Plant Physiol. (1989) 90, 867-8750032-0889/89/90/0867/09/$01 .00/0

Received for publication August 22, 1988and in revised form February 21, 1989

Electrolyte Leakage, Lipoxygenase, and Lipid PeroxidationInduced in Tomato Leaf Tissue by Specific and Nonspecific

Elicitors from Cladosporium fulvum'

Tobin L. Peever2 and Verna J. Higgins*

Department of Botany, University of Toronto, Toronto, Ontario M5S 1A 1 Canada

ABSTRACT

Glycoprotein nonspecific elicitor (NSE) and a specific elicitorpreparation from intercellular fluids (SE) of tomato (Lycopersiconesculentum Mill. cv Bonny Best or Potentate) infected with race2.4.5 of Cladosporium fuhvum Cooke [syn. Fulvia fulva (Cooke)Ciferri] were injected into cv Sonatine (resistant to race 2.4.5) tocompare electrolyte leakage, lipoxygenase activity, and lipid per-oxidation induced in response to these elicitors. Increased elec-trolyte leakage was induced by NSE or SE; the leakage due toNSE but not to SE was inhibited by the nonsteroidal antiinflam-matory drug (NSAID) piroxicam. Under normal photoperiod con-ditions, higher levels of lipoxygenase activity were detected 6hours after injection with either elicitor. This activity peaked by12 hours with both elicitors and declined to control levels by 24hours when visible necrosis could be detected. Both NSE andSE-induced lipoxygenase was inhibited by piroxicam in vitro.Lipid peroxidation in elicitor-treated tissue was also assayed at6, 12, and 24 hours after injection using the TBA test for malon-aldehyde. Increased peroxidation was detected in response toNSE or SE at 12 hours with similar values obtained at 24 hours.With plants incubated in the dark, lipoxygenase, and lipid perox-idation were similarly induced in SE-injected tissue whereasnecrosis induction by SE was light dependent.

The interactions between plant pathogens and their hostshave been shown in several instances to involve effects on theplasma membranes of the host cells. These effects includedepolarization (27, 33), altered H+ and K+ fluxes (1), electro-lyte leakage (2), and increases in lipid peroxidation (20-22)and can be different in incompatible and compatible inter-actions. Cell-free extracts of plant pathogens or 'elicitors'induce many of the same responses in the absence of thepathogen (28-30).

In addition to nonenzymic mechanisms ofmembrane dam-age such as oxygen radicals, there are several enzymes capableof breaking down the lipid component of membranes includ-ing lipases and lipoxygenases. The activity of these enzymesincreases following inoculation with pathogens or treatmentwith elicitors (14, 25, 26). Although the subcellular locationof lipoxygenase, its regulatory control, and synthesis are

' Supported by the Natural Sciences and Engineering ResearchCouncil of Canada.

2 Present address: Department of Plant Pathology, Cornell Univer-sity, Ithaca, NY 14853.

867

poorly understood (15), its activity has been detected inthe tissues of numerous plant species and appears to in-crease under conditions of stress such as pathogenesis andsenescence.The objective of this study was to determine if the specific

(8, 9) and nonspecific elicitors (7, 24) of necrosis proposed tobe involved in the interaction between Cladosporium fulvumCooke [syn. Fulvia fulva (Cooke) Ciferri] and tomato actthrough a similar effect on host plasma membranes. Initialtests involved the effect of various oxygen radical scavengersand of NSAIDs,3 proposed inhibitors of lipoxygenase, onelicitor-induced electrolyte leakage. We report here the differ-ential effect of the NSAID piroxicam on electrolyte leakageinduced by specific and nonspecific elicitors and subsequentinvestigation of the involvement of lipoxygenase and lipidperoxidation in these effects.

MATERIALS AND METHODS

Growth of Plants

Tomato (Lycopersicon esculentum L.) seeds of cv Sonatine,Bonny Best, and Potentate were obtained from de RuiterSeeds Co., Columbus, OH, McKenzie Seed Co. Ltd. Brandon,Manitoba, Canada, and R. A. Brammall, Simcoe Horticul-tural Experimental Station, Simcoe, Ontario, Canada, respec-tively, and germinated in a standard soilless mix (Pro-MixBX, Premier Peat Moss Ltd., Chemin Temiscouta, Rivieredu Loup, Quebec, Canada). Plants were transplanted at 2weeks of age and maintained in growth chambers with a 14 hphotoperiod at 15,000 lux (200 ,uE * m-2 s'), 85% RH, andday and night temperatures of 23 and 21°C. Treated plantswere incubated under the same conditions. Plants were usedat 5 to 6 weeks of age for all experiments.

