Receptor-Like Activity Evoked by Extracellular ADP inArabidopsis Root Epidermal Plasma Membrane1

Vadim Demidchik, Zhonglin Shang, Ryoung Shin, Renato Colaco, Anuphon Laohavisit,Sergey Shabala, and Julia M. Davies*

Department of Biology, University of Essex, Colchester CO4 3SQ, United Kingdom (V.D.); College of LifeScience, Hebei Normal University, Shijiazhang 050016, Hebei, China (Z.S.); Riken Plant Science Center,Yokohama City, Kanagawa 230–0045, Japan (R.S.); Department of Plant Sciences, University of Cambridge,Cambridge CB2 3EA, United Kingdom (R.C., A.L., J.M.D.); and School of Agricultural Science, University ofTasmania, Hobart, Tasmania 7001, Australia (S.S.)

Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellularATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channelactivation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activatenet Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermalprotoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application andrepeated application evoked a smaller current. Such response speed and densitization are consistent with operation ofequivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identifiedin higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species.While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx.Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

Extracellular purine nucleotides have been shownto be involved in the regulation of plant cell viability,membrane permeability, immunity, symbiosis, stressresponses, and growth (Lew and Dearnaley, 2000; Tanget al., 2003; Chivasa et al., 2005, 2009; Kim et al., 2006,2009; Roux and Steinebrunner, 2007; Wu et al., 2007;Riewe et al., 2008a; Wu and Wu, 2008; Yi et al., 2008;Demidchik et al., 2009; Govindarajulu et al., 2009; Kimet al., 2009; Clark et al., 2010a, 2010b; Tanaka et al.,2010a, 2010b; Terrile et al., 2010; Tonon et al., 2010).Purine nucleotide release from plant cells may occurthrough wounding, exocytosis, or through the activityof plasmamembrane (PM) ATP-binding cassette trans-porters (Thomas et al., 2000; Kim et al., 2006). Growthpoints are hotspots of extracellular ATP (Kim et al.,2006) while touch-stimulated ATP release is mediatedby heterotrimeric G-protein activity (Weerasinghe et al.,2009). Levels of extracellular ATP and ADP can be reg-ulated by the concerted action of ecto-apyrases, phos-

phatases, and adenosine nucleosidases with retrieval ofadenine (the ultimate hydrolytic product) by PM purinepermeases (Komoszynski, 1996; Steinebrunner et al.,2003; Wu et al., 2007; Riewe et al., 2008a, 2008b;Govindarajulu et al., 2009; Clark et al., 2010b; Lianget al., 2010). Manipulation of ecto-apyrase levels re-sults in abnormal growth in Arabidopsis (Arabidopsisthaliana), cotton (Gossypium hirsutum), and potato (So-lanum tuberosum; Wu et al., 2007; Riewe et al., 2008a;Clark et al., 2010a, 2010b) while nodulation by Brady-rhizobium japonicum is impaired in ecto-apyrase-deficientsoybean (Glycine max; Govindarajulu et al., 2009).Current models suggest that poise of the cell’s statebetween death, stress adaptation, and growth involvessignaling governed by the level of extracellular ATP(Chivasa et al., 2005, 2009; Roux and Steinebrunner,2007; Wu et al., 2007; Wu and Wu, 2008; Kim et al.,2009; Clark et al., 2010a, 2010b; Terrile et al., 2010;Tonon et al., 2010).

The mechanism of purine nucleotide perception isunclear as higher plant genomes do not encode forhomologs of animal PM purinoceptors (Kim et al.,2006; Fountain et al., 2008). The latter are either cou-pled to heterotrimeric G proteins or are ionotropicreceptors (activated by nanomolar or even 0.1 mM

ATP) that permit Ca2+ influx to transduce the purinenucleotide signal (for review, see Khakh and North,2006). It remains possible that plants evolved struc-turally different systems for purine sensing to animalsbut antagonists of animal purinoceptors are effectiveagainst plant extracellular ATP responses. Despite the

1 This work was supported by the Leverhulme Trust (grant no.F/09 741/C), the Biotechnology and Biological Sciences ResearchCouncil, the University of Cambridge Frank Smart Trust andBrookes Fund, the Royal Society (China Incoming Fellowship), theAustralian Research Council, and The Monsanto Company.

* Corresponding author; e-mail jmd32@cam.ac.uk.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Julia M. Davies (jmd32@cam.ac.uk).

www.plantphysiol.org/cgi/doi/10.1104/pp.111.174722

Plant Physiology�, July 2011, Vol. 156, pp. 1375–1385, www.plantphysiol.org � 2011 American Society of Plant Biologists 1375

apparent absence of classical purinoceptors in higherplants, extracellular purine nucleotides can cause tran-sient elevations of cytosolic free Ca2+ ([Ca2+]cyt), incommon with animals (Demidchik et al., 2003a, 2009;Jeter et al., 2004; Tanaka et al., 2010b). In Arabidop-sis roots, extracellular ATP most likely triggers intra-cellular Ca2+ release, resulting in stimulation of PMNADPH oxidase activity (Demidchik et al., 2009). Theresultant reactive oxygen species (ROS) activate a PMCa2+ influx pathway that governs a transcriptionalresponse. In Arabidopsis root hairs, extracellular ATPalso causes intracellular ROS accumulation by activat-ing NADPH oxidases (Clark et al., 2010b). Leaves alsosupport extracellular ATP-induced transcription viaPM NADPH oxidases (Song et al., 2006). Nitric oxideis now also established as a product of extracellularpurine nucleotide perception. Nitric oxide evolutionby plants has been observed on application of extra-cellular ATP or extracellular ADP and linked to influxof Ca2+ or production of phosphatidic acid (Foresiet al., 2007; Wu and Wu, 2008; Reichler et al., 2009;Clark et al., 2010b; Sueldo et al., 2010; Tonon et al.,2010).

