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JXB Advance Access published online on November 17, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern267
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© The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

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

R. Errakhi1,2 *, A. Dauphin1 *, P. Meimoun1, A. Lehner1 {dagger}, D. Reboutier1, P. Vatsa3, J. Briand1, K. Madiona1, J. P. Rona1, M. Barakate2, D. Wendehenne3, C. Beaulieu4 and F. Bouteau1,{ddagger}

1LEM (EA 3514), Université Paris Diderot-Paris7, 2, place Jussieu, F-75251 Paris cedex 05, France
2Laboratoire de Biologie et Biotechnologie des Microorganismes, Faculté des Sciences-Semlalia, BP 2390, 40001 Marrakech, Maroc
3UMR INRA 1088, CNRS 5184, Université de Bourgogne, Plante-Microbe-Environnement, Dijon, France
4GRBA, Département de Biologie, Université de Sherbrooke, Québec, Canada JIK 2RI

{ddagger} To whom correspondence should be addressed: E-mail: francois.bouteau{at}univ-paris-diderot.fr

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


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pathogenicity of various Streptomyces scabies isolates involved in potato scab disease was correlated 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 thaxtomin A. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements 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 prerequisite 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
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Plants are constantly exposed to pathogens and have evolved a diversity of responses in order to withstand these attacks. Recognition and perception of a pathogen or their derived-elicitors by plant cells lead to modulation of the defence-signalling pathways. The inducible defence responses include the production of reactive oxygen species (ROS), the modulation of ion fluxes, an increase of cytosolic [Ca2+], the activation of mitogen-activated protein kinases (MAPKs) and the expression of defence-related genes that are involved in the production of various metabolites as well as pathogenesis-related proteins showing antimicrobial properties (Nürnberger and Scheel, 2001; Garcia-Brugger et al., 2006). In addition, plants often induce a hypersensitive response (HR) characterized by a localized cell death often associated with disease resistance (Lam, 2004). The HR is a form of programmed cell death (PCD) that is thought to kill the pathogens and/or to limit their spread (Heath, 2000). HR is a genetically controlled process that displays apoptosis-like features such as cell shrinkage, chromatin condensation, and DNA cleavage into inter-nucleosomal fragments (Lam, 2004). It requires gene expression and metabolic activities (Lam, 2004).

As in animal cells, a perturbation of Ca2+ homeostasis in plant seems to be a prerequisite to PCD (Davis and Distelhorst, 2006; Lecourieux et al., 2006). Changes in [Ca2+]cyt are rapid and have been reported in response to various microbial phytotoxins and elicitors (Reddy, 2001; White and Broadley, 2003). They have also been associated with 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 of a Ca2+ channel (OsTPC1) in elicitor-induced defence responses and in hypersensitive cell death in rice. Recent data have also illustrated the role of cyclic nucleotide-gated ion 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, numerous studies led to the conclusion that the activation of defence responses depends on Ca2+ influxes from the apoplast into the cytosol of plant cells (see Lecourieux et al., 2006, for a review). Notably, elicitor-induced uptake of Ca2+ from the extracellular medium was shown to be required for the controlled generation of H2O2 (Pugin et al., 1997; Keller et al., 1998; Hu et al., 2004), the activation of MAPK pathways (Link et al., 2002; Garcia-Brugger et al., 2006), the activation of defence-related genes (Lecourieux et al., 2002), and production of phytoalexin (see Lecourieux et al., 2006, for a review). For instance, Lecourieux et al. (2002) showed that suppression of the sustained [Ca2+]cyt increase in cryptogein-treated tobacco cells suppressed the accumulation of transcripts corresponding to phenylalanine ammonia lyase (PAL), a key enzyme of the phenylpropanoid 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 of pathogen, TXT has been shown to induce common scab-like disease symptoms and localized cell death, when it is applied to developing tubers or roots (Lawrence et al., 1990). A mutation within TXT biosynthesis genes rendered a S. scabies strain non-pathogenic (Healy et al., 2000). Their pathogenicity is thus directly correlated with their ability to produce TXT. Thaxtomin A has been shown to inhibit cellulose synthesis, suggesting that the cell wall is one of the main targets of TXT (Fry and Loria, 2002; Scheible et al., 2003). Recent studies have shown that TXT stimulates H+ efflux across the plasma membrane (PM) and a short-lived Ca2+ influx in the roots of different species (Tegg et al., 2005). This Ca2+ influx is inhibited by La3+, a PM Ca2+ channel inhibitor (Tegg et al., 2005). Thaxtomin A also induces a cell death which depends on active gene transcription and de novo protein synthesis and that displays PCD features (Duval et al., 2005). However, TXT-induced cell death appeared atypical since it does not involve typical defence responses associated with the HR: ROS production, alkalization, and activation 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 events related to plant responses to pathogens and, in particular, to determine whether a short-lived Ca2+ influx could be involved in the atypical HR cell death induced by TXT in Arabidopsis thaliana. Electrophysiological molecular approaches and a Ca2+ assay were used on A. thaliana suspension-cultured cells which is a convenient material for studying early physiological events induced by pathogens (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 that TXT-induced Ca2+ influx is an early signalling cascade event necessary to achieve defence responses including cell death.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture conditions
Arabidopsis thaliana L. (ecotype Columbia) suspension cells were grown in Gamborg medium (pH 5.8). They were maintained at 24±2 °C, under continuous white light (40 µmol photons m–2 s–1) and continuous shaking (gyratory shaker) at 120 rpm. Suspensions were subcultured weekly using 1:10 dilution. All experiments were performed at 22±2 °C using log-phase cells (4 d after subculture).

