Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells...

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Research

Blackwell Science Ltd

Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells involve cAMP

and Ca

2

+

Laurence V.

Bindschedler, Farida

Minibayeva

1

, Sarah L.

Gardner

2

, Chris

Gerrish, Dewi R.

Davies and G. Paul

Bolwell

Division of Biochemistry, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK; Present address:

1

Institute of

Biochemistry and Biophysics, PO Box 30, Kazan 420503, Russia;

2

School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

Summary

• Modulators of cAMP, calcium and G proteins were used to treat bean (

Phaseolusvulgaris

) cells before addition of an elicitor from

Colletotrichum lindemuthianum

inorder to elucidate the early steps of signal transduction leading to the production ofthe apoplastic oxidative burst.• Hydrogen peroxide production by elicited bean cells was monitored with luminol-or xylenol-orange-based assays.• Pretreatment with forskolin, dibutyryl cAMP or the Ca

2

+

ionophore A23187enhanced the production of reactive oxygen species (ROS). The Ca

2

+

channelblocker, verapamil, and the calmodulin antagonist W7 led to a decreased oxidativeburst and cancelled the dibutyryl cAMP effect. The production of ROS was increasedby cholera toxin (CTX), an activator of G proteins.• Thus, an increase of cytosolic calcium ([Ca

2

+

]

cyt

) mediated through an increasedlevel of cAMP is required for ROS production. The data support a role for G proteinsand cAMP in extracellular alkalinization and Ca

2

+

influx, possibly in the provision ofa reductant, which with the extracellular peroxidase, are required for the apoplasticoxidative burst.

Key words:

Phaseolus vulgaris

; Leguminoseae; oxidative burst; hydrogen peroxide;cAMP; calcium.

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Author for correspondence:

G. P. Bolwell

Tel:

+

44 1784 44 35 53

Fax:

+

44 1784 43 43 26 Email: [email protected]

Received:

26 February 2001

Accepted:

21 March 2001

Introduction

Reactive oxygen species (ROS) are rapidly produced in plantsas a defence response to pathogen attack (Wojtaszek &Bolwell, 1997; Bolwell, 1999). There are a number ofpotential sources of ROS. In many species, it is thought thatthe superoxide anion is the first ROS produced, originatingat the plasma membrane from an NADPH oxidase (Lamb& Dixon, 1997) analogous to the system that occurs inneutrophils, or from an NADH-dependent superoxidesynthase (Auh & Murphy, 1995). However, in elicitor treatedFrench bean cells hydrogen peroxide appears to originatefrom a cell wall peroxidase in a three component systemrequiring ion fluxes leading to extracellular alkalinization, andthe release of a reductant (Bolwell

et al.

, 1995; Robertson

et al.

, 1995; Bolwell, 1996; Bolwell

et al.

, 1999). Critical tothe evidence leading to this hypothesis are the observationsthat the NADH/NAD ratio and ATP level transiently fall, atthe same time as an increase in oxygen uptake occurs, whilethe NADPH level remains constant (Robertson

et al.

, 1995;Bolwell, 1996). In addition, the oxidative burst in French beancells is inhibited by potassium cyanide (KCN), whereas it isinsensitive to cyanide in mammalian cells. Moreover, its relativeinsensitivity to diphelynene iodonium (DPI), a well-knowninhibitor of the NADPH-oxidase, favours the hypothesis ofan apoplastic peroxidase generating the ROS in French bean(Bolwell

et al.

, 1998). The particular peroxidase isoformresponsible for the apoplastic oxidative burst has been purifiedcloned and expressed, and the reaction modelled

in vitro

asreported in a preliminary manner in Bolwell

et al

. (1999).

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The earliest signalling events involved in the oxidative burstare beginning to be elucidated. The increase in cytosolic Ca

2

+

,which in some tissues occurs within seconds of elicitation, isthought to be a primary signal essential for the subsequentdown-stream events. Down-stream events following theincrease in cytosolic calcium include the production of ROS(Chandra & Low, 1997; Rajasekhar

et al.

, 1999; Blume

et al

.,2000; Grant

et al

., 2000) and the induction of defence-relatedgenes such as those encoding PR proteins and those involvedin phytoalexin production (Blume

et al.

, 2000). In the case ofan infection with an avirulent pathogen, this results in theestablishment of a local programmed cell death (PCD) at thesite of infection (Grant

et al.

