Malate-Permeable Channels and Cation Channels Activated by Aluminum in the Apical Cells of ...

14 Pages·2001·0.31 MB·English

Malate-Permeable Channels and Cation Channels
Activated by Aluminum in the Apical Cells of
1
Wheat Roots
Wen-Hao Zhang*, Peter R. Ryan, and Stephen D. Tyerman
School of Biological Sciences, The Flinders University of South Australia, G.P.O. Box 2100, Adelaide, South
Australia 5001, Australia (W.-H.Z., S.D.T.); and Commonwealth Scientific and Industrial Research
Organization Plant Industry, G.P.O. Box 1600, Canberra, Australian Capital Territory 2601, Australia (P.R.R.)
Aluminum (Al31)-dependent efflux of malate from root apices is a mechanism for Al31 tolerance in wheat (Triticum
aestivum). The malate anions protect the sensitive root tips by chelating the toxic Al31 cations in the rhizosphere to form
non-toxiccomplexes.Activationofmalate-permeablechannelsintheplasmamembranecouldbecriticalinregulatingthis
malate efflux. We examined this by investigating Al31-activated channels in protoplasts from root apices of near-isogenic
wheatdifferinginAl31toleranceatasinglelocus.Usingwhole-cellpatchclampwefoundthatAl31stimulatedanelectrical
currentcarriedbyanioneffluxacrosstheplasmamembraneintheAl31-tolerant(ET8)andAl31-sensitive(ES8)genotypes.
Thiscurrentoccurredmorefrequently,hadagreatercurrentdensity,andremainedactiveforlongerinET8protoplaststhan
forES8protoplasts.TheAl31-activatedcurrentexhibitedhigherpermeabilitytomalate22thantoCl2(P /P $2.6)and
mal Cl
wasinhibitedbyanionchannelantagonists,niflumateanddiphenylamine-2-carboxylicacid.InET8,butnotES8,protoplasts
an outward-rectifying K1 current was activated in the presence of Al31 when cAMP was included in the pipette solution.
ThesefindingsprovideevidencethatthedifferenceinAl31-inducedmalateeffluxbetweenAl31-tolerantandAl31-sensitive
genotypes lies in the differing capacity for Al31 to activate malate permeable channels and cation channels for sustained
malate release.
When aluminum (Al) is solubilized in acid soils to Using near-isogenic lines of wheat that differ in
thephytotoxicspeciesAl31itbecomesamajorfactor Al31toleranceatasinglelocus,Delhaizeetal.(1993a,
limiting crop growth and yield (Foy et al., 1978; 1993b) showed that 10 times more malate was re-
Kochian,1995).Anumberofplantspeciesandgeno- leased from the root apices of an Al31-tolerant line
types within species exhibit an inheritable tolerance than from an Al31-sensitive line when exposed to
toAl31.Twostrategiesthathavebeenidentifiedthat toxiclevelsofAl31.AsimilarAl31-activatedeffluxof
allowplantstotolerateAl31aretheexclusionoftoxic malate has been found in other Al31-tolerant wheat
Al31 from the root apex by releasing Al31-chelating genotypes(Basuetal.,1994;Ryanetal.,1995b;Pellet
ligands such as organic acids and phosphate, or by etal.,1996).Additionofmalatetoanutrientsolution
releasing OH2 to increase external pH; and the de- containing a toxic concentration of Al31 significantly
toxificationofAl31onceithasenteredthecytoplasm improves the growth of Al31-sensitive wheat geno-
by chelation and/or sequestration to less Al31- types (Delhaize et al., 1993b; Ryan et al., 1995b).
sensitive compartments (Taylor, 1991; Delhaize and Taken together, these results suggest that one mech-
Ryan, 1995; Kochian, 1995; Ma, 2000). Several Al31- anism for Al31 tolerance in wheat relies on the Al31-
tolerant plant species and genotypes exhibit Al31- activated exudation of malate from the root apices.
dependent exudation of organic acids from their Theorganicanionsprotecttheplantsbychelatingthe
roots. For instance, the efflux of malate is stimulated toxic Al31 cations in the rhizosphere to form non-
toxic complexes.
fromwheat(Triticumaestivum;Delhaizeetal.,1993b;
Malate exists predominantly as the divalent anion
Basu et al., 1994; Pellet et al., 1996), citrate from
inthecytoplasm,andmovementofmalateoutofthe
maize, snapbean, and Cassia tora (Miyasaka et al.,
root cells is an energetically passive process because
1991; Pellet et al., 1995; Ma et al., 1997), and oxalate
of the large negative electrical potential difference
from buckwheat and taro (Ma and Miyasaka, 1998;
across the plasma membrane. Thus, Al31-stimulated
Zheng et al., 1998). Organic acid release is restricted
to the root apices, which is the critical zone for Al31 malateeffluxislikelytobemediatedbyactivationof
an anion channel permeable to malate in the plasma
stress (Ryan et al., 1993).
