Plant Physiol. (1978) 61, 561-566

Corn Leaf Isoperoxidase Reaction to Mechanical Injury andInfection with Helminthosporium maydisEFFECTS OF CYCLOHEXIMIDE

Received for publication July 19, 1977 and in revised form October 27, 1977

HELENA BIRECKADepartment of Biological Sciences, Union College, Schenectady, New York 12308

MICHAEL 0. GARRAWAYDepartment ofPlant Pathology, The Ohio State University, Columbus, Ohio 43210

ABSTRACT

Mechanical injury or infection with Helmintosporium maydis race T orO en proxidase acti in leaves of two corn ibreds which differin their susceptibility to the fungal race T. Increases in act were foundin the soluble fraction extracted from tissues with 20 mm phosphate buffer(pH 6), and in the ionicafUy bound fraction extracted fromn wail debris with0.6 to 1 M NaCa; the covalently bound wai peroxidase fractin wasunaffected. Mechanical injury and infection with either race enhanced thesame distinctive cathodic isoforms present in the soluble fraction or in boththe soluble and lonicaUy bound fractons.

During the firt 20 to 22 hours after ioculation no differences werefound in the enzym enhancement in relaton either to the corn line orfungal race. During the second day, the weaker the infection symptom,the greater was the peroxidase encement. The race T-nfected suscep.tibe ibred sbowed no Increase or a decrease in the enzyme actvity,whereas in most cases both brweds Infected with race 0 exhibited a furtherperoxidase e mt.

Pretreatment of heathy leaves or leaf discs with cyclhexide atconcentratons which did not cause a loss of turgor had no effect onperoxidase enhancement. However, in tssues Inoculated with either therace T or 0, cyclohexmidle, at concentrations whi did not affect themechanical Injurywinduced Increase In peroxidase actvity, did hibit theenzyme enhancement and did cause a loss of turgor.The observed changes in Isoperoxidase activtes (a) confirm the as-

sumpion that the Infection-induced enhancement of this enzyme restsfrom a nonspecific response to injury, (b) support the postulation that theoften observed snuall or no Increases in peroxidase activity to compatiblehosts are a consequence of metabolic diorders rather than the cause of alower resistance; and (c) hdficate that cyclobeximide as a protein sthesisinhbltor may increase the compatibit hetween the bost and pathogn,enhane mDetabolic disorders in ioculated tissues, and in coneqeinterfere with the nonspecific cell response to Injury.

Actnomycin D did not affect the mechankal Injury- or infecton-inducedperoxidase enhancement. Neither did ethylene at 5 to 80 pII have asignificant effect on the enzyme actity in healthy, injured, or Infectedtiues.

infected with the fungus. The same isoperoxidases (IPs) whichreacted to infection reacted also to mechanical injury. Such asimilarity was also found between mechanically injured and to-bacco mosaic virus-infected tobacco leaves (5). The similarities inthe reaction to mechanical and viral or fungal injuries wereobserved not only in the distinctive IPs that increased in activity,but also in their distribution between cell protoplast and walls,thus supporting the assumption that the infection-induced perox-idase enhancement may result from a nonspecific response toinjury. Without eliminating the possibility of peroxidase contri-bution to the plant protection against a parasite, we postulatedthat the often observed relatively small increases in the enzymeactivity in compatible hosts may be a consequence of metabolicdisorders rather than the cause of a lower resistance.Vance et al. (23, 24) have recently reported that attempted

penetration of Helminthosporwum avenae into leaf discs of a non-compatible host, reed canarygrass, was accompanied by an in-crease in peroxidase activity, in particular in the walls at thepenetration sites, and in wall lignification. Exposure of the inoc-ulated discs to cycloheximide solutions at 10 or 25 itg/ml for 18 hrresulted in inhibition of peroxidase enhancement and lignin for-mation and in penetration of the fungus into the leaf tissue. Theauthors have suggested that the resistance mechanism involves aninduction of cathodic IPs in the challenged tissue.

This report represents a continuation of previous studies on theFunk corn N inbred and its male sterile counterpart and dealswith the IP reaction to two H. maydis races, T and 0, differingfrom each other in the production of the host-specific T toxin.The great compatibility between the Tms inbred and the fungalrace T is thought to be due to the susceptibility of the hostorganelle membranes to the T toxin (2, 19). Both Tms and Ninbreds exhibit a low susceptibility to the race 0, nonproducer ofthe toxin. Special attention has been paid to the effect of cyclo-heximide on peroxidase enhancement induced by mechanicalinjury and infection. In view of the implication of ethylene inplant pathogenesis (20), its effect on peroxidase has also beeninvestigated.