In this study, the effects of extracellular ADP on iontransport at the plant PM have been examined. Extra-cellular ADP elevates [Ca2+]cyt in both roots and leavesof Arabidopsis and it can be equally or more effectivethan extracellular ATP (Demidchik et al., 2003a, 2009;Jeter et al., 2004; Tanaka et al., 2010b). In common withextracellular ATP, extracellular ADP can elicit oscilla-tions of [Ca2+]cyt in Arabidopsis seedlings (Tanakaet al., 2010b). At the physiological level, extracellularADP stimulates nodule development (Govindarajuluet al., 2009) and was shown to stimulate root hairelongation (Lew and Dearnaley, 2000). In commonwith extracellular ATP, it is now known that extracel-lular ADP has a biphasic effect on cotton fiber and roothair elongation with high concentrations being inhib-itory (Clark et al., 2010a, 2010b). Arabidopsis rootepidermal cells have been used here as these are wellcharacterized electrophysiologically and are amongthe first cell types to sense the pathogen attack andenvironmental change that could involve purine sig-naling. By using a fast-perfusion system in patch-clampelectrophysiology experiments, extracellular ADP hasbeen delivered to the extracellular PM face of proto-plasts within 1 s, permitting the fastest resolution todate of rapid activation of plant ion transport eventsby an extracellular purine nucleotide. These events ap-pear to be independent of heterotrimeric G proteins.Epidermal protoplasts expressing the [Ca2+]cyt re-porter aequorin have been used to explore further theability of extracellular ADP to evoke [Ca2+]cyt elevation.In further contrast to extracellular ATP, extracellularADP does not elicit intracellular ROS accumulation.Using whole roots, it has also been possible to resolveextracellular ADP-induced net Ca2+ and K+ fluxes; highconcentrations of ATP but not ADP evoke net Ca2+ efflux.Overall the results show rapid activation of PM Ca2+—and K+—conductances by extracellular ADP, with

initial events supported by PM ionotropic receptor-like activity. The differential responses of intracellularROS and net Ca2+ flux to extracellular ADP and ex-tracellular ATP could provide the basis for distinctsignaling outcomes.

RESULTS

Extracellular ADP Activates a PM Ca2+

Influx Conductance

To establish whether the PM could respond to extra-cellular ADP, protoplasts from mature epidermal rootcells were assayed using the whole-cell patch-clampconfiguration to resolve activity of channel populationsas a function of applied membrane voltage and extra-cellular agonist. Application of 20 mM extracellular ADPto protoplasts (using conventional peristaltic pumpdelivery to the recording chamber) transiently activateda hyperpolarization-activated (i.e. activated by increas-ingly negative voltage) Ca2+-permeable conductance(Fig. 1A), very similar to that characterized in responseto 20 mM extracellular ATP in this cell type (Demidchiket al., 2009). The concentration used is representative ofwounding levels (Song et al., 2006). The increase ininwardly directed current at hyperpolarized (insidenegative) voltage was observed 1 to 3 min after extra-cellular ADP addition (a similar time course to the ex-tracellular ATP response reported by Demidchik et al.,2009; Shang et al., 2009) and was evident over thephysiological voltage range for this cell type (Maathuisand Sanders, 1993). Mean 6 SE current-voltage (IV)relationships are shown in Figure 1B; negative voltagesare physiologically relevant. At 2200 mV, extracellularADP induced a peak mean 6 SE current increase from2163 6 36 pA to 2382 6 63 pA at 2200 mV (n = 4);current then declined over a 5 to 15 min period.Negative current signifies influx of positive charge intothe cytosol or efflux of negative charge. The peak cur-rent response is not significantly different (Student’st test) to that found for 20 mM extracellular ATP(Demidchik et al., 2009).

Rapid, Reversible, and Desensitizing Response to

Extracellular ADP at the PM

Having found that the PMwas electrically responsiveto extracellular ADP on slow perfusion and long-termexposure, a fast-perfusion system was used to deliverextracellular ADP to the PM within 1 s. In contrast toexperiments using peristaltic pump delivery of theagonist to the recording chamber (with a waiting timefor flow from the chamber edge to the protoplast), thefast-perfusion system used a primed multichannel de-livery pipette placed near the protoplast (describedfurther in “Materials and Methods”). In these experi-ments, the pipette solution (PS; equivalent to the cyto-sol) contained a nonhydrolyzable GDP analog (0.5 mM

GDPbS) to minimize G-protein-mediated channel acti-vation (Li and Assmann, 1993). In preliminary experi-

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ments, this increased the K+ efflux current, as describedby Li and Assmann (1993), indicative of effectiveG-protein inhibition. It has been proposed that extracel-lular purine nucleotides can relay a signal via hetero-trimeric G proteins in plants (Song et al., 2006; Rouxand Steinebrunner, 2007; Wu and Wu, 2008), so theirinhibition could delineate possible direct extracellularADP effects on a receptor-like channel. Experimentswereperformed within the first 20 to 30 min of recording sothat extracellular ADP-activated currents could be dis-tinguished from the time-dependent developmentof the constitutive hyperpolarization-activated Ca2+-permeable conductance. The latter becomes the dominantCa2+ conductance in this cell type after approximately1 h (Demidchik et al., 2002).Fast perfusion proved problematic as in approxi-

mately 80% of trials (65 out of 85), the recording

configuration was disturbed or lost by distortion of theprotoplast. Given the high failure rate, a higher con-centration of extracellular ADP (0.3 mM) was used inthese trials to ensure activation. In the remaining 15trials in which seal resistance was unaffected by fastperfusion, extracellular ADP evoked a rapid and re-versible change in PM conductance (Fig. 2A). With thePM clamped at a holding potential (HP) of 290 mV,fast perfusion with control bathing solution (BS) waswithout effect (Fig. 2A, top trace). Addition of 0.3 mM

extracellular ADP caused a downward current deflec-tion, consistent with entry of positive charge into theprotoplast (Fig. 2A, middle trace). Current activationoccurred immediately after exposure to extracellularADP. Fast removal of extracellular ADP resulted inimmediate recovery to control conditions. As can beseen from the middle trace of Figure 2A, a repeatedapplication of 0.3 mM extracellular ADP to the sameprotoplast again caused rapid and reversible currentactivation. However in this case, recovery to baselinecurrent began in the presence of extracellular ADP.This was typical of all protoplasts where extracellularADP activation was observed.