Thaxtomin A production
Thaxtomin A was purified from oat bran broth cultures of S. scabies as previously described (Goyer et al., 1998). In summary, cultures of S. scabies in oat bran broth were incubated on a rotary shaker at 30 °C for 7–8 d. Supernatant was extracted twice with an equal volume of ethyl acetate. The solvent phase containing TXT was concentrated by evaporation and purified by thin layer chromatography on glass plates precoated with 0.25 mm Silica Gel 60. Yellow compounds with an RF of 0.27 were eluted from silica using chloroform-methanol (7:3, v/v). Thaxtomin A was quantified by HPLC using a Varian LC5500 liquid chromatograph equipped with Water's C18 column (10 µm particle size, 3.9x300 mm). Controls with methanol were systematically performed for each experiment and failed to induce significant responses.

Cell viability assay
Cell death was quantified using the fluorescein diacetate (FDA) spectrofluorimetric method as previously described by Reboutier et al. (2007). A. thaliana cell suspensions were collected and washed by filtration in a suspension buffer containing 175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, and 10 mM HEPES (H10 medium) adjusted to pH 5.8 (with KOH). One millilitre of cell suspension (0.1 g FW) was incubated in the presence of TXT and/or with the appropriate pharmacological effectors. After incubation, 500 µl of the suspension was diluted in 1.5 ml of H10 medium in a quartz cuvette (final cell density was 1.105 cells ml–1). Cells were gently stirred and FDA was added at a final concentration of 12 µM. Fluorescence production was monitored over a 120 s period time using a Hitachi F-2000 spectrofluorimeter. The slope of fluorescence production, corresponding to the esterase activity, was calculated for each treatment, and directly compared with non-treated cells. Linearity between the percentage of dead cells and FDA detected esterase activity was verified by melting different amounts of control living cells and heated dead cells. A 100% esterase activity corresponds to about 90% living cells (10% of dead cells are generally present in control conditions as revealed by Evans Blue staining of control cells, see below). A 0% esterase activity corresponds to 100% of dead cells. Cell death was calculated using the formula: % of increase in cell death=(slope of treated cells/slope of non-treated cells)x100. Experiments were repeated three times for each condition.

Cell viability was also quantified using Evans Blue staining. One millilitre of cell suspension was incubated in their culture medium in the presence of TXT and/or with the appropriate pharmacological effectors. Then, cells (50 µl) were incubated for 5 min in 1 ml phosphate buffer pH 7 supplemented with Evans Blue to a final concentration of 0.005%. Cells that accumulate Evans Blue were considered dead. At least 1000 cells were counted for each independent treatment.