, 2000). A number of Ca

2

+

effectsare thought to be mediated via calcium-dependent proteinkinases (CDPK), a family of serine/threonine protein kinases,though the activation of the NADPH oxidase does not appearto require direct activation by a CDPK (Romeis

et al.

, 2000).However, it is possible that Ca

2

+

has a direct effect on theNADPH oxidase, since there is evidence that the plant enzymehas an N–terminal sequence with two Ca

2

+

binding EF-handmotifs (Keller

et al.

, 1998; Torres

et al.

, 1998). Nevertheless,protein kinase inhibitors such as K252a or staurosporineinhibit the oxidative burst (Guo

et al.

, 1998; Piedras

et al.

,1998; Tenhaken & Rübel, 1998; Cazalé

et al.

, 1999; Rajasekhar

et al.

, 1999), thus supporting a role for a protein kinase cascadein the regulation of this phenomenon. Beside the early calciuminflux into the cytosolic compartment, a rapid efflux of K

+

and Cl

–

and extracellular alkalinization of elicited cell cultureshas also been observed (Scheel, 1998; Fellbrich

et al.

, 2000).Extracellular alkalinization appears to be essential for theapoplastic oxidative burst (Bolwell

et al.

, 1995).There is emerging evidence for a role of cAMP as a second-

ary signal in plants (Assmann, 1995; Bolwell, 1995). In par-ticular, a transient increase in the concentration of cAMP inresponse to elicitor has been demonstrated in French bean(Bolwell, 1992), carrot (Kurosaki

et al.

, 1993) and

Medicagosativa

(Cooke

et al.

, 1994). Despite this, it has not been estab-lished that cAMP action is mediated via protein kinase A asit usually is in mammalian cells (Bolwell, 1995; Sopory &Munshi, 1998; Newton

et al.

, 1999), and in carrot, cAMP seemsto be involved in regulating Ca

2

+

fluxes via a cross-talk mech-anism (Kurosaki

et al.

, 1993). Moreover, cAMP has beenshown to activate K

+

channel activity in the mesophyll cells of

Vicia faba

, possibly via a cAMP-regulated protein kinase (Li

et al.

, 1994) and this effect appears to be specific to this cyclicnucleotide. More recently it has been shown in tobacco pro-toplasts expressing aequorin that the cytoplasmic level of Ca

2

+

increases within seconds after addition of dibutyryl cAMP ordibutyryl cGMP, which are the cell-permeable derivatives ofthe cyclic nucleotides (Volotovski, 1998; Volotovski

et al.

,1998). Others have reported identification and characteriza-tion of a new gene family in

Arabidopsis

which shares featureswith cyclic nucleotide-gated channels from animals andinward-rectifying K

+

channels from plants (Kohler

et al.

,

1999). A plant cyclic nucleotide-gated cation channel,AtCNGC2, from

Arabidopsis

has been cloned and its functioncharacterized (Leng

et al.

, 1999). By analogy with similaranimal cation channels, AtCNGC2 is suggested to be involvedin signal transduction by allowing a flux of Ca

2

+

, K

+

and otherions in the presence of cAMP or cGMP. Furthermore, the

Arabidopsis

dnd1 gene (defence, no death) has been isolatedfrom a mutant line that failed to produce a hypersensitiveresponse to the avirulent

Pseudomonas

pathogens and thisgene has been found to encode the same ion channel protein,AtCNGC2 (Clough

et al.

, 2000). Arazi

et al

. (2000) have iso-lated a tobacco plasma channel protein with a high affinity forcalmodulin and a highly conserved cyclic nucleotide-bindingdomain. A transgenic tobacco, expressing a dominant-actingcalmodulin, hyper-activates a calmodulin-dependent NADkinase and leads to an increased and more rapid productionof ROS, an increased alkalinization of the medium and theinitiation of a more rapid PCD (Harding

et al.

, 1997; Harding& Roberts, 1998). Taken together, there is growing evidencefor the involvement of cAMP in signal transduction andcross-talk between the various pathways of plants respondingto the attack by avirulent pathogens. The role of the cyclicnucleotide in Ca

2

+

signalling is most likely to regulate theflux of the cation into the cytosol and thus the activity ofcalmodulin or related proteins such as CDPK, which arecalcium-sensors transducing Ca

2

+

signals.We have previously shown that cAMP is transiently elevated

in response to elicitor in bean cells (Bolwell, 1992). Moreover,the induction of phenylalanine ammonia-lyase (PAL) ismodulated by the addition of either forskolin, an activator ofadenylyl cyclase or by dibutyryl cAMP, a cell-permeable deriv-ative of cAMP (Bolwell

et al.

