ORIGINAL ARTICLE

Isak B. Gerber Æ Dana Zeidler Æ Jorg Durner

Ian A. Dubery

Early perception responses of Nicotiana tabacum cells in responseto lipopolysaccharides from Burkholderia cepacia

Received: 19 June 2003 / Accepted: 29 September 2003 / Published online: 6 November 2003� Springer-Verlag 2003

Abstract Lipopolysaccharides (LPS) are cell surfacecomponents ofGram-negative bacteria and, asmicrobe- /pathogen-associated molecular patterns, have diverseroles in plant–microbe interactions, e.g. LPS are able topromote plant disease tolerance through activation ofinduced or acquired resistance. However, little is knownabout the mechanisms of signal perception and trans-duction in response to elicitation by these bio-active li-poglycans. The present study focused on theinvolvement of LPS isolated from the outer cell wall ofthe Gram-negative bacterium Burkholderia cepacia(strain ASP B 2D) in the molecular mechanisms andcomponents involved in signal perception and trans-duction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Thepurified LPSB.cep. was found to trigger a rapid influx ofCa2+ into the cytoplasm of aequorin-transformed to-bacco cells. An oxidative burst, concomitant with theproduction of reactive oxygen and nitrogen species wasmeasured by chemiluminescence and fluorescence. Theseearly perception responses were accompanied by K+/H+ exchange and alkalinization of the extracellularmedium. Through the use of various inhibitors of theoxidative burst reaction, as well as scavengers of pro-duced radicals, the biochemical basis of the cellular re-sponse to LPSB.cep. elicitation was dissected, elucidatedand compared to that induced by a yeast elicitor. Theseresults suggest that LPSB.cep. interacts with tobacco cells

in a manner different from the response elicited by yeastelicitor.

Keywords Cytosolic calcium influx Æ 2¢,7¢-Dihydrodi-chlorofluorescein-diacetate Æ Lipopolysaccharide ÆNicotiana Æ Oxidative burst reaction Æ Reactive oxygen/nitrogen species

Abbreviations DDC Diethyldithiocarbamate Æ DMSODimethyl sulfoxide Æ DPI Diphenylene iodonium ÆH2DCF-DA 2¢,7¢-Dihydrodichlorofluorescein-diacetate Æ LPS Lipopolysaccharides Æ NAC N-Acetyl-L-cysteine Æ PTIO 2-Phenyl-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide Æ ROS Reactive oxygenspecies Æ YE Yeast elicitor

Introduction

Plants have evolved an effective range of defensive toolsand are resistant towards most pathogens that they areconfronted with in their environment, as they are eithernot hosts for a particular pathogen, or they are hostscontaining resistance genes which allow them to recog-nize specifically distinct pathogen races (Scheel 1998).Plant resistance responses include non-host- and host- orrace/cultivar-specific resistance responses. In host resis-tance, gene-for-gene interactions take place, in whichpathogen invasion is recognized by proteins encoded byplant disease resistance (R) genes that interact withspecific pathogen-derived avirulence (Avr) gene prod-ucts. In addition to gene-for-gene recognition mediatedby R and Avr genes, non-host resistance is achievedthrough the recognition of specific pathogen or plant cellwall-derived signal molecules. These elicitors are low-molecular-weight compounds that are either synthesizedas such, or are liberated from polymeric precursorsduring infection (Somssich and Hahlbrock 1998). Thesedifferent elicitors, such as glycoproteins, peptides, oli-gosaccharides and lipopolysaccharides (LPS), have great

Planta (2004) 218: 647–657DOI 10.1007/s00425-003-1142-0

I. B. Gerber Æ I. A. Dubery (&)Department of Biochemistry, RAU-University,Auckland Park,2006 Johannesburg, South AfricaE-mail: iad@na.rau.ac.zaFax: +27-11-4892401

D. Zeidler Æ J. DurnerInstitute of Biochemical Plant Pathology,GSF—National Research Center for Environment and Health,85764 Oberschleissheim,Germany

structural variety. Complex and largely unresolved per-ception systems exist for these elicitors on the plant cellsurface that activate multiple intracellular defense sig-naling pathways and defense responses involving cyto-solic Ca2+ and H+ ions, reactive oxygen species,jasmonate, salicylic acid, ethylene, and protein phos-phorylation.

The effects of Gram-negative bacterial LPS on mam-malian and insect cells have been well documented. LPSare amphipathic, tripartite molecules consisting of ahydrophobic lipid A portion, a core hetero-oligosac-charide and a repetitive hydrophilic O-antigen polysac-charide region. The lipid A component of LPS has beenattributed to activation of innate host defense systems inboth invertebrate and vertebrate cells via analogouspathways. LPS have also been shown to activate thesynthesis of antimicrobial peptides in Drosophila, as wellas the production of immunoregulatory, inflammatoryand cytotoxic molecules in humans (Schromm et al. 2000;Dow et al. 2000). The lipid A portion of LPS represents ahighly specific indicator or pathogen-associated molec-ular pattern (PAMP) for infection of eukaryotic organ-isms by Gram-negative bacteria. LPS can bind to theToll-like receptor 4, which is capable of recognizingPAMPs via extracellular leucine-rich repeat domains,leading to transduction of ligand-specific signal percep-tion and initiation of an intracellular signaling cascadeand activation of innate immune responses (Alexanderand Rietschel 2001; Nurnberger and Brunner 2002).

In contrast to the well-documented effects of LPS onmammalian cells, their effects on plant cells have beenfar less studied and much remains to be elucidated aboutthe perception of LPS by plants and the subsequentactivation of defense-related responses or the inductionof plant resistance to pathogens in the form of systemicacquired resistance (SAR) and induced systemic resis-tance (ISR). However, evidence is fast emerging impli-cating bacterial LPS in the stimulation of defense-relatedplant responses and enhancement of the plant�s responseto subsequent pathogen attack by pre-treatment withLPS. Newman et al. (2000, 2002) reported that LPScould trigger defense-related responses in plants, whileresistance-related responses such as the synthesis of thesignal molecule salicylate and the oxidative burst are nottriggered. In contrast, Meyer et al. (2001) showed thatLPS from the phytopathogen Xanthomonas campestrispv. campestris could induce an oxidative burst reactionin tobacco cell cultures, while Albus et al. (2001) re-ported the suppression of a YE-induced oxidative burstin alfalfa after treatment with LPS from the symbioticsoil bacterium Sinorhizobium meliloti. These differencesin LPS-mediated responses might be related to theconformation assumed by the molecule (Schromm et al.2000).

