An early Ca2+ influx is a prerequisite to thaxtomin A-induced cell death in Arabidopsis thaliana cells

Journal of Experimental Botany, Vol. 59, No. 15, pp. 4259–4270, 2008

doi:10.1093/jxb/ern267 Advance Access publication 17 November, 2008

RESEARCH PAPER

An early Ca2+ influx is a prerequisite to thaxtomin A-inducedcell death in Arabidopsis thaliana cells

R. Errakhi1,2,*, A. Dauphin1,*, P. Meimoun1, A. Lehner1,†, D. Reboutier1, P. Vatsa3, J. Briand1, K. Madiona1,

J. P. Rona1, M. Barakate2, D. Wendehenne3, C. Beaulieu4 and F. Bouteau1,‡

1 LEM (EA 3514), Universite Paris Diderot-Paris7, 2, place Jussieu, F-75251 Paris cedex 05, France2 Laboratoire de Biologie et Biotechnologie des Microorganismes, Faculte des Sciences-Semlalia, BP 2390, 40001Marrakech, Maroc3 UMR INRA 1088, CNRS 5184, Universite de Bourgogne, Plante-Microbe-Environnement, Dijon, France4 GRBA, Departement de Biologie, Universite de Sherbrooke, Quebec, Canada JIK 2RI

Received 10 September 2008; Revised 3 October 2008; Accepted 6 October 2008

Abstract

The pathogenicity of various Streptomyces scabies

isolates involved in potato scab disease was corre-

lated with the production of thaxtomin A. Since

calcium is known as an essential second messenger

associated with pathogen-induced plant responses

and cell death, it was investigated whether thaxtomin

A could induce a Ca2+ influx related to cell death and

to other putative plant responses using Arabidopsis

thaliana suspension cells, which is a convenient model

to study plant–microbe interactions. A. thaliana cells

were treated with micromolar concentrations of thaxto-

min A. Cell death was quantified and ion flux variations

were analysed from electrophysiological measure-

ments with the apoaequorin Ca2+ reporter protein and

by external pH measurement. Involvement of anion

and calcium channels in signal transduction leading to

programmed cell death was determined by using

specific inhibitors. These data suggest that this toxin

induces a rapid Ca2+ influx and cell death in A. thaliana

cell suspensions. Moreover, these data provide strong

evidence that the Ca2+ influx induced by thaxtomin A

is necessary to achieve this cell death and is a pre-

requisite to early thaxtomin A-induced responses:

anion current increase, alkalization of the external

medium, and the expression of PAL1 coding for a key

enzyme of the phenylpropanoid pathway.

Key words: Calcium, cell death, ion channel, plant pathogen,

Streptomyces, thaxtomin A.

Introduction

Plants are constantly exposed to pathogens and haveevolved a diversity of responses in order to withstandthese attacks. Recognition and perception of a pathogen ortheir derived-elicitors by plant cells lead to modulation ofthe defence-signalling pathways. The inducible defenceresponses include the production of reactive oxygenspecies (ROS), the modulation of ion fluxes, an increaseof cytosolic [Ca2+], the activation of mitogen-activatedprotein kinases (MAPKs) and the expression of defence-related genes that are involved in the production ofvarious metabolites as well as pathogenesis-related pro-teins showing antimicrobial properties (Nurnberger andScheel, 2001; Garcia-Brugger et al., 2006). In addition,plants often induce a hypersensitive response (HR)characterized by a localized cell death often associatedwith disease resistance (Lam, 2004). The HR is a form ofprogrammed cell death (PCD) that is thought to kill the

* Both authors contributed equally to this work.y Present address: Laboratoire de Glycobiologie et Transport chez les Vegetaux, FRE CNRS 3090, IFRMP23, Universite de Rouen, Mont Saint Aignan,France.z To whom correspondence should be addressed: E-mail: [email protected]: 9-AC, 9-anthracene carboxylic acid; BAPTA, 1.2-bis(o-aminophenoxy)ethane-N,N,N#,N#-tetraacetic acid; [Ca2+]cyt, cytosolic calciumconcentration; FDA, fluorescein-diacetate; gli, glibenclamide; FW, Fresh weight; HR, hypersensitive response; IAA, indole acetic acid; KORC, K+ outwardrectifying current; PM, plasma membrane; ROS, reactive oxygen species; TXT, thaxtomin A; Vm, plasma membrane potential.

ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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pathogens and/or to limit their spread (Heath, 2000). HRis a genetically controlled process that displays apoptosis-like features such as cell shrinkage, chromatin condensa-tion, and DNA cleavage into inter-nucleosomal fragments(Lam, 2004). It requires gene expression and metabolicactivities (Lam, 2004).As in animal cells, a perturbation of Ca2+ homeostasis in