membrane of root apical cells. The observations that
Al31-activated efflux of malate from wheat roots
1ThisstudywasfundedbytheAustralianResearchCouncil. (Ryanetal.,1995a)andoxalatefrombuckwheatroots
*Correspondingauthor;[email protected]; (Zheng et al., 1998) is sensitive to several anion-
fax[phone]. channel blockers are in line with this proposition.
PlantPhysiology,March2001,VDool.w1n2l5o,apdepd. 1fr4o5m9– o1n4 7N2o,vwewmwbe.pr l2a3n,t p20h1y8si o- lP.ourbglis©he2d0 0b1y Awmwwer.ipclaanntSpohcyiseitoyl.oorfgP lantPhysiologists 1459
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Zhangetal.
1
Malate content in root tissues and the activities of anions and K (Schroeder, 1995). A similar mecha-
enzymes involved in malate synthesis (phosphoenol- nismmayexplaintheAl31-activatedeffluxofK1and
pyruvatecarboxylaseandmalatedehydrogenase)are malate from Al31-tolerant wheat roots, but support-
not significantly different between wheat genotypes ive electrophysiological data are lacking. Outward
thatdifferinAl31-inducedmalateefflux(Ryanetal., and inward rectifying K1 channels have been char-
1995a). Thus, it appears that the capacity of efflux acterizedintheplasmamembraneofwheatrootcells
rather than synthesis of malate accounts for the dif- (Schachtman et al., 1991; Findlay et al., 1994; Gas-
ference in malate efflux between the Al31-tolerant smann and Schroeder, 1994; Skerrett and Tyerman,
and -sensitive genotypes. 1994),butAl31hasbeenshowntobeapotentantag-
Anion channels have been characterized in the onist of both these channels (Gassmann and Schroe-
plasma membranes of a range of different plant cells der, 1994; Ryan et al., 1997). Therefore, the mecha-
where they are known to be involved in several nism underlying the Al31-dependent efflux of K1
important cellular functions. These include turgor fromAl31-tolerantwheatgenotypesremainsunclear.
regulation, stomatal movement, nutrient acquisition, Onepossibilitynotexaminedpreviouslyiswhethera
and control of membrane potential (Tyerman, 1992;
freely diffusible molecule in the cytosol, which is
Schroeder, 1995; Ward et al., 1995; Barbier-Brygoo et 1
required for K channel activity in the presence
al.,2000).Twotypesofanionchannelsintheplasma ofAl31,islostfromtheprotoplastduringthewhole-
membrane have been identified in protoplasts de-
cellexperimentsbydilutionintothepipettesolution.
rived from wheat roots. One is the outwardly recti-
There are several candidate molecules of a size that
fying anion channel that is activated at membrane
would readily be perfused from the cytoplasm and
potentials more positive than the equilibrium poten-
that may be required for channel activation, but one
tial for the permeant anion (Skerrett and Tyerman, 1
thathasbeenshowntoactivateK -outwardchannels
1994). This channel has been suggested to mediate
2 2 in mesophyll cells of broad bean is cAMP (Li et al.,
uptake of Cl and NO into the root cells in the
3 2 1994).
presence of high concentrations of external Cl and
2 Inthepresentstudyweusedthewhole-cellpatch-
NO (Skerrett and Tyerman, 1994). The second an-
ion3channel is activated by Al31 (Ryan et al., 1997) cdliafmferpintgecihnniAqul3e1-atonldernaneacre-iasotgaensiicnglilneelsocoufsw(hDeeal-t
and is observed in the plasma membrane of proto-
plasts isolated from root tips of Al31-tolerant geno- hanaiizoensectoaull.d, 1ca9r9r3ya)cutrorednettsetrhmrionueghwAhel3t1h-earctmivaaltaetde
types of wheat. This channel is activated specifically
by Al31, inhibited by the anion channel blockers anion channels. We looked for differences in activa-
tion of the anion channel currents between the near
niflumic acid and 5-nitro-2-(3-phenylpropyl amino)-
isogenic wheat lines, and we investigated the effect
benzoic acid, and remains active for long periods
1
provided Al31 is present in the bathing solution of cAMP on the activity of K currents.