Increasing amounts of evidence are emerging toprove the effectiveness of LPS from non-pathogenicplant growth-promoting rhizobacteria (PGPR) as bio-logical control agents to stimulate plants to developdefensive barricades in the absence of pathogens and/or

barriers that are induced upon contact with pathogensto confer ISR in the plants. PGPR-mediated ISR is notcommonly associated with pathogenesis-related (PR)proteins, indicating that PR-proteins are not a pre-req-uisite for the induction of resistance (van Loon et al.1998). LPS extracted from an endophytic strain ofBurkholderia cepacia have been shown to have a pro-tective effect on the Nicotiana tabacum–Phytophthoranicotianae interaction when the plants were pre-treatedwith LPS and subsequently inoculated with zoospores ofthe pathogen. In addition, the LPS were found to inducePR-proteins and indicated an enhanced defensivecapacity due to LPS-preconditioning of the plants(Coventry and Dubery 2001). LPS pre-treatment alsopotentiated the expression of genes encoding PR pro-teins upon subsequent bacterial inoculation (Dow et al.2000). Taken together, these results indicate that LPSinduce quite specific alterations in plant gene expressionand defense- and resistance-related responses, and sug-gest the existence of an important signaling and responsesystem in plant–pathogen interactions that could be partof a broad-spectrum defense mechanism against patho-gens.

B. cepacia is a member of the Burkholderia speciesthat are common inhabitants of the rhizosphere ofimportant crop plants and are considered beneficialorganisms because of their ability to control plant dis-eases (Cao et al. 2001). In this report, the effects of LPSfrom an endophytic strain of B. cepacia on the signalperception and signal transduction responses in Nicoti-ana tabacum cell suspension cultures are described, withspecial attention to the induction of intracellular Ca2+

influxes, extracellular alkalinization, and production ofreactive oxygen and nitrogen species during the oxida-tive burst.

Materials and methods

Plant cell cultures and culture conditions

Nicotiana tabacum L. cv. Samsun cell-suspension cultures wereestablished from seeds obtained from the ARC Research Institutefor Tobacco and Cotton, South Africa, and were grown at 25�C inthe dark in Murashige and Skoog (1962) medium containing0.25 mg l)1 kinetin (Sigma) and 0.50 mg l)1 2,4-dichlorophenoxy-acetic acid (Sigma) on a shaker at 120 rpm. All experiments wereperformed using cells in the logarithmic growth phase, 3–5 daysafter sub-cultivation. Aequorin-transformed tobacco cell cultures(cv. Xanthi; (Plant Biosciences, Norwich, UK) were similarlymaintained.

Extraction and purification of Burkholderia cepacialipopolysaccharides (LPSB.cep.)

An endophytic strain of Burkholderia cepacia, (ASP B 2D), wascultured for 10–14 days at 25�C in sterile liquid nutrient broth(BioLab) at 16 g l)1 on an orbital shaker at 110 rpm. LPS wereextracted from B. cepacia cultures using an adaptation of thephenol–water method described by Westphal and Jann (1965).After cultivation as described previously, the bacteria were centri-fuged at 10,000 g for 10 min and the sediment was washed with

648

150 ml of saline solution (0.9% sodium chloride). The bacteriawere harvested by lyophilization. Lyophilized bacteria (20 g) weresuspended in 350 ml of distilled water at 65–68�C. An equal volumeof 90% phenol, preheated to 65–68�C, was added while stirring.The mixture was kept at 65�C for 15 min and then cooled on ice to10�C. The emulsion was centrifuged at 10,000 g for 30 min, and theupper water phase removed. The lower phenol phase and theinsoluble residue in the interphase were treated again with 350 mlof distilled water at 65–68�C as described above. The water-phaseextracts were combined and concentrated to 100 ml under reducedpressure using a rotary evaporator at 35–40�C. This concentratedsolution was dialyzed (MWCO=12,000) for 3–4 days with fourchanges of distilled water. The dialyzed solution was centrifuged at10,000 g for 20 min and lyophilized. For further purification of thecrude extract, 0.5 g extract was dissolved in 20 ml distilled water.RNase (0.05 mg, Roche) was added and the solution was incubatedat 37�C for 2 h. An equal volume of 90% phenol was added, thesolution vortexed and centrifuged at 10,000 g for 15 min. Theupper water layer was removed and dialyzed against distilled waterfor 1 day, with three changes of water. The dialyzed solutioncontaining the purified LPS was lyophilized and the mass deter-mined. The average yield of LPS was 10% of the total startingweight of lyophilized B. cepacia. No contaminating protein couldbe detected in the pure LPS fractions and the banding pattern uponelectrophoretic analysis was identical to that previously presented(Coventry and Dubery 2001).

Extraction and purification of yeast elicitor

Yeast elicitor (YE) was prepared from baker�s yeast (Saccharo-myces cerevisiae) as described by Meyer et al. (2001). The YE wasdissolved in distilled water at the desired concentrations for allsubsequent experiments involving YE.

Aequorin luminescence-dependent Ca2+ influx measurements

All of the aequorin-transformed tobacco cell cultures (cv. Xanthi)were studied between 16 and 30 h after transfer to fresh media,when the cells responded maximally to elicitor stimulation butdisplayed no activated characteristics in the absence of elicitation.In vivo reconstitution of aequorin was carried out by addingcoelenterazine dissolved in ethanol to 1 ml of suspension-culturedaequorin-transformed tobacco cells at a final concentration of5 lM (Chandra and Low 1997). The cells were incubated with thecoelenterazine for 6 h in the dark on a shaker at 120 rpm. Controlcells were treated similarly with an equivalent volume (42 ll ml)1

cells) of ethanol alone. Luminescence measurements were madeusing a Luminometer 1250 (BioOrbit, Turku, Finland) calibratedto an internal standard of 10 mV. Coelenterazine-treated aequorin-transformed cell cultures (0.5 ml) were transferred to a luminom-eter reaction vial and luminescence was measured every 0.1 s forthe duration of the experiment to permit accurate evaluation of thekinetics of the induced Ca2+ transient. The Ca2+-channel blocker,LaCl3, was dissolved in dimethyl sulfoxide (DMSO) and added tocells at a final concentration of 5 mM, 30 min before the start ofmeasurements, while elicitors (YE and LPS, 100 lg ml)1 each) andthe Ca2+-ionophore A23187 (10 lM final concentration in DMSO)were added at the times indicated in the various figures. At the endof the experiment, all the unconsumed aequorin was discharged byinjecting 250 ll of a solution containing 10% Nonidet P-40 and100 mM CaCl2 into the reaction vial to determine the total lightoutput by the cells.

Luminol-chemiluminescence assay of the oxidative burst reaction

H2O2-dependent chemiluminescence of luminol was performed aspreviously described (Gerber and Dubery 2002). N. tabacum (cv.Samsun) cell suspensions were diluted in pre-incubation medium

[3% w/v sucrose in 0.25· Murashige and Skoog (1962) mediumcontaining 6% w/v Ficoll 400] to a final concentration of 0.2 g ml)1

medium and incubated for 3–5 h. Aliquots of these suspensions(200 ll each) were mixed with 500 ll of 50 mM potassium phos-phate buffer (pH 7.9), and 100 ll of 1.2 mM luminol in potassiumphosphate buffer (pH 7.9), as well as 100 ll of either LPS or YE(100 lg ml)1 final concentration) in a chemiluminescence reactionvial, and transferred to the BioOrbit 1250 luminometer, calibratedas described above. The reaction was started by the addition of100 ll of 10 mM potassium ferricyanide and the H2O2-dependentchemiluminescence produced by the cell cultures was measuredcontinuously over 60 min. Control experiments were treated withpre-incubation medium instead of elicitor.