, 1991). The present work showsthat treatment of cultured bean cells with forskolin anddibutyryl cAMP similarly enhances the production of activeoxygen species in response to elicitor and that this effect canbe explained by the influence of the cyclic nucleotide on theconcentration of cytosolic Ca

2

+

. The possible involvement ofG-protein in the induction of cAMP biosynthesis will also bediscussed. The data add further support for the model previ-ously presented (Bolwell, 1999).

Materials and Methods

Chemicals

All chemicals were purchased from Sigma (Poole, Dorset, UK).

Cell cultures and elicitation

Cell cultures of French bean (

Phaseolus vulgaris

L.) weremaintained as described previously (Dixon & Lamb, 1979).Twenty ml of 5-d-old bean cell cultures were transferred toa 100-ml beaker and agitated on a rotating platform at180 rpm. The oxidative burst modulators were added and

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cells incubated for an optimized period of time prior toaddition of the elicitor, unless stated differently. The optimumpreincubation periods were determined to be 30 min forverapamil and dibutyryl cAMP, 5 min for forskolin, W7 andA23187 and 60 min for cholera toxin (CTX). Verapamil anddibutyryl cAMP were dissolved in water, W7 and A23187 inDMSO and forskolin in ethanol. CTX was reconstituted inTBS (50 mM Tris, 150 mM NaCl pH 7.2) containing 10 mMDTT and activated for 1 h at 37

°

C immediately before use.As a control, cells were incubated with the correspondingsolvent or buffer as used for the modulator tested. An elicitorpreparation of the fungal bean pathogen

Colletotrichumlindemuthianum

at a final concentration of 30 µg ml

–1

glucoseequivalents was used for the elicitation as described by Dixon& Lamb (1979).

Detection of Hydrogen peroxide and other ROS

H

2

O

2

production by elicited bean cells was specificallymonitored using a luminol-based assay described by Bolwell

et al

. (1998). ROS production was also monitored withxylenol orange, where hydroperoxides are reduced by ferrousions in acid solution forming a ferric product-xylenol orangecomplex, detected spectrophotometrically at 560 nm (Gray

et al.

, 1999). Because of their instability, solutions werefreshly prepared: 25 mM FeSO

4

and 25 mM (NH

4

)

2

SO

4

were dissolved in 2.5 M H

2

SO

4

. One millilitre of this solutionwas added to 100 ml of 125 µM xylenol orange (Sigma)and 100 mM sorbitol. The reagent remained stable for 6–8 h.At different time points after elicitation 300 µl of cells wereremoved and transferred to a microfuge tube, quickly cen-trifuged (15 s at maximum speed) and 100 µl of the super-natant was immediately added to 1 ml of xylenol orangereagent. The reaction mixture was incubated 30 min at roomtemperature before measuring the absorbance at 560 nmagainst a blank containing 100 µl water. For both methods,H

2

O

2

measurements were expressed as relative values with100% corresponding to the maximum of the elicited control.

Results

Comparison of the luminol and xylenol orange assays to detect ROS in elicited bean cell cultures

In a study of the effects of various pharmacological agentson the oxidative burst in response to elicitor, agonistsand antagonists of cAMP, G proteins and Ca

2

+

flux wereinvestigated. The results shown are for a single batch of cellsbut are typical of at least three independent experiments basedon different batches of cells. There was some variation in thetiming of the peak of the oxidative burst from batch to batchof cells but the degree of inhibition or activation by theeffectors studied was similar within replicates. Some of theagents used are sparingly soluble in water and therefore were

dissolved in DMSO or ethanol. The pretreatment of beancells with 0.05% v/v DMSO for 5 min before the addition of

C. lindemuthianum

elicitor reduced by 50% the productionand/or determination of hydrogen peroxide as measured withthe luminol chemiluminesence method (Fig. 1b) but in allexperiments, the appropriate control was used as a comparison.Furthermore, in such cases, the total ROS content was measuredwith the xylenol orange assay to confirm the influence(agonist or antagonist) of the compound on the oxidative

Fig. 1 (a) Comparison of the luminol and xylenol orange assays to detect H2O2 in elicited bean cell cultures, detection of the oxidative burst in bean cells elicitated with a Colletotrichum lindemuthianum elicitor preparation. H2O2 was estimated with a luminol-chemiluminescence based method (open circles), whereas the xylenol orange method detects general reactive oxygen species (ROS) production (closed circles). (b) Effect of solvents on the detection of the oxidative burst. DMSO or ethanol was added 5 min before elicitation to give 0.05% v/v final concentration. The maximal amounts of H2O2 detected during the oxidative burst for the different samples are compared to the maximum of an elicited control (expressed as 100%) in absence of solvent as measured with the luminol assay (open squares) or the xylenol orange assay(closed squares).