Aequorin luminescence measurements
Cytoplasmic Ca2+ variations were recorded with A. thaliana cell suspensions expressing the apoaequorin gene. (Brault et al., 2004; Bouizgarne et al., 2006). For calcium measurement, aequorin was reconstituted by the overnight incubation of cell suspensions in Gamborg medium supplemented by 30 g l–1 sucrose and 2.5 µM native coelenterazine. Cell culture aliquots (500 µl in Gamborg medium) were transferred carefully into a luminometer glass tube, and the luminescence counts were recorded continuously at 0.2 s intervals with a FB12-Berthold luminometer. Treatments were performed by pipette injection of 20 µl containing the effectors. At the end of each experiment, the residual aequorin was discharged by the addition of 500 µl of a 1 M CaCl2 solution dissolved in 100% methanol. The resulting luminescence was used to estimate the total amount of aequorin in each experiment. Calibration of calcium measurement was performed by using the equation: pCa=0.332588(–logk)+5.5593, where k is a rate constant equal to luminescence counts per second divided by the total remaining counts (Knight et al., 1996).

Electrophysiology
Cells were impaled in the culture medium with borosilicate capillary glass (Clark GC 150F) micropipettes (resistance: 50 M{Omega} when filled with 600 mM KCl). The main ion concentrations in the medium after 4 d were 9 mM K+, and 11 mM Formula (Bouizgarne et al., 2006). Individual cells were voltage-clamped using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA) as previously described by Bouizgarne et al. (2006); and Reboutier et al. (2002, 2005, 2007).

Extracellular pH measurements
Extracellular pH was measured directly in the medium (Brault et al., 2004; Bouizgarne et al., 2006). The experiments were run simultaneously in 6x10 ml flasks (control and tests) each containing 1 g FW for 5 ml of suspension medium under continuous orbital shaking (60 rpm). For each condition, the pH ranged between 5.6 and 5.8. Simultaneous changes in pH were measured by using ELIT 808 ionometer with pH-sensitive combined electrodes functioning in parallel.

H2O2 measurement
H2O2 release in the medium culture was quantified by measuring the chemiluminescence of luminol reacting with H2O2 (Bouizgarne et al., 2006). A. thaliana cells were prepared as described for FDA measurement (0.1 g FW ml–1) in H10 medium. Briefly, 1.5 ml of the cell suspension was inoculated with 1 or 10 mM TXT. At different times, 200 µl of the medium were added to 600 µl phosphate buffer (50 mM, pH 7.9) prior to the addition of 100 µl luminol 1.1 mM (and 100 µl K3[Fe(CN)6] 14 mM). Chemiluminescence was monitored every 5 min with a FB12-Berthold luminometer (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 were first stained with the mitochondrial membrane potential probe JC-1 by incubating 2 ml of cell suspensions for 15 min (24 °C in the dark) with 2 µg ml–1 JC-1 (3 µM). JC-1 was provided by Molecular Probes Inc. (Eugene, OR, USA) and was dissolved and stored according to the manufacturer's instructions. Cells were then treated with 1 µM valinomycin (Sigma, Steinheim, Germany), a drug known to affect mitochondrial membrane potential or with 10 µM TXT. Cells were subjected to analysis using a Hitachi F-2000 spectrofluorimeter. The excitation wavelength used was 500 nm. Fluorescence signals were collected using a band pass filter centred at 530 nm and 590 nm.

RT-PCR analysis of gene expression
Four-day-old cells were treated with TXT, harvested, and frozen in liquid nitrogen. Total RNAs were prepared with the GeneluteTM Mammalian Total RNA Kit (Sigma). RNAs were treated by the Deoxyribonuclease I kit (Sigma). Total RNAs were quantified with a spectrophotometer and their integrity was checked on denaturing agarose gel. Total RNAs (2 µg) were converted into first-strand cDNA by using the SuperscriptTM II Rnase H– Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) with oligo(dT)22. One µl of cDNA samples was amplified in 20 µl PCR mixture. Specific primers 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 ACG GGC TCA CAA TCT; AOX1a forward: TTT TCC GAT TTG AAA CAA TGA TGA, reverse: CCC AAT AGC TCG CGA TTC CTT TAT). Control PCR was performed using the housekeeping gene EF1A4 (Bouizgarne et al., 2006). Thermal cycling conditions comprised an initial denaturation step at 94 °C fo 2 min, followed by 34 cycles or by 26 cycles for EF1A4, of 94 °C for 30 s, 55 °C for 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 by ethidium bromide fluorescence. Representative results from three independent experiments are shown.