In previous studies (4) two corn inbreds with normal (N)' andTexas male sterile (Tms) cytoplasm, which differ in their suscep-tibility to Helminthosporium maydis Nisikado and Miyake race T,showed no significant differences in the peroxidase reaction when

' Abbreviations: N: normal; Tms: Texas male sterile; IP: isoperoxidase.

MATERIALS AND METHODS

The plants were grown on one-fourth Hoagland solution in thegreenhouse. The fourth and fifth leaves from 3- to 4-week-oldplants were used in all experiments. After detachment and rinsingwith H20 leaf laminae were sprayed with H. maydis race T or 0spore suspensions (2,000-3,000 spores/ml) in H20 containing 0.1%(v/v) Tween 40. Mechanical injury was inflicted by rubbingdetached laminae with carborundum followed by rinsing. Theintact (control) or injured laminae were sprayed with H20 con-

561

BIRECKA AND GARRAWAY

taining 0.1% Tween 40. In some experiments the detached leaveswere placed with their cut end in H20, actinomycin D, cyclohex-imide, or horseradish peroxidase solutions for 210 min underlaboratory conditions. The immersed leafportion was then blottedwith filter paper and cut off before appropriate inoculation. Thelaminae were stapled to a sheet of filter paper and were kept inthe dark in a water vapor-saturated atmosphere at 28 C.

In experiments with ethylene, the control and inoculated lami-nae were placed separately in hermetically sealed water vapor-saturated conical flasks into which C2H4 was injected using asyringe. In some treatments 0.25 M mercuric perchlorate solutionwas also added. Pea seedlings, known to react to C2H4 withperoxidase enhancement, were placed together with corn leaves inone of the experiments. After exposure, ethylene present in the airas well as that absorbed by Hg2(CI04)2 solutions were determinedusing gas chromatography.Two leaves/replicate and three replicates/treatment were used

in all experiments.Leaf discs 8 mm in diameter were cut with a cork borer. The

discs were exposed in Petri dishes to H20 or actinomycin D orcycloheximide solutions containing traces of Tween 40. Afterexposure they were rinsed, blotted, placed on moist filter paper inPetri dishes, inoculated, and transferred to a thermostat at 28 C.The possible peroxidase leakage into the paper wasasd afterexposure.

Immediately after sampling the tissue was weighed and trans-ferred to a deep freeze. Procedures related to peroxidase extractionwere similar to those previously used (7). Attempts to isolate freeperoxidase from cell walls, including intercellular spaces, by infil-trating fresh tissue with water were unsuccessful. Therefore, theprotoplast enzyme fraction extracted with 20 mm buffer (pH 6)included the free fraction present in walls.

Extraction of the ionically bound fraction from wall debris with0.6 M NaCl yielded the same results as did extraction with I MNaCl; therefore, the weaker solution was used in most experi-ments.

In contrast to pea internode peroxidase (9), the soluble fractionfrom corn leaf homogenates did not show a significant increase inactivity when 20 mm buffer (pH 8.9) or 100 mm buffer (pH 6) wassubstituted for 20 mm buffer (pH 6). Thus, no marked artifactslikely occurred during separation of this fraction.

Peroxidase determination, electrophoresis, and IP snning

were carried out as previously (3). Prior to electrophoresis, theNaCl extracts were diluted in order to avoid changes in themobility of IPs due to hih salt concentration. In some casesappropriate amounts of NaCl were added to the buffer-solublefractions to enable comparative analysis of the IP spectrum. Therelative differences between treatments in the enzyme activity inextracts before electrophoresis were similar to those found aftersummation of individual IP activities following electrophoresis.This indicates that the obtained data were not affected by freeinhibitors or activators present in the extracts, provided that theirmobility was different from that of the IPs. The IP patterns didnot reveal the presence of the maize mitochondrial Cyt c whentested by pretreating the gels with H202 and KCN solutions (12).No statistical analyses were carried out, but where differences

are described as significant, all replicates differed from all repli-cates in the appropriate controls or treatments.