Culture of Fungi

An isolate of C. fulvum race 0 was obtained from the culturecollection, Department of Botany, University of Toronto,Toronto, Ontario, Canada, and an isolate of race 2.4.5 wasobtained from P. J. G. M. de Wit, Agricultural University,Wageningen, The Netherlands. Cultures were maintained insterile soil culture at 4°C, plated out on V8 juice agar, andtransferred a maximum of three times prior to use. Spore

3Abbreviations: NSAIDs, nonsteroidal antiinflammatory drugs;NSE, nonspecific elicitor; SE, specific elicitor; CIF, control intercel-lular fluids; TBA, thiobarbituric acid; ConA, concanavalin A.

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Plant Physiol. Vol. 90, 1989

suspensions prepared from plates of the sporulating pathogen(7-14 d after transfer) were used to inoculate modified Friesmedium (24) for the production ofNSE or to inoculate plantsfor the production of SE from intercellular fluids.

Preparation of NSE

NSE was prepared from culture filtrates of C. fulvum race0 or race 2.4.5. The fungus was grown for 21 d under a 12 hphotoperiod at 1000 lux ( 14 uE* m-2 - s-') at 25°C on modifiedFries medium according to the method of Lazarovits andHiggins (24). Fungal mycelium and culture medium werefiltered through glass wool and then through Whatman No. 1filter paper under vacuum. Crude culture filtrates of race 2.4.5were concentrated to 5% of their original volume using anAmicon ultrafiltration apparatus and PM- 10 membrane (mo-lecular cut-off 10,000 D) and stored at -20°C. Crude culturefiltrates of race 0 were further purified by affinity chromatog-raphy on ConA Sepharose according to the method of Laza-rovits et al. (23). This was followed by gel chromatographyusing Sephacryl S-200 Superfine (Pharmacia, bed volume 1.6x 39 cm packed in distilled water). The sample (each 2-5 mgglucose equivalents) was eluted with distilled water (flow rate0.7 ml - min-') and 4.2 mL fractions collected. Active fractionsusually eluted about fractions 4 to 9 (mannoside from theConA elution began to be eluted in about fraction 15) andwere pooled and stored at -20°C. Carbohydrate levels in thepooled fractions were determined using the assay of Duboiset al. (13) with D-glucose as the standard. NSE (race 0) wasused at 100 ug ml-' glucose equivalents for the lipoxygenaseand lipid peroxidation experiments and NSE (race 2.4.5) at40 ug-mL-' glucose equivalents for the electrolyte leakageexperiments.

Preparation of Specific Elicitor

SE intercellular fluids were prepared from tomato cv BonnyBest or Potentate (no known genes for resistance to C. fulvum)infected with race 2.4.5 of C. fulvum according to the methodof de Wit and Spikman (8). Leaflets were spray inoculatedwith (1 x 106. ml-') conidia and harvested 10 to 14 d laterwhen sporulation was evident over the entire abaxial surfaceof the leaflets. Leaflets were infiltrated in vacuo, placed inspecially designed centrifuge tubes (8), and centrifuged at1650g (5°C, 30 min). Leaflets of healthy, uninoculated cvBonny Best or Potentate grown in the same chamber as theinfected plants were used to produce CIF. These leaflets wereharvested, infiltrated, and centrifuged at the same time as theleaflets from infected plants. Prior to use, all intercellularfluids were centrifuged at 1 500g (5°C, 10 min), dialyzed (2000mol wt cutoff, 5C, 20 h), and stored frozen at -20°C. SE andCIF were used at 50% of their original concentration for theelectrolyte leakage experiments and at the original concentra-tion for the lipoxygenase and lipid peroxidation experiments.

Electrolyte Leakage Experiments

Opposite leaflets of several tomato cultivars carrying differ-ent genes for resistance to C. fulvum were injected with NSEand water or with SE and CIF treatments, and the electrolyte

leakage from these leaflets was measured according to themethod of Lazarovits and Higgins (24). Entire leaflets wereinjected with 1 to 2 mL of each treatment using 27 gaugeneedles and 1 mL disposable syringes. The NSAIDs piroxicam(4-hydroxy-2-methyl-3-[pyrid-2-yl-carbamoyl]-2H- 1,2-ben-zothiazine 1,1-dioxide) and ibuprofen (alpha-methyl-4-[2-methylpropyl] benzeneacetic acid) were obtained from SigmaChemical Co., St. Louis, MO, and solubilized in hot ethanol,cooled, and added to the NSE and SE treatments to a finalconcentration of 100 ,M (in 1% ethanol) before injection intoopposite leaflets from those injected with NSE or SE alone(in 1% ethanol). Injected plants were incubated for 5 h in thegrowth chamber after which time the plants were removedand 21 to 28 discs were cut from each injected leaflet with a9 mm corkborer. Cut discs were immediately floated adaxialside up on 50 mL of low conductivity water (Millipore Milli-Q system, Bedford MA) in a glass Petri plate. The water wasreplaced after 5 min, and the discs were turned over. Afterfloating a further 5 min, the discs were placed adaxial side upin 25 mL Erlenmeyer flasks containing 5 mL of low conduc-tivity water with an equal number of discs dispersed overthree or four flasks. The flasks were then placed on a wrist-action shaker and the 'time zero' conductivity measurementsmade after the first 2 min of shaking. Conductivity measure-ments made after the first 2 min of shaking. Conductivitymeasurements were made after 0.5 h and every hour thereafterfor 3 h. Net conductivity values were calculated by subtractingthe time zero values from the actual values measured 1 to 3h after cutting the discs.