Figure 1. Extracellular ADP activates a PM influx conductance in rootepidermal protoplasts. A, Effect of 20 mM extracellular ADP on whole-cell currents from a representative protoplast, recording conditionswere as used previously by Demidchik et al. (2009). B, Mean 6 SE IVrelationships of control and peak ADP peak response (n = 4). Control BSwas as described in “Materials and Methods” (20 mM CaCl2, 0.1 mM

KCl, and Na+ equimolar to test BS, pH 6). PS comprised (mM): 40K-gluconate, 10 KCl, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (free Ca2+ adjusted to 100 nM with 0.4 CaCl2), pH 7.2adjusted with Tris/MES.

Figure 2. Rapid activation of epidermal PM cation conductances byextracellular ADP. The results are from whole-cell recordings onprotoplasts from the root mature epidermis. A, Top trace: two sequen-tial applications of control BS (green bars) to a protoplast from themature epidermis failed to elicit a current; HP across the PM was 290mV inside negative. Middle trace (same protoplast): two sequentialapplications to the same protoplast of 0.3 mM ADP (red bars) by fastperfusion evoked inwardly directed current (downward deflection).Baseline current was recovered on removal of the ADP stimulus.Bottom trace (different protoplast); inhibiting effect of 0.3 mM Gd3+,added together with ADP in the second application. BS was as in Figure1. Test BS included 0.3 mM NaADP 6 0.3 mM GdCl3 as indicated. PSincluded 0.5 mM GDPbS. B, Current in response to voltage ramp (40-slong started from +100 mV) recorded from a single protoplast, approx-imately 1 min after the addition of control BS (green), 0.3 mM ADP (red),or 0.3 mM ADP with 0.3 mM Gd3+ (blue). Solutions as in A.

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Mean 6 SE values of extracellular ADP-evokedcurrents at HP = 290 mV are given in Table I. Inexperimental set A, the first application of 0.3 mM

extracellular ADP (event 1) caused a mean 89% in-crease in inwardly directed current. After removal ofthe stimulus and recovery to baseline current, a secondapplication of 0.3 mM extracellular ADP (event 2)caused a mean 54% increase. All differences are sta-tistically significant (Student’s t test; P , 0.05). Inexperimental set B, event 1 (0.3 mM extracellular ADP)evoked amean 101% increase in inwardly directed currentat HP = 290 mV; in event 2, application of 0.3 mM extra-cellularADPand 0.3mMGd3+ to the sameprotoplasts onlycaused a mean 11% increase. Thus, the greater part of theextracellular ADP-induced current was sensitive to thecation channel blocker, Gd3+. To explore cation channelactivity further, IV relationships were determined.

Extracellular ADP Increases PM Ca2+ Influx and K+

Efflux Conductance

As Figure 2B shows, fast delivery of 0.3 mM extra-cellular ADP increased both inwardly and outwardlydirected currents across the PM of a single protoplast,resulting in a positive shift of reversal potential (Erev)away from the equilibrium potentials for K+ and Cl2

(EK =2158 mV; ECl =236mV) and toward that for Ca2+

(ECa = 155 mV). The mean Erev of the extracellular ADP-induced current (estimated by subtracting the controlIV from that determined in the presence of extracellularADP; Fig. 3, inset) was 4.7 6 8.4 mV (n = 4), indicatingCa2+ permeation. Extracellular ADP approximatelydoubled the Gd3+-sensitive inward current (Figs. 2Band 3) over the physiological voltage range for this celltype (2100 to 2150 mV; Maathuis and Sanders, 1993);thus, Ca2+ could be delivered to the cell to act as a signal

in response to the extracellular purine nucleotide. Forexample, from the mean response shown in Figure 3, at2120 mV inward Ca2+ current increased from2696 23mA m22 to 2159 6 25 mA in response to 0.3 mM

extracellular ADP (n = 4; mean 6 SE). This large Gd3+-sensitive Ca2+ influx current helps explain an extracel-lular ADP-induced elevation in whole root and rootepidermal protoplast [Ca2+]cyt found in aequorin lumi-nometry assays (Demidchik et al., 2003a, 2009). In ourexperimental conditions only K+ efflux (50 mM K+ in thePS) could mediate the outward current (Shabala et al.,2006). Both inward and outward currents were alsosensitive to the cation channel blocker Gd3+. It may bethat both Ca2+ influx and K+ efflux conductances werecaused by the activation of one cation channel type orthat Ca2+ influx activated K+ efflux.

Extracellular ADP Elevates Root Epidermal [Ca2+]cyt

Epidermal protoplasts were isolated from the ma-ture epidermis of plants constitutively expressingcytosolic apoaequorin to examine extracellular ADP-induced [Ca2+]cyt elevation. Previously, such proto-plasts were found to sustain transient [Ca2+]cyt elevationsin response to 0.1 mM extracellular ATP, its poorlyhydrolyzable analog adenosine 5#-(a,b-methylene)triphosphate (abATP), and ADP. All these were inhib-ited by Gd3+ and the purinoceptor antagonist, suramin(Demidchik et al., 2009). AMP (as an ADP breakdownproduct) up to 1 mM has been shown to be ineffective(Demidchik et al., 2009). A test concentration of 0.1 mM

ADP was used in this study as it is a feasible concen-tration to simulate a wound response (Song et al.,2006) and was found to cause Ca2+ current activationin four out of nine whole-cell patch-clamp trials us-ing slow perfusion (data not shown). Application of0.1 mM extracellular ADP at pH 6.0 caused a transientincrease in [Ca2+]cyt (Fig. 4A). Overall the mean peak

Table I. The effect of sequential extracellular ADP addition on PMcurrent

Control values were taken 1 to 3 s immediately preceding theapplication of ADP. Event 1 refers to the first application of ligand andEvent 2 to the subsequent application to the same protoplast. Record-ing conditions as in Figure 1A. HP = 290 mV.

