activation by fatty acids of the production of active oxygen species by tobacco cells

Activation by fatty acids of the production of active oxygen speciesby tobacco cells

Yves Mathieu1, Marie-Aude Rouet-Mayer1, Hélène Barbier-Brygoo, Christiane Laurière *

Institut des Sciences du Végétal, CNRS, UPR 2355, 1, avenue de la terrasse, 91198 Gif s/Yvette cedex, France

Received 9 October 2001; accepted 18 December 2001

Abstract

Among the different transduction steps leading from hypoosmotic stress to oxidative burst in suspension-cultured tobacco (Nicotianatabacum cv Xanthi) cells, phospholipase activation was evidenced. Using thin layer chromatography and phospholipase inhibitors theinvolved lipase was strongly suggested to be a phospholipase A2 (EC 3.1.1.4). Fatty acids like arachidonate and linolenate stimulated theoxidative response and prevented its inhibition by a phospholipase inhibitor, confirming the physiological relevance of the phospholipaseaction. A production of active oxygen species by plasma membrane vesicles was demonstrated, using two different probes. The producingsystem characterized in vitro was NADPH-dependent, strongly depressed by iodonium diphenyl and activated by fatty acids like theoxidative response assayed in vivo. Several other anionic amphiphiles like SDS were able to mimic the activation of the oxidative responseby fatty acids, both in vivo and in vitro, suggesting that negative charges may be involved in the action mode of fatty acids. Inversely, acationic detergent was an efficient inhibitor of the hypoosmotically induced oxidative burst and the inhibition was fully reversed by SDS.The possible identification of the active oxygen synthase involved in hypoosmotic signalling with a plasma membrane-located NADPHoxidase is discussed. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Active oxygen species; Fatty acids;Nicotiana tabacum; Osmotic stress; Phospholipase; Quinacrine

1. Introduction

The oxidative burst, a rapid and transient generation ofactive oxygen species like superoxide anion and hydrogenperoxide, is one of the earliest responses of plants topathogenic infections. Activation of this response is acentral component of a highly integrated signal system andhas been known for more than 15 years[22]. More recently,induction of oxidative burst by physical stimuli like me-

chanical or osmotic stresses was demonstrated in soybean[40] and tobacco[10] cell suspensions. Activation of theoxidative response by hypoosmotic stress needed openingof Ca2+ channels and anion effluxes, as well as phosphory-lation events[10]. Activation of mitogen-activated protein(MAP) kinases by hypoosmotic stress of tobacco cellsuspensions was also described and suggested to be on theway to the oxidative burst response[9].

The most controversial and challenging issue in studieson the oxidative burst is the question of the molecularidentification of the activated oxygen producing system.Two main possibilities have been presented for the origin ofoxidative burst[6]. The first one involves an homologue ofthe neutrophil NADPH oxidase located in the plasmamembrane and sensitive to the iodonium compounds diphe-nylene iodonium (DPI) or iodonium diphenyl (IDP). Severalgenes encoding homologues of the mammalian respiratoryburst oxidase gp91 subunit have been cloned in plants[17,36] and the expression of oneArabidopsis homologuewas up-regulated by elicitation with harpin[14]. Recently,an O2

.– synthase using NADPH and showing similar phar

Abbreviations: AOS, active oxygen species; BPB, 4-bromophenacylbromide; CDMEA, cetyldimethylethylammonium bromide; DPI, dipheny-lene iodonium; HRP, horseradish peroxidase; IDP, iodonium diphenyl;MAP kinase, mitogen-activated protein kinase; NBD-C6, 2-(6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-hexanoic acid); NBD-C6-HPC,2-(6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine; OG, oligogalacturonides; PLA2, phospholi-pase A2 (phosphatide 2-acylhydrolase); PMA, phorbol 12-myristate 13-acetate; SOD, superoxide dismutase; TLC, thin layer chromatography

* Corresponding author. Fax +33-1-69-82-37-68.E-mail address: [email protected] (C. Laurière).1 YM and MAR contributed equally to this work.

Plant Physiol. Biochem. 40 (2002) 313–324

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macological properties with the animal oxidase was solubi-lized from bean plasma membrane [37]. The second possi-bility relies on recent evidence for the operation of apH-dependent cell-wall peroxidase, which may be blockedby KCN, in the oxidative response induced in plant–bacte-rial interactions. In elicitor-treated bean suspension cells, anessential factor for the appearance of a burst of reactiveoxygen species appears to be a transient alkalinization of theapoplast. The wall-bound peroxidase would be able toproduce H2O2 at neutral pH in the presence of a reductant[5]. In lettuce infected by Pseudomonas syringea, hydrogenperoxide production was more sensitive to inhibitors ofperoxidase than to inhibitors of the NADPH oxidase [3]. Inaddition to these two dominant models, several othermechanisms of active oxygen species (AOS) generationwere also assumed, like a DPI-sensitive NAD(P)H quinonereductase complex activated by calcium [25].

Several major difficulties in the study of systems gener-ating active oxygen species may be underlined. First,distinct mechanisms, differing in their sensitivities to iodo-nium compounds, were evidenced as involved in the gen-eration of oxidative burst by comparative biochemistrystudies [5,32]. Secondly, the amount of O2

.– produced byhorseradish peroxidase (HRP) was shown to be decreased inthe presence of DPI [5]. In addition, the classical peroxidaseinhibitors KCN and NaN3 have significant H2O2 scavengingproperties limiting the interpretation of results [2]. A crucialdifficulty also resides in the lack of a reliable and specificassay of O2

.– synthase activity, different assays possiblyleading to different results according to the probe used [30].As a consequence of these difficulties, only a few cell-freedata are available, although recent studies indicated thegeneration of active oxygen species in the plasma mem-brane [5,32,33].

