizations of anion channels havebeen performed on cells from cereal plant roots.From the epidermal and cortical cells of maize andbarley root tips, inwardly rectifying anion channels,activated specifically by extracellular Al31 and perme-able to both inorganic (Ryan et al., 1997; Pineros andKochian, 2001) and organic (Kollmeier et al., 2001;Zhang et al., 2001) anions have been reported. Asa consequence of their activation by Al31, ALAACs arethought to represent the pathway for organic-acidefflux from cereal roots, which is responsible forchelating and detoxifying extracellular Al31. ARACactivity is distinct from the ALAACs in that it is notmodulated by Al31, La31, or Cu21 (data not shown)and is not permeable to organic-acid anions. Further-more, ALAACs display S-type anion channel charac-teristics, i.e. slow deactivation kinetics and inwardlyrectifying current voltage relationship. Finally,ALAACs have relatively large conductance of be-tween 27 and 144 pS compared to the small unitaryconductance values recorded for ARAC.

Anion efflux channels have also been characterizedin the xylem parenchyma cells of barley roots (Kohlerand Raschke, 2000; Kohler et al., 2002) where they areproposed to mediate anion transport into the xylemvessels. In these studies, three anion efflux channeltypes were identified. X-SLAC was found in only 7%of cells and displayed R-type current voltage relation-ships but displayed slow (S-type) deactivation kinetics(in the range of tens of seconds). The more prevalentchannel types, X-QUAC and X-IRAC, exhibited S-typecurrent voltage relationships. Thus to date, this studyrepresents the only record, to our knowledge, of anR-type anion channel reported in higher plant roots.

Regulation of ARAC

ARAC is regulated by voltage (see above) andcytosolic factors. This study indicates that SO4

22 isa potent activator of ARAC and, in the absence ofintracellular SO4

22, ARAC exhibited rundown. Thus,the loss of cytosolic factors necessary for ARACactivity could be compensated for by intracellularSO4

22. The basis of this channel regulation is unknown,but it is proposed to reflect the binding of SO4

22 to anintracellular regulatory site (Frachisse et al., 1999). Inthis study, stable SO4

22 efflux currents could be re-corded using 1 mM intracellular sulfate, suggestingthat the binding constant of such a site would be lessthan 1 mM. Furthermore, ARAC activity recorded inoutside-out patches was also stable, suggesting thatthe binding site could be an integral part of thechannel.

We also observed that ARAC whole-cell currentmagnitude increased by approximately 7-fold within10 min after obtaining the whole-cell configuration,indicating that intracellular SO4

22 activated or recruit-ed inactive plasma membrane anion channels inArabidopsis epidermal root cells. Interestingly, TableI shows that all cells exhibited SO4

22 efflux currents,





but only approximately 50% of cells exhibited NO32 or

Cl2 efflux. This result was initially surprising becauseit was expected that the same channel type wasresponsible for SO4

22, NO32, and Cl2 efflux (which is

likely; see review by White and Broadly, 2001) andtherefore the proportion of cells exhibiting anion effluxcurrents should be independent of the intracellularanion chosen as the charge carrier. The explanation forthis anomaly is that ARACs are present in all epider-mal root cells, but a significant proportion of these cellsare in a quiescent state in which ARAC is inactive. Thisis consistent with Figure 4 in which ARAC activity isabsent immediately after obtaining the whole-cellconfiguration (i.e. ARAC is inactive) but becomesapparent over several minutes. Thus, cells exhibitingCl2 or NO3

2 efflux currents probably reflect the pro-portion of cells in which ARAC is active in planta.

The above observations indicate that ARAC is understrong posttranslational regulation in planta. Thephysiological significance of this is unknown, but itprobably reflects the fact that the epidermal root cellsused in this study represent a variety of cell types (e.g.atrichloblast and trichloblast cells and cells from theroot elongation zone) that have different physiologicalroles and demands as influenced by the range ofdifferent extracellular environments individual rootcell types will encounter (see below). The basis of thisposttranslational regulation was not investigated inthis study but could reflect the absence of a cytosolicfactor on which channel activity is dependent (seeabove); candidates could include cytosolic calcium,kinases or phosphatases, pH or nucleotides—all ofwhich have been shown to regulate anion channelactivity in plant cells (for review, see White andBroadly, 2001).