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burst. Figure 1(a) shows the equivalence of both methodsused to determine the amplitude of the response in controlelicited cell cultures. By contrast to luminol, there was noeffect on ROS measurement by the xylenol orange assay witheither DMSO or ethanol (Fig. 1b). Nevertheless, the sameconclusions could be drawn when cells were treated withagonists or antagonists and compared with the appropriatecontrol, whatever assay was used as shown as an example inFig. 2(a,b). Therefore only the data obtained with the luminolassay is presented in figures subsequent to Fig. 2(a,b).

The effect of forskolin on the apoplastic oxidative burst and pH

Forskolin, a cell-permeable derivative and a potent activatorof adenylyl cyclase has been used extensively in mammaliansystems to increase intracellular cAMP and to study cAMP-dependent physiological effects. Forskolin activates mammalianadenylyl cyclases and probably the plant homologues sinceforskolin (1- and 100 µM) has previously been shown toelevate cAMP levels transiently in cultured bean cells within15 min of the addition of the diterpene (Bolwell, 1992). Theaddition of a range of forskolin concentrations to bean cells5 min before elicitation resulted in an enhanced productionof H

2

O

2

as measured with the luminol chemiluminescencemethod (Fig. 2a). Ten µM forskolin gave the maximum effectwith more than a twofold increase when compared with theethanol control. In the absence of elicitor forskolin was unableto stimulate an oxidative burst indicating that the agonistmodulates the signalling mechanism involved rather thandirectly triggering the response. The experiment was initiallydone with DMSO as a carrier solvent and despite theinhibitory effect of DMSO (Fig. 1b), the same inductioneffect could be observed (data not shown). Since addition ofethanol (0.05%

−

0.1% v/v) may still have interfered with thelevel of hydrogen peroxide as measured with luminol comparedwith a control without ethanol, the experiment was repeatedusing the xylenol orange assay to detect ROS. A similarincrease of the oxidative burst was observed with the additionof 1-, 10- or 100 µM forskolin (Fig. 2b) except that with thismethod the burst observed with 10 µM forskolin wasnoticeably prolonged. In this case ethanol had no significanteffect on the oxidative burst (data not shown).

The data suggest that activation of an adenylyl cyclase isa possible part of a signalling pathway modulating the pro-duction of hydrogen peroxide, which is compatible with theenhanced production of ROS by forskolin in elicited beancells and correlated with an increased level of cAMP (Bolwell,1992). Moreover, 100 µM forskolin induced alkalinization ofthe apoplasm, elevating the extracellular pH of 0.5 units from6.35 to 6.85 (Fig. 2c). Nevertheless, forskolin may also act atdifferent levels, other than activating the adenylyl cyclase,since the cAMP homologue, dibutyryl cAMP, had no effecton the pH change (below).

Fig. 2 Effect of the adenylyl cyclase activator forskolin on the apoplastic oxidative burst and pH. Bean cells were incubated for 5 min with 0 µM (open circles, elicited control), 1 µM (closed triangles), 10 µM (closed squares), 100 µM (closed circles) forskolin before addition of the elicitor and with 100 µM (open squares) forskolin in the absence of elicitor (nonelicited control). Reactice oxygen species (ROS) production was evaluated with (a) luminol-chemiluminescence specific for H2O2 detection; (b) xylenol orange assay, measuring the absorbance at 560 nm (c) pH of the culture medium of elicited bean cells pretreated (closed circles) or not pretreated (open circles, elicited control) for 5 min with 100 µM forskolin before the elicitor was added.