(Ryan et al., 1997). These characteristics are compa-
rablewiththeAl31-inducedmalateeffluxfromintact
roots(Delhaizeetal.,1993b;Ryanetal.,1995a),lead- RESULTS
ingRyanetal.(1997)toproposethattheanionchan-
To investigate whether the Al31-activated anion
nel they characterized mediates the malate efflux
stimulated by Al31. However, the question as to channel identified previously (Ryan et al., 1997) is
whether the Al31-activated anion channel was actu- permeable to malate, similar experiments to those
reported by Ryan et al. (1997) were performed with
ally permeable to malate remained unanswered.
Moreover, if the Al31-activated anion channel is re- a pipette solution that contained malate as the main
anion. To suppress the initially large background
sponsible for malate efflux, some differences would
1 1
K currents,tetraethylammonium(TEA )wasused
be expected in channel activity, selectivity, or gating
properties between the Al31-tolerant and -sensitive as the main cation in the pipette. Several types of
wheat genotypes. This information is important for currents measured in the initial sealing solution
understanding the physiology of Al31-tolerance, as were present in protoplasts isolated from ES8 and
well as for identifying the genes that confer Al31 ET8 wheat root apices. These include a time-
tolerance in wheat. dependent and instantaneous inward current and a
In wheat the malate efflux stimulated by Al31 is small instantaneous outward current. A time-
accompanied by enhanced K1 efflux from the root dependent outward current was often observed
1
apices (Ryan et al., 1995a). This allows a net flux of when K was used as a main cation in the pipette
malate that does not decrease the external pH. solution. These currents resembled those time-
1
Present models for the mechanism of stomatal clo- dependent inward and outward K and non-
sure suggest that the activation of an anion channel selective cation currents that have been character-
intheplasmamembraneoftheguardcelldepolarizes ized previously (Findlay et al., 1994; Skerrett and
the membrane past the equilibrium potential for po- Tyerman, 1994; Tyerman et al., 1997) and were not
tassium (E ), and leads to a coordinated efflux of examined in the present study.
K
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Al31-ActivatedMalateChannelsinWheatRootCells
Al31-Activated Inward Currents in ET8 and theinwardcurrentwassimilarforbothlines(2Alin
ES8 Protoplasts Fig. 2B). For some of the ET8 and ES8 protoplasts,
addition of 50 mm AlCl (free Al31 activity, {Al31} 5
3
For ET8 and ES8 protoplasts positive and negative 18 mm) to the bathing solution elicited (after a delay)
voltage-pulses activated small inward and outward atime-dependentinwardcurrentuponnegativevolt-
currents. The inward currents did not display any agepulses(exampleresponsesshowninFig.1,Band
appreciable time dependence in the control bathing E). During the delay and in protoplasts that did not
solution(Fig.1,AandD).Theaveragemagnitudeof showaresponse,theinwardcurrentwasofteninhib-
Figure 1. Al31-activated inward currents recorded for whole-cell patches of an ET8 (A–C), and ES8 (D–F), protoplast.
Superimposedcurrenttracesinresponsetovoltage-pulsesrangingfrom2167to173mV(ET8)orfrom2186to184mV
(ES8)before(AandD),and40minafter(BandD)theadditionof50mMAlCl3.CandD,Theinitialcurrentwasusedto
constructcurrent-voltagecurvesfortheprotoplastsincontrolsolution(E)andaftervarioustimesofexposureto50mMAlCl3.
Thepipettesolutioncontained40mMmalate,1mMCaCl2,2mMMgSO4,2mMNa2ATP,10mMEGTA,and10mMHEPES
(N-2-hydroxyethylpiperazine-N-2-ethanesulphonicacid),pH7.2with110TEAOH.
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Zhangetal.
currents measured in control solution decreased in-
significantly over 60 min (P 5 0.07) for ET8 and ES8
protoplasts.Thedifferencebetweenthetwolinesisa
result of a greater proportion of ET8 protoplasts
showing an Al31-activated inward current (occur-
rencerate39%inET8,11%inES8)combinedwiththe
activatedcurrentbeinglargerandoccurringwithina
shorter delay time for ET8 protoplasts than for ES8
protoplasts (Table I).
The Al31-activated current in ET8 protoplasts
showedastronginwardrectification(Fig.1,BandC)
andremainedactiveforaslongasAlCl waspresent
3
in the bathing solution. The current versus voltage
(I-V) curves show that activation of the inward cur-
rent was accompanied by a positive-going shift in
reversal potential (from 218 to approximately 160
mV in Fig. 1C). About 70% of ET8 protoplasts that
showed the Al31-activated inward current re-
sponded within 10 min of exposure to AlCl . The
3
mean delay time for activation of the inward current
was 9.1 min for ET8 (Table I).