RESULTS

Six experiments were carried out with leaves inoculated withH. maydis immediately after detachment. The infection symptomsduring the first 18 to 24 hr after inoculation did not revealsigificant differences in relation to the inbred line or fungal race.In the later period, Tms inbred leaves infected with the race Tshowed large oblong lesions and flaccidity, whereas lesions in theN inbred were much smaller and the laminae remained turgescent.Race 0 caused pinpoint lesions and a visual decrease in Chl inboth inbreds; the laminae remained turgescent except for oneexperiment.Of six experiments, three, two, and one yielded results repre-

sented by experiments I, II, and III, respetively, in Table I. Totalperoxidase activity and its distribution between fractions weresimilar in both inbreds. Within the 1st day after inocuation, theinfection-induced enzyme enhancement showed no differences inrelation to the inbred or fungal race. During the following day asigificant further increase in the activities ofthe soluble and wallioncally bound fractions was observed in the race T-infected Ninbred in all experiments. However, no increase or even a decreasein the enzyme activities was found in the race T-infected Tmsinbred in four of six experiments. In the same period, the race 0-

Table I. Effect of Mechanical Injury and Infection with Helminthosporium maydis Races T and 0 on Peroxidase Activity in Detached Leaf Laminae of Tms and N

Cytoplasm Corn Inbreds.

The leaves were sprayed with H. maydis spore suspensions (2,000-3,000 spores/ml) in H20 containing 0.1% (v/v) Tween 40. Mechanical injury was inflicted byrubbing the laminae with Carborundum followed by rinsing. The laminae were kept in the dark at 28 C.

Experiment I Experiment II Experiment III

Imme- 20 hr after treatment 44 hr after treatment Inme- 48 hr after Imme- 22 hr after 48 hr after2

Peroxidase1 diately diately treatment diately treatment treatmentafter Control Mech. Infected Control Mech. Infected after Control Infected after Control Infected Control Infecteddetach- injury with race injury with race detach- with race |detach- with race with racement T 0 T 0 ment T 0 ment T 0 T 0

A A/min-g fresh wt

Tmis inbred

Soluble 115 130 238 182 190 150 248 169 253 130 152 203 253 137 150 190 205 156 134 257

Wall bound

Ionically 35 40 60 48 47 43 88 66 90 37 42 91 108 38 43 54 64 47 58 138

Covalently 20 18 23 25 27 16 25 25 25 18 21 26 25 17 19 22 23 21 24 26

Total 170 188 321 255 264 209 361 260 368 185 215 320 386 192 212 266 292 224 216 421

N inbred

Soluble 122 132 250 185 188 145 234 233 273 127 155 208 268 138 147 203 194 161 259 200

Wall bound

Ionically 32 35 53 50 53 40 95 90 102 35 37 97 105 35 39 55 59 44 119 58

Covalently 17 20 20 27 25 20 24 26 25 20 22 24 25 18 20 21 24 22 22 24

Total 171 187 323 262 266 205 353 349 400 182 214 329 398 191 206 279 277 227 400 282

1The soluble fraction includes protoplast isoperoxidases and those extracted from wallsand intercellular spaces with 20 mM buffer, pH 6.0.

2In this experiment, the race 0-infected N inbred leaves were heavily infected and flaccid.

562 Plant Physiol. Vol. 61, 1978

ISOPEROXIDASES, INFECTION, AND CYCLOHEXIMIDE

infected leaves of both inbreds showed a continued increase in

peroxidase activity, except for the N inbred leaves in experimentIII when they were heavily infected and flaccid. In most cases, 44to 48 hr after inoculation the Tms and N inbreds infected withrace 0 did not differ from each other in the enzyme activity.

Peroxidase reaction to mechanical injury induced by carborun-dum was similar to that observed in previous studies (4) when theinjury was induced by cutting the laminae into 8-mm segments.Both inbreds showed a similar enzyme enhancement in the solubleand ionically bound fractions. Neither mechanical injury norinfection caused significant changes in the activity of peroxidasecovalently bound to the walls.

Electrophoretic analysis of extracts from healthy, mechanicallyinjured, or race T-infected leaves of both inbreds invealed thepresence of the same six cathodic and four anodic IP bands in thesoluble fraction and four cathodic and two anodic IPs in the

563

ionically bound wall fraction. The infection- or mechanical injury-induced enhancement was due mainly to an increase in the activityof the same three cathodic IPs, C2, C3, and especially C5. No newIP bands were detected. Thus, the pattern of IPs and their reactionto mechanical injury or infection were similar to those found inprevious studies (4). The IP spectrum in race 0-infected leavesdid not differ from that in leaves infected with race T as presentedby experiment III in Table II. It is worth noting that the significantdecrease of peroxidase activity in race T-infected Tms inbredleaves, observed during the 2nd day after inoculation in thisexperiment, was due to a decrease in almost all IPs in the solublefraction.