Injection of NSE or SE for Lipoxygenase and LipidPeroxidation Assays

Four leaflets (two adjacent pairs) of the third leaf of cvSonatine (resistant to C. fulvum race 2.4.5) were each com-pletely injected with distilled water, NSE, SE, or CIF asdescribed above. NSE and SE were injected on diagonallyopposite leaflets and distilled water and CIF controls injectedinto leaflets directly opposite these treatments, respectively.The concentrations of NSE and SE selected for the assayinduced necrosis at similar times (24 h) after injection. In-jected plants were normally incubated in the growth chamberwith continuous lighting and leaf samples taken at 6, 12, or24 h. For the experiments on light effects, plants were injectedwith CIF and SE and incubated in either constant light ordark conditions. Samples for determination of lipoxygenaseactivity and lipid peroxidation were taken at 12 h. Leafletpanels of the fourth leaf on the above plants were injectedwith CIF or SE, incubated in the dark or the light and observedfor necrosis at 16, 21, and 36 h after injection.

Extraction of Lipoxygenase from Tomato Leaflets

Lipoxygenase was extracted from uninjected and injectedtomato leaf tissue according to the method of Lupu et al.(25). Following injection and incubation, each leaflet (ap-proximately 0.25 g) was harvested, the midvein removed, andthe tissue homogenized in ice-cold 0.2 M sodium-phosphatebuffer (pH 6.5, 1% Triton X-100) using a Potter-Elvejemhomogenizer. The homogenate was centrifuged at 10,000g

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C. FULVUM ELICITOR-INDUCED EFFECTS ON TOMATO

(5°C, 20 min) and the supernatants (hereafter termed 'leafextracts') were immediately flash frozen in dry ice and storedfor up to 7 d at - 200C before use.

Total protein in the leaf extracts was determined using theBio-Rad protein assay (Bio-Rad Laboratories, Richmond,CA) based on the dye-binding assay of Bradford (5) withsoybean lipoxygenase (Sigma L-7127) as the protein standard.Amount of protein in the extracts rather than fresh weightsof the extracted leaflets was used to standardize both lipoxy-genase activity and lipid peroxidation in the extracts as itappeared to give a more accurate estimation of the efficiencyof extraction.

Lipoxygenase Assays

Lipoxygenase activity in leaf extracts was measured usingthe polarographic method described by Grossman and Zakut(17) and the spectrophotometric assay of Ben-Aziz et al. (4).The polarographic assay is based upon oxygen uptake bylipoxygenase in a linoleic acid solution as measured by anoxygen electrode. A 2.5 mL aliquot of substrate solutioncontaining 8.0 x 10-4 M linoleic acid (cis-9, cis-12 octadeca-dienoic acid), 0.025% Tween 20 in 0.2 M sodium-phosphatebuffer (pH 6.5) was equilibrated with air at 250C in an oxygenelectrode apparatus (Yellow Springs Instruments, YellowSprings, OH) attached to a strip chart recorder. The electrodewas inserted into the substrate solution and a steady trace wasset at 100% air saturation for 3 min. Fifty ,L of leaf extracts(in a total volume of 500 ,uL with water) was added to thesubstrate solution with a syringe, and the initial rate ofoxygenuptake was recorded from two separate curves. Lipoxygenaseactivities were expressed as ,umol 02,* min-' - mg-' protein usingthe standard oxygen content of air-saturated water at 250C(0.258 Amol.mL-' water).

Lipoxygenase activity in leaf extracts was also measuredspectrophotometrically at 234 nm using linoleic acid (cis-9,cis- 12 octadecadienoic acid) as the substrate. Lipoxygenasecatalyzes the addition of oxygen to cis -1,4 unsaturated fattyacids producing conjugated linoleate hydroperoxides whichabsorb at 234 nm (32). The method of Ben-Aziz et al. (4),based on a modification of the assay of Tappel (32), was usedto determine lipoxygenase activities in leaf extracts. One mLof substrate solution containing 2.28 x 10-4 M linoleic acid(Sigma L-1376) and 0.25% Tween 20 in 0.2 M citrate-phos-phate buffer (pH 6.5) was added to quartz cuvettes in aBeckman Acta C3 spectrophotometer. The spectrophotome-ter was zeroed and 5 to 15 ,uL leaf extract was added to thesample cuvette with thorough mixing. Increases in absorbanceat 234 nm (25°C) were followed for 15 min and rates ofincrease were calculated from the initial linear portion of atleast two separate curves. Activity was expressed as change inabsorbance at 234 nm * min-' mg-' protein.