In contrast to ET8 protoplasts, addition of Al31 to
ES8 protoplasts generally inhibited the background
inward currents. Inward currents such as that shown
inFigure1Ewereobservedinonly11%ofprotoplasts
after prolonged exposure to Al31 (mean delay was 36
min,TableI).Theaveragemaximumamplitudeofthe
Figure 2. Effects of Al31 on the inward current in ET8 and ES8 Al31-induced inward current in ES8 protoplasts was
protoplasts.A,Changesinrelativeinwardcurrentfollowingaddition only slightly larger than that measured prior to
of 50 mM AlCl3. The currents were collected from all protoplasts additionofAl31(Fig.2B).Thereversalpotentialofthe
examinedandnormalizedtotheinitialcurrentforavoltagepulseto Al31-activatedinwardcurrentforES8protoplastswas
2180 mV measured before addition of AlCl . The mean initial
3 often less positive than that for ET8 protoplasts (Fig.
currentdensityinthecontrolsolutionat2180mVwas222.664.4
1F; Table I), but this was not statistically significant.
mAm22(n584)forET8protoplastsand223.165.2mAm22(n5 AnotherdifferencetoET8protoplastsisthattheAl31-
38) for ES8 protoplasts. B, Mean initial current density from only
thoseprotoplastsexhibitinganAl31-activatedinwardcurrent.Values activated inward current in ES8 protoplasts was not
representthemeanofthemaximumcurrentsmeasuredat2180mV sustained in the presence of constant Al31 in the bath
after addition of 50 mM AlCl3. Data are mean 6 SE (number of solution (Fig. 1F).
protoplasts).
The Al31-Activated Inward Current Was Inhibited by
ited. Figure 2A shows the mean inward currents
Niflumate and Diphenylamine-2-Carboxylic Acid (DPC)
relative to those before addition of AlCl for all ET8
3
and ES8 protoplasts tested at 2, 10, and 20 min after It has previously been shown that malate efflux
the addition of 50 mm AlCl . On average, ET8 proto- from intact ET8 roots was inhibited by the anion
3
plasts increased inward current, whereas ES8 de- channel inhibitors niflumate (Ryan et al., 1995a) and
creased inward current after the addition of AlCl . DPC (T. Kataoka, A. Stekelenbury, E. Delhaize, and
These changes in current are not due to non-specifi3c P.R. Ryan, unpublished data). The Al31-activated in-
time-dependentchangesbecausetheaverageinward wardcurrentwasinhibitedby100mmniflumate(Fig.
TableI. ComparisonoftheAl31-activatedinwardcurrentintheES8andET8genotypes
OccurrencerateisthepercentageoftotalcellsmeasuredinwhichAl31activatedaninwardcurrent.
DelayreferstotheaveragetimeelapsedbetweentheadditionofAl31andtheactivationofaninward
current.Currentdensity(I )referstothemaximumcurrentafterAl31additionmeasuredat2180mV.
m
ThereversalpotentialfortheAl31-activatedinwardcurrentisalsoshown(E ).Thevaluesarethemean
rev
andSEMwiththeno.ofprotoplastsexaminedinthebrackets.
Genotype OccurrenceRate Delay I E
m rev
% min mAm22 mV
ET8 39(109) 9.161.0(43) 268.867.6(43) 35.463.9(43)
ES8 11(44) 36.066.8(5) 227.565.7(5) 24.269.1(5)
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Al31-ActivatedMalateChannelsinWheatRootCells
3, A and B) and by 10 mm DPC (Fig. 3, D and E). The longer than 10 min (data not shown). This observa-
current-voltage curves shown in Figure 3C (niflu- tionsuggeststhatDPCmayhaveothereffectsonthe
mate) and Figure 3F (DPC) show the average re- plasma membranes under these conditions.