Statistics
Significant differences between treatments were determined by the Mann and Whitney test and P values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
TXT-induced cell death is dependent on Ca2+ influx
The rate of cell death was quantified using Evans Blue coloration (Fig. 1A, C) or the FDA method (Fig. 1B, C) 24 h post-treatment. It was first confirmed that Streptomyces scabies induces cell death of A. thaliana suspension cells (Fig. 1A). TXT itself is also able to induce cell death as previously reported (Duval et al., 2005). This cell death was accompanied by cell plasmolysis (Fig. 1A), a hallmark of programmed cell death. This cell death appeared to be dose dependent (Fig. 1B). Thirty per cent of cells were dead upon addition of micromolar concentrations of TXT and more than half of the cells were dead at higher concentrations (10–20 µM). The cell death plateau was reached within 10 h for 10 µM of TXT (Fig. 1C). The Ca2+ influx induced by TXT was then confirmed in our model by using cultured cells expressing apoaequorin addressed in the cytosol. Thaxtomin A induced a rapid increase in [Ca2+]cyt of about 200 nM over 30 s (Fig. 2A). This short-lived increase in [Ca2+]cyt could be inhibited by La3+, Gd3+ or BAPTA (Fig. 2A, B). This increase 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 were recorded over 30 min with aequorin-expressing cells upon the addition of TXT (data not shown). The effect of TXT has been tested on cell death in the presence or absence of PM Ca2+ channel inhibitors (La3+ or Gd3+) or the Ca2+ chelator BAPTA. Although the pretreatment of A. thaliana cells with Gd3+ or with BAPTA induced a slight increase in cell death, the TXT-induced cell death significantly decreased after such pretreatments (Fig. 2C), indicating that the influx of Ca2+ is an upstream event in the signalling pathway leading to TXT-induced cell death.


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

 

Figure 2
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  		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 derived from Arabidopsis seedlings transformed by the apoaequorin gene upon 10 µM TXT addition, alone or after pretreatment with the Ca2+ channel inhibitors La3+ or Gd3+ (0.5 mM) or the calcium chelator BAPTA (1 mM). (B) Mean values of {Delta}[Ca2+]cyt. Data correspond to the means of five independent experiments. Addition of 0.06% methanol (equivalent 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 µM TXT-induced cell death detected by the vital staining Evans Blue method after 6 h. The data correspond to the means of three replicates during one experiment and the error bars correspond to standard errors. Data are representative of at least three independent experiments. * Significantly different from the TXT treatment, P <0.05.

 
TXT induces a transient depolarization of plasma membrane due to K+ and anion channel regulation
The effect of TXT on anion and K+ fluxes (some of the earliest signalling events detectable in plant–pathogen interactions) was tested further by electrophysiology. In our control conditions, the value of the resting membrane potential (Vm) of A. thaliana suspension cells was –47±5 mV (n=26) which is close to those recorded in previous studies (Reboutier et al., 2002, 2005, 2007; Bouizgarne et al., 2006). TXT induced a rapid depolarization of the cell PM reaching its maximal value within 1 min. This membrane potential (Vm) variation was concentration-dependent and transient, although not fully reversed (Fig. 3A, B). Previous electrophysiological studies and pharmacological analysis identified two main ion channel currents in the PM of A. thaliana cells: a K+ outward rectifying current (KORC) and an anion current, which display the main hallmarks of slow anion channels (Reboutier et al., 2002). TXT induced a concentration-dependent decrease of KORC reaching about 60% at 10 µM (Fig. 3C, D, E) from a mean control value of 0.5±0.1 nA (n=23). This KORC decrease was fully reversed upon repolarization of the cells (Fig. 3E). Thaxtomin A also induced a concentration-dependent increase in anion current reaching 150% (Fig. 3F, G, H) from a mean control value of –0.38±0.05 nA (n=22). Upon repolarization of the cells the anion current level decreased but remained slightly higher than the control level recorded before TXT addition (Fig. 3H). The transient regulation of these currents certainly explains the observed transient depolarization.