Physiological Significance of ARAC

Plasma membrane anion efflux channels in plantsplay a number of fundamental roles in plant cellbiology and root cell physiology. Osmoregulation hasbeen best studied in guard cells, in which stomatalpore closure is initiated by salt loss effected principallyby the opening of anion channels. Epidermal root cellsare likely to experience large variations in the osmoticpotential of the soil solution requiring cells to osmo-regulate. Using Arabidopsis suspension cell cultures,Teodoro et al. (1998) showed that hypoosmotic shockinduced a Cl2 efflux, which was inhibited by the anionchannel blocker, A-9-C. By analogy with anion chan-nels in the guard cells, ARAC could initiate anionefflux in response to hypoosmotic conditions. Anionchannels are also thought to play a pivotal role incytosolic pH regulation in which they provide a shuntconductance to facilitate activation of H1 pumping(and hence removal of cytosolic H1) in response tocytosolic acidification (Johannes et al., 1998). Indeed,cytosolic pH is known to regulate Cl2 efflux in higherplants (Beffagna et al., 1997). Although the regulation

of ARAC by cytosolic pH has not been investigated inthis study, ARAC could mediate an anion efflux shuntconductance during acid stress in root cells.

Organic-acid efflux has been well documented fromhigher plant root cells, and in Arabidopsis, malate andcitrate efflux from the roots has been shown to benecessary for efficient phosphate acquisition (Naranget al., 2000) and the detoxification of Cu21 (Murphyet al., 1999). However, in the absence of measurableARAC-mediated citrate or malate efflux in this studyand the absence of ARAC regulation by phosphatesupply (see below) or by extracellular Cu21 (data notshown), it is unlikely that ARAC is responsible fororganic-acid efflux from Arabidopsis roots. Althoughit has received far less attention than organic-acidanions, inorganic anion efflux has also been recordedfrom higher plant roots, for example Cl2 efflux frombarley (Jackson and Edwards, 1966) and Arabidopsis(Lorenzen et al., 2004) and SO4

22 efflux from carrot(Cram, 1983) and tomato roots (Lopez et al., 2002). Thephysiological significance of these fluxes is not alwaysclear, but they may be important in preventing thetoxic accumulation of anions in the cytosol of rootcells, particularly when extracellular anion concentra-tion is high. ARAC probably mediates inorganic anionefflux; indeed, the gating properties of ARAC are wellsuited to preventing intracellular accumulation ofSO4

22 in that (1) increasing extracellular SO422 mod-

ulates the gating of ARAC such that it activates atmore negative membrane voltages (and thus increasesthe dynamic voltage range at which ARAC is active),and (2) accumulation of intracellular SO4

22 wouldfurther enhance ARAC activity.

Phosphate Starvation and Citrate Efflux

Phosphate is a major mineral nutrient required byplants, but it is one of the most immobile and in-accessible nutrients present in soils (Holford, 1997).Plant have evolved a variety of strategies to increasethe availability of phosphate from the soil solution,a key one being the secretion of organic acids tomobilize sparingly soluble forms of phosphate fromsoil solutions (Hoffland et al., 1992; Jones, 1998). Theroots of Arabidopsis and the closely related membersof the Brassica family have been shown to enhancemalate and citrate efflux in response to phosphatedeficiency (Narang et al., 2000). Since citrate andmalate exist predominately as tri- and divalent anionsin the cytoplasm, their movement out of root cells is anenergetically passive process due the large negativepotential difference across the plasma membrane.Thus, it is likely that citrate- and malate-permeablechannels mediate organic-acid efflux.