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The effect of dibutyryl cAMP on the apoplastic oxidative burst

Dibutyryl cAMP is a potent activator of protein kinase A.Dibutyryl cAMP is cell permeable and is also more resistantto degradation by phosphodiesterases than cAMP. Addition ofdibutyryl cAMP at a range of concentrations causedenhancement of H2O2 production but in this case the effectwas dependent on the preincubation time (Fig. 3a). When cellswere preincubated for 15- or 30 min with 10 µM dibutyrylcAMP up to threefold enhancement of ROS production wasfound, whereas addition of dibutyryl cAMP simultaneouslyor 60 min before the elicitation did not provoke any increased

level of hydrogen peroxide production. Presumably, theoptimal preincubation time reflects the permeability of thebean cells to the membrane-permeable cyclic nucleotide,the conversion of the dibutyryl cAMP to cAMP by intra-cellular esterase activity, and its relative stability to degradationby phosphodiesterases in the cell. Therefore, in furtherinvestigation, cells were preincubated for 30 min with 1-, 10-or 100 µM dibutyryl cAMP. A maximal twofold increasein the oxidative burst was observed when cells were pre-treated for 30 min with 10 µM dibutyryl cAMP (Fig. 3b).Unlike forskolin, the increase in the oxidative burst causedby dibutyryl cAMP was not correlated with a significantdifference in the pH change in response to dibutyryl cAMPcompared with that in the ethanol control (data not shown).There was also no detectable oxidative burst when dibutyrylcAMP was applied in the absence of elicitor (data not shown).These data complement the observations obtained withforskolin, thus reinforcing the possibility that throughactivation of adenylyl cyclase, cAMP is a component ofthe signalling pathway leading to the production of apop-lastic ROS.

The effect of verapamil on the apoplastic oxidative burst

While there is no evidence that cyclic nucleotides directly activateprotein kinases PKA or protein kinase G (PKG) in plants cells,there is accumulating evidence that both cAMP and cGMP maytrigger ion fluxes via cyclic nucleotide-gated ion channels,including Ca 2+ and K+. The Ca2+ channel blocker, verapamil(100 µM) inhibited the apoplastic oxidative burst when thecells were preincubated with the blocker for 30 or 60 minbefore addition of the elicitor, whereas added simultaneously,verapamil delayed but did not inhibit the appearance of H2O2(Fig. 4a). In the absence of elicitor, verapamil did not inducean oxidative burst (data not shown). Consequently, cells werepreincubated with verapamil for 30 min before adding theelicitor to study the dose effect of this antagonist. Verapamilat 1 µM reduced the production of H2O2 by 60% and at10 µM and 100 µM gave a 75% inhibition (Fig. 4b).

The effect of A23187 on the apoplastic oxidative burst

The ionophore A23187 has been used frequently to examinethe role of intracellular Ca2+ in signal transduction. Additionof A23187 (1 µM) at the same time as the elicitor caused aconsiderable increase in the oxidative burst compared to aDMSO control (data not shown). This effect was greaterwhen cells were preincubated for 5 min with the ionophore.A reduced effect was found when the cells were preincubatedfor 15 min before the addition of the elicitor and the effect ofthe ionophore virtually disappeared after 60 min preincuba-tion (data not shown). Therefore, to study the influence ofconcentration of the ionophore on the production of ROS,

Fig. 3 Effect of dibutyryl cAMP on the apoplastic oxidative burst. H2O2 was detected with luminol. (a) Bean cells were incubated with 10 µM dibutyryl cAMP before addition of the elicitor and compared to an untreated but elicited control (open circles). The preincubation time with the cyclic nucleotide was 15 min (closed squares), 30 min (closed circles) or 60 min (closed trianlges). (b) Bean cells were incubated for 30 min with 0 µM (open circles, elicited control), 1 µM (closed triangles), 10 µM (closed squares), 100 µM (closed circles) dibutyryl cAMP before addition of the elicitor.


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cells were preincubated for 5 min with A23187 before theaddition of elicitor. There was a fivefold increase of the oxidativeburst at 50 µM A23187 (Fig. 5). This further confirms thatthe oxidative burst is modulated by an increase in cytosolicCa2+. It should be noted that the ionophore alone, in theabsence of elicitor, did not cause a significant production ofhydrogen peroxide, suggesting that regulatory events otherthan an influx of Ca2+ are required for the oxidative burst.