Figure 3
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  		Fig. 3. (A–H) Thaxtomin A-induced depolarization and ion current regulation of A. thaliana cells. (A) Typical transient depolarization observed in response to increasing concentration of TXT. (B) Mean values of the amplitude of the plasma membrane potential changes recorded at maximal depolarization (grey bar) and after repolarization (black bar) upon TXT addition. (C) KORC measured under control conditions and after adding 10 µM TXT in the culture medium. Voltage pulses were +80 mV. Holding potential was Vm. (D) Current–voltage relationships. The KORC amplitudes at steady-state were measured for membrane potentials ranging from –200 to +80 mV before (filled circles) and after TXT addition at maximal depolarization (open circles) and after repolarization (filled inverted triangles). (E) Mean steady-state values of KORC recorded for a +80 mV voltage pulses at maximal depolarization (grey bar) and after repolarization (black bar). (F) Anion currents measured under control conditions and after adding 10 µM 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 (open circles) and after repolarization (filled inverted triangles). (H) Mean steady-state values of anion current recorded at –200 mV at maximal depolarization (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 ±SD of at least five independent experiments. * Significantly different from the control, P<0.05.

 
Pretreatment of the cells with the Ca2+ channel inhibitor La3+ and Gd3+ or with the Ca2+ chelator BAPTA abolished the TXT-induced depolarization (Fig. 4A) and the modulation of KORC and anion currents (Fig. 4B, C) indicating that the influx of Ca2+ is also an upstream event when compared to TXT-induced anion and K+ channels regulation leading to depolarization.


Figure 4
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  		Fig. 4. (A, B) Inhibition of thaxtomin A-induced depolarization and ion current regulation by Ca2+ channel inhibitors or Ca2+ chelator. (A) Running membrane 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 to TXT effect. (B) Absence of KORC variation 1 min after addition of 10 µM TXT after La3+ pretreatment. (C) Absence of anion current variation 1 min after the addition of 10 µM TXT in the same conditions. Protocols were as illustrated in the figure. Data are representative of four independent experiments.

 
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 anion currents, the effect of two structurally unrelated anion channel inhibitors, 9-AC and glibenclamide (gli), effective on A. thaliana cell anion currents (Fig. 5A), was tested on TXT-induced cell death. The anion channel inhibitors alone induced a slight cell death (Fig. 5B) as previously reported (Reboutier et al., 2005). However, the TXT-induced cell death recorded in the presence of TXT supplemented with 9-AC and gli did not decrease (Fig. 5B) suggesting that the anion current increase is not an upstream event in the signalling pathway leading to TXT-induced cell death.


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

 
TXT induces a biphasic modulation of external pH
The effect of TXT on the external medium pH of A. thaliana cell cultures was investigated. Indeed, changes in the pH value might reflect TXT-induced H+ flux modulation through the PM. TXT at 10 µM induced a slight acidification reaching a maximal value of –0.038±0.006 upH (n=17) during the first 30 min (Fig. 6A), as previously reported with roots of different species (Tegg et al., 2005). This acidification was followed by a large alkalization (Fig. 6A) reaching 0.48±0.06 upH (n=6) after 4 h (Fig. 6B). The alkalization induced by TXT was decreased in the presence of the calcium channel inhibitors La3+ and Gd3+ as well as in the presence of BAPTA (Fig. 6B), indicating that Ca2+ influx is involved in pH modification. The effect of the phytohormone indole acetic acid (IAA) on external medium pH was tested further because TXT, which is structurally related to IAA, has been suggested to compete with IAA on the PM receptor (Tegg et al., 2005). As IAA was described to stimulate H+-ATPase activity directly in some cases (Kim et al., 2001), it was tested if it could counteract the TXT-induced alkalization. Treatment with IAA did not significantly modify the external medium pH (data not shown). The addition of IAA with TXT did not modify the TXT-induced biphasic modulation of external pH (Fig. 6B), the alkalization reaching 0.46±0.07 upH (n=6) after 4 h, suggesting that there is no competition between the two molecules at this level.