Root epidermal cells fromArabidopsis plants grownin phosphate-free conditions possessed citrate (andmalate) efflux currents, which exhibited channel-likeactivation/deactivation kinetics and voltage depen-dence (i.e. similar to that exhibited by ARAC-medi-

Diatloff et al.




ated anion efflux currents). This raised the possibilitythat either (1) phosphate starvation induced a citrate-and malate-permeable channel activity distinct fromARAC, or (2) citrate and malate conductance throughARAC is small and beyond the resolution of the patchclamp technique but that phosphate starvation up-regulated the ARAC activity to levels that allow citrateand malate efflux currents to be resolved. However,the absence of any significant regulation of ARACactivity by phosphate supply (Fig. 7D) suggests thatphosphate starvation induces a novel channel activitydistinct fromARAC activity and permeable to organic-acid anions. Thus, we refer to the phosphate-regulatedARAC as PR-ARAC.In some experiments, Cl2 was omitted from the

pipette media to avoid the contamination of citrateefflux currents by Cl2 efflux. From a technical view-point, Ag/AgCl electrodes (which were used in thisstudy) ideally require the presence of high millimolaramounts of Cl2 in the pipette solution. However, inthis study, junction potentials could be offset by theamplifier, and no drift in the junction potential duringthe experiment was evident, suggesting that the Ag/AgCl electrodes were stable in this study. Possiblereasons for this are that only small currents (,50 pA)were recorded using citrate-containing pipette solu-tions and that there was sufficient contaminant Cl2

(calculated to be at least 0.3 mM from the reportedimpurities in the citric acid) for reliable stable opera-tion of the Ag/AgCl electrodes. Taking intracellularCl2 to be 0.3 mM, ECl was set at 2305 mV; thus, theinward currents shown in Figure 7A could not be theresult of Cl2 efflux.PR-ARAC differs significantly from other channels

from higher plant roots that show a degree of perme-ability to organic-acid anions. First, PR-ARAC hassignificantly greater selectivity for organic-acid anionsover Cl2 compared to that reported for ALAACs frommaize root tips (Pcit/PCl 5 0.18 and Pmal/PCl 5 0.25;Kollmeier et al., 2001) and wheat roots (Pmal/PCl 5 2.6;Zhang et al., 1991). Second, the ALAACs from maizeand wheat exhibit S-type voltage dependence (i.e.inwardly rectifying current voltage relationships),whereas PR-ARAC exhibited R-type voltage depen-dence. Finally, ALAAC activity is dependent on thepresence of extracellular Al31 and does not exhibitrundown, even in excised outside-out patches (Pinerosand Kochian, 2001). However, the rapid rundown ofPR-ARAC indicates that its activity is probably de-pendent on cytosolic factors that are washed outduring the patch clamp experiment.The low frequency of occurrence for PR-ARAC

currents indicates that relatively few epidermal rootcells were active in organic-acid anion efflux in re-sponse to phosphate starvation. It is expected that thesites of organic-acid efflux in the root system willprobably reflect sites of active phosphate uptake. In-deed, organic-acid efflux has been shown to be a keyfactor influencing the phosphate acquisition efficiencyof Arabidopsis (Narang et al., 2000). The completion of

the Arabidopsis genome has revealed nine membersof the phosphate transport family, Pht1, which arethought to mediate the uptake of phosphate from thesoil solution (Mudge et al., 2002). Promoter analysisof Pht1 family members revealed complex and cell-specific expression in response to phosphate starvation(Mudge et al., 2002). For example, Pht1:2 phosphate-induced expression was limited to mainly the roothairs (or trichloblast cells of the root epidermis) butwas absent in the epidermal atrichloblast cells and theroot elongation zones and root tip. Thus, the pattern ofPht1;2 expression only partially overlaps with the cell-specific GFP expression of the N9093 line used in thisstudy; hence, many of the cells used in this study maynot have been active in phosphate uptake. Therefore, itis possible that the low frequency of occurrence of PR-ARAC reflects the isolation of protoplasts from rootepidermal cells, which were not directly mediatingphosphate uptake.

In summary, we have characterized a novel anionconductance in the plasma membrane of epidermalcells from Arabidopsis roots, which is permeable tocitrate and malate and regulated by extracellularphosphate supply. It is likely that the channels un-derlying this conductance are involved in phosphateacquisition and represent the pathway for organic-acidanion efflux associated with phosphate nutrition inArabidopsis roots.

MATERIALS AND METHODS

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

mmol21 m22 s21 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.

Confocal Microscopy

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 manipu-

lation.