In Vitro Inhibition of Lipoxygenase by Piroxicam

The NSAID piroxicam was also tested for ability to inhibitlipoxygenase activity in elicitor-injected leaf extracts meas-ured by the spectrophotometric assay. Leafextracts from NSEand SE-injected cv Sonatine incubated in the light wereprepared as described above and added to the substrate solu-

tion with 100 to 300 ,M piroxicam (1-3% ethanol) and mixedthoroughly. Rates oflipoxygenase activity were calculated andcompared to rates obtained with controls consisting ofextractsplus 1-3% ethanol. The concentration of piroxicam requiredfor 50% inhibition of activity in each extract was calculatedusing four concentrations of Piroxicam.

Substrate Specificity of Lipoxygenase

The specificity of the lipoxygenase activity in leaf extractsfor substrates with the cis- 1 ,4-pentadiene structure was testedusing oleic acid. Oleic acid (cis-9, octadecanoic acid) is a C18fatty acid similar to linoleic acid but with a single doublebond at position 9 and therefore is not a substrate for lipox-ygenase (32). Buffered substrate solutions of both oleic andlinoleic acids were prepared identically as described for thespectrophotometric assay. Lipoxygenase activity using the twosubstrates was compared in leaf extracts from NSE injectedtomato incubated for 12 hours after injection.

pH Dependence of Lipoxygenase Activity

Linoleic acid solutions buffered to various pH levels wereused to determine the pH optimum of the enzyme extractedfrom NSE and SE-injected tissue. Linoleic acid solutionswere prepared as described for the spectrophotometric assayusing 0.2 M citrate-phosphate buffer to pH values of 3.0, 3.5,4.0, 5.0, 6.0, 6.5, and 7.0 or with 0.05 M Tris HC1 buffer topH values of 7.0, 8.0, and 9.0 and lipoxygenase activitiesdetermined.

Lipid Peroxidation Assay

Levels of lipid peroxidation in elicitor-treated leaf tissuewere measured using the thiobarbituric acid test for malon-aldehyde (10). A 67 gL aliquot of leaf extract was mixed with933 ,L 20% TCA + 0.5% thiobarbituric acid in 1.5 mLplastic centrifuge tubes and incubated at 95°C for 30 min.The tubes were then cooled immediately in an ice-bath andcentrifuged for 10 min (10,000g, 5°C). Following centrifuga-tion, the specific absorbance of the product and the nonspe-cific, background absorbance were read at 532 and 600 nm,respectively, using 20% TCA + 0.5% TBA as the referencestandard. After subtracting the nonspecific from the specificabsorbance, the net absorbance at 532 nm was expressed interms of protein in the extracts.

Statistical Analyses

Comparisons of differences observed in the electrolyte leak-age, lipoxygenase, and lipid peroxidation experiments werecompleted using the parametric t test and the nonparametricU-test programs of the statistical software STATSEASE writ-ten by Bryan Clarke, Department of Genetics, University ofNottingham, U.K.

RESULTS

Electrolyte Leakage Assay

Electrolyte leakage from NSE and SE-injected leaflet tissuewas assayed by following increases in conductivity over 3 h.

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Plant Physiol. Vol. 90, 1989

The race/cultivar specificity of the elicitors was reproducedby the electrolyte leakage assay (results not shown). NSEinduced leakage in both resistant and susceptible tomatocultivars, whereas SE induced leakage only in those cultivarsresistant to the race of C. fulvum used to produce the SE andnot in susceptible cultivars. Electrolyte leakage data for NSEand SE-injected Sonatine and their respective controls (Fig.1) displayed an immediate enhancement of leakage. Levels ofleakage from elicitor-treated leaf tissue varied from approxi-mately 7 to 20 micro-Siemens (,gS) with each separate batchof plants used. Small differences in growth conditions of theplants may be responsible for this variability. Mean leakagevalues from three separate experiments comparing NSE-in-jected leaflets to leaflets injected with NSE plus piroxicam or

a

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TIME ( hours after cutting discs)Figure 1. a, Effect of NSE from Cladosporium fulvum race 2.4.5 on

electrolyte leakage from cv Sonatine leaflets; b, Effect of SE fromintercellular fluids of tomato inoculated with race 2.4.5 on electrolyteleakage from cv Sonatine leaflets. NSE and SE (solid lines) or waterand CIF (dashed lines) were injected into attached leaflets and, after5 h incubation, leaf discs were cut from injected tissue and floatedon 5 mL of water in 25 mL flasks (8 discs/flask). Values (with barsshowing standard deviation) are mean conductivity readings of fourreplicates taken 0-3 h after cutting the discs.

ibuprofen all showed significant differences in leakage be-tween leaflets injected with NSE alone and those injected withNSE plus piroxicam or ibuprofen (Table I). The degrees ofinhibition of leakage were approximately 54 and 29%, respec-tively, for NSE plus piroxicam and NSE plus ibuprofen. Aspiroxicam was the most effective inhibitor, it was used in allsubsequent experiments. No significant difference was de-tected between leaflets injected with SE plus piroxicam andwith SE alone in three separate experiments (Table I). Theoxygen radical scavengers a-tocopherol (1 mM) and dabco (10mM) failed to inhibit either NSE- or SE-induced electrolyteleakage (results not shown). Piroxicam did not inhibit NSEor SE-induced necrosis when injected into tomato leafletpanels (results not shown).