In plant pathogen interactions, a few studies have beendevoted to the role of phospholipases in soybean suspensioncells. Activation of phospholipase C during elicitation ofoxidative burst was demonstrated [24]. Phospholipase A(PLA) stimulation was also suggested to be important insome elicitor-induced oxidative bursts, although other elici-tors may operate through PLA-independent signalling path-ways [11]. On the other hand, an important role of phos-pholipases A and C in the transduction events that promoteosmotic regulation was documented in guard cells andpulvinar motor cells [12]. In both systems, signallinginvolving phospholipase C induces a reduction in cellturgor. By contrast, activation of PLA2 mediates an increaseof cell turgor in both guard cells and pulvinar motor cells.

In this study, the involvement of enzymatic fatty acidrelease, most probably due to phospholipase A2, in theactivation of oxidative burst induced by hypoosmotic stresswas demonstrated. The possibility that the tobacco generat-ing system induced by osmotic stress may be activated byfatty acids was investigated both on tobacco cells and onplasma membrane preparations. To validate the results, twodifferent probes to assay the activated oxygen production

were used both in vivo and in vitro, with a common probeat the two levels. The action mode of fatty acids wasinvestigated, as well as the physiological relevance of thelipase action. The similarities observed between in vivo andin vitro situations suggested that the oxidative burst gener-ating system may be located at the plasma membrane level,allowing further characterization of this redox component.

2. Results

2.1. Hypoosmotic stress induces phospholipase A2

activation

To evaluate the effect of hypoosmotic stress on phospho-lipase activity, suspension-cultured tobacco cells were trans-ferred to an isoosmotic medium or an hypoosmotic medium,and the kinetics of phospholipase activation were assayedby fluorimetry, using NBD-C6-HPC as a probe (Fig. 1A).After the hypoosmotic stress, significant increase of phos-pholipase activity was observed. However, in the absence ofany change in medium osmolarity (Iso), a continuousincrease in fluorescence was observed, suggesting a high

Fig. 1. Phospholipase A2 activation in response to osmotic stress in tobaccocell suspensions. The substrate NBD-C6-HPC was added for 25 min toaliquots of cell suspension previously equilibrated in isoosmotic medium,before transfer at zero time in isoosmotic (Iso) or hypoosmotic (Hypo)medium. At the indicated times, fluorescence of the extracellular mediumwas directly determined (A) or extracellular components were first sepa-rated by TLC, before quantification of the fluorescence of the NBD-C6spot, identified by comparison with a standard (B). Fluorescence isexpressed relative to the highest fluorescence value (hypoosmotic sampleafter 38 or 40 min) taken as 100. Between 2 to 40 min of signal action, thestandard errors were as following (taking the hypooosmotic sample as 100in each experiment): Iso: 66.5 ± 5.4 (n = 10)(A) and 66.9 ± 18 (n = 2) (B).

314 Y. Mathieu et al. / Plant Physiol. Biochem. 40 (2002) 313–324

level of endogenous phospholipase activity. To discriminatebetween several phospholipase activities which may lead toan increase in fluorescence, the nature of the releasedfluorescent product was determined using thin layer chro-matography (TLC) and lipid standards. A spot correspond-ing to the free fatty acid NBD-C6 was visualized and thecorresponding fluorescence was quantified, at time intervalsafter cell transfer to hypoosmotic or isoosmotic medium(Fig. 1B). Similar time courses of fluorescence increaseswere observed, indicating an activation of PLA by hypoos-motic stress. The fatty acid NBD-C6 is present in the sn-2position of the substrate, suggesting that the hydrolysis ofNBD-C6-HPC is due to a PLA2. Alternatively, it cannot becompletely excluded that the involved enzyme may be alipase cleaving the two fatty acids. Interestingly, linoleicand linolenic acid dominate the sn-2 position of plantphospholipids, and are equivalent to arachidonic acid inanimals [29]. Among the available PLA2 inhibitors, mol-ecules known as efficient on one class only of animal PLA2

were discarded, due to the lack of information on plantPLA2 structures. The first characterized PLA2s from a planttissue are structurally related to the animal secretory PLA2s[35]. Two PLA2 inhibitors, 4-bromophenacyl bromide(BPB) and quinacrine, were chosen and their effects on thehypoosmotically induced release of the free fatty acidNBD-C6 were tested (Table 1). Both inhibitors were able toclearly inhibit the PLA2 activity, with a better efficiency of500 µM BPB, in comparison to 2 mM quinacrine, which hasa low efficiency at lower concentration.

2.2. Hypoosmotically induced oxidative burst is preventedby two phospholipase inhibitors

Hypoosmotically induced oxidative response was ob-served, using scopoletin to monitor H2O2 production (Fig.2). As previously reported [10], a production of H2O2, toolow to produce its accumulation in the extracellular me-dium, was also observed in isoosmotic conditions. This lowproduction, quite variable from one cell sample to another,is due to the mechanical stimulation of cells [10].