The effect of W7 on the apoplastic oxidative burst

W7 is known to be an inhibitor of calmodulin-dependentproteins and calmodulin-dependent regulatory events includingthe activation of calmodulin dependent kinases and plantCDPKs. Therefore its mode of action should be to blockcalcium-dependent effects. In the present study the effect of

W7 was found to be concentration dependent with an 80%inhibition at 100 µM when the cells were preincubated withthe inhibitor for 5 min before the addition of elicitor (Fig. 6).Preincubation time did not influence the effect of W7 on thereduction of the ROS production. Only 25% inhibition wasobserved with a 10-µM concentration. These data are consistentwith previous work on the effect of W7 on a CDPK fromFrench bean where it was observed that the I50 for W7 on theFrench bean CDPK was 400 µM (unpublished results).

Fig. 4 Effect of the calcium channel blocker verapamil on the apoplastic oxidative burst. H2O2 was detected with luminol. (a) Bean cells were incubated with 100 µM verapamil before addition of the elicitor and compared with an untreated elicited control (open squares). The preincubation time with verapamil was 0 min (closed squares), 30 min (closed circles) or 60 min (closed triangles). (b) Bean cells were incubated for 30 min with 0 µM (open circles, elicited control), 1 µM (closed triangles), 10 µM (closed circles), 100 µM (closed squares) verapamil before addition of the elicitor.

Fig. 5 Effect of the ionophore A23187 on the apoplastic oxidative burst. H2O2 was detected with luminol. A23187 was added to the bean cells 5 min before Colletotrichum lindemuthianum elicitor to give a final concentration of 0 µM (open circles, elicited control), 1 µM (closed triangles), 10 µM (closed squares), 50 µM (closed circles). The effect of 10 µM A23187 on the production of ROS was also evaluated in nonelicited cells (open squares, nonelicited control).

Fig. 6 Effect of the calmodulin antagonist W7 on the apoplastic oxidative burst. H2O2 was detected with luminol. Bean cells were treated with 0 µM (open circles, elicited control), 10 µM (closed squares) or 100 µM (closed circles) W7 5 min before the addition of Colletotrichum lindemuthianum elicitor.


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Interaction of dibutyryl cAMP and W7 in modulating the apoplastic oxidative burst

In the experiment shown in Fig. 7, W7 at 100 µM stronglyinhibited the oxidative burst compared to an elicited control.Furthermore, the enhanced production of ROS observedwhen dibutyryl cAMP was added 30 min before the elicitorwas also inhibited by W7 added at the same time. How-ever, this inhibition was not to the same extent as in theabsence of the cyclic nucleotide. This is consistent with theobservation that alkalinization alone is sufficient to inducea modest oxidative burst (Bolwell et al., 1995). Full potenti-ation of the oxidative burst may therefore require partic-ipation of a CDPK, possibly in the formation of or release ofa reductant. In any case, these results may indicate that cAMPacts upstream of a CDPK or other calmodulin-dependentproteins.

Reversal of the effect of dibutyryl cAMP by verapamil

Verapamil, the Ca2+ channel blocker, inhibited hydrogenperoxide production in response to elicitor by about 70% inthis particular experiment whilst dibutyryl cAMP increasedthe response as observed previously. Preincubation of the cellswith verapamil in combination with dibutyryl cAMP, beforeaddition of the elicitor resulted in a strong inhibition of theoxidative burst response (Fig. 8). With these data we mayspeculate that the action of cAMP to enhance the oxidative burstis mostly mediated through the activation of ion channels.

The effect of CTX on the oxidative burst

CTX is known to activate Gs proteins by inhibiting theGTPase activity of the α subunit, keeping it in the activeform. Application of high concentrations of CTX such as4 µg ml−1 or 0.4 µg ml−1 did not affect the elicitation or evenreduced the amount of ROS produced, probably due tocytotoxic effects (data not shown). Nevertheless, the lowestconcentration, 0.04 µg ml−1, induced an increased productionof hydrogen peroxide (Fig. 9). It is worth mentioning that inthe absence of elicitor, a small increase in H2O2 correspondingto 6% of the elicited control could be detected 20 min afteraddition of 4 µg ml−1 CTX, suggesting that activation of Gproteins alone can induce a small oxidative burst (data notshown). Since cells were incubated with CTX for 1 h beforeaddition of the elicitor, the absence of a stimulation of theoxidative burst at high CTX concentrations might be causedby the small initial burst observed with the CTX alone, leavingthe cells transiently refractory to a subsequent elicitation.