Detection of reactive oxygen species (ROS) produced duringthe oxidative burst reaction using fluorescence microscopy

To analyze ROS production by fluorescence microscopy, tobaccosuspension cells (cv. Xanthi) were incubated in loading buffer(10 mM Tris/KCl, pH 7.2) with 2¢,7¢-dihydrodichlorofluorescein-diacetate (H2DCF-DA) at a final concentration of 10 lM (addedfrom a 10 mM stock in DMSO) for 10 min. Subsequently, the cellswere transferred to a microscope slide. The slides were placed underthe microscope (Zeiss Axioskop, equipped with standard FITCemission filters), and treated with LPS (100 lg ml)1). Fluorescencepictures were taken with a Canon Powershot G2 digital camera.

H2DCF-DA fluorescence microplate assay of oxidative burstreaction products

The procedure of using H2DCF-DA fluorescence to measure theoxidative burst reaction in phagocytic cells (Rosenkranz et al. 1992),was adapted andmodified to measure the oxidative burst reaction ofelicitor-challengedN. tabacum cell-suspension cultures in automaticfluorometric multiwell microplate assays (Gerber and Dubery2003). N. tabacum (cv. Samsun) cells were diluted in pre-incubationmedium [3% w/v sucrose in 0.25· Murashige and Skoog (1962)medium] to a final concentration of 0.2 g ml)1 medium and incu-bated for 3–5 h at room temperature on a shaker at 120 rpm.Aliquots of these suspensions (1 ml) were subsequently treated withYE or LPS at final concentrations of 100 lg ml)1 each, except forthe YE and LPS concentration-dependent induction of the oxida-tive burst. The induced cell suspensions (200 ll) were transferred tothe wells of a 96-well black fluorescence plate (Labsystems) and20 ll of a 1 mMH2DCF-DA stock in DMSOwas added to obtain afinal concentration of 100 lM H2DCF-DA. Control cells weremock-treated with 100 ll pre-incubation medium instead of elicitor.The fluorescence resulting from the oxidative burst reaction pro-duced by the cells was measured continuously for 1 h on a micro-plate fluorometer (Fluoroskan Ascent; Labsystems) at an excitationwavelength of 485 nm and emission wavelength of 538 nm. Theresulting fluorescence was expressed as relative ROS production.

Inhibition of the oxidative burst and scavenging of reactive oxygen/nitrogen intermediates

The concentration-dependent inhibition of the oxidative burst wasanalyzed by the addition of diphenylene iodonium (DPI; finalconcentrations of 5 lM, 10 lM, 20 lM); and diethyldithiocarba-mate (DDC; final concentrations of 25 mM, 50 mM, 70 mM). DPIand DDC were added to the tobacco cells 1 h prior to inductionwith LPS. The effects of the radical scavengers N-acetyl-L-cysteine(NAC; final concentrations of 250 lM, 500 lM, 750 lM and1 mM] and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(PTIO; final concentration of 200 lM), on the elicitor-inducedoxidative burst were also assessed. NAC was added in combinationwith LPS and PTIO 1 h prior to elicitation by LPS. All of theabove-mentioned inhibitors were dissolved in DMSO.

649

Analysis of the role of Ca2+ in the elicitor-induced oxidative burstreaction

N. tabacum (cv. Samsun) cell cultures were pre-incubated as de-scribed previously and treated with 10 lM of the Ca2+-ionophoreA23187 from a 1 mg ml)1 stock in DMSO, or with 5 mM of theCa2+-channel blocker LaCl3. The Ca2+-ionophore was added tothe cells in combination with LPS (100 lg ml)1), while the Ca2+-channel blocker LaCl3 was added to the cells 30 min prior toelicitation by LPS, and the H2DCF-DA fluorescence assay wasused to monitor ROS production over 60 min.

Extracellular alkalinization measurements

N. tabacum (cv. Samsun) cell-suspension cultures were incubated in4-ml aliquots in 0.2 g ml)1 pre-incubation medium in open vials onan orbital shaker at 120 rpm for 3 h. The pH before elicitation wasconstantly monitored to ensure that the observed elicitor-inducedalkalinization response was in fact due to elicitor treatment and notother stress factors or external components. LPS was then added tothe cells at a final concentration of 100 lg ml)1 and the extracel-lular pH of the culture medium was measured with a combinedglass pH electrode over 60 min. The pH electrode was attached to a�PC Turtle� (Hanna Instruments), which was used as a data col-lector to register pH readings once a minute for the duration of theexperiment. Control cells were treated with 1 ml pre-incubationmedium alone.

Results

LPSB.cep. elicits an intracellular Ca2+ influx

The Ca2+-ionophore A23187 was added, at a finalconcentration of 10 lM, to aequorin-transformed to-bacco cells reconstituted with coelenterazine (5 lM), inorder to assess the effect of a rapid Ca2+ influx on thechemiluminescence output. The Ca2+-ionophore wasadded after 1 min of measurements as indicated by thearrow in Fig. 1a, line i. The total light output of thecoelenterazine-treated cells was determined by theaddition of 250 ll of a cell permeabilization solutioncontaining 10% Nonidet P-40 and 100 mM CaCl2, in aseparate experiment, after 1 min of measurements(Fig. 1a, line ii). This cell permeabilization experimentwas performed at the end of each treatment of the cellswith YE and LPS to verify that the return of the mea-sured chemiluminescence to basal levels was not due todepletion of the coelenterazine-reconstituted aequorinprotein, and in all cases it was found that only a smallfraction of the intracellular aequorin had been con-sumed after elicitor treatment.

The addition of YE to the cells resulted in animmediate increase in chemiluminescence, representingan increase in intracellular Ca2+, with a sharp peak of7.4 mV from the resting value of 2 mV (Fig. 1b). After areturn to near basal levels, a second and much largerCa2+ peak reaching a maximum of 44 mV after 4 minwas observed, and declined back to lower values of9 mV over the next 25 min. Such a bi-phasic responsewas also observed upon treatment of the cell with LPS asshown in Fig. 1b. An initial increase in cytosolic Ca2+

with a sharp peak of 7.7 mV from resting values of 2 mV

was observed to return to basal levels after seconds,similar to the YE-induced response. However, the sec-ond peak for the LPS-induced response started 3 minafter the first peak and reached a much lower maximumof 5.8 mV after 9 min of LPS elicitation. This peak de-clined slowly over the next 10 min back to near basallevels of 3 mV. Comparison of the levels of cytosolicCa2+ increase induced by YE and LPS shows that YE isa much stronger or potent inducer of a cytosolic Ca2+

influx than LPS. The addition of the Ca2+-channelblocker, LaCl3 (5 mM), 30 min before elicitor treatmentresulted in complete abolition of the influx of Ca2+ intothe cytoplasm upon treatment of the cells with YE andLPS. Integration of the chemiluminescence responsesobtained by elicitation of the cells by YE and LPS, andthe inhibition of these reactions by LaCl3, indicated a95% and 94% reduction in intracellular Ca2+ levels forthe YE- and LPS-induced responses, respectively.