sponsetotheinhibitorsandindicatethatinhibitionis
not voltage dependent. At 2186 mV, 100 mm niflu- Al31-Activated Inward Current Carried by
mateinhibitedtheinitialcurrentby61%613%(n5
Malate Efflux
4), and 10 mm DPC inhibited the current by 51% 6
14% (n 5 3). A relatively large “spiky” inward cur- The pipette solution in most of the experiments
2
rentwasoftenobservedatthemostnegativevoltage contained 40 mm malate and a small amount of Cl
2
levels when the DPC concentration was greater than (2–4mm).InclusionofCl wasnecessarybecausethe
20 mm and/or the protoplasts were exposed to DPC Ag/AgClhalf-cellrequiresastableCl2concentration
Figure 3. Effects of anion channel antagonists on the Al31-activated inward current in ET8 protoplasts. A and D, Al31-
activatedcurrentcausedbyadditionof50mMAlCl3.Superimposedcurrenttracesinresponsetovoltagepulsesrangingfrom
2186to184mVat30-mVintervalsfromaholdingpotentialof24mV.Additionof100mMniflumate(B)or10mMDPC
(E) in the presence of 50 mM AlCl3. Current-voltage curves using initial currents in the absence and presence of 100 mM
niflumate(C)or10mMDPC(F).Dataaremean6SEMfromfourprotoplasts(niflumate)andthreeprotoplasts(DPC).
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Zhangetal.
2
to reduce the junction potentials. The reversal po- increasing the external Cl concentration to 50 mm
tential of the Al31-activated inward current under (Fig. 4C). A shift in reversal potential could not be
bi-ionic conditions could normally be used to deter- discerned even though E was shifted from 240 to
mine the relative permeability of the underlying 281 mV by increasing thCel external Cl2 concentra-
anion channels to malate22 and Cl2. However, tion (Fig. 4D). Identical results were observed for
malate cannot be added to the bathing solution in four protoplasts and the mean reversal potentials in
these experiments because the continued activation 10 and 50 mm external Cl2 were 39.6 6 6.6 and
of the current requires the presence of Al31 in the 37.8 6 9.1 mV, respectively.
bathing solution (Ryan et al., 1997). The addition of In a second method to test malate permeability the
malate also inhibits the Al31-activated inward cur- Ag/AgCl half-cell was replaced with a platinum
rent by chelating external Al31 (data not shown). electrode and Cl2 was omitted from the pipette so-
Therefore, three methods were used to determine lution altogether. Under these experimental condi-
whether the Al31-activated anion channel was per- tions, addition of Al31 still activated an inward cur-
meable to malate. The first method involved chang- rent that was comparable with those measured
2
ing the external Cl concentration after the inward previously(Fig.5).Onlyasingleresultwasobtained
current was activated by Al31, and following the using this method.
shift in reversal potential (the Al31 concentration To measure the relative malate22 to Cl2 perme-
was altered to maintain {Al31} constant). Figure 4 ability ratio, accurate reversal potentials needed to
shows the response of an ET8 protoplast where the be obtained and background currents not contribut-
addition of Al31 to the bath activated an inward ing to the Al31-activated anion currents need to be
current (Fig. 4, A and B) and shifted the reversal subtracted. Subtracting the I-V curve measured in
potential from 18 mV to more positive than 170 the control solution from the I-V curve measured
mV (Fig. 4D). The magnitude and time-dependence after addition of Al31 is one way to remove the
of the inward current was relatively unaffected by background currents from the Al31-activated cur-
Figure4. EffectofexternalTEAClconcentrationontheAl31-activatedinwardcurrentinanET8protoplast.Currenttraces
evokedbyvoltagepulsesbetween174and2166mVin20-mVintervalsfromaholdingpotentialof226mVbefore(A)
and8minafter(B)exposureto50mMAlCl3.C,Thebathsolutionwaschangedfrom10mMTEACl,0.2mMCaCl2,and50
mM AlCl3 ({Al31} 5 18 mM, pH 4.0) to 50 mM TEACl, 0.2 mM CaCl2, and 110 mM AlCl3 ({Al31} 5 17 mM, pH 4.0). D,
Current-voltagecurvesusingtheinitialcurrentsfromAthroughC.Pipettesolutioncontained40mMmalicacid,2mMCaCl2,
2mMMgSO4,2mMNa2ATP,10mMEGTA,10mMHEPES,and110mMTEAOH,pH7.2.
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Al31-ActivatedMalateChannelsinWheatRootCells
niflumate or DPC in 10 mm external TEACl and
assuming that the anion current was the only elec-
trogenic transport inhibited by these compounds.
1
Outward K channels are blocked by niflumate
(Garrill et al., 1996), but in the present experiments
1
TEA was the only cation in the pipette solution.
When the average reversal potential of the net cur-
rents (45 6 12 mV, n 5 6) was used the P 22/P 2
mal Cl
is 2.6. With this permeability ratio it would be ex-
2
pected that a 5-fold increase in external Cl would
shift the reversal potential more negative by 23 mV.