Discussion

The data presented here are part of a study of modulatorsof the elicitor-induced oxidative burst in French bean cellscarried out in order to elucidate mechanisms of signaltransduction. Initial screening of potential pharmacologicalagents has been described (Bolwell et al., 1999). The selectionwas partly based on those known to be active in modulatingother aspects of the elicitation response, namely the inductionof PAL and the accumulation of phytoalexins and cell wall

Fig. 7 Interaction of dibutyryl cAMP and W7 in modulating the apoplastic oxidative burst. H2O2 was detected with luminol. Bean cells were pretreated for 30 min before the addition of Colletotrichum lindemuthianum elicitor. The pretreatment was: no addition (open circles, elicited control), 10 µM dibutyryl cAMP (closed circles), 10 µM dibutyryl cAMP and 100 µM W7 (closed squares), 100 µM W7 (closed triangles).

Fig. 8 Reversal of the effect of dibutyryl cAMP by verapamil in the modulation of the apoplastic oxidative burst. H2O2 was detected with luminol. Bean cells were pretreated for 30 min before the addition of Colletotrichum lindemuthianum elicitor. The pretreatment was: no addition (open circles, elicited control), 10 µM dibutyryl cAMP (closed circles), 10 µM dibutyryl cAMP and 10 µM verapamil (closed squares), 10 µM verapamil (closed triangles).



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phenolics (Bolwell et al., 1991). These fell into three groups,modulators of calcium mobilization, ionophores affecting pHand K+ exchange and modulators of G-proteins and cAMPmetabolism. This paper particularly focused on the modula-tion of the appearance of ROS in cultured French bean cellsby compounds known to influence [Ca2+]cyt in various organ-isms and/or the level of intracellular cAMP.

The Ca2+ channel blocker verapamil, which modulatesmovement of Ca2+ into cells through voltage-sensitivecalcium channels in mammalian cells, is also known to exertinhibitory effects on Ca2+ influx in plant cells (Volotovski,1998). In the present study, verapamil inhibited the elicitor-induced production of ROS, suggesting that at least some ofthe events triggered by the elicitor were mediated via an extra-cellular Ca2+ flux into the cytosol. The involvement of cal-cium in events upstream of the oxidative burst was confirmedby the increase in ROS production in the presence of theionophore A23187. It has also been shown that an increase incytoplasmic calcium is required for production of ROS intobacco cells (Chandra & Low, 1997) and Arabidopsis (Grantet al., 2000). In parsley protoplasts, the oxidative burst is pre-ceded by an influx of Ca2+ and efflux of K+ and Cl– (Blumeet al., 2000; Fellbrich et al., 2000).

cAMP is now considered to be a potent secondarymessenger in plants (Bolwell, 1992; Kurosaki & Nishi, 1993;Kurosaki et al., 1993; Cooke et al., 1994; Assmann, 1995;Bolwell, 1995; Newton et al., 1999). The likely increase inendogenous cAMP when bean cells were treated with for-skolin, or the exogenous application of the cAMP homologue,potentiated an increased oxidative burst in the presence of theColletotrichum elicitor, indicating a putative role for cAMP inthe transduction of a signal leading to ROS production. This

is reinforced by the transient increase in cAMP observed inelicited bean cells within 15 min (Bolwell, 1992), this beingwithin the time frame corresponding to the maximum level ofhydrogen peroxide production.

In bean, the induction of PAL leads to the de novo synthesisof isoflavonoid phytoalexins and wall bound phenolic com-pounds. Elicitation of PAL with the extracts of C. lindemuth-ianum was increased in the presence of the Gs proteinactivator CTX and of the adenylyl cyclase activator forskolin(Bolwell et al., 1991). In the present study, the amount ofROS generated in the presence of these two compounds wasalso increased. Thus, this indicates that an early event such asthe oxidative burst and a later event such as the induction ofPAL probably both require common steps in the early eventsof the signalling pathway involving a G protein and cAMP.Suramin, a potent G-protein inhibitor, blocked the produc-tion of ROS in elicited soybean cell cultures down-stream ofthe activation of protein kinases (Rajasekhar et al., 1999).There is emerging evidence that small-GTP binding pro-teins of the Rac family can trigger ROS production and HR(Kawasaki et al., 1999; Hassanain et al., 2000). Expressionof cholera toxin in planta led to elevated resistance to pathogen(Beffa et al., 1995), which indicates that G protein activationmight be an ubiquitous feature for the initialization of mech-anisms involved in the establishment of resistance and pro-duction of an oxidative burst.