Protoplast Isolation

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 kg21 with sorbitol. The chopped





tissue was agitated at 30�C for 90 min. The digest was filtered using 50-mm

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). After the Suc gradient step, clean

protoplasts were resuspended in 5 mL Solution A and centrifuged at 60g for

5 min. Protoplasts were resuspended in 1 mL of Solution A and stored on ice.

Electrophysiology

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). Cells were perfused in a chamber consisting of a thin

glass base to which protoplasts adhered loosely. Electrodes were pulled from

borosilicate-glass capillaries (Kimax 51, Kimax Products, NJ) to give resistan-

ces of less than 25 MV in sealing solution (see below). Using sealing solution,

seals .10 GV were regularly achieved. To reduce pipette capacitance, elec-

trodes were coated by dipping the pipette tip into a 50% (w/w) mixture of

mineral oil and Parafilm (American National Can, Greenwich, CT). An Ag/

AgCl reference electrode was connected to the bath via a 3-M KCl/agar salt

bridge. Whole-cell capacitance and series resistance were compensated for by

the amplifier. Access resistance was monitored during experiments and was

less than 20 MV. Before analog-to-digital conversion, the voltage signals

representing clamp currents were low-pass filtered at 5 kHz. Outside-out

patches were obtained from the whole-cell configuration by pulling away the

pipette from the protoplast. Data were digitized at 2 kHz and filtered at 300 Hz

for analysis. All data were acquired by pClamp 8.0 (Axon Instruments) and

analyzed using either pClamp 8.0 or FigP (version 2.2, Biosoft, Cambridge,

UK). Liquid-junction potential was corrected for in all experiments as de-

scribed by Neher (1992). Tip potentials were measured at the end of an

experiment by measuring the potential change when the pipette tip was

broken. Only experiments in which the tip potential was less than 4 mV were

used. Ion equilibrium potentials were calculated after correction for ionic

activities (as calculated by GEOCHEM-PC; Parker et al., 1995). Variation in

data is presented as the SE of the mean.

Solutions

Giga-V seals were formed in a sealing solution containing 10 mM CaCl2,

5 mM MgCl2, 10 mM MES, adjusted to pH 6.0 with Tris-base and adjusted to

700 mosmol kg21 using sorbitol. After obtaining a whole-cell configuration

this solution was replaced by SBS, which contained 5 mM LaCl3, 10 mM CaCl2,

5 mM MgCl2, 10 mM MES, adjusted to pH 6.0 with Tris-base and adjusted to

700 mosmol kg21 using sorbitol; all currents were recorded in this unless

otherwise stated. Standard intracellular (pipette-filling) solution (25 mM

Cs2SO4, 2 mM MgCl2, 10 mM HEPES, 1 mM MgATP, 2 mM EGTA adjusted to

pH 7.2 with Tris base and 720 mosmol kg21 using sorbitol) was used in all

experiments unless otherwise stated.

Received May 25, 2004; returned for revision July 16, 2004; accepted July 16,

2004.

LITERATURE CITED

Barbara JG, Stoeckel H, Takeda K (1994) Hyperpolarization-activated

inward chloride current in protoplasts from suspension-cultured carrot

cells. Protoplasma 180: 136–144

Beffagna N, Romani G, Meraviglia G, Pallini S (1997) Effects of abscisic

acid and cytoplasmic pH on potassium and chloride efflux in Arabidopsis

thaliana seedlings. Plant Cell Physiol 38: 503–510

Cho MH, Spalding EP (1996) An anion channel in Arabidopsis hypocotyls

activated by blue light. Proc Natl Acad Sci USA 93: 8134–8138

Colcombet J, Thomine S, Guern J, Frachisse JM, Barbier-Brygoo H (2001)

Nucleotides provide a voltage-sensitive gate for the rapid anion channel

of Arabidopsis hypocotyl cells. J Biol Chem 276: 36139–36145

Cram WJ (1983) Characteristics of sulfate transport across the plasma-

lemma and tonoplast of carrot root cells. Plant Physiol 72: 204–211

Demidchik V, Bowen HC, Maathuis FJM, Shabala SN, Tester MA, White

PJ, Davies JM (2002) Arabidopsis thaliana root non-selective cation

channels mediate calcium uptake and are involved in growth. Plant J

32: 799–808

Diatloff E, Geger D, Shang L, Hedrich R, Roberts SK (2004) Differential

regulation of K1 channels in Arabidopsis epidermal and stelar root cells.