Thefindingabove(Fig.4)thatthereversalpotential
did not change significantly under these conditions
may indicate that the permeability ratio increased
2
with increasing external Cl concentration. In an
alternate manner, it may suggest that the value for
PmAall2t2h/oPuCgl2hoAf 2l3.16 uinsdaerepsottiemnatteasntthaegorneiasltraotfioC. a21
channels in wheat and Arabidopsis root cells (Pin˜-
eros and Tester, 1995; Huang et al., 1996; Kiegle et
al., 2000), the possibility that the Al31-activated in-
ward current corresponded in part to an increase in
Ca21influxwasalsotested.Theinwardcurrentwas
first activated by Al31 and then the external Ca21
concentration was increased from 0.2 to 20 mm (ad-
justing [Al31] to maintain {Al31} constant). Under
these conditions the inward current was reduced
slightly,andwasnotincreased,indicatingthatCa21
influx was probably not contributing to the current
(data not shown). These separate lines of evidence
arguethattheAl31-activatedinwardcurrentlargely
corresponds to malate efflux.
Al31 Activation of a K1 Outward Current in the
Presence of cAMP
Ryan et al. (1995a) have shown that the Al31-
activated malate efflux from root apices of ET8
wheatwasaccompaniedbyasimultaneouseffluxof
1 1
K ions. Time-dependent K outward currents are
Figure5. Al31-activatedinwardcurrentforawhole-cellpatchofan prominent in ET8 and ES8 proto1plasts at depolar-
ET8protoplastwiththepipettesolutioncontainingnoCl2.Current ized membrane voltages when K was included in
traces elicited by voltage pulses ranging from 2180 to 60 mV in the pipette solution (Fig. 6A). As reported previ-
1
controlsolution(A)and2180to90mVafter5minfromadditionof ously(Ryanetal.,1997),thetime-dependentK out-
50mMAlCl3tothebathsolution.C,Current-voltagecurvesofinitial ward current in ET8 and ES8 protoplasts was mark-
currentsshowninAandB.Pipettesolutioncontained40mMmalic edly inhibited when the protoplasts were exposed to
acid,2mMCaSO4,2mMMgSO4,2mMNa2ATP,10mMEGTA,10 Al31(Fig.6,B–D).However,when0.5mmcAMPwas
mMHEPES,and110mMTEAOH,pH7.2. included in the pipette solution, ET8 protoplasts
showed the following response sequence as shown
by the example in Figure 7. To begin with, Al31
1
rent, assuming that only the anion channels are acti- inhibited a K outward current (Fig. 7, A and B).
vated and no other electrogenic transport is affected Then after about 10 min, depending on the proto-
byAl31.Thereversalpotentialofthisnetcurrentwas plast, a K1 outward current returned. This outward
used in the modified Goldman-Hodgkin-Katz equa- currentcouldbeobservedbypositive-goingvoltage
tion (Lewis, 1979) to estimate P 22/P 2. The cal- pulses for at least 60 min in different protoplasts
culated P 22/P 2 from the mexapl erimCelnt without (Fig. 8A). The voltage dependence of the steady-
2 mal Cl
Cl present in the pipette solution was 8.4. state outward current that re-activated was similar
Reversal potentials were also obtained by sub- to the initial current in the control solution as indi-
tracting the I-V curves before and after addition of cated by the I-V curves (Fig. 7D). However, there
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Zhangetal.
Figure6. EffectsofAl31onK1outwardcurrentinawhole-cellpatchofanET8protoplast.A,Superimposedcurrenttraces
activatedbypulsesrangingfrom2191to179mVat20-mVintervalsfromaholdingpotentialof2111mVintheabsence
of Al31 (A), and after 2 (B) and 30 min (C) of exposure to 50 mM AlCl3 (pH 4.0). D, Steady-state current-voltage curves
obtainedbeforeandafteradditionof50mMAlCl3tothebath.Pipettesolutioncontained40mMmalicacid,2mMCaCl2,
2mMMgCl2,2mMNa2ATP,10mMEGTA,10mMHEPES,and90mMKOH,pH7.2.Controlsolutioncontained1mMKCl
and0.2mMCaCl2,pH4.0.