Figure 6
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  		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 cell suspensions treated with 10 µM TXT. (B) Changes in external pH after treatment with 10 µM TXT or on cell pretreated with Gd3+ or La3+ (0.5 mM), BAPTA (1 mM) or IAA (10 µM). The data correspond to the means of six replicates and error bars correspond to standard errors. (C) Time-course of H2O2 accumulation in the external medium without treatment (control), after TXT treatment (1 µM and 10 µM) 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 µM valinomycin (used as positive control) or 10 µM TXT, monitored using JC-1 as a mitochondrial membrane potential probe. The fluorescence associated to JC-1 was measured after 15 min and 3 h for control cells, cells treated with 1 µM valinomycin or with 10 µM TXT. * Significantly different from the control, P <0.05.

 
The alkalization of the external medium (Fig. 6B) could be linked to decrease of PM-H+-ATPase activity and/or to the activation of PM-NADPH-oxidases leading to the generation of H2O2 (Pugin et al., 1997). As the absence of ROS production in response to TXT had previously been reported (Duval et al., 2005), the effect of TXT (up to 10 µM) on luminol-mediated chemiluminescence was tested to check if H2O2 was released into the culture medium. In this previous study the authors did not record any alkalization of the cell medium as observed in our conditions. Hypoosmotic shock, used as a positive control, largely increased H2O2 production. TXT failed to induce H2O2 production in the culture medium (Fig. 6C). Seeing that mitochondria are known to be a putative pathogen-derived phytotoxin or elicitor target (Krause and Durner, 2004; Bouizgarne et al., 2006; Errakhi et al., 2008), the effect of TXT on mitochondrial potential was checked to assess whether a putative decrease in ATP level leading to a decrease of H+-ATPase activity could explain the alkalization of the medium. Experiments have been carried out by using the JC-1 fluorochrome, which is known to be incorporated and accumulated specifically in plant mitochondria (Simeonova et al., 2004). Valinomycin (1 µM), used as a positive control, induced a decrease of this potential within 15 min, when 10 µM TXT did not modify this potential after 3 h (Fig. 6D).

TXT-induced expression of defence response genes
Early defence responses are often concomitant with an increase in the expression of defence-related genes. To investigate the effects of Ca2+ influx on TXT-induced responses at the molecular level, the accumulation of several gene transcripts known to be accumulated during HR and corresponding to different classes of so-called defence genes was analysed by RT-PCR: VDAC and AOX1a, also known as hypersensitive-related (HSR) genes (Lacomme and Roby, 1999); PAL1, which encodes phenyl ammonia-lyase, a key enzyme of the phenylpropanoid biosynthetic pathway; PR1 (pathogenesis-related) and PDF1.2 involved, respectively, in the classical SA and JA or ET-defence signalling pathways. Increases in mRNA levels after treatment of the cells with 10 µM TXT for 4 h were only detected for PAL1 (Fig. 7A). This treatment did not lead to the accumulation of the transcripts of the other genes. Treatment of the cells with 0.5 mM La3+ and 10 µM TXT decreased the induction of PAL1 when compared with TXT treatment (Fig. 7B). This result confirms the hypothesis that Ca2+ could be implicated in the signalling pathway involved in the response to TXT.