Plant Cell Environ 27: 980–990

Dietrich P, Hedrich R (1998) Anions permeate and gate GCAC1, a voltage-

dependent guard cell anion channel. Plant J 15: 479–487

Frachisse JM, Colcombet J, Guern J, Barbier-Brygoo H (2000) Character-

ization of a nitrate-permeable channel able to mediate sustained anion

efflux in hypocotyl cells from Arabidopsis thaliana. Plant J 21: 361–371

Frachisse JM, Thomine S, Colcombet J, Guern J, Barbier-Brygoo H (1999)

Sulfate is both a substrate and an activator of the voltage-dependent

anion channel of Arabidopsis hypocotyl cells. Plant Physiol 121: 253–261

Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved

patch clamp techniques for high resolution current recording from cells

and cell-free membrane patches. Pflugers Arch 391: 85–100

Hedrich R, Busch H, Raschke K (1990) Ca21 and nucleotide dependent

regulation of voltage dependent anion channel in the plasma membrane

of guard cell. EMBO J 9: 3889–3892

Hedrich R, Marten I (1993) Malate-induced feedback regulation of plasma

membrane anion channels could provide CO2 sensor to guard cells.

EMBO J 12: 897–901

Hoffland E, van den Boogaard R, Nelemans J, Findenegg G (1992)

Biosynthesis and root exudation of citric and malic acids in phosphate-

starved rape plants. New Phytol 122: 675–680

Holford ICR (1997) Soil phosphorus: its measurement and its uptake by

plants. Aust J Soil Res 35: 227–239

Jackson PC, Edwards DG (1966) Cation effects on chloride fluxes and

accumulation levels in barley roots. J Gen Physiol 50: 225–241

Johannes E, Crofts A, Sanders D (1998) Control of Cl- efflux in Chara

corallina by cytosolic pH, free Ca21, and phosphorylation indicates a role

of plasma membrane anion channels in cytosolic pH regulation. Plant

Physiol 118: 173–181

Jones DL (1998) Organic acids in the rhizosphere: a critical review. Plant

Soil 205: 25–44

Keller BU, Hedrich R, Raschke K (1989) Voltage dependent anion channels

in the plasma membrane of guard cells. Nature 341: 450–453

Kiegle E, Gilliham M, Haseloff J, Tester M (2000) Hyperpolarisation-

activated calcium currents found only in cells from the elongation zone

of Arabidopsis thaliana roots. Plant J 21: 225–229

Kohler B, Raschke K (2000) The delivery of salts to the xylem: three types

of anion conductance in the plasmalemma of the xylem parenchyma of

roots of barley. Plant Physiol 122: 243–254

Kohler B, Wegner LH, Osipov V, Raschke K (2002) 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. Plant J 30: 133–142

Kollmeier M, Dietrich P, Bauer CS, Horst WJ, Hedrich R (2001) Aluminum

activates a citrate-permeable anion channel in the aluminum-sensitive

zone of the maize root apex. A comparison between an aluminum-

sensitive and an aluminum-resistant cultivar. Plant Physiol 126: 397–410

Lewis BD, Spalding EP (1998) Nonselective block by La31 of Arabidopsis

ion channels involved in signal transduction. J Membr Biol 162: 81–90

Lopez J, Bell CI, Tremblay N, Dorais M, Gosselin A (2002) Uptake and

translocation of sulphate in tomato seedlings in relation to sulphate

supply. J Plant Nutr 25: 1471–1485

Lorenzen I, Aberle T, Plieth C (2004) Salt stress-induced chloride flux:

a study using transgenic Arabidopsis expressing a fluorescent anion

probe. Plant J 38: 539–544

Mudge SR, Rae AL, Diatloff E, Smith FW (2002) Expression analysis

suggests novel roles for members of the Pht1 family of phosphate

transporters in Arabidopsis. Plant J 31: 341–353

Murphy AS, Eisinger WR, Shaff JE, Kochian LV, Taiz L (1999) Early

copper-induced leakage of K1 from Arabidopsis seedlings is medi-

ated by ion channels and coupled to citrate efflux. Plant Physiol 121:

1375–1382

Narang RA, Bruene A, Altmann T (2000) Analysis of phosphate acquisi-

tion efficiency in different Arabidopsis accessions. Plant Physiol 124:

1786–1799

Neher E (1992) Correction for liquid junction potentials in patch clamp

experiments. Methods Enzymol 207: 123–131

Diatloff et al.