The effect of the two inhibitors of PLA2 activity, quina-crine or BPB, on oxidative response was evaluated. Onlypartial inhibition of H2O2 production by quinacrine wasobserved, although BPB completely prevented the oxidative

response induced by transfer of the cells in isoosmoticmedium. In addition to its ability to efficiently inhibit PLA2

activity, quinacrine was also reported to prevent O2.– syn-

thase activity in plants [37]. To know if quinacrine may actdirectly on the AOS-producing system as a flavin inhibitor,its effect on the oxidative response induced by an elicitor ofdefence reaction, oligogalacturonides, was tested (Fig. 3A).For quinacrine concentrations ranging from 0.5 to 3 mM, noinhibition of the oxidative response induced by oligogalac-turonides was observed, although progressive inhibition wasobserved in the case of the hypoosmotic signal (Fig. 3B).This indicates that quinacrine does not act by inhibition ofthe AOS-producing system and suggests that oligogalactu-ronide signalling does not imply phospholipase for theinduction of oxidative burst. To validate this hypothesis, theaction of oligogalacturonide and hypoosmotic signals on thephospholipase activity was evaluated. Fluorescence increasedue to PLA2 activity between 2 and 40 min of signal action

Table 1Inhibition of PLA2 activity by BPB and quinacrine in tobacco cells. The substrate NBD-C6-HPC was added for 25 min to aliquots of cell suspensionpreviously equilibrated in isoosmotic medium, before transfer at zero time in isoosmotic or hypoosmotic medium in the presence of 500 µM BPB or 2 mMquinacrine or in the absence of any inhibitor (control). The inhibitors were also added during the last 10–15 min of the equilibration time. The increase influorescence due to PLA2 activity was monitored from 15 to 40 min after transfer of the cells. The fluorescence of the free fatty acid NBD-C6 was determinedafter TLC analysis. Similar qualitative results were obtained in two (quinacrine) or three (BPB) independent experiments and the quantification of one ofthem is reported here. The highest fluorescence value (hypoosmotic sample without inhibitor) was taken as 100

Control BPB Quinacrine

Fluorescence (relative units) Fluorescence (relative units) Inhibition (%) Fluorescence (relative units) Inhibition (%)

Isoosmotic 41 11 73 19 53Hypoosmotic 100 26 74 51 49

Fig. 2. Inhibition by quinacrine and BPB of the oxidative burst induced byhypoosmotic stress in tobacco cell suspensions. Aliquots of cell suspensionwere equilibrated for 3 h in isoosmotic medium before transfer at zero timein isoosmotic (Iso) or hypoosmotic medium (Hypo) or the same mediacontaining BPB (500 µM) or quinacrine (2 mM). The inhibitors were alsoadded during the last 10–15 min of the equilibration time. H2O2 productionwas measured using scopoletin fluorescence. FW, fresh weight. At 15 min,the standard errors were as following (taking the hypoosmotic sample as100 in each experiment): Iso: 28.3 ± 12.2 (n = 2); Iso+quina: 15.7 ± 11.0(n = 2); Hypo+quina: 25.0 ± 3.6 (n = 2 ); Iso+BPB: 11.0 ± 1.5 (n = 2);Hypo+BPB: 11.0 ± 1.5 (n = 2).

Y. Mathieu et al. / Plant Physiol. Biochem. 40 (2002) 313–324 315

allowed confirmation of the absence of phospholipaseactivation by oligogalacturonides (hypoosmotic sample wastaken as 100): oligogalacturonide signal: 67.9 ± 3.1; isoos-motic signal: 66.5 ± 5.4 (means ± SE for n = 10). Compari-son between oligogalacturonide and hypoosmotic signalswas also achieved using the other phospholipase A inhibitorBPB (Fig. 3C, D). Both oxidative responses were prevented,suggesting that BPB may act on other molecular targets inaddition to its efficiency to inhibit phospholipase A. It mayalso be noticed that the low phospholipase activity occur-ring in the presence of BPB (Table 1) was not accompaniedby an oxidative response (Fig. 3D), suggesting that aminimal fatty acid release is necessary to induce the H2O2

production.

2.3. Fatty acids stimulate the oxidative responseand prevent the inhibition by a phospholipase inhibitor

To confirm the physiological role of the phospholipaseactivity on the cellular oxidative response, the action of twofatty acids, arachidonate and linolenate, on the AOS pro-duction was evaluated. Using scopoletin, significant stimu-lation of the oxidative response was observed, in compari-son to cells transferred in isoosmotic conditions (Fig. 4A).The fatty acid-induced response was, like the AOS-producing system induced by hypoosmotic stress [10],

highly susceptible to IDP (Fig. 4A). This suggests that thesame AOS-producing system is stimulated by both hypoos-motic stress and fatty acids. Dose–response curves for thetwo fatty acids (Fig. 4B) display 2- to 3-fold enhancementof the production for fatty acid concentrations ranging from50 to 300 µM. To further examine the assumption thatquinacrine prevents the oxidative burst by inhibition of thePLA2-mediated fatty acid release, the effect of quinacrineon the oxidative response induced by linolenic acid wastested using epinephrine (Fig. 5). In the presence of quina-crine, a significant enhancement of the adrenochrome for-mation by fatty acids was still observed (C18:3+quina andquina curves). This stimulation is similar to the enhance-ment observed in the absence of quinacrine (C18:3 andcontrol curves), indicating that the stimulation by fatty acidis resistant to quinacrine action.

2.4. A NADPH-dependent production of AOS by plasmamembrane is strongly depressed by IDP and activatedby fatty acids

It was previously shown that the hypoosmotically in-duced AOS-producing system of tobacco cells wasNADPH-dependent [10]. The possibility that a redox com-ponent, using NADPH for the production of AOS may workin the plasma membrane was investigated (Fig. 6A).

Fig. 3. Effect of quinacrine (A, B) and BPB (C, D) on the oxidative responses induced either by oligogalacturonides (OG) or by hypoosmotic stress (Hypo).OG, oligogalacturonides were added at zero time to cells previously equilibrated in their culture medium. Hypo, cells previously equilibrated in isoosmoticmedium were transferred at zero time in hypoosmotic medium. For both signals, increasing concentrations (symbols of increasing sizes) of quinacrine (0.5,1, 2, 3 mM) or BPB (100, 200, 350, 500 µM) were tested. The corresponding assay deprived of inhibitor was called control in each case (Ctl; bold line).The inhibitors were also added during the last 10–15 min of the equilibration time. H2O2 production was measured using scopoletin fluorescence. In somecases, the available amount of scopoletin was fully oxidized as early as 15 min after the stimuli. At 14 min, the standard errors were as following (takingthe control sample as 100 in each experiment): OG + 3 mM quinacrine: 100 ± 7 (n = 2); Hypo + 3 mM quinacrine: 40 ± 3.2 (n = 2); OG + 500 µM BPB:30 ± 16 (n = 2); Hypo + 500 µM BPB: 10 ± 13 (n = 2).