Fig. 1a,b Effect of elicitor treatment on the intracellular Ca2+

levels of coelenterazine-treated aequorin-transformed tobacco(Nicotiana tabacum) cells. a Cell permeabilization experimentindicating the maximum light output of the coelenterazine-treatedaequorin-transformed tobacco cells. (i) 10 lM of the Ca2+-ionophore A23187 (m) was added to the cells after 1 min. Anincrease in chemiluminescence from 2.5 mV to 155 mV wasobserved. (ii) The maximum light output of these cells was assessedby the addition of 250 ll of 10% Nonidet P-40 and 100 mM CaCl2(n) at time 1 min. The maximum light output of the cells wasmeasured at 454 mV from a resting value of 2 mV. b IntracellularCa2+ influx induced by YE (100 lg ml)1; n) or LPS (100 lg ml)1;d) The elicitors were added to the cells after 1 min (arrow) and theincrease in cytosolic Ca2+ was monitored over 30 min by takingmeasurements every 0.1 s. Inhibition of the YE and LPS-inducedCa2+ pulse was evaluated by the addition of 5 mM LaCl3 (m) tothe cells 30 min before addition of YE or LPS. Control cellsreceived pre-incubation medium alone (¤)

650

Recognition of LPSB.cep. triggers an oxidative burstreaction

The LPS- and YE-induced oxidative burst reactions intobacco cell cultures were monitored over 60 min usingH2O2-dependent chemiluminescence of luminol. TheLPS-induced oxidative burst started 5 min after LPSaddition and reached a maximum (±SE) of 1.24 lMH2O2 (±0.2 lM) after 10 min and showed a delayedreturn to near basal levels after 40 min (Fig. 2). The YE-induced oxidative burst started slightly before 5 minafter YE addition, reached a maximum of 4.11 lMH2O2 (±0.27 lM) at about 10 min, and returned tonear basal levels after 40 min. The addition of the H2O2-degrading enzyme, catalase (300 U ml)1), to the LPS-and YE-induced reactions almost completely abolishedthe measured oxidative burst reactions. Catalase inhib-ited the LPS-induced oxidative burst by 91% and theYE-induced reaction by 97% (data not shown).

Detection of the oxidative burst by fluorescencemicroscopy

Tobacco suspension cells were loaded with H2DCF-DA(10 lM), placed on a microscope slide featuring anincubation well and analyzed with epifluorescencemicroscopy. Figure 3 shows a bright-field image of a cellcluster after loading with H2DCF-DA and subsequentincubation in fresh loading buffer, but before elicitationwith LPS (100 lg ml)1). Addition of LPS during imageacquisition resulted in a rapid burst of fluorescence,indicative of a massive ROS production. Previously, wehad used confocal laser scanning microscopy to definesubcellular localization of nitric oxide in tobacco leaves(Foissner et al. 2000). Using H2DCF-DA, we could notidentify distinct subcellular sites of ROS-production(data not shown). ROS (and especially H2O2) are highlydiffusible molecules and are not even contained withinthe cells at later time points (data not shown). Fur-thermore, H2DCF-DA or other fluorescein derivativesmight preferentially accumulate in specific cellularcompartments. H2DCF-DA is a single-wavelengthprobe, and no adjustments can be made for differentialaccumulation of the probe within the cell. Thus, detailedanalyses of the LPS-induced oxidative burst, includinginhibition studies, were done with the microplate fluo-rometer.

H2DCF-DA fluorescence microplate assay of oxidativeburst products

H2DCF-DA fluorescence was used to measure accu-mulated ROS produced by the oxidative burst reactionof elicitor-challenged N. tabacum cell suspension cul-tures. The effect of increasing LPS concentrations addedto the tobacco cell cultures was analyzed to determinethe lowest LPS concentrations that would yield maxi-mum ROS production. ROS production due to con-centrations less than 50 lg ml)1 LPS did not result in

Fig. 2 Luminol-chemiluminescence measurements of the oxidativeburst reaction by tobacco cells after elicitation by LPS (m) or YE(n) at final concentrations of 100 lg ml)1 each. The experiment wasperformed with Ficoll 400 in the reaction medium to keep the cellsin suspension for the entire time course of the reaction (60 min),giving a much more accurate determination of the oxidative burst.Control cells (¤) were treated with pre-incubation medium insteadof elicitor

Fig. 3 Time course of theoxidative burst as detected byintracellular H2DCF-DAfluorescence in tobacco cellsafter LPS stimulation. Tobaccosuspension cells were loadedwith H2DCF-DA (10 lM),washed, treated with LPS(100 lg ml)1) and examined byfluorescence microscopy.Shown are a brightfield imageof a cell cluster, and a time-lapse series for the first 9 min ofthe oxidative burst

651

ROS levels appreciably higher than basal ROS levels ofcontrol cells. An initial sharp increase in ROS produc-tion from 50–100 lg ml)1 LPS was observed, afterwhich no notable increase occurred between concentra-tions of 200–400 lg ml)1 (Gerber and Dubery 2003). AnLPS concentration of 100 lg ml)1 was sufficient to in-duce high levels of ROS in the cell cultures. In referenceto these results, and results previously obtained byCoventry and Dubery (2001), as well as a possible toxiceffect that higher LPS concentrations might have on thecells, all further experiments utilized LPS at100 lg ml)1.

The assay was also used to dissect various aspects ofthe tobacco cell cultures� interaction with YE and LPS.This included measurements of the maximum produc-tion of ROS by YE- and LPS-induced tobacco cells,inhibition of the oxidative burst by various compoundsand the role of calcium during the production of theoxidative burst.

Inhibition of the LPSB.cep.-induced oxidative burst

The data obtained from the concentration-dependentinhibition of the YE-induced oxidative burst by di-phenylene iodonium (DPI), diethyldithiocarbamate(DDC) and the reduction of ROS levels by N-acetyl-L-cysteine (NAC) were used as a reference to obtain thevarious concentrations of these inhibitors giving maxi-mum inhibition/suppression of ROS production. Inhib-itor concentrations giving maximal suppression of theYE-induced oxidative burst were 20 lM DPI and70 mM DDC (data not shown). The experiments wererepeated with LPS (100 lg ml)1) using these above-mentioned concentrations of DPI and DDC.

The maximum ROS production of the LPS-induced,DPI treated cell cultures was substantially lower thanthe basal levels of ROS of the control experiments,indicating almost total inhibition of ROS production byDPI (Fig. 4). Although substantial suppression of LPS-induced ROS production by DDC was observed, thelevels of ROS were still higher than that of the controlexperiments (Fig. 4).