plant seems to be a prerequisite to PCD (Davis andDistelhorst, 2006; Lecourieux et al., 2006). Changes in[Ca2+]cyt are rapid and have been reported in response tovarious microbial phytotoxins and elicitors (Reddy, 2001;White and Broadley, 2003). They have also been associatedwith an induction of HR cell death (Levine et al., 1996;Grant et al., 2000; Lecourieux et al., 2002). Recently,Kurusu et al. (2005) have demonstrated the involvement ofa Ca2+ channel (OsTPC1) in elicitor-induced defenceresponses and in hypersensitive cell death in rice. Recentdata have also illustrated the role of cyclic nucleotide-gatedion channels (CNGC) permeable to Ca2+ in plant defence(Yoshioka et al., 2006) and in cell death (Clough et al.,2000; Balague et al., 2003). Less specifically, numerousstudies led to the conclusion that the activation of defenceresponses depends on Ca2+ influxes from the apoplast intothe cytosol of plant cells (see Lecourieux et al., 2006, fora review). Notably, elicitor-induced uptake of Ca2+ fromthe extracellular medium was shown to be required for thecontrolled generation of H2O2 (Pugin et al., 1997; Kelleret al., 1998; Hu et al., 2004), the activation of MAPKpathways (Link et al., 2002; Garcia-Brugger et al., 2006),the activation of defence-related genes (Lecourieux et al.,2002), and production of phytoalexin (see Lecourieuxet al., 2006, for a review). For instance, Lecourieux et al.(2002) showed that suppression of the sustained [Ca2+]cytincrease in cryptogein-treated tobacco cells suppressed theaccumulation of transcripts corresponding to phenylalanineammonia lyase (PAL), a key enzyme of the phenyl-propanoid pathway.Thaxtomin A (TXT) is a nitrated dipeptide phytotoxin

produced by all plant-pathogenic Streptomyces species(King et al., 1991; Loria et al., 1997). In the absence ofpathogen, TXT has been shown to induce common scab-like disease symptoms and localized cell death, when it isapplied to developing tubers or roots (Lawrence et al.,1990). A mutation within TXT biosynthesis genesrendered a S. scabies strain non-pathogenic (Healy et al.,2000). Their pathogenicity is thus directly correlated withtheir ability to produce TXT. Thaxtomin A has beenshown to inhibit cellulose synthesis, suggesting that thecell wall is one of the main targets of TXT (Fry and Loria,2002; Scheible et al., 2003). Recent studies have shownthat TXT stimulates H+ efflux across the plasma mem-brane (PM) and a short-lived Ca2+ influx in the roots ofdifferent species (Tegg et al., 2005). This Ca2+ influx isinhibited by La3+, a PM Ca2+ channel inhibitor (Tegget al., 2005). Thaxtomin A also induces a cell death which

depends on active gene transcription and de novo proteinsynthesis and that displays PCD features (Duval et al.,2005). However, TXT-induced cell death appeared atypi-cal since it does not involve typical defence responsesassociated with the HR: ROS production, alkalization, andactivation of the ethylene/jasmonate or salicylic acid (SA)pathways (Duval et al., 2005).The aim of this study was to investigate the role of the

TXT-induced influx of Ca2+ on physiological eventsrelated to plant responses to pathogens and, in particular,to determine whether a short-lived Ca2+ influx could beinvolved in the atypical HR cell death induced by TXT inArabidopsis thaliana. Electrophysiological molecularapproaches and a Ca2+ assay were used on A. thalianasuspension-cultured cells which is a convenient materialfor studying early physiological events induced bypathogens (Atkinson et al., 1996; Wendehenne et al.,2002; Duval et al., 2005; Bouizgarne et al., 2006;Reboutier et al., 2007), in order to provide evidence thatTXT-induced Ca2+ influx is an early signalling cascadeevent necessary to achieve defence responses includingcell death.

Materials and methods

Cell culture conditions

Arabidopsis thaliana L. (ecotype Columbia) suspension cells weregrown in Gamborg medium (pH 5.8). They were maintained at2462 �C, under continuous white light (40 lmol photons m�2 s�1)and continuous shaking (gyratory shaker) at 120 rpm. Suspensionswere subcultured weekly using 1:10 dilution. All experiments wereperformed at 2262 �C using log-phase cells (4 d after subculture).

Thaxtomin A production

Thaxtomin A was purified from oat bran broth cultures of S. scabiesas previously described (Goyer et al., 1998). In summary, culturesof S. scabies in oat bran broth were incubated on a rotary shaker at30 �C for 7–8 d. Supernatant was extracted twice with an equalvolume of ethyl acetate. The solvent phase containing TXT wasconcentrated by evaporation and purified by thin layer chromatog-raphy on glass plates precoated with 0.25 mm Silica Gel 60. Yellowcompounds with an RF of 0.27 were eluted from silica usingchloroform-methanol (7:3, v/v). Thaxtomin A was quantified byHPLC using a Varian LC5500 liquid chromatograph equipped withWater’s C18 column (10 lm particle size, 3.93300 mm). Controlswith methanol were systematically performed for each experimentand failed to induce significant responses.