Procedure Mean 6 SE Maximal Currents

mA m22

Set AEvent 1 (n = 4)Control 240.63 6 3.450.3 mM ADP 276.59 6 4.95

Event 2 (n = 3)Control 236.74 6 2.000.3 mM ADP 256.51 6 5.3

Set BEvent 1 (n = 3)Control 248.36 6 19.830.3 mM ADP 297.01 6 12.90

Event 2 (n = 3)0.3 mM ADP 243.4 6 15.230.3 mM ADP + 0.3 mM Gd3 248.33 6 21.30

Figure 3. Extracellular ADP activates Ca2+ influx and K+ efflux inepidermal protoplasts. Mean 6 SE IV relationships for control condi-tions and in the presence of 0.3 mM extracellular ADP, n = 4; solutionsas in Figure 2. Current values were taken from voltage ramps applied1 to 3 min after application of control BS or activation by ADP. Insert:difference IV relationship (control currents were subtracted from thevalues obtained after ADP activation).

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[Ca2+]cyt increase caused by this extracellular ADP con-centration was 0.43 6 0.02 mM [Ca2+]cyt (n = 52), a valuecomparable to that reported previously for protoplastsfrom this cell type in response to 0.1 mM ADP or ATP(Demidchik et al., 2009). Extracellular ADP was addedas its Na+ salt; NaCl as a control is not effective as anelicitor in this assay (Demidchik et al., 2009). The ex-tracellular ADP-induced increase depended on Ca2+

influx as depletion of external Ca2+ from 1 mM to 5 mM

in a paired test reduced the extracellular ADP-inducedincrease by 84% (0.1 mM extracellular ADP, 1 mM Ca2+;0.386 0.04 mM [Ca2+]cyt, n = 3: 0.1 mM extracellular ADP,5 mM Ca2+; 0.06 6 0.05 mM [Ca2+]cyt, n = 4). An appli-cation of 0.1 mM extracellular ADP was used as thestandard stimulus in further trials.The peak [Ca2+]cyt response to extracellular ADP

was insensitive to the G-protein inhibitor pertussistoxin (200 ng mL21; Alloisio et al., 2004; extracellularADP; 0.346 0.04mM [Ca2+]cyt, n = 6: extracellular ADP +pertussis toxin; 0.34 6 0.02 mM [Ca2+]cyt, n = 11). Thecation channel blocker, Gd3+, is already known toinhibit the extracellular ADP-induced elevation of[Ca2+]cyt in these epidermal protoplasts (Demidchiket al., 2009). TEA+ (10 mM), a K+ channel blocker, sig-nificantly inhibited the [Ca2+]cyt elevation by 25% (ex-tracellular ADP; 0.63 6 0.02 mM [Ca2+]cyt, n = 6: ADP +TEA+; 0.476 0.03mM [Ca2+]cyt, n = 6; P, 0.001, ANOVA).This suggests that K+ channels may be involved inchanging membrane voltage regulating driving forcefor the Ca2+ influx. Depolarizing the PM with 80 mM

K+ (Demidchik et al., 2002) significantly reduced the[Ca2+]cyt response by 55%, consistent with reduction ofCa2+ influx at depolarized voltage in patch-clamp trials(1 mM K+; 0.55 6 0.03 mM [Ca2+]cyt, n = 5: 80 mM K+;0.25 6 0.01 mM [Ca2+]cyt, n = 4; P , 0.001). The effect ofpH was tested against the standard application of0.1 mM extracellular ADP, revealing a clear neutral pHoptimum (0.57 6 0.02 mM [Ca2+]cyt, n = 4) and a sig-nificantly enhanced response at alkaline pH compared

to physiological apoplastic pH 5 to 6 (Fig. 4B; P ,0.001). Nevertheless, at pH 5, elevation of [Ca2+]cyt re-mained significant (control [Ca2+]cyt baseline pH 5,0.116 0.02 mM; [Ca2+]cyt peak, 0.446 0.03 mM, n = 5; P,0.001). In separate tests (using a plate reader ratherthan a luminometer), although absolute values of peak[Ca2+]cyt were lower, the pattern of a greater responseat neutral pH rather than pH 6 was retained for 0.1 mM

ADP and also 0.1 mM ATP (Fig. 4C, n = 4; P , 0.05).Response toATPwas significantly greater than toADP atboth pH. The response to the poorly hydrolyzable ADPanalog ADPbS [adenosine 5#-O-(2-thiodiphosphate)]was not significantly different to the ADP responsewhile abATP evoked significantly lower responses thanATP at both pH (n = 4; P , 0.05). Thus part of the ATPresponse may involve hydrolysis products but the ADPresponse does not.

Extracellular ADP Does Not Increase Intracellular Levelsof ROS

In Arabidopsis roots, extracellular ATP causes ac-cumulation of intracellular ROS via activation of thePM NADPH oxidase AtRBOHC (Demidchik et al.,2009). At the whole-root level, extracellular ADP wasapplied under the same conditions used by Demidchiket al. (2009) for intracellular ROS imaging [using ester-loaded 5-(and-6-)-chloromethyl-2’,7’-dichlorohydro-fluorscein diacetate] in response to extracellular ATP.Solutions were designed to ensure that extracellular[Ca2+] remained constant at 0.1 mM throughout exper-imental treatments as purine nucleotides are potentCa2+ chelators. Chelation of extracellular Ca2+ can trig-ger extracellular superoxide anion production by roots(Mortimer et al., 2008). Here, extracellular ATP wasfound again to cause intracellular ROS accumulation(Fig. 5A). In contrast, extracellular ADP did not resultin significant accumulation of intracellular ROS (Fig. 5,A and B). Previously a response was seen by 15 s after

Figure 4. Transient [Ca2+]cyt elevation by extra-cellular ADP in epidermal protoplasts. A, Repre-sentative trace of [Ca2+]cyt response in matureepidermal protoplasts evoked by 0.1 mM ADP,recorded using a luminometer. Discharge denotesthe addition of 2 M CaCl2 and 20% (v/v) ethanolto enable [Ca2+]cyt estimation. B, pH dependenceof the epidermal protoplast [Ca2+]cyt response.Mean6 SE peak [Ca2+]cyt response to 0.1 mM ADPwith variable extracellular pH. Results are fromfour to five independent trials. C, pH dependenceof the epidermal protoplast mean6 SE peak [Ca2+]cytresponse in separate trials using a plate reader.The effects of 0.1 mM ADP, ATP, ADPbS, or abme-ATP were tested at pH 6 or 7 (n = 4).