It is not possible to generalize about the mechanism ofrecognition, signalling and production of ROS in all plantsystems undergoing elicitation by a particular elicitor. Forinstance, the origin of hydrogen peroxide is not ubiquitous. Insome systems, it seems to be dependent on a plasma mem-brane NADPH oxidase and in others, like bean, hydrogenperoxide is generated by an apoplastic peroxidase (Bolwellet al., 1995, 1999). Likewise, there is still a lack of the informa-tion that is required to be able to describe any complete sig-nalling pathway triggering ROS production. For instance, inthe bean system nothing is known about elicitor recognitionthrough putative receptor(s) and whether such receptor(s)would be directly coupled to a G protein that activates an ade-nylyl cyclase leading to an increase in cAMP. It is also possiblethat G proteins may act down-stream of protein kinases assuggested by Rajasekhar et al. (1999). Nevertheless, there isgood evidence that an adenylyl cyclase is activated since thelevel of cAMP increased after elicitation of bean cells (Bolwell,1992). cAMP may directly act on cyclic-nucleotide gatedchannels and induce an increase in [Ca2+]cyt and alkalinizationof the medium. A G protein α-subunit may also activateplasma membrane Ca2+ channels (Aharon et al., 1998). It isimprobable that the cAMP signal is mediated through cAMP-dependent protein kinases (PKA), since no plant PKA hasbeen purified to homogeneity so far and no sequence withsufficient similarity to ascribe to a PKA has been found in theArabidopsis genome. Although Ca2+ may directly activate theNADPH oxidase without a direct requirement for a CDPK

Fig. 9 Effect of the Gs protein activator cholera toxin (CTX) on the apoplastic oxidative burst. H2O2 was detected with luminol. Bean cells were treated with 0 µg ml−1 (open circles, elicited control) or 0.04 µg ml−1 (closed circles) activated CTX 60 min before the addition of Colletotrichum lindemuthianum elicitor.


© New Phytologist (2001) 151: 185–194 www.newphytologist.com

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(Romeis et al., 2000), its role in the apoplastic oxidative burstis obscure. Activation of G proteins and an enhancement ofcAMP levels may be related to the opening of ion channelsand elevated alkalinization leading to an increased apoplasticburst similar to that seen with K+ ionophores. A target forCa2+ influx would be CDPKs, as it has been observed that aspecific form appears to be involved in the phosphorylation ofPAL following elicitation (Allwood et al., 1999). The effect ofW7 would indicate a possible role of CDPKs in the apoplasticoxidative burst but it is difficult to reconcile its role with anaction on the ion influxes or the extracellular peroxidase. Ifthere is an involvement of these kinases, it is likely to be on theprovision of the reductant and/or lipid activators, whichwould constitute the third element of the three-componentsystem required for the apoplastic production of hydrogenperoxide (Bolwell et al., 1998). A CDPK was shown to bephosphorylated in elicited tobacco cells but it does not have adirect role on the NADPH oxidase-mediated oxidative burst,judged on the basis of the effect of W7 (Romeis et al., 2000).

Despite previous scepticism about cAMP as a secondarymessenger in plants, here we have a growing body of evidenceof its role in triggering the oxidative burst and later eventssuch as the induction of PAL. Nevertheless, the role of cGMPin this system should also be investigated, since it has beenshown to be a secondary messenger in the nitric oxide (NO)pathway (Dürner et al., 1998; Bolwell, 1999). It has not beenshown that cGMP is specific for NO production and does nottrigger the production of hydrogen peroxide in plants. Inmammals, it has been shown that both, cAMP and cGMP canstimulate cyclic nucleotide-gated cation channels leading toan influx of calcium (Qiu et al., 2000). Probably a similarsystem exists in plants since both cyclic nucleotides inducedan increase of Ca2+ in tobacco (Volotovski, 1998). Moreover,cyclic nucleotide-gated cation channels have been cloned inArabidopsis (Leng et al., 1999; Clough et al., 2000) and tobacco(Arazi et al., 2000). In elicited Arabidopsis cells, NO has beenshown to be produced much earlier than H2O2 and cGMPappears to be required for the induction of NO-dependentPCD (Clarke et al., 2000). Thus, the specificity of cyclicnucleotides in signalling the production of different activatedoxygen species in plants constitutes a new field of investigation.

Acknowledgements

FM was the recipient of a NATO fellowship through theRoyal Society (UK). GPB and DRD gratefully acknowledgefunding from the Leverhulme Trust for the work performedby SLG.

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