Cell viability assay

Cell death was quantified using the fluorescein diacetate (FDA)spectrofluorimetric method as previously described by Reboutieret al. (2007). A. thaliana cell suspensions were collected andwashed by filtration in a suspension buffer containing 175 mMmannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, and 10 mM HEPES(H10 medium) adjusted to pH 5.8 (with KOH). One millilitre of cellsuspension (0.1 g FW) was incubated in the presence of TXT and/orwith the appropriate pharmacological effectors. After incubation,500 ll of the suspension was diluted in 1.5 ml of H10 medium in

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a quartz cuvette (final cell density was 1.105 cells ml�1). Cells weregently stirred and FDA was added at a final concentration of12 lM. Fluorescence production was monitored over a 120 s periodtime using a Hitachi F-2000 spectrofluorimeter. The slope offluorescence production, corresponding to the esterase activity, wascalculated for each treatment, and directly compared with non-treated cells. Linearity between the percentage of dead cells andFDA detected esterase activity was verified by melting differentamounts of control living cells and heated dead cells. A 100%esterase activity corresponds to about 90% living cells (10% of deadcells are generally present in control conditions as revealed byEvans Blue staining of control cells, see below). A 0% esteraseactivity corresponds to 100% of dead cells. Cell death wascalculated using the formula: % of increase in cell death¼(slope oftreated cells/slope of non-treated cells)3100. Experiments wererepeated three times for each condition.Cell viability was also quantified using Evans Blue staining. One

millilitre of cell suspension was incubated in their culture mediumin the presence of TXT and/or with the appropriate pharmacologicaleffectors. Then, cells (50 ll) were incubated for 5 min in 1 mlphosphate buffer pH 7 supplemented with Evans Blue to a finalconcentration of 0.005%. Cells that accumulate Evans Blue wereconsidered dead. At least 1000 cells were counted for eachindependent treatment.

Aequorin luminescence measurements

Cytoplasmic Ca2+ variations were recorded with A. thaliana cellsuspensions expressing the apoaequorin gene. (Brault et al., 2004;Bouizgarne et al., 2006). For calcium measurement, aequorin wasreconstituted by the overnight incubation of cell suspensions inGamborg medium supplemented by 30 g l�1 sucrose and 2.5 lMnative coelenterazine. Cell culture aliquots (500 ll in Gamborgmedium) were transferred carefully into a luminometer glass tube,and the luminescence counts were recorded continuously at 0.2 sintervals with a FB12-Berthold luminometer. Treatments wereperformed by pipette injection of 20 ll containing the effectors. Atthe end of each experiment, the residual aequorin was discharged bythe addition of 500 ll of a 1 M CaCl2 solution dissolved in 100%methanol. The resulting luminescence was used to estimate thetotal amount of aequorin in each experiment. Calibration ofcalcium measurement was performed by using the equation:pCa¼0.332588(–logk)+5.5593, where k is a rate constant equal toluminescence counts per second divided by the total remainingcounts (Knight et al., 1996).

Electrophysiology

Cells were impaled in the culture medium with borosilicatecapillary glass (Clark GC 150F) micropipettes (resistance: 50 MXwhen filled with 600 mM KCl). The main ion concentrations in themedium after 4 d were 9 mM K+, and 11 mM NO�

3 (Bouizgarneet al., 2006). Individual cells were voltage-clamped using anAxoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA)as previously described by Bouizgarne et al. (2006); and Reboutieret al. (2002, 2005, 2007).

Extracellular pH measurements

Extracellular pH was measured directly in the medium (Braultet al., 2004; Bouizgarne et al., 2006). The experiments were runsimultaneously in 6310 ml flasks (control and tests) each contain-ing 1 g FW for 5 ml of suspension medium under continuous orbitalshaking (60 rpm). For each condition, the pH ranged between 5.6and 5.8. Simultaneous changes in pH were measured by using ELIT808 ionometer with pH-sensitive combined electrodes functioningin parallel.

H2O2 measurement

H2O2 release in the medium culture was quantified by measuringthe chemiluminescence of luminol reacting with H2O2 (Bouizgarneet al., 2006). A. thaliana cells were prepared as described for FDAmeasurement (0.1 g FW ml�1) in H10 medium. Briefly, 1.5 ml ofthe cell suspension was inoculated with 1 or 10 mM TXT. Atdifferent times, 200 ll of the medium were added to 600 llphosphate buffer (50 mM, pH 7.9) prior to the addition of 100 llluminol 1.1 mM (and 100 ll K3[Fe(CN)6] 14 mM). Chemilumi-nescence was monitored every 5 min with a FB12-Bertholdluminometer (signal integrating time of 0.2 s).

Mitochondrial membrane potential measurement

A. thaliana cells were prepared as described for FDA measurement(0.1 g FW ml�1) in H10 medium. Before treatment, the cells werefirst stained with the mitochondrial membrane potential probe JC-1by incubating 2 ml of cell suspensions for 15 min (24 �C in thedark) with 2 lg ml�1 JC-1 (3 lM). JC-1 was provided byMolecular Probes Inc. (Eugene, OR, USA) and was dissolved andstored according to the manufacturer’s instructions. Cells were thentreated with 1 lM valinomycin (Sigma, Steinheim, Germany),a drug known to affect mitochondrial membrane potential or with10 lM TXT. Cells were subjected to analysis using a HitachiF-2000 spectrofluorimeter. The excitation wavelength used was500 nm. Fluorescence signals were collected using a band pass filtercentred at 530 nm and 590 nm.