Scavenging of reactive oxygen/nitrogen species

The effect of NAC on the H2DCF-DA fluorescence as-say of the LPS-induced oxidative burst in tobacco cellcultures was examined over 60 min. Simultaneousaddition of NAC (1 mM) and LPS (100 lg ml)1) to thecells resulted in a drastic reduction of the measurablelevels of ROS to levels well below that of the controlexperiments (Fig. 4).

Possel et al. (1997) employed H2DCF-DA fluores-cence to determine the specificity and sensitivity of thisfluorescent marker for various compounds oxidizingdichlorofluorescein (H2DCF) and reported that H2DCFwas much more sensitive to peroxynitrite (OONO))

oxidation than to any other compound tested. Theirfindings prompted an investigation in the present studyinto the detection of nitric oxide (NO) during elicitationof tobacco cells by LPS, using the H2DCF-DA fluores-cence assay. The effect of the NO-scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO,200 lM), on the YE- and LPS-induced oxidative/NObursts was assessed. PTIO is a stable radical scavengerfor NO as well as OONO), and has a substantialinhibitory activity against the biological actions of thesereactive nitrogen species (Pfeiffer et al. 1997). PTIO re-duced the maximum ROS production during the YE-induced oxidative burst by 97% (data not shown) andthe LPS-induced ROS production by 95% (Fig. 4),indicating that NO plays a major role in contributing tothe measured fluorescence during the H2DCF-DA fluo-rescence assay.

Ca2+ influx is required for the LPSB.cep.-inducedoxidative burst

The role of Ca2+ in signal transduction leading to anoxidative burst signal was examined in N. tabacum cellcultures by using a modulator of Ca2+ entry and aCa2+-channel blocker. The Ca2+-ionophore A 23187(10 lM) was added to the cells in combination with ei-ther YE or LPS and the effect on the oxidative burst wasmeasured using the H2DCF-DA fluorescence assay. Itwas found that treatments with the Ca2+-ionophoreadded to the cells in combination with either YE (datanot shown) or LPS (Fig. 5) had little effect on the elic-itor-induced oxidative burst. In contrast, addition of thegeneral Ca2+-channel blocker LaCl3 (5 mM) inhibited

Fig. 4 Suppression/inhibition of the LPS-induced oxidative burstreaction by the superoxide dismutase inhibitor, DDC, the NADPHoxidase inhibitor, DPI, the ROS scavenger NAC, and the nitricoxide scavenger, PTIO. N. tabacum cell cultures were pre-incubatedwith DDC (70 mM), DPI (20 lM) or PTIO (200 lM) for 1 h priorto elicitation by LPS (100 lg ml)1). NAC (1 mM) was added to thecells in combination with LPS. The H2DCF-DA fluorescence of theoxidative burst was continually measured for 1 h on a microplatefluorometer at excitation/emission wavelengths of 485 nm/538 nm.The maximum relative ROS production at time period 60 min isindicated. Mean values ± SE (n=9)

652

the elicitor-stimulated oxidative burst reaction. Inhibi-tion of the LPS-induced oxidative burst reaction byLaCl3 only started to reach levels lower than the LPS-induced reaction alone after 35 min and the ROS levelsdeclined over the next 25 min to levels close that ofcontrol experiments.

LPSB.cep.-induced extracellular alkalinization

Treatment of the tobacco cell cultures with 100 lg ml)1

LPS resulted in an LPS-induced pH burst that started ata pH of 5.7 and reached a maximum pH of 6.0 after10 min. This pH was maintained over the next 10 minbefore declining by only 0.1 pH units to a pH value of5.9, which was maintained for the duration of theexperiment with very slight fluctuations (Fig. 6). An

extracellular pH increase was also observed upontreatment of the cells with YE (100 lg ml)1). YE-treat-ment of the cells resulted in a rapid pH burst from aresting pH value of 5.7 to a maximum pH of 6.3 within10 min. After maintaining this value for a further10 min, the pH started to decline to almost basal levelsover the next 30 min and remained at this pH (5.8) forthe duration of the experiment. This extracellular alka-linization, due to a K+ efflux and H+ influx across thecellular membrane, is one of the earliest cellular re-sponses involved in signal perception and signal trans-duction leading to or associated with the oxidative burst(Otte et al. 2001). The constitutive pH level of the cul-ture medium always averaged 5.7±0.1. Control cellswere treated with pre-incubation medium alone andshowed only very slight variation in pH during the entireexperiment.

Discussion

Ca2+ influx occurs in response to LPSB.cep. elicitation

Ca2+ fluxes are known to be involved in stimulus–re-sponse pathways and signal transduction of regulatoryprocesses in plants (Trewavas and Knight 1994). Plantstransformed with the Ca2+-sensitive luminescent pro-tein, aequorin, have been exploited to quantitate intra-cellular Ca2+ fluxes accompanying diverse stimuli,including elicitors (Knight et al. 1991; Chandra and Low1997; Trewavas 2000). The amplitude, duration, fre-quency and location of the Ca2+ signal, as well as theinteractions of Ca2+ with Ca2+-responsive proteinssuch as calmodulin and signaling pathways, direct thedownstream signaling events (Trewavas 2000).

Suspension-cultured aequorin-transformed tobaccocells were employed to examine the effect of LPS and YEon intracellular Ca2+ levels and the involvement of theseelicitor-induced Ca2+ influxes in the production of ROSduring the oxidative burst, since cytosolic Ca2+ has beenshown to be a major second messenger in the onset ofthe oxidative burst and other defense reactions (Knightet al. 1991; Mithofer et al. 1999). The addition of LPSand YE to the cells resulted in the induction of a bi-phasic response for both elicitors; however, the kineticsof their individual responses differed considerably. Thefirst peak is thought to represent the influx of extracel-lular Ca2+ into the cytoplasm and the second peakrepresent the release of Ca2+ into the cytoplasm frominternal stores in the organelles (Cessna and Low 2001).

The effect of LPS from the phytopathogen Xantho-monas campestris pv. campestris (LPSX.c.c.) and YE, onthe level of cytoplasmic Ca2+ in transgenic aequorin-transformed tobacco cells was analyzed by Meyer et al.(2001) and a similar pattern of cytosolic Ca2+ increaseafter YE-elicitation was observed in this study. In con-trast to this YE-induced response, Meyer et al. (2001)found that the addition of LPSX.c.c induced a long-duration Ca2+ signal. This Ca2+ signal showed no

Fig. 5 The role of Ca2+ during the elicitor-induced oxidative burstreaction. The Ca2+-ionophore A23187 (10 lM; m) and the Ca2+-channel blocker LaCl3 (5 mM; x) were added to suspension-cultured N. tabacum cells and the cells were subsequently treatedwith 100 lg ml)1 LPS. The effect of the Ca2+-ionophore andCa2+-channel blocker on the relative ROS production of elicitor-treated tobacco cells was monitored over 60 min with the H2DCF-DA microplate assay. Relative ROS production by cells inducedwith LPS alone (n) is also indicated. Control cells (¤) were treatedwith pre-incubation medium alone. LaCl3 was added to the cells30 min before elicitation with LPS

Fig. 6 Extracellular pH increase of the culture medium of N.tabacum cells after elicitation by YE (¤) or LPS (n) at 100 lg ml)1

each. Control cells (m) were treated with pre-incubation mediumalone. The extracellular pH increase was measured with acombined glass electrode over 60 min after elicitation. Resultsrepresent the average of at least three independent experiments

653

transient, but fluctuated slightly at a lower magnitudethan that induced by YE. Similar results were obtainedupon treatment of cells with LPSB.cep., regarding theonset time of the response and the fact that the responsewas much lower than the YE-induced intracellular Ca2+

levels; however, an initial Ca2+ transient was observedafter LPSB.cep.-elicitation of the cells. Moreover, Ca2+

pulses trigger downstream signaling events that includethe activation of Ca2+-regulated protein kinases, whichare required for the stimulation of the oxidative burst(Cessna and Low 2001).