Parker DR, Norvell WA, Chaney RL (1995) GEOCHEM-PC: a chemical

speciation program for IBM and compatible computers. In RH Loeppert,

AP Schwab, S Goldberg, eds, Chemical Equilibrium and Reaction

Models, Special Publication 42. Soil Science Society of America,

Madison, WI, pp 253–269

Pineros MA, Kochian LV (2001) A patch-clamp study on the physiology of

aluminum toxicity and aluminum tolerance in maize: identification and

characterization of Al31-induced anion channels. Plant Physiol 125:

292–305

Roberts SK, Tester M (1995) Inward and outward K1-selective currents in

the plasma membrane of protoplasts from maize root cortex and stele.

Plant J 8: 811–825

Roelfsema MRG, Levchenko V, Hedrich R (2004) ABA depolarizes guard

cells in intact plants, through a transient activation of R- and S-type

anion channels. Plant J 37: 578–588

Ryan PR, Skerrett M, Findlay GP, Delhaize E, Tyerman SD (1997)

Aluminum activates an anion channel in the apical cells of wheat roots.

Proc Natl Acad Sci USA 94: 6547–6552

Schmidt C, Schroeder JI (1994) Anion selectivity of slow anion channels in

the plasma-membrane of guard-cells: large nitrate permeability. Plant

Physiol 106: 383–391

Schroeder JI, Keller BU (1992) Two types of anion channel currents in

guard cells with distinct voltage regulation. Proc Natl Acad Sci USA

89: 5025–5029

Skerrett M, Tyerman SD (1994) A channel that allows inwardly directed

fluxes of anions in protoplasts derived from wheat roots. Planta 192:

295–305

Teodoro AE, Zingarelli L, Lado P (1998) Early changes of Cl2 efflux and H1

extrusion induced by osmotic stress in Arabidopsis thaliana cells. Physiol

Plant 102: 29–37

Tester M (1988) Blockade of potassium channels in the plasmalemma of

chara corallina by tetraethylammonium, Ba21, Na 1 and Cs1. J Membr

Biol 105: 77–85

Thomine S, Lelievre F, Boufflet M, Guern J, Barbier-Brygoo H (1997)

Anion-channel blockers interfere with auxin responses in dark-grown

Arabidopsis hypocotyls. Plant Physiol 115: 533–542

Thomine S, Zimmermann S, Guern J, Barbier-Brygoo H (1995) ATP-

dependent regulation of an anion channel at the plasma membrane of

protoplasts from epidermal cells of Arabidopsis hypocotyls. Plant Cell

7: 2091–2100

Very AA, Davies JM (2000) Hyperpolarization-activated calcium channels

at the tip of Arabidopsis root hairs. Proc Natl Acad Sci USA 97: 9801–9806

White PJ, Broadley MR (2001) Chloride in soils and its uptake and

movement within the plant: a review. Ann Bot (Lond) 88: 967–988

Zhang WH, Ryan PR, Tyerman SD (2001) Malate-permeable channels and

cation channels activated by aluminum in the apical cells of wheat roots.

Plant Physiol 125: 1459–1472

Zimmermann S, Thomine S, Guern J, Barbier-Brygoo H (1994) An anion

current at the plasma-membrane of tobacco protoplasts shows ATP-

dependent voltage regulation and is modulated by auxin. Plant J 6:

707–716





Characterization of Anion Channels in the Plasma Membrane of ... · plasma membrane of hypocotyl...

Documents

Transcript of Characterization of Anion Channels in the Plasma Membrane of ... · plasma membrane of hypocotyl...