RT-PCR analysis of gene expression

Four-day-old cells were treated with TXT, harvested, and frozen inliquid nitrogen. Total RNAs were prepared with the Genelute�Mammalian Total RNA Kit (Sigma). RNAs were treated by theDeoxyribonuclease I kit (Sigma). Total RNAs were quantified witha spectrophotometer and their integrity was checked on denaturingagarose gel. Total RNAs (2 lg) were converted into first-strandcDNA by using the Superscript� II Rnase H– Reverse TranscriptaseKit (Invitrogen, Carlsbad, CA, USA) with oligo(dT)22. One ll ofcDNA samples was amplified in 20 ll PCR mixture. Specificprimers were used for PR1, PDF1.2a, and PAL1 (Bouizgarne et al.,2006). VDAC and AOX1a primers were designed (VDAC forward:CCT GCC CCT GGA CTG AAA GTT, reverse: CAG TCG ACGGGC TCA CAA TCT; AOX1a forward: TTT TCC GAT TTG AAACAA TGA TGA, reverse: CCC AAT AGC TCG CGA TTC CTTTAT). Control PCR was performed using the housekeeping geneEF1A4 (Bouizgarne et al., 2006). Thermal cycling conditionscomprised an initial denaturation step at 94 �C fo 2 min, followedby 34 cycles or by 26 cycles for EF1A4, of 94 �C for 30 s, 55 �Cfor 30 s, 72 �C for 1 min 30 s, and ending with 72 �C for 10 min.PCR products were loaded on gel electrophoresis and visualized byethidium bromide fluorescence. Representative results from threeindependent experiments are shown.

Statistics

Significant differences between treatments were determined by theMann and Whitney test and P values <0.05 were consideredsignificant.

Results

TXT-induced cell death is dependent on Ca2+ influx

The rate of cell death was quantified using Evans Bluecoloration (Fig. 1A, C) or the FDA method (Fig. 1B, C)

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24 h post-treatment. It was first confirmed that Streptomy-ces scabies induces cell death of A. thaliana suspensioncells (Fig. 1A). TXT itself is also able to induce cell deathas previously reported (Duval et al., 2005). This cell deathwas accompanied by cell plasmolysis (Fig. 1A), a hallmarkof programmed cell death. This cell death appeared to bedose dependent (Fig. 1B). Thirty per cent of cells weredead upon addition of micromolar concentrations of TXTand more than half of the cells were dead at higherconcentrations (10–20 lM). The cell death plateau wasreached within 10 h for 10 lM of TXT (Fig. 1C). TheCa2+ influx induced by TXT was then confirmed in ourmodel by using cultured cells expressing apoaequorinaddressed in the cytosol. Thaxtomin A induced a rapidincrease in [Ca2+]cyt of about 200 nM over 30 s (Fig.2A). This short-lived increase in [Ca2+]cyt could beinhibited by La3+, Gd3+ or BAPTA (Fig. 2A, B). Thisincrease was probably due to an influx through PM Ca2+

channels as reported for root cells by Tegg et al. (2005).No further significant increases in [Ca2+]cyt wererecorded over 30 min with aequorin-expressing cellsupon the addition of TXT (data not shown). The effect ofTXT has been tested on cell death in the presence orabsence of PM Ca2+ channel inhibitors (La3+ or Gd3+) orthe Ca2+ chelator BAPTA. Although the pretreatment ofA. thaliana cells with Gd3+ or with BAPTA induceda slight increase in cell death, the TXT-induced celldeath significantly decreased after such pretreatments

(Fig. 2C), indicating that the influx of Ca2+ is anupstream event in the signalling pathway leading toTXT-induced cell death.

TXT induces a transient depolarization of plasmamembrane due to K+ and anion channel regulation

The effect of TXT on anion and K+ fluxes (some of theearliest signalling events detectable in plant–pathogeninteractions) was tested further by electrophysiology. Inour control conditions, the value of the resting membranepotential (Vm) of A. thaliana suspension cells was –4765mV (n¼26) which is close to those recorded in previousstudies (Reboutier et al., 2002, 2005, 2007; Bouizgarneet al., 2006). TXT induced a rapid depolarization of the cellPM reaching its maximal value within 1 min. Thismembrane potential (Vm) variation was concentration-dependent and transient, although not fully reversed(Fig. 3A, B). Previous electrophysiological studies andpharmacological analysis identified two main ion channelcurrents in the PM of A. thaliana cells: a K+ outwardrectifying current (KORC) and an anion current, whichdisplay the main hallmarks of slow anion channels(Reboutier et al., 2002). TXT induced a concentration-dependent decrease of KORC reaching about 60% at10 lM (Fig. 3C, D, E) from a mean control value of0.560.1 nA (n¼23). This KORC decrease was fullyreversed upon repolarization of the cells (Fig. 3E).Thaxtomin A also induced a concentration-dependent

Fig. 1. (A–C) Effect of thaxtomin A on A. thaliana suspension cell viability. (A) Light micrographs of A. thaliana cells co-cultured withStreptomyces scabies (strain EF35) or treated with 10 lM thaxtomin A (TXT) for 24 h and stained with Evans Blue before observation. (B) Effect ofincreasing concentrations of TXT on FDA estimated cell viability expressed in cell death increase after 24 h of treatment. ‘C’ corresponds to thecontrol cells without treatment and ‘A’ to cells killed by anoxia. * Significantly different from the control, P <0.05. (C) Time-course of the cell deathincrease estimated with FDA (filled circles) or vital staining by Evans Blue (open squares) upon 24 h treatment with 10 lM TXT. Data correspond tothe means of at least five independent experiments.