Exposure of detached laminae (Table III) or leaf discs (TableIV) to actinomycin D at 20 jig/ml for 210 min or at 10 ,ug/ml for120 min, respectively, had no effect on peroxidase enhancementdue to detachment and exposure in the dark, cork borer-induced

Table II. Effect of infection with Helminthosporium maydis Races T and 0 on activity of soluble andwall ionically bound isoperoxidases in leaf laminae of tms cytoplasm corn inbred

Experiment III (see Table I)

22 hr after treatment 48 hr after treatmentImmePdiately

after Infected with race Infected with racedetachment T O Control T O

Isoperoxidases Soluble I Wall Soluble I Wall Soluble I Wall Soluble I Wall Soluble I Wall Soluble I Wall

A A/min.g fresh wt

Cathodic 6 1

C6 trace 2 3 8 4 8 1 2 3 7 6 10

C5 1 14 20 16 23 23 1 17 12 18 28 70

C4 8 19 16 19 8 31

C3 26 41 46 32 30 76

C2 11 2 13 8 17 10 13 5 10 9 18 31

Cl 21 10 19 10 22 13 19 12 13 12 19 12

Total 67 28 115 42 128 54 85 36 76 46 178 123

Anodic

Al-A4 69 10 75 12 77 11 71 11 58 12 79 15

1 A similar isoenzyme pattern was found in the N cytoplasm inbred.

Table III. Effect of actinomycin D and cycloheximide on activity of combined soluble and wall ionicallybound peroxidase in detached healthy and infected leaf laminae of tms cytoplasm corn inbred

Detached intact laminae were placed with their cut end in H20, actinomycin D, cycloheximide, orhorseradish peroxidase solutions for 3.5 hr. After exposure the imersed leaf portion was blotted andcut off. The laminae after inoculation were placed in darkness at 28 C.

Experiment I Experiment II

Leaves Control Actinomycin D Cycloheximide Horseradish Control Cycloheximide

|H20 | 20 pg/ml 4 pig/mll 10 pg/ml peroxidase H20 40 )g/ml

& A/min*g fresh wt

immediately after treatment

Healthy 167 162 164 160 245 142 145

48 hr after treatment 24 hr after treatment

Healthy 207 210 207 195 291 170 175

Infected, race T 390 398 373 332 498 275 227

Infected, race 0 500 485 491 380 555

1The activity of the horseradish peroxidase solution was 825

hA/min-ml. In a separate group of laminae immersed

in a methyl red solution, the dye was spread throughout the laminae at the end of exposure.

Plant Physiol. Vol. 61, 1978

BIRECKA AND GARRAWAY Plant Physiol. Vol. 61, 1978

Table IV. Effect of actinomycin D and cycloheximide on activity of combined soluble and wall ionically boundperoxidase in control and infected leaf discs of tms cytoplasm corn inbred

Leaf discs 8 mm in diam were floated on water or actinomycin D) or cycloheximide solutions for 30 to120 min. After exposure, the discs were rinsed, blotted, inoculated, and placed in darkness at 28 C.

11zxperFmenu A 11 bxper3umenL 11

Exnoaure- min-

a A/min-g fresh wt

immediately after exposure

170

48 hr after exposure 24 hr after

145

272

181

367

265

190

370

mechanical injury, or infection within the first 48 hr. Nor didcycloheximide at 4,g/ml affect the enzyme enhancement indetached healthy or infected laminae. At 10 or 40 ,ug/ml, cyclo-heximide had no effect on peroxidase increases in detachedhealthy laminae, but in the infected ones enzyme enhancementwas diminished by 30 to 45%.

Cutting injury induced a significant increase of peroxidaseactivity in noninoculated leaf discs of both inbreds. Infection withrace T or 0 caused a further significant enzyme enhancement,except for race T-infected Tms inbred leaf discs 48 hr afterinoculation; the latter revealed even a lower peroxidase activitythan did the corresponding control discs.

Pretreatment with cycloheximide at 2 or 4 ,ug/ml for 30 min didnot interfere with the mechanical injury-induced increases inenzyme activity (Table V) or its distribution between the analyzedfractions (Table VI). However, at 8 ,ug/ml, cycloheximide in-hibited peroxidase enhancement by 75 to 80%. As shown in TableIV, pretreatment of noninoculated discs with the inhibitor at 10,ug/ml for 30 min or at 4, 10 or 25 ,ug/ml for 120 min caused adecrease in the enzyme activity. The sensitivity of peroxidaseenhancement to cycloheximide in the infected discs was signifi-cantly higher than that in the healthy ones. Pretreatment with theinhibitor at 2 to 4,g/ml for 30 min partially or completelyprevented the increase in enzyme activity; and at 8 Ag/ml, cyclo-heximide decreased the activity to 40 to 50% of the initial level.The effects of cycloheximide were revealed in the inhibition of