LPSB.cep. induces an oxidative burst

The oxidative burst can be detected based on the H2O2-dependent chemiluminescence of luminol (Roswelll andWhite 1978). The chemiluminescence during LPS- andYE-elicitation of tobacco cells was due to the productionof extracellular H2O2 during an oxidative burst reaction,as indicated in Fig. 2. Addition of catalase to the reac-tions reduced the measured chemiluminescence for boththe LPS- and YE-elicited cells by more than 90%. Cat-alase cannot cross the cell membrane and can only de-grade/decompose extracellular H2O2, and since H2O2

production is a major component of the oxidative burstreaction, it can be concluded that LPS and YE inducesubstantial oxidative burst reactions in tobacco cellcultures. The results are consistent with H2O2 beinggenerated initially in the apoplast with subsequent rapidmovement into the cytoplasm.

Detection of the oxidative burst reaction products

2¢,7¢-Dihydrodichlorofluorescein-diacetate is a non-po-lar compound that readily diffuses into cells where it ishydrolyzed by intracellular esterases to the non-fluo-rescent derivative 2¢,7¢-dihydrodichlorofluorescein(H2DCF), which is polar and trapped within the cells. Inthe presence of intracellular oxidants, inter alia H2O2,this compound is oxidized to the highly fluorescentcompound 2¢,7¢-dichlorofluorescein (DCF; Keston andBrandt 1965). Treatment of tobacco cells with cryptog-ein resulted in a fast ROS burst as detected by H2DCF(Allan and Fluhr 1997). Here, elicitation of tobacco cellsby LPS revealed strong H2DCF fluorescence, indicatingthe presence of ROS (Fig. 3). The overall time courses ofROS after cryptogein and LPS activation were almostidentical, with the earliest responses detectable withinthe first 3 min and full activation between 6 and 12 min(Fig. 3; Allan and Fluhr 1997)

Cumulative ROS production due to the oxidativeburst in YE- and LPS-elicited tobacco cell-suspensioncultures was monitored and recorded non-destructivelyfor time-course experiments. Analysis of the oxidativeburst and ROS production in this manner lends itself todissection of the signal transduction pathways involvedin the interaction of the N. tabacum cells with the YE

and LPS elicitors, because of its ease of assay and rapiddetermination of the effects of ROS scavengers (NAC),inhibitors of ROS production (DPI and DDC), and thenitric oxide/peroxynitrite scavenger (PTIO) on the oxi-dative burst.

The origin of the LPSB.cep.-induced oxidative burst

YE appears to be a more potent elicitor of the oxidativeburst in tobacco cells than LPSB.cep., as indicated bycomparison of the maximum levels of ROS productionfor the YE and LPS-induced oxidative burst reactions,e.g. in Fig. 4. Consequently, the data obtained from theinhibition of the YE-induced oxidative burst with DPI(an inhibitor of mammalian NADPH oxidases; O�Don-nel et al. 1993), DDC (an inhibitor of superoxidedismutases; Heikkila et al. 1976), and NAC (a scavengerof free radicals), were used as a reference to obtain thevarious concentrations of these inhibitors that wouldalso give maximum inhibition/suppression of the LPS-induced ROS production (Gerber and Dubery 2003).The experiments were repeated with LPS, using theinhibitor concentrations that gave maximum inhibitionof the YE-induced oxidative burst.

Tobacco cells were pre-incubated with DPI and DDCfor 1 h prior to elicitation by LPS, while NAC was ad-ded to the cells in combination with LPS (Fig. 4). DDCsuppressed LPS-induced ROS production by 40%, whileDPI and NAC suppressed ROS production by 83% and95%, respectively, to levels well below that of the controlexperiments (Table 1).

The difference in ROS production between LPS-in-duced and DPI pre-treated cell cultures indicates thatsuperoxide anion radicals, the reaction product of aputative NADH oxidase, are indeed a ROS componentcontributing greatly to the fluorescence reaction. H2O2 istherefore not solely responsible for the measured ROS,but O2

)Æ, and possibly other ROS species as well. Theaddition of catalase, which can only act extracellularly,also did not appreciably decrease the levels of ROSproduction for the YE- and LPS-induced oxidativebursts (data not shown), thus further supporting theabove statement.

Since ROS production was not completely abolishedby the addition of DPI and DDC, it indicates that bothH2O2 and O2

)Æ are being produced during the YE- andLPS-induced oxidative burst reactions in N. tabacum cell

Table 1 Summary of the inhibition of the YE- and LPS-inducedoxidative bursts in Nicotiana tabacum cells by various inhibitors/scavengers, as determined by the H2DCF-DA fluorescence assay

Elicitor % Inhibition by:

NAC DPI DDC PTIO(1 mM) (20 lM) (70 mM) (200 lM)

YE 97.5 90 80.5 97LPS 95 83 40 95

654

cultures, and that both of these compounds act as oxi-dants of H2DCF to the fluorescent DCF. It is incorrectto assume that this fluorescence probe reacts only withH2O2, as Hempel et al. (1999) found that H2DCF acts asa detector for a broad range of intracellular oxidizingreactions, including peroxynitrite (ONOO)). Recentevidence has indicated that NO is released by plant cellsalong with ROS during the oxidative burst. This �NOburst� drives a diffusion-limited reaction with O2

)Æ togenerate OONO). Although peroxynitrite is incapableof inducing hypersensitive cell death, NO can reactsynergistically with H2O2 to trigger hypersensitive celldeath (Delledonne et al. 2001). These findings encour-aged further investigations, in the present study, into theproduction of NO by the LPS-induced tobacco cells(Fig. 4). The 95% inhibition of the LPS-induced ROSproduction by PTIO suggests that LPS induces NOproduction in addition to ROS, thus generatingONOO). This ONOO) constitutes a major fraction ofthe intracellular oxidants, and in addition, this reactionreduces the amount of O2

)Æ and hence H2O2, available toreact with the H2DCF, thus lowering the measured flu-orescence. Evidence was recently supplied to support anNO burst, reminiscent of the oxidative burst in tobaccocells, in response to elicitation by cryptogein (Foissneret al. 2000). The overall time courses of ROS produc-tion, as observed in this study, and NO production aftercryptogein elicitation were almost identical. Synchro-nized bursts suggest a concerted action of ROS and NO(McDowell and Dangl 2000). To our knowledge, this isthe first report of a possible NO burst in plant cells inresponse to elicitation by bacterial LPS.