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increase in anion current reaching 150% (Fig. 3F, G, H)from a mean control value of –0.3860.05 nA (n¼22).Upon repolarization of the cells the anion current leveldecreased but remained slightly higher than the controllevel recorded before TXT addition (Fig. 3H). The transientregulation of these currents certainly explains the observedtransient depolarization.Pretreatment of the cells with the Ca2+ channel inhibitor

La3+ and Gd3+ or with the Ca2+ chelator BAPTAabolished the TXT-induced depolarization (Fig. 4A) andthe modulation of KORC and anion currents (Fig. 4B, C)indicating that the influx of Ca2+ is also an upstream eventwhen compared to TXT-induced anion and K+ channelsregulation leading to depolarization.Anion current increases were reported to be a necessary

event to achieve cell death by the elicitor cryptogein(Gauthier et al., 2007) and oxalic acid (Errakhi et al.,2008). As TXT induced a transient increase in anioncurrents, the effect of two structurally unrelated anionchannel inhibitors, 9-AC and glibenclamide (gli), effectiveon A. thaliana cell anion currents (Fig. 5A), was tested onTXT-induced cell death. The anion channel inhibitorsalone induced a slight cell death (Fig. 5B) as previouslyreported (Reboutier et al., 2005). However, the TXT-induced cell death recorded in the presence of TXTsupplemented with 9-AC and gli did not decrease (Fig.5B) suggesting that the anion current increase is not anupstream event in the signalling pathway leading to TXT-induced cell death.

TXT induces a biphasic modulation of external pH

The effect of TXT on the external medium pH ofA. thaliana cell cultures was investigated. Indeed, changes inthe pH value might reflect TXT-induced H+ flux modula-tion through the PM. TXT at 10 lM induced a slightacidification reaching a maximal value of –0.03860.006upH (n¼17) during the first 30 min (Fig. 6A), aspreviously reported with roots of different species (Tegget al., 2005). This acidification was followed by a largealkalization (Fig. 6A) reaching 0.4860.06 upH (n¼6)after 4 h (Fig. 6B). The alkalization induced by TXT wasdecreased in the presence of the calcium channelinhibitors La3+ and Gd3+ as well as in the presence ofBAPTA (Fig. 6B), indicating that Ca2+ influx is involvedin pH modification. The effect of the phytohormoneindole acetic acid (IAA) on external medium pH wastested further because TXT, which is structurally relatedto IAA, has been suggested to compete with IAA on thePM receptor (Tegg et al., 2005). As IAA was describedto stimulate H+-ATPase activity directly in some cases(Kim et al., 2001), it was tested if it could counteract the

Fig. 2. (A–C) Effects Ca2+ influx inhibitors on cytosolic Ca2+

concentration and cell death in A. thaliana cell suspensions. (A)Changes in [Ca2+]cyt were measured by using cell suspensions derivedfrom Arabidopsis seedlings transformed by the apoaequorin gene upon10 lM TXT addition, alone or after pretreatment with the Ca2+ channelinhibitors La3+ or Gd3+ (0.5 mM) or the calcium chelator BAPTA(1 mM). (B) Mean values of D[Ca2+]cyt. Data correspond to the meansof five independent experiments. Addition of 0.06% methanol (equiva-lent to methanol added with TXT) failed to induce an increase in[Ca2+]cyt (not shown). (C) Effect of a pretreatment with La3+ or Gd3+

(0.5 mM each) or BAPTA (1 mM) on 10 lM TXT-induced cell deathdetected by the vital staining Evans Blue method after 6 h. The datacorrespond to the means of three replicates during one experiment and

the error bars correspond to standard errors. Data are representative of atleast three independent experiments. * Significantly different from theTXT treatment, P <0.05.



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TXT-induced alkalization. Treatment with IAA did notsignificantly modify the external medium pH (data notshown). The addition of IAA with TXT did not modifythe TXT-induced biphasic modulation of external pH (Fig.6B), the alkalization reaching 0.4660.07 upH (n¼6) after4 h, suggesting that there is no competition between thetwo molecules at this level.The alkalization of the external medium (Fig. 6B) could

be linked to decrease of PM-H+-ATPase activity and/or tothe activation of PM-NADPH-oxidases leading to thegeneration of H2O2 (Pugin et al., 1997). As the absence ofROS production in response to TXT had previously beenreported (Duval et al., 2005), the effect of TXT (up to

10 lM) on luminol-mediated chemiluminescence wastested to check if H2O2 was released into the culturemedium. In this previous study the authors did not recordany alkalization of the cell medium as observed in ourconditions. Hypoosmotic shock, used as a positive con-trol, largely increased H2O2 production. TXT failed toinduce H2O2 production in the culture medium (Fig. 6C).Seeing that mitochondria are known to be a putativepathogen-derived phytotoxin or elicitor target (Krause andDurner, 2004; Bouizgarne et al., 2006; Errakhi et al.,2008), the effect of TXT on mitochondrial potential waschecked to assess whether a putative decrease in ATPlevel leading to a decrease of H+-ATPase activity could