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extracellular ATP application (0.01–1 mM) with 0.1 mM

extracellular ATP causing over a 3-fold increase insignal intensity (Demidchik et al., 2009). Here, thesame concentration range for extracellular ADP underidentical conditions did not evoke intracellular ROSproduction, even over an extended observation pe-riod. Under control conditions, mean6 SE fluorescenceintensity of single primary roots was 19 6 0.9 fluores-cence per pixel (n = 23) and there were no significantincreases on application of extracellular ADP (Tukey’smultiple comparison test; Fig. 5B). Separate trialsusing a different imaging system (Leica) and withextracellular Ca2+ maintained at 0.1 or 1 mM resulted inhigher control values than recorded previously, show-ing the variable nature of ROS accumulation (Fig. 5, Cand D). Extracellular ATP (1 mM) caused an increase influorescence intensity with 1 mM Ca2+ but 1 mM ADPdid not (Fig. 5, C and D; n = 11). Fluorescence intensitywith ADP was significantly lower than with ATP(Student’s t test; P , 0.02). Overall the results suggestthat the extracellular ADP/[Ca2+]cyt pathway is inde-pendent of intracellular ROS.

Extracellular ADP Regulates Net Ca2+ and K+ Fluxes fromExcised Roots

To test whether extracellular ADP can activate cat-ion fluxes in intact cells, we monitored net fluxes at theepidermis of excised roots using the MIFE system for

noninvasive microelectrode Ca2+- and K+-flux mea-surements. This permits fine spatio-temporal mappingof net fluxes. Constancy of extracellular Ca2+ (0.1 mM

Ca2+) ensured that changes in flux were due to extra-cellular ADP perception alone (rather than changes in[Ca2+]) and also aided electrode function. Applicationof extracellular ADP (Na+ salt) to the elongation zoneepidermis caused transient increases in net Ca2+ influx(i.e. more positive flux values) in a dose-dependentmanner (Fig. 6, A and B; n = 3–6 for 1 mM to 1 mM

extracellular ADP). A significant increase above basallevel was first detected at 3 mM (P , 0.02; Student’st test). Duration of transients was up to 4 min. Controlflux levels were recovered. In control experimentsfor the effect of Na+, 4 mM NaCl had no effect on netflux (n = 4).

Net Ca2+ influx in response to extracellular ADPwasless pronounced in mature zone epidermis and [ADP]below 10 mM did not evoked significant changes (Fig.6C, n = 3–7). The mature zone has been found previ-ously to be less receptive to exogenous ROS than theelongation zone (Demidchik et al., 2003b, 2009). Dif-ferences between elongation zone and mature zonewere clearer for extracellular ADP-induced K+ efflux(Fig. 6D), with elongation zone epidermis (Fig. 6E, n =3–6) supporting greater net K+ efflux than the maturezone (Fig. 6F, n = 3–7) across the concentration rangetested. Significant changes in K+ flux relative to basalwere observed at 1 mM ADP in both zones. Extracel-

Figure 5. Extracellular ADP does not cause intracellular ROS accumulation. Arabidopsis roots were ester loaded with 5-(and-6)-chloromethyl-2#,7#-dichlorodihydrofluorescein (CM-H2DCF) and exposed to extracellular ADP or ATP. A, Top sections, rootsbathed in medium (0.1 mM KCl, 0.1 mM CaCl2, pH 6.0) with 0, 0.1 mM, 1 mM extracellular ADP, or 0.1 mM extracellular ATP withfluorescence false color mapping applied using the same scaling used for the positive extracellular ATP response reported byDemidchik et al. (2009). Bottom sections, corresponding bright-field images. Scale bar = 2 mm. B, Mean 6 SE fluorescence perpixel for control (n = 23), 0.01mM extracellular ADP (n = 24), 0.1 mM extracellular ADP (n = 25), and 1mM extracellular ADP (n =23) exposure. C, In separate trials, roots were exposed to 1 mM ADP or ATP but with Ca2+ maintained at 1 mM. Scale bar = 0.2mm, fluorescence false color-mapping scale is shown on the right. D, Mean 6 SE fluorescence per pixel for 0.1 mM Ca2+ buffer(n = 8), 1 mM Ca2+ buffer (n = 17), 1 mM ADP with 1 mM Ca2+ (n = 11), 1 mM ATP with 1 mM Ca2+ (n = 11).

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lular AMP was found previously to be a very weakinducer of root Ca2+ influx (Demidchik et al., 2009) andseedling [Ca2+]cyt elevation (Tanaka et al., 2010b). Here,AMP did not elicit a K+ efflux response even at 1 mM,in either the elongation zone or mature epidermis(elongation zone control; 237 6 34 nmol m22 s21, n = 3:elongation zone + AMP, 231 6 26 nmol m22 s21, n = 3;mature zone control;2196 13 nmolm22 s21, n = 3: AMP,210 6 18 nmol m22 s21, n = 3). This suggests thatbreakdown of extracellular ADP to AMP would helpterminate the cell’s response.Extracellular ATP and its poorly hydrolyzable ana-

log adenosine 5#-(a,b-methylene)triphosphate (abATP)were also tested. The pattern of greater net Ca2+ and K+