Lipoxygenase Assays

NSE-lnjected Tomato

Lipoxygenase activities in water- and NSE-injected Sona-tine leaf tissue were measured both polarographically (Fig.2a), and spectrophotometrically (Fig. 2b). Water-injected con-trols incubated for 6, 12, or 24 h after injection had similarlevels of lipoxygenase activity indicating little variation be-tween plants. A large increase in levels of activity in the NSE-injected tissue was observed at 6 and 12 h incubation withlevels of activity not significantly different from the controlsat 24 h. No lipoxygenase activity could be detected in theNSE preparation itself when it was added directly to thespectrophotometric assay at a concentration higher thanwould be found in the tissue extracts. Activity in a heat-treated extract from NSE-injected leaf tissue was greatly re-

duced (0.036 A.A234 min-'. mg-' protein). Determination oflipoxygenase activity in two separate extracts from NSE-injected tomato using both linoleic and oleic acid as thesubstrate produced average activities of 0.703 and0.086AA234 -min-' mg-' protein, respectively.

SE-Injected Tomato

Lipoxygenase activities in extracts from uninjected Sona-tine leaf tissue or tissue injected with CIF or SE from inter-cellular fluids were measured both polarographically (Fig. 3a)

Table 1. Inhibition of NSE and SE-induced Electrolyte Leakage bythe NSAIDs Piroxicam and Ibuprofen

Opposite leaflets of cv Sonatine were injected with NSE, SE, NSEplus 100 gM piroxicam or ibuprofen; SE plus 100 uM piroxicam. Alltreatments contained 1% ethanol. Net conductivity values are means+ SD of three replicate experiments taken 3 h after the start of eachexperiment.

Treatment Net Conductivity Values % Inhibitionat 3 h of Leakage

AS %NSE 17.83 ± 2.05NSE + ibuprofen 12.73 ± 1.87 29NSE 17.40 ± 2.45NSE + piroxicam 8.00 ± 0.89 54SE 7.07 ± 0.59SE + piroxicam 7.70 ± 0.52 0

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C. FULVUM ELICITOR-INDUCED EFFECTS ON TOMATO

0.1

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6 12 24

INCUBATION TIME (h)

Figure 2. Lipoxygenase activity as measured by (a) polarographic or

(b) spectrophotometric assay in extracts from cv Sonatine leaf tissueinjected with water (dashed bar) or NSE (solid bar) and extracted at6, 12, and 24 h after injection. Values (with bars showing standarddeviation) represent the mean activity of two replicates in one exper-iment (a) or the mean activity in three separate experiments (b).

and spectrophotometrically (Fig. 3b). CIF-injected tissue ap-peared to contain higher levels of activity than the water-injected controls shown in Figure 2. Lipoxygenase levels inSE-injected tissue increased from 6 to 12 h after injection,then declined to control levels at 24 h. Extracts from unin-jected tissue had low levels of activity which were not signifi-cantly different from those ofthe water (Fig. 2) or CIF-injected(Fig. 3) controls. No lipoxygenase activity could be detectedin the intercellular fluid preparations themselves when theywere added directly to the polarographic assay system at a

concentration higher than would be found in the extractedtissue. The level of lipoxygenase activity in a heat treatedextract from SE-injected tissue was 0.058 AA234 min-'-mg-'protein.No increase in lipoxygenase activity over the control was

detected in extracts from cv Bonny Best injected with SE and

a

0

E

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0

0.8

b

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7

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> 0.2

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6 12 24 U

INCUBATION TIME (h)

Figure 3. Lipoxygenase activity as measured by (a) polarographic or(b) spectrophotometric assay in extracts from cv Sonatine leaf tissueinjected with CIF (vertically dashed bar) or SE from intercellular fluid(solid bar) extracted at 6, 12, and 24 h after injection. Values (withbars showing standard deviation) represent the mean activity of tworeplicates in one experiment (a) or the mean activity in three separateexperiments. Bars denoted by U (diagonally dashed bar) representextracts from uninjected Sonatine leaf tissue obtained at the sametime as the injected leaflets.

incubated for 12 h in the light, thus demonstrating the speci-ficity of lipoxygenase induction by SE.