Fig. 3. (A–H) Thaxtomin A-induced depolarization and ion current regulation of A. thaliana cells. (A) Typical transient depolarization observed inresponse to increasing concentration of TXT. (B) Mean values of the amplitude of the plasma membrane potential changes recorded at maximaldepolarization (grey bar) and after repolarization (black bar) upon TXT addition. (C) KORC measured under control conditions and after adding 10lM TXT in the culture medium. Voltage pulses were +80 mV. Holding potential was Vm. (D) Current–voltage relationships. The KORC amplitudesat steady-state were measured for membrane potentials ranging from –200 to +80 mV before (filled circles) and after TXT addition at maximaldepolarization (open circles) and after repolarization (filled inverted triangles). (E) Mean steady-state values of KORC recorded for a +80 mV voltagepulses at maximal depolarization (grey bar) and after repolarization (black bar). (F) Anion currents measured under control conditions and afteradding 10 lM TXT. Voltage pulses were –200 mV. Holding potential was Vm. (G) Current–voltage relationships. The current amplitudes (at 1.8 s)were measured for membrane potentials ranging from –200 to +80 mV before (filled circles) and after TXT addition at maximal depolarization (opencircles) and after repolarization (filled inverted triangles). (H) Mean steady-state values of anion current recorded at –200 mV at maximaldepolarization (grey bar) and after repolarization (black bar). The variations in the currents are given as a percentage with respect to the control level.Data correspond to mean values 6SD of at least five independent experiments. * Significantly different from the control, P<0.05.

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explain the alkalization of the medium. Experiments havebeen carried out by using the JC-1 fluorochrome, which isknown to be incorporated and accumulated specifically inplant mitochondria (Simeonova et al., 2004). Valinomycin(1 lM), used as a positive control, induced a decrease ofthis potential within 15 min, when 10 lM TXT did notmodify this potential after 3 h (Fig. 6D).

TXT-induced expression of defence response genes

Early defence responses are often concomitant with anincrease in the expression of defence-related genes. Toinvestigate the effects of Ca2+ influx on TXT-inducedresponses at the molecular level, the accumulation ofseveral gene transcripts known to be accumulated duringHR and corresponding to different classes of so-calleddefence genes was analysed by RT-PCR: VDAC andAOX1a, also known as hypersensitive-related (HSR) genes(Lacomme and Roby, 1999); PAL1, which encodes phenylammonia-lyase, a key enzyme of the phenylpropanoidbiosynthetic pathway; PR1 (pathogenesis-related) andPDF1.2 involved, respectively, in the classical SA and JAor ET-defence signalling pathways. Increases in mRNAlevels after treatment of the cells with 10 lM TXT for 4 hwere only detected for PAL1 (Fig. 7A). This treatment didnot lead to the accumulation of the transcripts of the othergenes. Treatment of the cells with 0.5 mM La3+ and 10 lMTXT decreased the induction of PAL1 when compared withTXT treatment (Fig. 7B). This result confirms the hypoth-esis that Ca2+ could be implicated in the signalling pathwayinvolved in the response to TXT.

Discussion

Thaxtomin A, a determinant factor of Streptomyces spppathogenicity (King et al., 1991; Loria et al., 1997; Healyet al., 2000) was recently shown to induce an atypicalPCD in A. thaliana (Duval et al., 2005). This phytotoxin

Fig. 4. (A, B) Inhibition of thaxtomin A-induced depolarization and ion current regulation by Ca2+ channel inhibitors or Ca2+ chelator. (A) Runningmembrane potential of a cell upon addition of TXT after a pretreatment with Gd3+ or La3+ (0.5 mM each) or BAPTA (1 mM). Dashed line referred toTXT effect. (B) Absence of KORC variation 1 min after addition of 10 lM TXT after La3+ pretreatment. (C) Absence of anion current variation1 min after the addition of 10 lM TXT in the same conditions. Protocols were as illustrated in the figure. Data are representative of four independentexperiments.

Fig. 5. (A, B) Effect of anion channel inhibitors on thaxtomin A-induced cell death. (A) Amplitude of anion channel inhibitor-induceddecrease in anion currents. Current amplitudes were measured after 1.8s of a –200 mV voltage pulse. The data correspond to means of fiveexperiments. * Significantly different from the control, P <0.05. (B)Effect of a pretreatment with anion channel inhibitors (9-AC or gli,40 lM and 10 lM, respectively) on 10 lM TXT-induced cell deathdetected by the Evans Blue vital staining method after 6 h. The datacorrespond to the means of three replicates during one experiment anderror bars correspond to standard errors. Data are representative of threeindependent experiments.



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also induces a [Ca2+]cyt increase depending on a calciuminflux from the external medium which could be inhibitedby La3+, a PM calcium channel inhibitor (Tegg et al.,2005). The aim of this work was to analyse the putativerole of this calcium influx in the TXT-induced cell deathobserved in A. thaliana-cultured cells (Duval et al., 2005)and in classically HR-associated responses. It was firstconfirmed by using A. thaliana cells expressing aequorinthat TXT induces a transient increase in cytosolic Ca2+

concentration. Then it was demonstrated that the TXT-induced increase in cytosolic Ca2+ and cell death weresignificantly reduced when the cells were treated withLa3+, Gd3+ or BAPTA, which are known to inhibit Ca2+

influx. The increase in [Ca2+]cyt is known to be a majorevent in numerous plant cell signal transduction pathways,but the appropriate physiological response to a givensignal is encoded by the amplitude, duration, frequency,and location of this calcium increase (McAinsh andHetherington, 1998; Lecourieux et al., 2005). The Ca2+

influx occurs a few seconds after TXT addition (Tegget al., 2005; this study) and peaks 1 or 2 min earlier thanother reported [Ca2+]cyt peaks for a range of plant species