NADPH addition to plasma membrane vesicles allowed anAOS formation, which was detected using epinephrine orcytochrome c probes. The superoxide dismutase- (SOD)dependent cytochrome c reduction, commonly used onneutrophil cells for instance, was unable to give satisfactoryquantification, probably due to the presence of superoxidetraps in the plasma membrane. One possibility is thedismutation of superoxide anions by SOD, which wasevaluated as 30–35 enzyme units/mg protein (data notshown). However, taking advantage of the cytochrome coxidation by hydrogen peroxide, a catalase-sensitive AOSformation was detected, but with a much lower sensibilitythan with epinephrine (Fig. 6A). The effect of IDP on theplasma membrane activity was evaluated using epinephrineand catalase-dependent cytochrome c oxidation (Fig. 6B).Clear inhibition of the NADPH-induced AOS formationwas observed in both assays, with about 50% inhibition by20 µM IDP. This result also suggests the occurrence of anIDP-resistant producing system in the plasma membranefraction.

Fig. 4. Stimulation of H2O2 production by the fatty acids arachidonate (C20:4) or linolenate (C18:3) in tobacco cell suspensions. Fatty acid was added atzero time when cells were transferred in fresh isoosmotic medium after equilibration. A, the corresponding untreated cells were called Iso. When present,the inhibitor IDP (20 µM) was added during the last 10–15 min of the equilibration time and after transfer. The fatty acid concentration used was 120 µMfor C18:3 and C20:4. H2O2 production was measured using scopoletin fluorescence. At 28 min, the standard errors were as following (taking the Iso sampleas 100 in each experiment): C18:3: 300 ± 114 (n = 2); C20:4: 220.4 ± 5.4 (n = 2); IDP: 23.7 ± 10.6 (n = 2); C18:3+IDP: 13.9 ± 10.8 (n = 2); C20:4+IDP:15.5 ± 1.3 (n = 2). B, H2O2 production after 28 min was reported; means ± SE of three independent experiments are reported.

Fig. 5. Fatty acid-induced oxidative response of tobacco cells in thepresence of quinacrine. C18:3 (200 µM), quinacrine (2 mM; quina) or bothproducts (C18:3+quina) were added at zero time to cells equilibrated inbuffered culture medium. H2O2 production was measured using epi-nephrine absorbance. At 60 min, the standard errors were as following(taking the control sample as 100 in each experiment): C18:3: 261 ± 68.8(n = 3); quina: 33.5 ± 4.2 (n = 3); C18:3+quina: 179 ± 29.9 (n = 3).

Fig. 6. Production of active oxygen species (AOS) by plasma membrane vesicles and inhibition by IDP. The reaction was induced by addition of NADPHto membrane proteins and the production was measured using either epinephrine (epi) or cytochrome c (cyt c) as probes. With cytochrome c, bothSOD-sensitive and catalase-sensitive productions were reported (A). B, Due to its better sensibility, the catalase-dependent cytochrome c oxidation was used.To increase the accuracy, assays were performed in the presence of 200 (epi) or 600 µM (cyt c) arachidonate, which increased the AOS formation (see Fig.7). Means ± SE of three independent experiments are reported.


The effect of arachidonate and linolenate on the NADPH-induced AOS formation by membranes was investigatedusing again as probes, epinephrine (Fig. 7A, C) and cyto-chrome c (Fig. 7B, D). Significant stimulation of theproduction was observed in both assays, after addition ofarachidonate or linolenate. The AOS production detectedwith both probes was highly dependent on the arachidonateamount (Fig. 7A, B) with, however, different dose–responsecurves. One possible reason for this discrepancy is thepresence of a high amount of catalase in the cytochrome cassay, shifting the fatty acid/protein ratio towards lowervalues. Differences between relative efficiencies of C20:4and C18:3 were also observed with the two probes, whenthe optimal concentration of arachidonate (200 and 600 µM,respectively, with epinephrine or cytochrome c) was used tocompare the two fatty acids (Fig. 7C, D).

2.5. Several other anionic amphiphile molecules mimicthe activation of the oxidative response by fatty acids

To characterize the action mode of fatty acids on theAOS-producing system, the effect of several other anionicamphiphiles was tested, both in vivo on tobacco cells and invitro on plasma membrane vesicles: myristate, SDS andtaurocholate (Fig. 8). The saturated fatty acid (C14:0) aswell as SDS efficiently stimulate the adrenochrome forma-tion on tobacco cells (Fig. 8A, B, respectively), contrary totaurocholate, which shows almost no action (Fig. 8C).

Again, the stimulated AOS production was clearly pre-vented by IDP (Fig. 8A, B). Very similar results wereobtained with the three anionic amphiphiles added in vitroon plasma membrane vesicles (Fig. 8D). Myristate and SDSstrongly stimulated (around 4-fold) the NADPH-inducedadrenochrome formation, but taurocholate had no signifi-cant effect. These results underline the coherence of theresults observed in vivo and in vitro and suggest that thepresence of negative charges may be important in the actionmode of fatty acids. To better analyse this importance ofnegative charges, an uncharged analogue of myristic acid(myristyl alcohol) was also tested. It was unable to induce astimulation of the oxidative response in cells (data notshown). The possible antagonist effect of a positivelycharged molecule was also investigated (Fig. 9). Thecationic molecule cetyldimethylethylammonium bromide(CDMEA) was able to inhibit the hypoosmotically inducedoxidative burst, and this inhibition was fully reversed bySDS. Interestingly, it can be noticed that SDS did not furtheractivate the hypoosmotically induced response, in agree-ment with a charge effect of released fatty acids duringhypoosmotic stress.