fluxes at the elongation zone was retained for bothagonists (Figs. 7 and 8). In the elongation zone, bothextracellular ATP and abATP up to 100 mM causedtransient net Ca2+ influx comparable to that evoked byextracellular ADP (Fig. 7, A–D). The threshold of thedetectable ATP response lay between 0.3 and 1 mM (n =6). However while at 300 mM and 1 mM extracellularADP promoted net Ca2+ influx, ATP and abATP causednet Ca2+ efflux (n = 5–17; Fig. 7, C and D). The meanpeak net Ca2+ efflux caused by 1 mM abATPwas almostdouble that caused by 1 mM ATP (Fig. 7, C and D). Atthe mature epidermis, while 1 mM ADP caused net Ca2+

influx, both 1 mM ATP and abATP again caused netCa2+ efflux with abATP eliciting the greater response(Fig. 7E; 1 mM ATP,256 21 nmol m22 s21, n = 8). ThusATP hydrolysis appears necessary to limit the Ca2+

efflux response. At both elongation zone and matureepidermis, both extracellular ATP and abATP causeddose-dependent net K+ efflux in common with ADP(Fig. 8). Thus while at high concentrations of ADPnet Ca2+ influx and K+ efflux appear as a coupledresponse, net Ca2+ flux is uncoupled from K+ efflux athigh concentrations of ATP or abATP.

DISCUSSION

Evidence is now accumulating that extracellularATP is a regulator of plant physiology and develop-ment (Lew and Dearnaley, 2000; Tang et al., 2003;Chivasa et al., 2005, 2009; Kim et al., 2006, 2009; Rouxand Steinebrunner, 2007; Wu et al., 2007; Riewe et al.,2008a, 2008b; Wu and Wu, 2008; Yi et al., 2008;Demidchik et al., 2009; Reichler et al., 2009; Clarket al., 2010a, 2010b; Tanaka et al., 2010a, 2010b; Terrileet al., 2010; Tonon et al., 2010). In common with ATP, itis likely that ADP is released on wounding and couldperhaps also be released with ATP by exocytosis or theactivity of ATP-binding cassette transporters (Thomaset al., 2000; Kim et al., 2006; Tanaka et al., 2010a).Extracellular ATP hotspots have been found in elon-gating root cells (Kim et al., 2006) and ADP may alsobe released there. This would be consistent with thefinding here that the Arabidopsis root elongation zoneepidermis supported a greater flux response to extra-cellular ADP than the mature zone. Such data support

Figure 6. Extracellular ADP-induced elevation of net Ca2+ influx and K+ efflux at the root epidermis. A, Response of elongationzone epidermal net Ca2+ flux to 3 mM, 100 mM, or 1 mM ADP, representative traces superimposed. Positive values signify influx. B,Mean6 SE net Ca2+ influx at the elongation zone epidermis (n = 3–6). C, Mean6 SE net Ca2+ influx at the mature epidermis (n =3–7). D, Response of mature and elongation zone epidermis to 300mM ADP, representative traces superimposed. Negative valuessignify efflux. E, Mean 6 SE net K+ efflux at the elongation zone epidermis (n = 3–6). F, Mean 6 SE net K+ efflux at the matureepidermis (n = 3–7). Basal assay solution comprised 0.1 mM KCl, 0.1 mM CaCl2, pH 6.0.

Receptor-Like Activity Evoked by Extracellular ADP

Plant Physiol. Vol. 156, 2011 1381

the premise that the sensitivity of different cells toextracellular purine nucleotides differs (Tanaka et al.,2010b). Suramin (a purinoceptor antagonist) inhibitsArabidopsis root elongation and extracellular ADP-induced [Ca2+]cyt elevation (Demidchik et al., 2009).Moreover, there is a dose-dependent effect of extra-cellular ADP on Arabidopsis root hair elongation(Clark et al., 2010b) further implicating extracellularADP in growth regulation. Plant extracellular apy-rases can hydrolyze ADP to regulate its extracellularlevels and so terminate any signal it could invoke(Komoszynski, 1996; Steinebrunner et al., 2003; Rieweet al., 2008a, 2008b; Tanaka et al., 2010a, 2010b).

Results here show that the epidermis is a contribu-tory cell type to the whole-root elevation of [Ca2+]cyt byextracellular ADP observed by Demidchik et al.(2003a) and the whole-seedling response, thought tobe generated largely by the root, observed by Tanakaet al., (2010b). Moreover, that protoplasts could sustain

the [Ca2+]cyt elevation demonstrates that the wall is notcritical for the response of this cell type. Previously, ithad been suggested that wall proteins capable ofphosphorylation intercept extracellular purine nucle-otides to evoke a cellular response (Chivasa et al.,2005). The results also show that extracellular ADP cangenerate a [Ca2+]cyt signal in the mature epidermisseemingly independently of heterotrimeric G-proteinactivity. Further studies on the epidermis now need tofocus on G-protein mutants.

The response speed of the PM to extracellular ADP inthe fast-perfusion patch-clamp trials here is consistentwith the receptor-like activity suggested by Lew andDearnaley (2000) to explain extracellular ADP-induceddepolarization of Arabidopsis root hair PM voltage.Previously, extracellular ADP-induced rapid elevationof [Ca2+]cyt in root epidermal protoplasts was inhibitedby 92% with the same concentration (0.3 mM) of Gd3+

used here to block PM influx currents (Demidchik et al.,2009). The very rapid response of the PM inward Ca2+

conductance to extracellular ADP (fast perfusion) andits block by 0.3 mM Gd3+ raises the possibility that Ca2+

influx is an initial event. In common with animalpurinceptor responses (Roberts and Evans, 2007), ex-tracellular ADP-induced current decayed in the contin-ued presence of the agonist and repeated application ofextracellular ADP to the epidermal PM resulted in alower level of current activation (Fig. 2A; Table I). Thus,a [Ca2+]cyt signal evoked by extracellular ADP releasecould be diminished by desensitization and terminatedby apyrase or phosphatase activity. This helps explainthe transient nature of the [Ca2+]cyt response and per-haps also that of the epidermal Ca2+ and K+ fluxes.Efflux of K+ could readily counter the depolarizingeffect of purine nucleotide-induced Ca2+ influx on PMvoltage and help return this membrane to a hyper-polarized state. The greater magnitude of the K+ effluxcompared to Ca2+ influx suggests that K+ efflux mayalso serve other purposes.