in response to various elicitors (Blume et al., 2000;Lecourieux et al., 2002). Duval et al. (2005) postulatedthat a rapid inhibition of cellulose synthesis can modifythe plant cell wall composition and organization that weresomehow perceived by the cell which, in turn, initiateda cell death programme. Tegg et al. (2005) furthersuggested that Ca2+ exchange with H+ in the apoplast ledto an acidification of the cell wall responsible for enzymeactivation and disruption of cellulose synthesis, position-ing the Ca2+ influx as an upstream event of the inhibitionof cellulose synthesis. As no further Ca2+ influx wasrecorded after the initial short-lived Ca2+ influx, it is likelythe most rapid event triggering the cascade of eventsleading to cell death. The regulation of K+ and anionchannels reported here were also initiated in less than1 min after TXT addition but after Ca2+ influx, sincepretreatment by La3+, Gd3+ and BAPTA avoided theTXT-induced ion channel regulation and subsequent PMdepolarization. The involvement of the KORC decrease inresponse to TXT, although probably involved in PMdepolarization, is not easy to link to cell death. Effec-tively, in most cases, an increase in K+ efflux, or in

Fig. 6. (A, B) Effect of thaxtomin A on external medium pH of A. thaliana cells. (A) Time-course of external pH variation of A. thaliana cellsuspensions treated with 10 lM TXT. (B) Changes in external pH after treatment with 10 lM TXT or on cell pretreated with Gd3+ or La3+ (0.5 mM),BAPTA (1 mM) or IAA (10 lM). The data correspond to the means of six replicates and error bars correspond to standard errors. (C) Time-course ofH2O2 accumulation in the external medium without treatment (control), after TXT treatment (1 lM and 10 lM) or after an hypo-osmotic shock.Results are the means of three independent experiments. (D) Mitochondrial membrane potential in cell suspensions in response to 1 lM valinomycin(used as positive control) or 10 lM TXT, monitored using JC-1 as a mitochondrial membrane potential probe. The fluorescence associated to JC-1was measured after 15 min and 3 h for control cells, cells treated with 1 lM valinomycin or with 10 lM TXT. * Significantly different from thecontrol, P <0.05.

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outward K+ currents, rather than a decrease were reportedin response to HR-inducing pathogens or elicitors (Atkinsonet al., 1996; Reboutier et al., 2007). The TXT-inducedincrease in anion channel current is in accordancewith previous studies suggesting that anion effluxes arepart of the early pathogen or elicitor induced responses(Jabs et al., 1997; Pugin et al., 1997). Several studiesreported a rapid elicitor-induced PM depolarization result-ing, at least in part, in the activation of anion channels(Pugin et al., 1997). Ca2+ influx was shown to bea prerequisite for the activation of PM anion channels inseveral systems (Jabs et al., 1997; Wendehenne et al.,2002). Moreover, a role for anion channels was alsoreported in cell death induction, since a rapid increase inanion currents seemed to be a prerequisite to achievecryptogein- and oxalic acid-induced cell death (Gauthieret al., 2007, Errakhi et al., 2008). Although the TXT-induced anion channel regulation was transient, and thuscould not be responsible for a massive anion efflux,a putative role for this current increase in a signallingpathway leading to TXT-induced cell death was checked.Such an increase in anion currents was reported toparticipate in the ABA triggered signalling pathwayleading to gene expression changes (Ghelis et al., 2000).Contrary to what has already been observed withcryptogein on tobacco cells (Wendehenne et al., 2002) oroxalic acid on A. thaliana cells (Errakhi et al., 2008), gliand 9-AC, two anion channel inhibitors previously shown

to be effective on A. thaliana cells (Brault et al., 2004;Reboutier et al., 2002, 2005), did not reduce the TXT-induced cell death after 6 h. Thus, the anion channelregulation, a downstream event of TXT-induced Ca2+

influx, did not seem to be involved in the pathway leadingto TXT-induced cell death.Concerning H+ fluxes, data obtained with TXT are

rather controversial. In our study, a biphasic regulation ofexternal pH was observed after TXT addition. A slightacidification was first observed, which is compatible withthe net H+ efflux recorded after TXT addition on variousstructures and plant types and suggested to be mediatedby the PM H+-ATPases (Tegg et al., 2005), and thena delayed and large alkalization of the medium. Theseresults are in contrast to the absence of alkalization whichhas been observed with tobacco or A. thaliana cells inprevious studies (Fry and Loria, 2002; Duval et al., 2005).Tegg et al. (2005) pointed out that the variability inexperimental conditions and in the material used foranalysing the external pH variations could explain thedifferences among bibliographic references. However, theobservation of an alkalization in response to TXT, led usto check for a putative production of ROS generation inresponse to TXT, since the absence of ROS productionwas concomitant with the absence of medium alkalizationin previous studies (Duval et al., 2005). Effectively,a [Ca2+]cyt increase was reported to be linked to H2O2