3. Discussion

PLA2 activation by hypoosmotic stress was stronglysuggested in tobacco cells, using a fluorescent probe and

Fig. 7. Stimulation by fatty acids of the active oxygen specie production induced by NADPH in plasma membrane vesicles. Arachidonate (C20:4) orlinolenate (C18:3) were added to the membrane fractions treated by NADPH. The AOS formation was measured using either epinephrine (A, C) orcytochrome c (B, D) as probes. The fatty acid concentrations used (C: 200 µM) and D: 600 µM) correspond to the optimal values observed in A and B.Means ± SE of three (A, B, C) or two (D) independent experiments are reported.


TLC (Fig. 1). Significant enhancement of PLA2 activity waspreviously reported in soybean suspension cells treated byharpin or Verticillium dahliae elicitor [11] and tomato leavesresponding to wounding, systemin or oligogalacturonides[31]. It may be noticed that, according to the plant modelused, oligogalacturonides may be efficient (tomato leaves)or inefficient (soybean or tobacco cell suspensions) inincreasing PLA2 activity. The PLA2 activity was clearly

inhibited by BPB and quinacrine (Table 1), allowing it to beshown that PLA2 action is involved in the occurrence of anhypoosmotically induced oxidative burst (Fig. 2). Quina-crine did not inhibit the oxidative response induced byoligogalacturonides (Fig. 3), suggesting that the activatedoxygen-generating system itself is not a target for theinhibitor. It also indicates that oligogalacturonides andhypoosmotic stress likely induce oxidative response through

Fig. 8. Comparison of the effects of several anionic amphiphiles on the production of AOS by tobacco cell suspensions (A, B, C) or by plasma membranevesicles (D). Myristate (C14:0, 150 µM), SDS (0.005%, w/v) or taurocholate (Tauro 1, 0.005%, w/v or tauro 2, 0.01%, w/v) were added at zero time to thecells previously equilibrated in their culture medium (A, B, C) or to the membrane proteins (D). AOS production of control cells or membrane proteinsdeprived of anionic amphiphile was also reported (Ctl). When present, IDP (20 µM) was added 10–15 min before zero time. AOS production was followedusing epinephrine as a probe both in vivo and in vitro. In A, B and C, the standard errors were as following (taking the control sample as 100 in eachexperiment): C14:0: 213 ± 78 (n = 6); IDP: 11.8 ± 4.5 (n = 3); C14:0+IDP: 18.3 ± 5.7 (n = 2); SDS: 320 ± 150 (n = 8); SDS+IDP: 28.6 ± 6.9 (n = 3); tauro(1): 102 ± 10 (n = 3); tauro (2): 127 ± 15 (n = 2). D, means ± SE of four independent experiments are reported.

Fig. 9. Inhibition of the oxidative response induced by hypoosmotic stress in tobacco cell suspensions by a cationic molecule, CDMEA and reversion of theinhibitory effect by anionic amphiphiles, C14:0 (A) or SDS (B). Aliquots of cell suspension were equilibrated for 3 h in isoosmotic medium before transferat zero time in hypoosmotic medium deprived of effector (Ctl), or the same medium containing CDMEA (27 µM), C14:0 (100 µM), SDS (173 µM), or twoproducts as indicated. When present after transfer, CDMEA was also added 10–15 min before transfer. AOS production was followed using epinephrineabsorbance. At 20 min, the standard errors were as following (taking the control sample as 100 in each experiment): C14:0: 105 ± 15 (n = 2); SDS: 115 ± 20(n = 3); CDMEA: 32 ± 23 (n = 5); C14:0+CDMEA: 92 ± 26 (n = 3); SDS+CDMEA: 87 ± 5 (n = 3).


different signalling pathways, although it cannot be com-pletely excluded that generating systems activated by eachsignal may be different. This probable divergence betweenthe transduction pathways leading to oxidative burst fromelicitor and osmotic signals reported here in tobacco iscoherent with the differences observed in soybean cells,where oligogalacturonides, contrary to two other elicitormolecules, were unable to induce PLA activity [11].

Very little is known in plants concerning both themolecular forms of PLA2 and their functions [29]. A fewactivities have been partially purified and characterized,which did not fall into one of the groups described inmammals. By contrast, mRNAs for putative PLA2 isoformswere recently isolated in rice, which correspond to lowmolecular weight enzymes, structurally related to the animalsecretory PLA2 [35]. Plasma membrane and microsome-associated PLA2 activities have also been reported, but noneof them have been purified yet. PLA2 or its products maystimulate several activities [29], among which H+ ATPase,K+ channel, protein kinases as well as one NADH oxidase,showing a low degree of detergent stimulation. Fatty acids,which are released in the apoplastic fluid after elicitation,are also able to stimulate the hydrogen peroxide productionby the model peroxidase from horseradish [4].

In mammalian cells, the essential requirement of cytoso-lic PLA2 for activation of the NADPH oxidase whichgenerates superoxide anions was demonstrated using PLA2

inhibitors [19] and a p85 cPLA2-deficient model cell line[13]. In the phorbol 12-myristate 13-acetate- (PMA) stimu-lated O2

.– generation, PLA2 and arachidonic acid appearedinvolved in the pathway which also includes protein kinaseC and MAP kinase [26]. Using the MAP kinase kinaseinhibitor PD 98059, Hazan et al. [18] indicated that theMAP kinase ERK (extracellular-signal-regulated kinase)mediated the activations of cytosolic PLA2 and NADPHoxidase stimulated by PMA.