Ca2+ is involved in the LPSB.cep.-induced oxidativeburst

Treatments with the Ca2+-ionophore had little or noeffect on the kinetics or the maximum levels of ROSproduction for the YE-induced (data not shown) or theLPS-induced oxidative bursts (Fig. 5) during the entire60 min of the experiment. In contrast, a very interestingphenomenon occurred when the cells were treated withthe general Ca2+-channel blocker, LaCl3. The additionof LaCl3 to the cells blocks the discharge of Ca2+ frominternal stores in the organelles and the influx of Ca2+

from external stores (Cessna and Low 2001). Elicitationof the oxidative burst by LPS in the presence of La3+

resulted in an initial production of ROS for the first25 min of the reaction, after which the levels starteddeclining over the next 10–15 min to ROS levels lowerthan that of the LPS-induced reaction alone, and furtherdeclining slowly over the next 20 min to levels stillslightly higher than the ROS levels of the control cells(Fig. 5). LaCl3 treatment of the cells in combination withYE-elicitation resulted in the same response (not shown).

The data presented here demonstrate that Ca2+

present in the cytoplasm at the onset of the reaction, issufficient to initiate the oxidative burst reaction, but

these Ca2+ levels need to be maintained in order tosustain the oxidative burst induced by YE and LPS. Itwould seem that as the cytoplasmic Ca2+ reacts with thecellular components necessary to induce or maintain theoxidative burst, the already low cytoplasmic Ca2+ con-centration is depleted because the Ca2+-channel blockerprevents further influx of Ca2+ into the cytoplasm frominternal or external stores. The prevention of an influx ofCa2+ into the cytoplasm from internal stores has anegative effect on the Ca2+-dependent componentsneeded for maintaining the LPS-induced oxidative burst.Ca2+ does not appear to be essential for the transduc-tion of all oxidative burst signals and other, simulta-neous parallel signaling events or factors, in addition toCa2+, can modulate the strength of an oxidative burstsignal (Chandra and Low 1997). In accordance, Cessnaand Low (2001) found that the oxidative burst does notdepend on the influx of external Ca2+, but is insteadgenerally mediated by the release of internal Ca2+ in amanner dependent on the proper function of kinases andanion channels, and that these Ca2+ pulses triggerdownstream signaling events including the activation ofcalcium-dependent protein kinases required for the oxi-dative burst.

LPSB.cep.-elicitation results in extracellularalkalinization

Several reports have been published regarding elicitor-induced alkalinization of the extracellular culture med-ium of plant cells (Felix et al. 1993, 1999; Pachten andBarz 1999; Albus et al. 2001). Otte et al. (2001) reportedthat inhibition of extracellular alkalinization after com-plete inhibition of the oxidative burst by DPI indicatesthat the elicitor-induced increase in extracellular pH ismainly based on proton consumption for O2

)Æ dismuta-tion to H2O2 and further suggested a simultaneousdeactivation of the plasma-membrane H+-ATPase dur-ing the oxidative burst and extracellular alkalinization.

In the present study, clear differences in the magni-tude and duration of the respective LPS- and YE-in-duced extracellular pH bursts were observed (Fig. 6).Although YE induced an extracellular pH burst ofgreater magnitude than LPS, the YE-induced burst ter-minated and declined back to almost basal pH levels ofthe control experiments, while the LPS-induced burstwas a more sustained and long-duration burst that didnot decline back to control pH levels for the entire timecourse of the experiment. During the measurement ofcytoplasmic Ca2+ influxes after YE and LPS (Fig. 1b)elicitation of the cells, it was also observed that the YE-induced influx of Ca2+ was of a greater magnitude thanthat induced by LPS, but the LPS-induced Ca2+ influxstarted later than that of YE. In addition, although theLPS-induced Ca2+ influx was lower than that inducedby YE, it was a more sustained, long-duration signalmaintaining low levels of Ca2+ for the duration of theexperiment.

655

In conclusion, results obtained in this study provideevidence that B. cepacia LPS has specific effects on thebiochemical perception systems involved in the interac-tion of plant cells with beneficial and/or pathogenicbacteria. The documented signal perception and trans-duction response to LPS contributes substantially tounderstanding the biochemical basis of the mechanism ofaction of LPS as an elicitor involved in the triggering ofplant defense responses and contributes towards relatingthe activation of mammalian innate immunity to similarresponses in plants. However, it is not yet known in whatform LPS is presented to the cells during plant–bacteriainteractions. LPS is either perceived intact as part of thebacterial surface, or released as micelles (Beveridge1999). In all probability, the first step in the signal per-ception and transduction of the LPS-induced defenseresponses in N. tabacum cells is the interaction of LPSwith a plant cell wall- or plasma membrane-boundreceptor or binding protein. Accumulating evidence frommammalian systems indicates that LPS interacts with atransmembrane receptor(s) responsible for signal trans-duction. Following LPS stimulation, Toll-like receptorsand a signaling complex of receptors are believed to beresponsible for transduction of the LPS signal throughan LPS-activation cluster (Triantafilou and Triantafilou2002). To date, no LPS receptors or binding complexeshave been identified in plant cells.

Acknowledgements We thank Dr. T. Nurnberger (Institute forPlant Biochemistry, Halle, Germany) for valuable advice andcooperation and Dr. J. Jones (John Innes Research Institute,Norwich, UK) for the aequorin-transformed cells. This researchwas supported by grants from the Deutsche Forschungsgemeins-chaft, DFG, SPP 1110, Innate Immunity (J.D.), the South AfricanNational Research Foundation, NRF, (I.A.D.) and the Volkswa-gen Foundation (I.A.D.).