In Vitro Inhibition of Lipoxygenase by Piroxicam

Lipoxygenase activity in extracts from both NSE and SE-injected leaf tissue was inhibited by piroxicam. The concen-tration required for 50% inhibition of activity was 1 5 gM forthe extract from SE-injected tissue and 133 ,M for the extractfrom NSE-injected tissue (results not shown).

pH Dependence of Lipoxygenase Activity

The pH dependence of lipoxygenase activity in extractsfrom light-incubated SE-injected and NSE-injected leaf tissuewas determined using substrate solutions buffered to severalpH values. Extracts from NSE and SE-injected tissue bothexhibited broad pH optima of 6 to 8. No difference in activitybetween the two types of buffer at pH 7.0 was detected.

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Plant Physiol. Vol. 90, 1989

Lipid Peroxidation

Lipid peroxidation in NSE and SE-injected leaf tissue was

measured using the TBA test for malonaldehyde. No signifi-cant differences in peroxidation between control- (water andCIF) and elicitor-injected (NSE and SE) tissue were observed6 h after injection, but at 12 and 24 h after injection, signifi-cantly higher levels of peroxidation were observed in elicitor-injected tissue (Table II). Lipid peroxidation in both water-injected and CIF-injected controls was more variable thanthat observed for controls in the lipoxygenase assays.

Effect of Light on SE-induced Lipoxygenase, LipidPeroxidation, and Necrosis

The induction of lipoxygenase, lipid peroxidation, andnecrosis in SE-injected Sonatine tissue from dark and lightincubated plants was examined due to the recent report of deWit et al. (9) on the effect of light on the induction of necrosis.Lipoxygenase activities in Sonatine leaf tissue injected withSE and incubated in the light or the dark for 12 h were

measured spectrophotometrically (Fig. 4). No significant dif-ferences in activity between extracts from dark and light-incubated plants could be detected. The pH optima for thelipoxygenase(s) extracted from both light and dark-incubatedtissue were similar for both treatments (results not shown).Values obtained for lipid peroxidation in SE-injected Sonatineincubated in the dark or the light were also not significantlydifferent from each other (Fig. 4).

In two separate experiments, leaflet panels (15-21 per treat-ment) were injected with SE or CIF and incubated in the darkor the light for 16, 21, or 36 h to test the effect of light on theinduction of necrosis. In one of the experiments (Fig. Sa),dark-incubated panels were photographed in the light at thethree observation times giving the panels approximately 45min total light exposure. In the second experiment, muchlower levels of necrosis were observed in the panels incubatedin the dark, without any photography, for up to 36 h afterinjection (Fig. 5b). High levels of necrosis were observed as

early as 16 h after injection in all of the SE-injected panelsincubated in the light (Fig. 5).

Table II. Lipid Peroxidation in NSE and SE-Injected Leaf Tissueafter 6, 12, and 24 h Incubation

Injections of distilled water, NSE, CIF, or SE were made intoopposite leaflets of cv Sonatine and incubated for 6, 12, or 24 hbefore extraction and quantification of lipid peroxidation using theTBA assay for malonaldehyde. Values ± SD of three replicates in oneexperiment.

Hours IncubationTreatment

6 12 24

net absorbance at 532 nm -mg-' proteinWater 0.169 ± 0.015 0.143 ± 0.011 0.113 ± 0.023NSE 0.185 ± 0.015 0.249 ± 0.013 0.259 ± 0.017CIF 0.141 ± 0.011 0.094 ± 0.018 0.200 ± 0.006SE 0.130 ± 0.026 0.208 ± 0.035 0.265 ± 0.007

J-%

I-

0

z

ae

F-

1W

120

a4I

0

D LLIPOXYGENASE

D LLIPID

PEROXIDATION

Figure 4. Lipoxygenase and lipid peroxidation in cv Sonatine tissueextracts injected with SE from intercellular fluid and incubated for 12h in the dark (D) or the light (L). Both lipoxygenase and lipid peroxi-dation activities were calculated as percentages of the activitiesobserved with CIF. Values (with bars showing standard deviation)represent means of three separate experiments.

DISCUSSION

The inhibition of NSE-induced electrolyte leakage in to-mato tissue by NSAIDs piroxicam and ibuprofen suggestedthe involvement of lipoxygenase in elicitor-mediated effects.Piroxicam and ibuprofen are reported to inhibit soybeanlipoxygenase in vitro (31) and to inhibit the accumulation ofphytoalexins in potato tubers (19). The failure of piroxicamto visibly affect the degree of necrosis induced by NSE indi-cated that lipoxygenase activity and necrosis induction may

be unrelated events, a possibility supported by the light/darkexperiments. Electrolyte leakage induced by specific elicitorsfrom intercellular fluids (SE) in cv Sonatine was not signifi-cantly affected by piroxicam, suggesting the possibility of a

different mechanism of membrane damage to that mediatedby NSE. However, direct determination of the activity oflipoxygenase after treatment of leaf tissue with either type ofelicitor and in vitro inhibition studies failed to explain thedifferent effects seen with piroxicam. NSE and SE are chem-ically unrelated elicitors (7, 9, 23), yet both induce electrolyteleakage, lipoxygenase, and lipid peroxidation. These responses

are probably general stress responses which are secondary toinitial recognition events controlling race/cultivar specificity.The NSAIDs may have had an effect on NSE-induced elec-trolyte leakage that was unrelated to lipoxygenase. Only one

study has demonstrated the inhibition of plant lipoxygenasesby these drugs in vitro (31) using soybean lipoxygenase type1 which has a pH optimum of 9.0 and adds oxygen primarilyto the C 13 position of linoleic acid (3). The lipoxygenase frompotato has a pH optimum of 6.5 and adds oxygen preferen-tially to the C9 rather than the C 13 position of linoleic acid