In spite of the importance of AOS production in manyaspects of plant cell physiology, few studies concern itsassay in plasma membrane fractions. This may be due inpart to the difficulties encountered with the chemilumines-cence of lucigenin or luminol [16], two O2

.– and H2O2

probes used these last years, and to the different resultsobtained according to the probe used [30]. Production ofAOS was, however, recently monitored in rose plasmamembrane fractions, where very faint signals were detectedusing nitroblue tetrazolium and lucigenin [30], and also intobacco and grapevine plasma membrane vesicles, usinglucigenin [32]. Interestingly, the O2

.– synthase of plasmamembrane from tobacco cells was sensitive to diphenyleneiodonium when NADPH, and not NADH, was used as anelectron donor [32]. In the present work, quantification ofactivated oxygen produced by plasma membrane vesicleswas achieved through two ways: adrenochrome formationfrom epinephrine and catalase-dependent oxidation of cyto-chrome c (Fig. 6). The first probe epinephrine was used tomonitor superoxide anion generation [7], but was also

shown to be oxidized by hydrogen peroxide in the presenceof HRP [1]. This ability of epinephrine to take into accountboth O2

.– and H2O2 was confirmed using in vitro modelsystems (data not shown): in the presence of xanthine andxanthine oxidase, fully SOD-sensitive formation of adreno-chrome was observed; addition of HRP resulted in anadditional adrenochrome formation, which was resistant toSOD action. This additional formation takes into accountthe H2O2 produced by the xanthine/xanthine oxidase sys-tem. Coherently, catalase-sensitive and SOD-resistant for-mation of adrenochrome was observed, using glucose oxi-dase in the presence of glucose and peroxidase.

Productions of AOS observed in hypoosmotically in-duced tobacco cells and in plasma membrane fractions wereboth NADPH-dependent, sensitive to low concentration ofIDP and activated by fatty acids ([10] and Figs. 4, 6, 7),suggesting that the producing system detected in vitro maycorrespond to the enzyme responsible for the oxidative burstinduced by the osmotic stress. The results observed withtwo different probes appeared coherent, both in vivo, usingscopoletin and epinephrine, or in vitro, using cytochrome cand epinephrine. Differences in the fatty acid concentrationneeded for full activation of the AOS-generating system,according to the probe used in vitro, can, however, benoticed (Fig.7A, B). It can be assumed that the higheramount of fatty acid necessary in the cytochrome c assay isdue to the presence of a catalase which modifies theprotein/fatty acid ratio. More generally, it can be noticedthat the efficiency of fatty acids slightly differ from oneprobe to another. On the contrary, similar efficiency ofC18:3 and C20:4 was observed using epinephrine, both invivo and in vitro (Figs. 4 and 7).

Hypoosmotically induced oxidative burst in tobacco cellsmay be compared to the oxidative response of mammaliancells due to the NADPH oxidase, which is also IDP-sensitive and enhanced by fatty acids. In tobacco plasmamembrane fractions, AOS production was detected in theabsence of cytosolic proteins, coherently to the recentresults concerning the plasma membrane of fully expandedleaves [33]. On the contrary, an absolute need of cytosolicproteins like p47 and p67 in the cell free assays wasreported for most animal cells. However, thyroid subunitgp91 of the NADPH oxidase, which presents a largeN-terminal domain with calcium binding sites very similarto the plant subunit, was recently reported to be deprived ofp47 binding sites [15]. In neutrophils, cis-unsaturated fattyacids, like arachidonate and linoleate, were shown as goodinducers of the NADPH oxidase [20]. The action mode offatty acids appeared to include the action of negativecharges, and other anionic amphiphiles like SDS werecommonly used on mammalian cell-free systems to inducethe superoxide anion production. On the contrary, otherdetergents such as lubrol, triton X100 and sodium cholatewere inactive [34], indicating that fatty acids and SDSmodulate NADPH oxidase by a non-detergent mechanism.Lipophilic and positively charged agents, like CTAB (cetyl


trimethylammonium bromide) inhibited the generation ofO2

.– and this inhibition was abrogated by negativelycharged, but not by non-ionic agents [27]. Quite similarresults were reported in the present study, with an efficientstimulation of the tobacco oxidative response by severalunsaturated and saturated fatty acids as well as SDS, anabsence of stimulation by detergents like taurocholate (Figs.4 and 8). On the other hand, the inhibitory effect of acationic molecule like CDMEA may be antagonized bynegatively charged molecules (Fig. 9), suggesting that thecharge content of the membrane may play an important rolein the regulation of the AOS-producing system. Interestingsimilarities may be also underlined between the O2

.– pro-ductions induced by hypoosmolarity in tobacco and mam-malian cells. In leukocytes, superoxide anion productionwas stimulated by hypotonic conditions in combination withprotein kinase C activators [21]. The swelling-induced O2

.–

generation in neutrophils was suppressed by lipophilicpositively charged agents and this swelling appeared to beaccompanied by a net increase in negative charges at theplasma membrane [27].

Other redox systems may be the targets of the fatty acidswhich activate the oxidative burst. Horseradish peroxidase[4] and plant NADH oxidase [8] activities are both stimu-lated by fatty acids. However, the relative efficiencies ofdifferent fatty acids to increase the H2O2 production by thehorseradish enzyme appear different from that describedhere for the AOS production by tobacco cells or plasmamembrane fractions. Concerning the NADH oxidase, whichpoorly uses NADPH, DPI effect and eventual ability togenerate AOS were not clearly studied. However, thepossibility of a coupling between this fatty acid activableoxidase and a DPI-sensitive quinone reductase constitutesan interesting clue. Production of AOS by a plasma mem-brane enzyme stimulated by quinones was clearly demon-strated [38] but the identity of this system with the DPI-sensitive NAD(P)H quinone reductase remains to bestudied. To further address these questions, the differenttools developed in the present study will be of great help inthe molecular identification of the producing O2

.– systeminvolved in the osmoregulation of tobacco cells.