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

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Thaxtomin A, a determinant factor of Streptomyces spp pathogenicity (King et al., 1991; Loria et al., 1997; Healy et al., 2000) was recently shown to induce an atypical PCD in A. thaliana (Duval et al., 2005). This phytotoxin also induces a [Ca2+]cyt increase depending on a calcium influx from the external medium which could be inhibited by La3+, a PM calcium channel inhibitor (Tegg et al., 2005). The aim of this work was to analyse the putative role of this calcium influx in the TXT-induced cell death observed in A. thaliana-cultured cells (Duval et al., 2005) and in classically HR-associated responses. It was first confirmed by using A. thaliana cells expressing aequorin that TXT induces a transient increase in cytosolic Ca2+ concentration. Then it was demonstrated that the TXT-induced increase in cytosolic Ca2+ and cell death were significantly reduced when the cells were treated with La3+, Gd3+ or BAPTA, which are known to inhibit Ca2+ influx. The increase in [Ca2+]cyt is known to be a major event in numerous plant cell signal transduction pathways, but the appropriate physiological response to a given signal is encoded by the amplitude, duration, frequency, and location of this calcium increase (McAinsh and Hetherington, 1998; Lecourieux et al., 2005). The Ca2+ influx occurs a few seconds after TXT addition (Tegg et al., 2005; this study) and peaks 1 or 2 min earlier than other 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) postulated that a rapid inhibition of cellulose synthesis can modify the plant cell wall composition and organization that were somehow perceived by the cell which, in turn, initiated a cell death programme. Tegg et al. (2005) further suggested that Ca2+ exchange with H+ in the apoplast led to an acidification of the cell wall responsible for enzyme activation and disruption of cellulose synthesis, positioning the Ca2+ influx as an upstream event of the inhibition of cellulose synthesis. As no further Ca2+ influx was recorded after the initial short-lived Ca2+ influx, it is likely the most rapid event triggering the cascade of events leading to cell death. The regulation of K+ and anion channels reported here were also initiated in less than 1 min after TXT addition but after Ca2+ influx, since pretreatment by La3+, Gd3+ and BAPTA avoided the TXT-induced ion channel regulation and subsequent PM depolarization. The involvement of the KORC decrease in response to TXT, although probably involved in PM depolarization, is not easy to link to cell death. Effectively, in most cases, an increase in K+ efflux, or in outward K+ currents, rather than a decrease were reported in response to HR-inducing pathogens or elicitors (Atkinson et al., 1996; Reboutier et al., 2007). The TXT-induced increase in anion channel current is in accordance with previous studies suggesting that anion effluxes are part of the early pathogen or elicitor induced responses (Jabs et al., 1997; Pugin et al., 1997). Several studies reported a rapid elicitor-induced PM depolarization resulting, at least in part, in the activation of anion channels (Pugin et al., 1997). Ca2+ influx was shown to be a prerequisite for the activation of PM anion channels in several systems (Jabs et al., 1997; Wendehenne et al., 2002). Moreover, a role for anion channels was also reported in cell death induction, since a rapid increase in anion currents seemed to be a prerequisite to achieve cryptogein- and oxalic acid-induced cell death (Gauthier et al., 2007, Errakhi et al., 2008). Although the TXT-induced anion channel regulation was transient, and thus could not be responsible for a massive anion efflux, a putative role for this current increase in a signalling pathway leading to TXT-induced cell death was checked. Such an increase in anion currents was reported to participate in the ABA triggered signalling pathway leading to gene expression changes (Ghelis et al., 2000). Contrary to what has already been observed with cryptogein on tobacco cells (Wendehenne et al., 2002) or oxalic acid on A. thaliana cells (Errakhi et al., 2008), gli and 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 channel regulation, a downstream event of TXT-induced Ca2+ influx, did not seem to be involved in the pathway leading to TXT-induced cell death.