References

Albus U, Baier R, Holst O, Puhler A, Niehaus K (2001) Sup-pression of an elicitor-induced oxidative burst reaction inMedicago sativa cell cultures by Sinorhizobium meliloti lipo-polysaccharides. New Phytol 151:597–606

Alexander C, Rietschel ET (2001) Bacterial lipopolysaccharidesand innate immunity. J Endotoxin Res 7:167–202

Allan AC, Fluhr R (1997) Two distinct sources of elicited reactiveoxygen species in tobacco epidermal cells. Plant Cell 9:1559–1572

Beveridge TJ (1999) Structures of Gram-negative cell walls andtheir derived membrane vesicles. J Bacteriol 181:4725–4733

Cao H, Baldini RL, Rahme LG (2001) Common mechanisms forpathogens of plants and animals. Annu Rev Phytopathol39:259–284

Cessna SG, Low PS (2001) Activation of the oxidative burst inaequorin-transformed Nicotiana tabacum cells is mediated byprotein kinase- and anion channel-dependent release of Ca2+

from internal stores. Planta 214:126–134Chandra S, Low PS (1997) Measurement of Ca2+ fluxes during

elicitation of the oxidative burst in aequorin-transformed to-bacco cells. J Biol Chem 272:28274–28280

Coventry HS, Dubery IA (2001) Lipopolysaccharides from Burk-holderia cepacia contribute to an enhanced defense capacity andthe induction of pathogenesis-related proteins in Nicotianatabacum. Physiol Mol Plant Pathol 58:149–158

Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal inter-actions between nitric oxide and reactive oxygen intermediatesin the plant hypersensitive disease resistance response. ProcNatl Acad Sci USA 98:13454–13459

DowM,NewmanM-A, von Roepenack E (2000) The induction andmodulation of plant defense responses by bacterial lipopolysac-charides. Annu Rev Phytopathol 38:241–261

Felix G, Regenass M, Boller T (1993) Specific perception ofsubnanomolar concentrations of chitin fragments by tomatocells: induction of extracellular alkalinization, changes in pro-tein phosphorylation, and establishment of a refractory state.Plant J 4:307–316

Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitiveperception system for the most conserved domain of bacterialflagellin. Plant J 18:265–276

Foissner I, Wendehenne D, Langebartels C, Durner J (2000) Invivo imaging of an elicitor-induced nitric oxide burst in to-bacco. Plant J 23:817–824

Gerber IB, Dubery IA (2002) Enhanced detection of chemilumi-nescence intensity during an oxidative burst reaction in sus-pension cultured Nicotiana tabacum cells through the use ofFicoll. Anal Biochem 306:152–154

Gerber IB, Dubery IA (2003) Fluorescence microplate assay for thedetection of oxidative burst products in tobacco cell suspen-sions using 2¢,7¢-dichlorofluorescein. Methods Cell Sci (in press)

Heikkila RE, Cabbat FS, Cohen G (1976) In vivo inhibition ofsuperoxide dismutase in mice by diethyldithiocarbamate. J BiolChem 251:2182–2185

Hempel SL, Buettner GR, O�Malley YQ,Wessels DA, Flaherty DM(1999) Dihydro-fluorescein diacetate is superior for detectingintracellular oxidants: comparison with 2¢,7¢-dichlorodihydro-fluorescein diacetate, 5 (and 6)-carboxy-2¢,7¢-dichloro-dihydro-fluorescein diacetate, and dihydrorhodamine 123. Free RadBiolMed 27:146–159

Keston AS, Brandt R (1965) Fluorometric analysis of ultramicroquantities of hydrogen peroxide. Anal Biochem 11:1–5

Knight MR, Campbell AK, Smith SM, Trewavas AJ (1991)Transgenic plant aequorin reports the effects of touch and coldshock and elicitors on cytoplasmic calcium. Nature 352:524–526

McDowell JM, Dangl JL (2000) Signal transduction in the plantimmune response. Trends Biochem Sci 25:79–82

Meyer A, Puhler A, Niehaus K (2001) The lipopolysaccharides ofthe phytopathogen Xanthomonas campestris pv. campestrisinduce an oxidative burst reaction in cell cultures of Nicotianatabacum. Planta 213:214–222

Mithofer A, Ebel J, Baghwat AA, Neuhaus-Url G (1999) Trans-genic aequorin monitors cytosolic calcium transients in soybeanchallenged with b-glucan or chitin elicitors. Planta 207:566–574

Murashige T, Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol Plant15:473–497

Newman MA, von Roepenack E, Daniels MJ, Dow JM (2000)Lipopolysaccharides and plant responses to phytopathogenicbacteria. Mol Plant Pathol 1:25–31

Newman MA, von Roepenack-Lahaye E, Parr A, Daniels MJ,Dow M (2002) Prior exposure to lipopolysaccharides potenti-ates expression of plant defenses in response to bacteria. Plant J29:487–495

Nurnberger T, Brunner F (2002) Innate immunity in plants andanimals: emerging parallels between the recognition of generalelicitors and pathogen-associated molecular patterns. CurrOpin Plant Biol 5:1–7

O�Donnel VB, Tew DG, Jones OTG, England PJ (1993) Studies onthe inhibitory mechanism of iodonium compounds with specialreference to neutrophil NADPH oxidase. Biochem J 290:41–49

Otte O, Pachten A, Hein F, Barz W (2001) Early elicitor-inducedevents in chickpea cells: functional links between oxidative burst,sequential occurrence of extracellular alkalinization and acidifi-cation, K+/H+ exchange and defense-related gene activation.Z Naturforsch Teil C 56:65–76

Pachten A, Barz W (1999) Elicitor-stimulated oxidative burst andextracellular pH changes in protoplast suspensions prepared

656

from cultured chickpea (Cicer arietinum L) cells. J Plant Physiol155:795–797

Pfeiffer S, Leopold E, Hemmins B, Schmidt K,Werner ER,Mayer B(1997) Interference of carboxy-PTIO with nitric oxide- and per-oxynitrite-mediated reactions. Free Rad Biol Med 22:787–794

Possel H, Noack H, Augustin W, Keilhoff G, Wolf G (1997) 2,7-Dihydrodichloro-fluorescein diacetate as a fluorescent markerfor peroxynitrite formation. FEBS Lett 416:175–178

Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, KnappW, Zlabinger GJ (1992) A microplate assay for the detection ofoxidative products using 2¢,7¢-dichlorofluorescein-diacetate.J Immunol Methods 156:39–45

Roswell DF, White EH (1978) The chemiluminescence of luminoland related hydrazides. Methods Enzymol 57:409–483

Scheel D (1998) Resistance response physiology and signal trans-duction. Curr Opin Plant Biol 1:305–310

Schromm AB, Brandenburg K, Loppnow H, Moran AP, KochMHJ, Rietschel ET, Seydel U (2000) Biological activities oflipopolysaccharides are determined by the shape of their lipid Aportion. Eur J Biochem 267:2008–2013

Somssich IK, Hahlbrock K (1998) Pathogen defense in plant-s—a paradigm of biological complexity. Trends Plant Sci3:86–90

Trewavas A (2000) Signal perception and transduction. In:Buchanan B, Gruissem W, Jones R (eds) Biochemistry andmolecular biology of plants. American Society of Plant Physi-ologists, Rockville, USA, pp 930–986

Trewavas AJ, Knight M (1994) Mechanical signaling, calcium andplant form. Plant Mol Biol 26:1329–1341

Triantafilou M, Triantafilou K (2002) Lipopolysaccharide recog-nition: CD14, TLRs and the LPS-activation cluster. TrendsImmunol 23:301–304

Van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemicresistance induced by rhizosphere bacteria. Annu Rev Phyto-pathol 36:453–483

Westphal O, Jann K (1965) Bacterial lipopolysaccharides: Extrac-tion with phenol-water and further applications of the proce-dure. Methods Carbohydr Chem 5:83–91

657