4. Methods

4.1. Plant cell culture

Tobacco cells (Nicotiana tabacum cv Xanthi) were cul-tured in B5 Gamborg medium with 1 µM 2,4-D and 60 nMkinetin in constant light. Suspension cells were used after 4or 5 d subculturing with 60–100 mg fresh weight ml–1 celldensity.

4.2. Chemicals

All chemicals were purchased from Sigma-Aldrich, ex-cept the probe NBD-C6-HPC which was from MolecularProbes Europe (Leiden, The Netherlands). Stock solutionsof IDP chloride (up to 150 mM), scopoletin (500 mM) andBPB (500 mM) were prepared in DMSO. Stock solution ofepinephrine (20 mM) was prepared in water acidified withH2SO4 (12.7 mM final concentration). Stocks solutions ofpartially acetylated cytochrome c (1 mM), quinacrine(60 mM), HRP (43.3 nkatals µl–1), SOD from bovineerythrocytes (62 u µl–1) and catalase from bovine liver(3.57 µkatals µl–1) were prepared in water. The fatty acids,linolenic acid, sodium arachidonate and myristate wereprepared in absolute ethanol, then diluted in water forappropriate concentration. NBD-C6 (3.4 mM) was dis-solved in ethanol:water (1:1, v/v). Stock solution of NBD-C6-HPC (1.3 mM) was prepared in absolute ethanol, thendiluted in water (ethanol/water 1:1, v/v). Oligogalactur-onides were prepared by limited hydrolysis of homo polyga-lacturonic acid (sodium salt). The hydrolysis (2 g polyga-lacturonic acid in 200 ml acidified water, pH 3.2) wasperformed for 10 h. The acid hydrolysate was dialysed,neutralized and concentrated under vacuum in order toobtain a powder. The oligogalacturonide preparation con-tains a mixture of oligomers (degree of polymerization from1 to 20).

4.3. Osmotic stress

Osmolarity was monitored using a freezing point os-mometer (Roebling, Berlin, Germany) on 100 µl aliquots.Cells were washed and equilibrated for 3–4 h after reparti-tion in aliquots in an ion-poor medium, which is isoosmoticto the culture medium (160 mOsm), containing 10 mMMes–Tris pH 5.2, 1 mM CaSO4 and 150 mM sucrose.Afterwards, extracellular medium was replaced by the samevolume of either hypooosmotic medium (10 mM Mes–TrispH 5.2, 1 mM CaSO4, sucrose-free) leading to 40 mOsmfinal osmotic strength or fresh isoosmotic medium forcontrol cells. Alternatively, osmotic stress was induced in aslightly different way. Cells were adjusted to 160 mOsmwith sucrose and buffered with 10 mM Mes–Tris pH 5.2.After 3–4 h equilibration of the cell suspension aliquots,extracellular medium was replaced by the same volume ofhypoosmotic or isoosmotic mediums described above.

4.4. Other cell treatments

In absence of osmotic stress, cells were also used directlyafter equilibration for 1 h in buffered (50 mM Mes–Tris pH5.2) culture medium. For the comparison of oxidativeresponses induced by elicitor and osmotic signals, oligoga-lacturonides (25 µg ml–1) were added to the cells afterwashing and equilibration in isoosmotic medium.


4.5. Phospholipase A2 assay

Phospholipase A2 activity was monitored using NBD-C6-HPC (excitation wavelength 470 nm, emission wave-length 540 nm) as the fluorescent substrate and followingthe increase in fluorescence on release of NBD-C6 [28]. Toeach aliquot of 6 ml cell suspension, 23 µl of a 1.3 mMstock solution of NBD-C6-HPC in ethanol/water (1:1, v/v)was added (5 µM final concentration). During 25 min ofpreincubation, the substrate transferred spontaneouslyacross the cell wall to plasma membrane. After transfer atzero time to isoosmotic or hypoosmotic medium, 1 mlsamples of cell suspension were taken off at different times,filtered on empty Poly-Prep column (Bio-Rad) and thefluorescence of released NBD-C6 was measured in theextracellular medium.

4.6. TLC analysis

Release of NBD-C6 from the substrate NBD-C6-HPC byPLA2 was monitored by TLC, essentially as described byWittenauer et al. [39] for in vitro assay. Aliquots of 3 ml cellsuspension were prepared for each product assay at a giventime. The substrate NBD-C6-HPC (6.5 µM final concentra-tion) was added 25 min before isoosmotic or hypoosmotictransfer. At zero time, 2.4 ml equilibration medium wasreplaced by 2.4 ml of fresh isoosmotic or hypoosmoticmedium with or without inhibitor. At each time, 1 ml ofextracellular medium was removed from each aliquot andthe enzyme reaction was terminated by the addition of3.5 ml of a methanol/chloroform/heptane (1.42:1.25:1.0;v/v/v) mixture. Samples (1 ml) of the aqueous and organicphases were taken to dryness in a vacuum concentrator(Speed Vac; Fisher Bioblock, Illkirch, France) or under N2

stream, respectively. Residues were dissolved in 50 µlmethanol and spotted on Silica gel TLC plates (Whatman250 µm thick). The plates were developed in a solventsystem of chloroform/methanol/water (65:25:4, v/v/v). Af-ter development, NBD-C6-HPC and NBD-C6 spots werelocated with a UV lamp, scrapped from the plate and the gelsuspended in 2 ml of ethanol/water (1:1, v/v). After remov-ing the gel by centrifugation, fluorescence at 540 nm wasdetermined.