I I I I I T I- ---

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C. FULVUM ELICITOR-INDUCED EFFECTS ON TOMATO

.a

XI 361B 21 31O

Figure 5. Necrosis in panels of cv Sonatine leafletsinjected with SE from intercellular fluid and incubated inthe dark (solid bars) or the light (diagonally dashed bars)for 16, 21, or 36 h. In experiment 1 (a), the dark-incubatedpanels received approximately 45 min light exposure,while those in the second experiment (b) received ap-proximately 5 min light exposure. Necrosis was nottested (nt) at 21 h in the second experiment. Valuesrepresent necrosis ratings (where 0 = no necrosis and 3

_A/?nt _ s/z_ = complete necrosis) of 15 to 18 injected panels/treat-16Zg 21 36 ment.

INCUBATION TIME (h)

(16). The lipoxygenase induced in response to NSE and SEinjection and extracted from tomato in the present study hada broad pH optimum from 6 to 8 and was inhibited bypiroxicam in vitro. In the inhibition study involving soybeanlipoxygenase, it was reported that 154 and 575 jiM of piroxi-cam and ibuprofen, respectively, were required for 50% in-hibition of the enzyme in vitro (31). The concentration ofboth of these drugs used in the leakage experiments was 100jAM which caused a 50% reduction in electrolyte leakage withpiroxicam and 30% with ibuprofen. Approximately the sameconcentrations of piroxicam were required for 50% inhibitionoflipoxygenase activity in extracts from elicitor-injected tissuein vitro. The similar pattern of lipoxygenase induction, the invitro inhibition of lipoxygenase activity by piroxicam and thesimilar pH activity profiles in extracts from NSE and SE-injected tissue suggest that suppression of the NSE-inducedelectrolyte leakage response by NSAIDs is due to an effectunrelated to the inhibition of lipoxygenase.Treatment of cv Sonatine tomato leaf tissue with either the

nonspecific elicitor (NSE) or the specific elicitor from C.fulvum intercellular fluids (SE) induced the activity of lipox-ygenase as early as 6 h after injection. The induction oflipoxygenase with SE was specific to the incompatible inter-action as lipoxygenase was not induced in response to treat-ment of cv Bonny Best with SE. Both the polarographic andspectrophotometric assays for lipoxygenase showed similarpatterns of activity at the three times tested with the exceptionof NSE-treated extracts in the polarographic assay whichshowed an earlier peak of induction. Lipoxygenase activitypeaked at 6 or 12 h followed by a decline to control levels by24 h. Decreased levels at 24 h after injection corresponded tothe appearance of extensive necrosis in the injected areasunder the normal constant light regime. A similar observationwas made by Lupu et al. (25) who found that lipoxygenaseactivity induced in tobacco tissue in response to infection byErysiphe cichoracearum decreased during the onset of senes-cence. It was suggested that this decrease may be due toprotein denaturation or to the formation of an antioxidant-enzyme complex.The induction of lipoxygenase has been observed in wheat

leaf tissue in response to infection by an incompatible race or

to elicitors from Puccinia graminis (26) and in suspension-cultured tobacco cells treated with elicitors from Phytophthoraparasitica var nicotianae (14). In both of these studies, theelicitors used were produced from culture filtrates of the

pathogen grown in vitro. The NSE produced by C. fulvum isalso isolated from culture filtrates ofthe fungus grown in vitroand is nonspecific (with respect to race and cultivar) in termsof its elicitor activity (24). The induction of lipoxygenase bythe NSE from C. fulvum adds a third elicitor-plant system inwhich lipoxygenase has been detected. Lipoxygenase has alsobeen shown to increase in an incompatible bacterial-plantinteraction (22) coincident with the development of the hy-persensitive response (HR), lipid peroxidation, electrolyteleakage, and membrane depolarization (21).