Previously, extracellular ATP was found to stimu-late production of extracellular superoxide anion byArabidopsis etiolated hypocotyls (Tonon et al., 2010),and leaves (Song et al., 2006). In leaves, this wasthrough activation of PM NADPH oxidases (Songet al., 2006). Extracellular ADP stimulated leaf androot extracellular superoxide anion production by anunknown mechanism (Song et al., 2006; Demidchiket al., 2009). In roots of Arabidopsis, Medicago trunca-tula, and Phaseolus vulgaris extracellular ATP alsoresulted in accumulation of intracellular ROS, essen-tially hydrogen peroxide (H2O2; Kim et al., 2006;Cardenas et al., 2008; Demidchik et al., 2009; Clarket al., 2010b). In this study, and in contrast to extracel-lular ATP, extracellular ADP did not trigger accumu-lation of intracellular ROS. This is in agreement withthe findings of Kim et al. (2006) in which extracellularATP but not extracellular ADP triggered intracellularROS accumulation in M. truncatula root hairs. Cer-tainly, there is a precedent for triggers of Ca2+ influxresulting in distinct ROS responses. In Arabidopsis cell

Figure 7. Extracellular ATP- and abme-ATP-induced net Ca2+fluxes atthe root epidermis. A, Response of elongation zone epidermis to 1 to1,000 mM ATP, representative traces superimposed. B, Mean 6 SE netCa2+ influx at the elongation zone epidermis in response to 0.3 to 100mM ATP (n = 6–18). C, Mean 6 SE net Ca2+ flux at the elongation zoneepidermis in response to 100 to 1,000 mM ATP (n = 7–18). D, Mean6 SE

net Ca2+ flux at the elongation zone epidermis in response to abme-ATP (n = 3–7). E, Mean 6 SE net Ca2+ flux at the mature epidermis inresponse to abme-ATP (n = 3–5). Recording conditions as Figure 6.

Demidchik et al.

1382 Plant Physiol. Vol. 156, 2011

suspension cells, both hyper- and hypoosmotic stresstrigger Ca2+ influx but only hypoosmotic stress resultsin extracellular H2O2 accumulation (Beffagna et al.,2005). Extracellular superoxide anions (resulting fromperception of extracellular purine nucleotides) couldgenerate H2O2 that could then be translocated tothe cytosol by PM aquaporins (Bienert et al., 2007;Dynowski et al., 2008). One possible explanation forthe disparity between the intracellular H2O2 accumu-lation response to extracellular ATP and extracellularADP is that in the extracellular ADP pathway, H2O2translocation is blocked. This could be achieved byclosure and/or retrieval of H2O2-permeable aquapor-ins (Boursiac et al., 2008). Alternatively, extracellularADP perception could inhibit an intracellular ROS-generating system, stimulate an intracellular antioxi-dant activity, or extracellular ADP perception in theapoplast could result in suppression of extracellularROS production downstream of superoxide anions.Given the importance of ROS in plant cell signaling,these possibilities merit further investigation particu-larly in the context of nodule development. Extracel-lular ADP has been found to regulate this process thatis known to involve complex spatio-temporal changesin ROS (Chang et al., 2009; Govindarajulu et al., 2009).Whether ATP-dependent intracellular ROS accumu-lation is the cause of net Ca2+ efflux from the rootepidermis at high [ATP] now needs to be determinedas this may be relevant to the suppression of defenseresponses noted when exogenous ATP is increased(Chivasa et al., 2009).

The pH dependence of animal ionotropic purinore-ceptors varies with isoform (King et al., 1997; Wildmanet al., 1999; Wirkner et al., 2008). Here, the extracellularADP-induced [Ca2+]cyt response was operative over awide pH range but activity at more neutral to alkalinepH suggests that extracellular ADP (and ATP) sig-naling could operate during stress responses in whichthe cytosol acidifies and apoplastic pH elevates aboveits normal acid value, such as hypoosmotic stress orelicitor challenge (Mathieu et al., 1996; Pugin et al.,1997; Beffagna et al., 2005). Additionally, pH de-pendence of ADP- or ATP-induced Ca2+ and K+ fluxcould be relevant at growth points, such as the root hairapex, at which apoplastic pH oscillates and pH oscil-lations are linked to nitrogen availability (Monshausenet al., 2007; Bloch et al., 2011). Overall, extracellular ADPis competent to act as a trigger of [Ca2+]cyt-dependentsignaling with the potential to evoke responses dis-tinct to those produced by ATP. Its initial perceptionat the PM appears reliant on ionotropic receptor-likeactivity.

CONCLUSION

Perception of extracellular ADP at the root epider-mal PM is consistent with the operation of functionalequivalents of ionotropic purine nucleotide receptors.In contrast to extracellular ATP, downstream events donot include accumulation of intracellular ROS thusindicating disparate signaling pathways.

Figure 8. Extracellular ATP- and abme-ATP-induced net K+ fluxes at the root epidermis. A, Response of elongation zoneepidermis to 0.3 to 1,000 mM ATP, representative traces superimposed. B, Mean 6 SE net K+ efflux at the elongation zoneepidermis in response to 0.3 to 1,000 mM ATP (n = 6–37). C, Mean6 SE net K+ efflux at the mature epidermis in response to 10 to1,000 mM ATP (n = 4–13). D, Response of elongation zone and mature epidermis to 300 mM abme-ATP, representative tracessuperimposed. E, Mean6 SE net K+ efflux at the elongation zone in response to 3 to 1,000 mM abme-ATP (n = 3–7). F, Mean6 SE

net K+ efflux at the mature epidermis in response to 3 to 1,000 mM abme-ATP (n = 3–6). Recording conditions as Figure 6.