4.7. Oxidative burst assay

Activated oxygen production by tobacco cells was mea-sured using either scopoletin or epinephrine probes. Theoxidative quenching of scopoletin fluorescence (excitationwavelength 350 nm; emission 460 nm) was monitored. Tomeasure H2O2 production rate, scopoletin in DMSO(240 µM final concentration) and peroxidase (218 nkatal-s ml–1 final concentration) were added to 5 ml aliquots ofcell suspension previously equilibrated in buffered iso-

osmotic medium. Scopoletin was progressively oxidizedand the production of AOS was calculated from fluores-cence decrease using a calibration curve (0–220 µM) estab-lished in the presence of H2O2. Aliquots of medium weretaken off at various intervals of time and monitored byspectrofluorimeter SFM25 (Kontron Instruments,Montigny-le Bretonneux, France). The formation of acti-vated oxygen was also determined using oxidation ofepinephrine to the red product adrenochrome. Epinephrinewas added to the cells at zero time (final concentration700 µM). After different times, 350 µl samples of extracel-lular medium, with 10 µl of HCl 0.5 N added, were trans-ferred to a titration microplate and the absorbance moni-tored (Dynatech, Dynex, Issy-les-Moulineaux, France).Absorbance at 490 nm was measured and the adrenochromeformation was quantified, using a calibration curve estab-lished in the presence of HRP (218 nkatals ml–1) and H2O2

(0–400 µM). The absorption spectra of the oxidation prod-ucts of epinephrine by H2O2 in the presence of peroxidaseor O2

.– were identical, indicating that adrenochrome wasproduced in both cases. It was verified that the differentinhibitor molecules used did not modify H2O2 determina-tion assays in the way previously described [10]. However,in the presence of quinacrine, the slope of the calibrationcurve was slightly modified and this new curve was used forall assays containing quinacrine, using scopoletin or epi-nephrine.

4.8. Preparation of a plasma membrane-enriched fraction

Five-day suspension-cultured tobacco cells were washedthree times with a 58 mM sucrose, 20 mM KCl, 5 mMEDTA, 10 mM Mes–Tris pH 5.2 solution and filtered onglass filter. Cells (about 200 g fresh weight) were plasmoly-sed 20 min in prechilled medium containing 400 mM su-crose, 10 mM Mes–Tris pH 7.8, filtered and homogenized ingrinding buffer (1 g cell in 2 ml buffer) through a manualcrusher (five passages). The grinding buffer contained500 mM sucrose, 50 mM Tris, 10% glycerol (w/v), 10 mMNa2 EDTA, 10 mM EGTA, 0.6% PVP (MW 40 000; p/v),and also 5 mM DTT, 10 mM ascorbate, 2 mM PMSF, 0.5 µgml–1 leupeptin which were added just before use. The slurrywas centrifuged 10 min at 10 000 × g, then the supernatantwas centrifuged 36 min at 50 000 × g. The microsomalpellet was suspended in 330 mM sucrose, 5 mM phosphatebuffer pH 7.8 and subjected to two-phase partitioning usingthe PEG-Dextran [6.6% (w/w) of each polymer; 5 mM KCl]method of Larsson [23]. This method results in a prepara-tion containing a high proportion of right-side out vesicles.Plasma membrane pellet was suspended in 250 mM sorbi-tol, 20% glycerol (w/v), 10 mM Mes–Tris pH 6.5, aliquotedand stored at -80 °C until use. After freezing, mixture ofinside-out and right-side out vesicles is present and thisfreezing was preferred to the use of detergents like Brij 58,in order to study the effect of detergents on the AOSproducing system.


4.9. Production of active oxygen species by plasmamembrane vesicles

Two cytochrome c assays were used, measuring eitherthe SOD-dependent cytochrome c reduction or the catalase-dependent cytochrome c oxidation. 1) For the SOD-dependent activity, a standard assay mixture contained170 µl buffer (100 mM HEPES–Tris, 125 mM phosphate–Tris pH 7.8), 100 µM partially acetylated cytochrome c,1 mM NADPH, plasma membrane (10 or 15 µg proteins)and water to make a total volume of 370 µl. Four assaymixtures were prepared and measured simultaneously, withor without SOD (240 units) and controls without plasmamembrane. Increase in A550 in the absence of SOD wasaround 0.014 units of absorbance min–1. Absorbances weremeasured during 5–6 min with DU 70 spectrophotometer(Beckman). Calculation of specific activity assumed anabsorption coefficient of 27.7 mM–1 cm–1. 2) The catalase-dependent cytochrome c assay takes into account theoxidation by H2O2 of the reduced cytochrome c. In thisassay, SOD was added to all assays, in order to convert allsuperoxide anions in H2O2. In the presence of catalase, thereduction of cytochrome c (around 0.024 units of absor-bance min–1) is only due to the plasma membrane cyto-chrome c reductase. When catalase is absent, the assay takesinto account the oxidation of cytochrome c due to H2O2 andthus the cytochrome c reduction was reduced (around 0.012units of absorbance min–1). This difference of reduction ofcytochrome c by plasma membrane in the presence or theabsence of catalase was calculated. Assay mixture was likeabove, except that SOD was present and 25 µkatals ofcatalase (in 370 µl total volume) were added or not. Pro-duction of active oxygen species by plasma membranevesicles was also monitored by epinephrine. Assay mixturewas as previously described for the cytochrome c assay,except that cytochrome c was replaced by 700 µM epineph-rine and A480 was monitored with DU 70 spectrophotometer(Beckman) for 15–20 min. For all assays, a control sampledeprived of NADPH was run simultaneously. The adreno-chrome formation was quantified using an absorption coef-ficient of 4 mM–1 cm–1 [7]. In both cytochrome c andepinephrine assays, the highest slope of the time course ratewas used for calculations. Unless specified, one representa-tive experiment of at least two independent experiments wasillustrated in the figures.

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activation by fatty acids of the production of active oxygen species by tobacco cells

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