Intercellular fluids from compatible interactions of C. ful-vum and tomato contain race/cultivar SE of chlorosis andnecrosis (8). Injection of race 245 intercellular fluids into thetomato cultivar Sonatine (resistant to race 245) produced astrong necrotic reaction after 16 to 24 h in the light, but notin the dark. These fluids also induced lipoxygenase in a similarpattern to that observed with the NSE but with smallerincreases over the control values. Lipoxygenase, but not ne-crosis, was induced in the dark in response to these fluids, aresult which suggests that the production of necrosis requiresexposure to light and that the lipoxygenase response is sepa-rable from necrosis.The lipoxygenase extracted in the present study was active

from pH 6 to 8 which is in the range reported for lipoxygenasesextracted from other plants (16, 17, 32). No differences in pHactivity profiles between extracts from NSE and SE-injectedplants or between extracts from SE-injected plants incubatedin the light or the dark could be detected, suggesting thatsimilar lipoxygenase isozymes are induced in response to allof these treatments.The linoleic acid oxidizing activity in tissue extracts was

heat labile, indicating that the observed activity was due tooxidation of linoleic acid by proteins rather than to somenonenzymic mechanism. Lipoxygenase catalyzes the additionof oxygen to several molecules including fatty acids, esters,and alcohols that contain a cis- 1,4-pentadiene system (3).Substrates include linoleic and linolenic acids but not mono-unsaturated fatty acids such as oleic acid (16, 32). The lackof activity observed with oleic acid in the present studyprovides additional evidence that the enzyme in the extractsis a lipoxygenase.

Increased lipid peroxidation in Sonatine tissue injected witheither the NSE or SE was first detected 12 h after injectionand no further increases were detected at 24 h. Lipid peroxi-dation has also been detected in response to other fungal

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Plant Physiol. Vol. 90, 1989

elicitors (29), fungal toxins (6), and in incompatible bacterial-plant interactions (20, 21). The development of a hypersen-sitive response, lipid peroxidation or an increase in electrolyteleakage was reduced in a bacteria-plant interaction with theaddition of the free radical scavenger Tiron or the enzymesuperoxide dismutase, suggesting that oxygen radical specieswere involved in the initiation of this peroxidation (20, 22).The involvement ofthe superoxide radical in the incompatibleinteraction between Phytophthora infestans has been dem-onstrated by Doke (1 1, 12). This radical is generated by a

membrane-bound NADPH-dependent Cyt P-450 oxidase inincompatible interactions and in response to elicitor treat-ment. The cause of peroxidation in bean tissue treated withfungal elicitor was also suggested to involve activated oxygenspecies, but no data was presented to substantiate this hypoth-esis (29). The lipid peroxidation that they observed could alsohave been due to enzymic degradation of membrane lipidsby lipoxygenase, a possibility that was mentioned but notexplored. The involvement of oxygen radicals in lipoxygenaseand lipid peroxidation was not explored in detail in this studybut it is possible that they play a role in these responses.However, initial experiments using the oxygen radical scav-

engers a-tocopherol and dabco had no effect on electrolyteleakage induced by either elicitor.

Lipid peroxidation in elicitor-injected tissue was deter-mined using the TBA test for malonaldehyde, a widely usedassay for lipid peroxidation in plant tissues (6, 10, 21, 29).Malonaldehyde is formed in small amounts during the per-oxidation of lipids in vivo but the majority detected in theassay is produced from existing peroxides during the acidheating stage of the assay (18). It is for this reason that theactual amount of malonaldehyde measured in the assay was

not quantified with a malonaldehyde standard as it would notgive an accurate estimate of the malonaldehyde formed invivo.

Lipoxygenase and lipid peroxidation were both inducedwith SE injection when the plants were incubated in the lightor the dark but necrosis was only induced in plants incubatedin the light. de Wit et al. (9) have reported similar light effectson necrosis. The lipoxygenase and lipid peroxidation re-

sponses appeared to be specific for the incompatible interac-tion, as they were not induced with SE injection into thecompatible cultivar Bonny Best. This result suggests that theinduction of these responses by the SE in the dark are unlikelyto be due to some nonspecific effect of the SE on the hostcells. The induction of lipoxygenase and lipid peroxidationwithout the concomitant induction of necrosis is significantas it suggests that these phenomena are separable, a possibilityalso suggested by the experiments with the NSAIDs. Theroutine use of necrosis as the only assay for SE activity (8)may therefore be misleading if other responses are occurringin the absence of necrosis. It appears that the light requirementfor necrosis is minimal as the brief exposure during photog-raphy of the injected panels in one experiment was enoughto induce substantial necrosis compared to a repeat experi-ment in which the plants were left in more complete darkness.SE or products induced by it may be photosensitized in thelight and transfer energy to oxygen or other molecules tocreate radical species which are in turn necessary for the

production of necrosis (18). The light required for necrosis,however, appears not to be necessary for the induction oflipoxygenase and lipid peroxidation.

ACKNOWLEDGMENTS

The authors would like to thank Albert Tenuta for assistance withthe graphics and Dr. E. Blumwald for critically reading the manu-script.

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27. Pavlovkin J, Novacky A, Ullrich-Eberius CI (1986) Membranepotential changes during bacteria-induced hypersensitive re-action. Physiol Mol Plant Pathol 28: 125-135

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