appeared to be a greater proportion of time depen- DISCUSSION
dent current compared with initial current after the Al31-Activated Inward Current Is Carried by Malate
re-activation(comparewithFig.7,AandB).ForES8
protoplasts there was a sustained inhibition of Ryan et al. (1997) had previously shown that Al31
2
steady-state outward current when challenged with activatesaCl -permeableanionchannelintheplasma
external Al31, regardless of the presence or absence membrane of protoplasts derived from root apices,
of cAMP in the pipette solution (Fig. 8B). Although but not from mature roots, of an Al31-tolerant wheat
genotype.Inthepresentstudythepermeabilityofthe
the examples shown in Figures 7 and 8 do not show
Al31-activated anion channels to malate was exam-
any activation of inward current, we did observe
Al31-activated inward current in some protoplasts ined using protoplasts derived from root apex of
1 thesameAl31-tolerantgenotype(ET8)astheprevious
that also displayed K outward current. However,
study.Aninwardcurrentwasactivatedafteraddition
tinhisthweaksindeitfifcicsuoltf tKo1qouuantwtiafyrdberecactuisfieerofdseiamctiilvaarittiioens of 50 mm AlCl3 (pH 4.0) in approximately 40% of the
protoplasts examined when 40 mm malate was used
and inactivation of Al31-activated inward current.
as the main anion in the pipette solution. This is
1 1
By using TEA instead of K in the pipette solution
comparablewiththepreviousstudyinwhichapprox-
we could examine the effects of cAMP on the in- imately one-half the protoplasts exhibited an Al31-
ward current. The amplitude and kinetics of the activated inward current when Cl2 was the main
Al31-activated inward current did not appear to permeant anion (Ryan et al., 1997). Several character-
be changed significantly by the presence of cAMP istics of the Al31-activated inward current found in
in the pipette solution for ES8 or ET8 (data not thisstudyarecomparablewiththeAl31-activatedCl2
shown). permeable channel, including fast activation by hy-
1466 Downloaded from on November 23, 2018 - Published by www.plantphysiol.org Plant Physiol. Vol. 125, 2001
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Al31-ActivatedMalateChannelsinWheatRootCells
Figure7. EffectofcAMPpresentinthepipettesolutionontheresponseofK1outwardcurrentinawhole-cellpatchofan
ET8 protoplast. Superimposed current traces activated by voltage-pulses between 2191 and 179 mV at 30-mV intervals
fromaholdingpotentialof2111mVincontrolsolution(A),and2(B)and20min(C)afterexposureoftheprotoplastto
50mMAlCl3(pH4.0).D,Steady-statecurrent-voltagecurvesobtainedbeforeandafterexposureto50mMAlCl3(pH4.0).
Pipettesolutioncontained0.5mMcAMP,40mMmalicacid,2mMCaCl2,2mMMgCl2,2mMNa2ATP,10mMEGTA,10
mMHEPES,and90mMKOH,pH7.2.Controlsolutioncontained1mMKCland0.2mMCaCl2,pH4.0.
perpolarizing voltage-pulses, long-lasting activa- tive permeability determined from reversal poten-
tion in the presence of external Al31 (Fig. 1C), and tials. This can occur if the channel pore has binding
2
inhibitionbytheanionchannelantagonistniflumate sites with a higher affinity for malate than Cl .
(Fig. 3). However, the Al31-activated Cl2 current in Unlike the Al31-activated current with Cl2 as the
the whole-cell patch clamp configuration shown by main anion (Ryan et al., 1997), the Al31-activated
Ryan et al. (1997) was much noisier than we ob- currentwithmalateoftenexhibitedslowinactivation
served in the present study. Because the noise vari- at the most negative membrane voltages (Figs. 3 and
ance increases in proportion with the number of 5). The more negative-voltage pulses used to evoke
channels activated, with the square of the single the Al31-activated inward current in this study com-
channel current, and as the channel open probabil- pared with those used by Ryan et al. (1997) may
ity approaches 0.5, any combination of differences account for the difference. Alternatively, this differ-
in these characteristics caused by different experi- ence could be due to differences in channel gating
mental conditions could lead to the difference in properties caused by the presence of malate. The
noise characteristics. A higher single channel cur- presence of malate in the external medium can alter
2
rent for Cl than for malate may result from the anion channel gating characteristics in guard cells
2
higherconcentrationofCl (i.e.100mm)usedinthe (Hedrich and Marten, 1993). It is also possible that
pipette solution of the previous study compared the flux of malate induced by the more negative
withthemalate22concentration(i.e.40mm)usedin membrane voltages is sufficient to allow significant
the present study. There may also be a difference in malate accumulation in the unstirred layer around
the permeation properties of the two anions such theprotoplastsuchthattheeffectiveAl31concentra-
2
that malate permeates more slowly than Cl under tion adjacent to the cell membrane is reduced. This
an equivalent electrochemical gradient despite the would then reduce the degree of channel activation
channel showing the opposite in terms of the rela- during a pulse if the Al31 concentration goes below
Plant Physiol. Vol. 125, 2001 Downloaded from on November 23, 2018 - Published by www.plantphysiol.org 1467
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Zhangetal.
current is unlikely to result from the stimulation of
cation influx. Furthermore, in one experiment Al31
was shown to activate an inward current of similar
characteristics when the pipette solution contained
2
malate, but no Cl (Fig. 5). These findings show that
theAl31-activatedinwardcurrentismainlycarriedby
malate anions flowing out of the cell.