Receptor-Like Activity Evoked by Extracellular ADP

Plant Physiol. Vol. 156, 2011 1383

MATERIALS AND METHODS

Plant Growth

Arabidopsis (Arabidopsis thaliana Columiba-0; Columbia-0 expressing

apoaequorin under a 35s promoter; Demidchik et al., 2009) from laboratory

stock was grown vertically on full-strength Murashige and Skoog (Duchefa;

www.duchefa.com) medium with 1% (w/v) Suc and 0.3% (w/v) Phytagel

(Sigma; www.sigmaaldrich.com). Growth conditions were 22�C and 16 h day

length, 100 mmol m22 s21 irradiance.

Patch-Clamp Electrophysiology

Protoplasts were isolated from root mature epidermis (10–15 d plants)

using the protocol described by Demidchik et al. (2003b). Protoplasts used for

patch clamping were 20 6 1.5 mm in diameter. The PS contained (mM): 40

K-gluconate, 10 KCl, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic

acid (free Ca2+ adjusted to 100 nM with 0.4 CaCl2), pH 7.2 adjusted with Tris/

MES. The G-protein antagonist GDPbS (0.5 mM) was incorporated into the PS

as indicated. Control BS comprised (mM): 20 CaCl2, 0.1 KCl, 0.3 NaCl (varied

to supply equimolar Na+ to that in purine-containing BS), pH 6 with 2 Tris/4

MES. BS containing purine agonist was freshly prepared directly before

application and the pH of the solution was adjusted where necessary (Tris). BS

and PS were adjusted to 290 to 300 mOsM with D-sorbitol. Delivery of BS +

ADP to the recording chamber was either by gravity feed or by a fast-flow

system comprising an AutoMate perfusion system with four pinch valves,

ValveBank controller, and four-barrel delivery pipette (barrel diameter 0.1

mm; AutoMate Scientific Inc.). This system allowed switching to test BS

in 0.01 s. Average delivery time was less than 1 s. The delivery pipette was

placed at 30 to 40 mm from the protoplast surface and delivery speed was 0.5

to 1 mL s21. Two barrels of the delivery pipette were used—one for control BS

and another for purine-containing BS. Solution changes were controlled by the

programmable ValveBank controller. Recording equipment was as described

in Demidchik et al. (2003b). Ion activities were calculated using Geochem

(Parker et al., 1995). Liquid junction potentials were measured and corrected

as described previously (Demidchik et al., 2003b).

[Ca2+]cyt Measurement

The protocol described in Demidchik et al. (2007) was used to isolate

protoplasts from root mature epidermis of Arabidopsis (10–15 d old) express-

ing cytosolic (apo)aequorin (cauliflower mosaic virus 35S promoter) with the

modification that incubation with protoplasting enzymes was increased from

50 to 60 min. Protoplasts were suspended for 3 to 4 h (dark, 28�C) in recording

medium (mM: 1 CaCl2, 1 KCl, 1 MgCl2, 10 Glc; pH 6.0 adjusted by 2 Tris/4

MES; 290–300 mOsM with D-sorbitol) with 10 mM coelenterazine (NanoLight

Technology; www.nanolight.com) to match the protocol used by Demidchik

et al. (2009) for ATP and ADP studies. After washing, recording medium

(without coelentrazine) and standard luminometry procedures were used.

The discharging solution enabling estimation of [Ca2+]cyt contained 2 M CaCl2and 20% (w/v) ethanol. Luminescence was recorded using a luminometer or

FLUOstar plate reader.

Imaging of ROS

Imaging of intracellular ROS used 50 mM 5-(and-6-)-chloromethyl-2’,7’-

dichlorohydrofluorscein diacetate, acetyl ester (Molecular Probes; www.

invitrogen.com) with dye loading as described previously (Demidchik et al.,

2009). Assay medium was as described by Demidchik et al. (2009) and

comprised (mM): 0.1 KCl, 0.1 or 1 CaCl2, 4 MES, and 2 Tris base (pH 6.0).

Images of 5-d-old roots were acquired either with a Nikon SMZ 1500

microscope and a QImaging Retiga cooled 12-bit camera or a Leica M165

FC microscope and a Leica DFC310 FX 1.4-megapixel camera. Ca2+ was

maintained at 0.1 or 1 mM in the presence of purines by appropriate additions

of CaCl2 (calculated using MaxChelator and Geochem programs; Parker et al.,

1995; Patton et al., 2004).

MIFE Recordings of Net Cation Fluxes

Excised roots (5–15 d old) were fixed into a recording chamber by an agar

drop and immersed in 0.5 mL control solution comprising (in mM) 0.1 KCl, 0.1

CaCl2, 4 MES, 2 Tris base (pH 6.0; Demidchik et al., 2009). Chamber design

permitted rapid mixing of test solution and resumption of flux recording 20 to

30 s after addition of 0.5 mL test solution. Ca2+ was maintained at 0.1 mM in the

presence of purines by appropriate additions of CaCl2. Levels were confirmed

experimentally in a cell-free assay using a Ca2+-selective microelectrode.

Purines were added as Tris, Na+, or Li+ salts. NaCl or LiCl at equimolar

concentrations did not perturb K+ efflux (tested experimentally). Measure-

ments of mature epidermal flux were taken 1 to 1.5 mm from the root apex and

for the elongation zone, 100 to 150 mm from the apex (Demidchik et al., 2007).

Net Ca2+ influx and K+ efflux were determined using the noninvasive slowly

vibrating microelectrode (MIFE) technique. K+- and Ca2+-selective microelec-

trodes (external tip diameter approximately 3 mm) were made and calibrated

as described previously (Shabala and Shabala, 2002). Electrodes were cali-

brated before and after use. Agonists (tested up to 1 mM) had no effect on

calibration or electrode response time. The microelectrode was placed 20 mm

above the root surface and moved between two positions, 20 and 50 mm above

the root surface in a square wave manner (5-s half-cycle).

ACKNOWLEDGMENTS

We thank Daniel Schachtmann for advice and Adeeba Dark for help with

plant cultivation.

Received February 18, 2011; accepted May 6, 2011; published May 11, 2011.

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