production through the activation of NADPH-oxidase(Pugin et al., 1997; Keller et al., 1998; Hu et al., 2004)and to be involved in ROS-mediated cell death (Levineet al., 1996). These data confirmed that TXT did notinduce H2O2 production, but the possibility cannot beexcluded that other forms of ROS (superoxide anion orhydroxyl radical) were produced inside and/or outside thecell after TXT addition and participated in the cell deathprocess as previously discussed by Duval et al. (2005).Since the alkalization of the external medium could not beexplained by a generation of H2O2, it was ascertained ifa decrease of PM H+-ATPase activity could be responsi-ble for this alkalization. There was no decrease in the cellmitochondrial potential in response to TXT even after 3 h,suggesting that a depletion of ATP could neither beresponsible for the decrease of H+-ATPase activity, norfor the subsequent alkalization observed. These data are inaccordance with the absence of accumulation of HSR2and HSR3 in response to TXT. Effectively HSR2 encodesa mitochondrial voltage-dependent gated anion channel(VDAC) protein (Lacomme and Roby, 1999), known tobe involved in the cytosolic release of cytochrome cduring certain cell death but not in response to TXT(Duval et al., 2005), and HSR3 encodes an alternativeoxidase (AOX1a) which seemed to be a marker ofmitochondrial stress since it has been reported that itslevel increases during elicitor-induced cell death (Krauseand Durner, 2004; Vidal et al., 2007). All these

Fig. 7. (A, B) Effect of thaxtomin A and involvement of calcium influxon defence-related gene expression. (A) Effect of 4 h treatment with10 lM TXT on the expression of defence-related genes (PDF1.2, PR1,PAL1) and AtHSR2/VDAC and AtHSR3/AOX1a, markers of thehypersensitive response; C, control; TXT, thaxtomin A treatment(10 lM); gDNA, primer combinations tested on genomic DNA. (B)Effect of La3+ on PAL1 expression in response to 10 lM TXT. Cellswere preincubated with La3+ 0.5 mM for 15 min prior to the addition ofTXT.



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observations indicate that mitochondria are not a primarytarget of TXT, even if recent data pointed out thatmitochondrial permeability transition could precede plantPCD (Yu et al., 2002). To assess for a more direct effectof TXT on PM H+-ATPases, experiments were conductedin the presence of IAA. IAA is known to stimulate H+-ATPase activity (Kim et al., 2001) and is suggested tocompete with TXT for the PM receptor since these twomolecules are structurally related (Tegg et al., 2005). Theaddition of 10 lM IAA in the presence of 10 lM TXT didnot counteract the acidification nor the alkalization.Further studies are therefore required to understand theputative TXT modulation of PM H+-ATPases. Theregulation of H+ fluxes described in response to pathogenis also generally rather controversial, suggesting thateffects may be plant or tissue specific. In most cases, theelicitor-induced decrease in PM H+-ATPase activity orextracellular alkalization was reported (Atkinson et al.,1996; Bouizgarne et al., 2006; Osses and Godoy, 2006).However, different studies highlight an acidification ofthe extracellular medium (Malerba et al., 2003) or anincrease in PM H+-ATPase activity in response to elicitoror to phytotoxin (Vera-Estrella et al., 1994; de Boer,2002). Attempts to use PM H+-ATPase inhibitors such aserythrosin B or vanadate, which is known to be a potentinhibitor of harpin-induced HR cell death (He et al.,1993), in order to search for a putative role of the TXT-induced acidification on cell death were unsuccessful,probably because of their toxic effects even in the shortterm (not shown). As previously done for external pHmeasurements, competition experiments were conductedin the presence of IAA and TXT to see if IAA couldcounteract the TXT effect at the cell death level. Thepretreatment of the cell with IAA did not reduce TXT-induced cell death (not shown). However, the alkaliza-tion which occurred after the addition of TXT wasinhibited after a pretreatment with La3+, Gd3+, andBAPTA leading to the conclusion that the mechanism(s)responsible for alkalization is thus probably dependenton the Ca2+ influx. However, its precise role in thepathway leading to cell death remains unclear and needsmore investigations.As previously reported (Duval et al., 2005), the

expression of defence-related genes PR1 and PDF1.2 wasnot induced by TXT in Arabidopsis cells. TXT treatmentdid not result in an accumulation of ethylene (data notshown) indicating that the resulting PCD did not involvethe classical ET-defence signalling pathways. However,TXT induced the accumulation of PAL1, which is knownto participate in the induction of defence by modulatingthe synthesis of cell wall component and defence related-compounds such as phytoalexins (such as the coumarinscopoletin, Kai et al.,, 2006). Our data suggest theinvolvement of the TXT-induced Ca2+ influx in theinduction of the defence gene PAL1.

In conclusion, TXT-induced calcium influx is an earlyevent that is required for the activation of downstreamdefence responses and cell death. In addition, the lack ofdependency of this cell death to earlier Ca2+-dependentTXT-induced responses such as anion channel modulationfurther suggest that several pathways could be triggered inresponse to this early Ca2+ influx, which may be crucial inplant–Streptomyces interactions.

Acknowledgements

We are grateful to G Vidal (Centre de recherche Paul Pascal, CNRSUPR 8641, 33600 Pessac, France) and to Professor JM Farrant(University of Cape Town, Department of Molecular and CellularBiology) for critical reading of the manuscript. This work wassupported by funds from the MESR (Ministere delegue al#Enseignement Superieur et a la Recherche) to EA 3514, fromPRAD 04/02, and AUF 63-01PS615.

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An early Ca2+ influx is a prerequisite to thaxtomin A-induced cell death in Arabidopsis thaliana cells

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