Anion channels characterized in the plasma mem-
brane of other cell types and from other species ex-
hibitaP /P oflessthan1.Forexample,theguard
mal Cl
cell slow anion channel has a P /P value of 0.24
mal Cl
(Schmidt and Schroeder, 1994), which is similar to
the quick activating anion channel identified in bar-
ley xylem parenchyma cells (Ko¨hler and Raschke,
2000). In contrast, the channels that account for the
Al31-activated inward current in wheat roots are
morepermeabletomalate22thantoCl2withaP /
mal
P of 2.6. The large variability of these measure-
Cl
ments, and the estimated P /P of 8.4 from one
mal Cl
experiment,indicatethatthepermeabilityratiocould
even be higher than 2.6. Because we have measured
only whole cell currents, we cannot dismiss the pos-
sibilitythatmorethanonetypeofanionchannelwas
activatedbyAl31andperhapswithdifferentrelative
permeabilities. Nevertheless, at least one type of the
anion channels present must have very high malate
permeability, which is novel for plasma membrane
anion channels. Malate-permeable anion channels
have been identified in the tonoplast of Kalanchoe
digremontiana(Cheffingsetal.,1997)andArabidopsis
and the estimated P /P for the latter example is
mal Cl
Figure8. Time-dependentchangesinoutwardcurrentforwholecell 3.5 (Cerana et al., 1995). An anion channel in the
patches of ET8 (A) and ES8 (B) protoplasts in the presence and plasma membrane of Arabidopsis hypocotyl cells
absence of cytoplasmic cAMP. The current was normalized to the wasrecentlyshowntobemorepermeabletodivalent
initial current value at 179 mV measured in control solution. The SO 22 than to Cl2 (P /P 5 2.0), but the perme-
m1d8aVt4a.i6anrec6omne1tar9on.l76somSlEuAMti2o(n2numw(nbase5r1o9f17p1.1r)ot6foopr2la9Es.St8s8).mCaAunrdmre2nEt2Td8(ennps5riotyt1oa7pt)l1aas7nts9d, aFbrai4lcihtyistsoemeatlaatle.,2219w9a9Ss)O.m4TuhcCehllAowl31er-a(cPtmivaal/tePdCli5nw0.a0r3d;
currentexhibitedstronginwardrectificationandvir-
respectively.
tually no outward current could be detected at volt-
ages as positive as 90 mV (compare with Figs. 1 and
the saturation concentration of the “receptor.” Chlo- 3). This rectification could be explained, in part, by
ride efflux would not be expected to do this because the absence of malate in the bath solution and the
of the minimal effect that Cl2 has on the Al31 activ- corresponding very positive equilibrium potential
ity. It is interesting in this respect to note that Al31 for malate22 anions.
induced an inactivating Cl2 current in Al31-tolerant
maize root tips (Pin˜eros and Kochian, 2001). Similar
kinetics have been observed for anion channels in
Activation of the Inward Current Is Different in
guardcells(LinderandRaschke,1992;Schroederand
ET8 and ES8
Keller,1992),tobaccosuspensioncells(Zimmermann
et al., 1994), xylem parenchyma cells (Ko¨hler and Consistent differences were observed between the
Raschke,2000),andArabidopsishypocotylcells(Fra- ET8andES8genotypesintheactivationofthemalate
chisse et al., 2000). current by Al31. The response was observed about
Several approaches were used to identify the ions four times more frequently in ET8 protoplasts than
contributingtotheAl31-activatedinwardcurrent.The for ES8 protoplasts and the maximum current den-
reversal potential of the Al31-activated current was sitywas2.5timesgreaterinET8protoplasts(TableI).
always much more positive than E and surprisingly Activation of the inward current in ES8 protoplasts
did not shift when E was variedC(Flig. 4). Moreover, occurred after a longer exposure to Al31 and, when
Cl
no increases in the current amplitude were observed activation did occur, the inward current was rela-
when the external concentrations of TEA1 or Ca21 tively short-lived compared with ET8 (Fig. 1, C and
wereraised,indicatingthattheAl31-activatedinward F). Since these two genotypes are virtually identical
1468 Downloaded from on November 23, 2018 - Published by www.plantphysiol.org Plant Physiol. Vol. 125, 2001
Copyright © 2001 American Society of Plant Biologists. All rights reserved.