Concerning H+ fluxes, data obtained with TXT are rather controversial. In our study, a biphasic regulation of external pH was observed after TXT addition. A slight acidification was first observed, which is compatible with the net H+ efflux recorded after TXT addition on various structures and plant types and suggested to be mediated by the PM H+-ATPases (Tegg et al., 2005), and then a delayed and large alkalization of the medium. These results are in contrast to the absence of alkalization which has been observed with tobacco or A. thaliana cells in previous studies (Fry and Loria, 2002; Duval et al., 2005). Tegg et al. (2005) pointed out that the variability in experimental conditions and in the material used for analysing the external pH variations could explain the differences among bibliographic references. However, the observation of an alkalization in response to TXT, led us to check for a putative production of ROS generation in response to TXT, since the absence of ROS production was concomitant with the absence of medium alkalization in 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 (Levine et al., 1996). These data confirmed that TXT did not induce H2O2 production, but the possibility cannot be excluded that other forms of ROS (superoxide anion or hydroxyl radical) were produced inside and/or outside the cell after TXT addition and participated in the cell death process as previously discussed by Duval et al. (2005). Since the alkalization of the external medium could not be explained by a generation of H2O2, it was ascertained if a decrease of PM H+-ATPase activity could be responsible for this alkalization. There was no decrease in the cell mitochondrial potential in response to TXT even after 3 h, suggesting that a depletion of ATP could neither be responsible for the decrease of H+-ATPase activity, nor for the subsequent alkalization observed. These data are in accordance with the absence of accumulation of HSR2 and HSR3 in response to TXT. Effectively HSR2 encodes a mitochondrial voltage-dependent gated anion channel (VDAC) protein (Lacomme and Roby, 1999), known to be involved in the cytosolic release of cytochrome c during certain cell death but not in response to TXT (Duval et al., 2005), and HSR3 encodes an alternative oxidase (AOX1a) which seemed to be a marker of mitochondrial stress since it has been reported that its level increases during elicitor-induced cell death (Krause and Durner, 2004; Vidal et al., 2007). All these observations indicate that mitochondria are not a primary target of TXT, even if recent data pointed out that mitochondrial permeability transition could precede plant PCD (Yu et al., 2002). To assess for a more direct effect of TXT on PM H+-ATPases, experiments were conducted in the presence of IAA. IAA is known to stimulate H+-ATPase activity (Kim et al., 2001) and is suggested to compete with TXT for the PM receptor since these two molecules are structurally related (Tegg et al., 2005). The addition of 10 µM IAA in the presence of 10 µM TXT did not counteract the acidification nor the alkalization. Further studies are therefore required to understand the putative TXT modulation of PM H+-ATPases. The regulation of H+ fluxes described in response to pathogen is also generally rather controversial, suggesting that effects may be plant or tissue specific. In most cases, the elicitor-induced decrease in PM H+-ATPase activity or extracellular alkalization was reported (Atkinson et al., 1996; Bouizgarne et al., 2006; Osses and Godoy, 2006). However, different studies highlight an acidification of the extracellular medium (Malerba et al., 2003) or an increase in PM H+-ATPase activity in response to elicitor or to phytotoxin (Vera-Estrella et al., 1994; de Boer, 2002). Attempts to use PM H+-ATPase inhibitors such as erythrosin B or vanadate, which is known to be a potent inhibitor 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 short term (not shown). As previously done for external pH measurements, competition experiments were conducted in the presence of IAA and TXT to see if IAA could counteract the TXT effect at the cell death level. The pretreatment of the cell with IAA did not reduce TXT-induced cell death (not shown). However, the alkalization which occurred after the addition of TXT was inhibited after a pretreatment with La3+, Gd3+, and BAPTA leading to the conclusion that the mechanism(s) responsible for alkalization is thus probably dependent on the Ca2+ influx. However, its precise role in the pathway leading to cell death remains unclear and needs more investigations.

As previously reported (Duval et al., 2005), the expression of defence-related genes PR1 and PDF1.2 was not induced by TXT in Arabidopsis cells. TXT treatment did not result in an accumulation of ethylene (data not shown) indicating that the resulting PCD did not involve the classical ET-defence signalling pathways. However, TXT induced the accumulation of PAL1, which is known to participate in the induction of defence by modulating the synthesis of cell wall component and defence related-compounds such as phytoalexins (such as the coumarin scopoletin, Kai et al.,, 2006). Our data suggest the involvement of the TXT-induced Ca2+ influx in the induction of the defence gene PAL1.

In conclusion, TXT-induced calcium influx is an early event that is required for the activation of downstream defence responses and cell death. In addition, the lack of dependency of this cell death to earlier Ca2+-dependent TXT-induced responses such as anion channel modulation further suggest that several pathways could be triggered in response to this early Ca2+ influx, which may be crucial in plant–Streptomyces interactions.


   Acknowledgements
 
We are grateful to G Vidal (Centre de recherche Paul Pascal, CNRS UPR 8641, 33600 Pessac, France) and to Professor JM Farrant (University of Cape Town, Department of Molecular and Cellular Biology) for critical reading of the manuscript. This work was supported by funds from the MESR (Ministère délégué à l'Enseignement Supérieur et à la Recherche) to EA 3514, from PRAD 04/02, and AUF 63-01PS615.


   Footnotes
 
* Both authors contributed equally to this work. Back

{dagger} Present address: Laboratoire de Glycobiologie et Transport chez les Végétaux, FRE CNRS 3090, IFRMP23, Université de Rouen, Mont Saint Aignan, France. Back


   Abbreviations
 
9-AC, 9-anthracene carboxylic acid; BAPTA, 1.2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; [Ca2+]cyt, cytosolic calcium concentration; FDA, fluorescein-diacetate; gli, glibenclamide; FW, Fresh weight; HR, hypersensitive response; IAA, indole acetic acid; KORC, K+ outward rectifying current; PM, plasma membrane; ROS, reactive oxygen species; TXT, thaxtomin A; Vm, plasma membrane potential.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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