distinctive IP enhancement, in their distribution between thesoluble and wall fractions, and in a decrease in the activity ofanodic isoforms which reacted insignificantly to mechanical injuryor infection. In infected discs pretreated with cycloheximide at 8Ag/ml, the decrease in activity of cathodic and anodic IPs wasrevealed mainly in the soluble fraction.During the 24 to 48-hr exposure on moist filter paper, peroxi-

dase leakage from untreated or cycloheximide-treated healthydiscs was negligible. Some leakage was observed from cyclohexi-mide-treated, infected discs. However, it amounted only to 3%and 6% of the total activity in the diseased tissues pretreated withcycloheximide at 2 and 8 ,ug/ml, respectively.No significant differences were found in the activities of the

wall covalently bound peroxidase fraction in relation to actino-mycin D, cycloheximide, cutting injury, or infection.

In all cases when cycloheximide caused a decrease in peroxidaseactivity, infected as well as control discs showed a loss of turgor.The latter was also observed in control tissues exposed to the

90

68

65

82

65

58

245

278

288

232

203

215

exposure

140

98

140

145

142

150

Table V. Effect of cycloheximide on activitv of combined soltuble and wallionically bound peroxidase in control and infected leaf discs of

and N cvtoplasm corn inbred

Leaf discs 8 mm in diam were floated on water or cycloheximide solutionsfor 30 min; after exposure thev were treated as indicated in Table IV.

Tmn inbred lI N inbred

Cycloheximide. j!g/ml

Leaf discs 0 2 4 8 1l 0 2 4 8

/ A/ming fresh wt

immediately after exposure

Control 163 11 160148 hr after exposure

Control 320 310 290 200 300 298 278 190

Infected, race 0 392 282 165 67 418 300 205 75

inhibitor at 8 jig/ml, in which only the enhancement ofthe enzymeactivity was inhibited. No significant differences in the visualsymptoms of infection between cycloheximide-treated and un-treated, detached leaves or leafdiscs were noticed during sampling.The effects of ethylene at 5, 40, and 80 p1/1 on detached leaves

of both inbreds were studied in three experiments. In two experi-ments, C2H4 did not affect peroxidase in any treatment. In oneexperiment, healthy and race-O-infected leaves exposed to C2H4at 80 p1/1 revealed consistently higher, by 10 to 15%, enzymeactivities as compared with those found in corresponding leavesexposed to air in the presence or absence of mercuric perchlorate.Under the same conditions, pea leaves and stems showed 100 to150% higher peroxidase activities than did the controls. No effectof C2H4 on the severity of infection could be observed. Gaschromatography revealed evolution ofethylene by diseased leaves.

DISCUSSION

The problem of peroxidase involvement in plant resistance topathogens has been controversial for a long time (11). Varietaldifferences in the compatibility to pathogens have been related tovarietal differences in peroxidase or specific IP activities. Theinfection-induced enzyme enhancement has been thought to pro-tect the host against the pathogen. On the other hand, in manycases an increase in peroxidase activity was not accompanied byan increase in the plant resistance.

564

Leaf discs

Control

Control

Infected, race T

Infected, race 0

r.]VU ,_U .LLI

F.wn r iman t T

ISOPEROXIDASES, INFECTION, AND CYCLOHEXIMIDE

Table VI. Effect of cycloheximide on soluble and wall ionically bound isoperoxidases in control and race0-infected leaf discs of N cytoplasm corn inbred1

48 hr after exposureImmediatelyafter exposure Cycloheximide, pig/ml

° 0 2 4 8

Isoperoxidases Soluble| Wall Solublel Wall Soluble lWall Soluble Wall Soluble | Wall

is A/min-g fresh wt

Control discs

Cathodic 57 27 129 83 122 84 118 75 89 35

Anodic 68 8 77 11 79 12 74 11 59 7

Total 125 35 206 94 201 96 192 86 148 42

Infected discs

Cathodic 199 126 139 88 96 41 23 15

Anodic 81 11 63 10 58 10 31 6

Total 280 137 202 98 154 51 54 21

1 A similar IP distribution was found in Tms inbred leaf discs.

Since degradation of the cell wall by pathogen-secreted poly-saccharide-degrading enzymes plays a basic role in pathogenesis,wall IPs with an affinity to appropriate substrates for ligninsynthesis may be of major importance. However, a pathogen-induced enhancement of lignin synthesis does not necessarilyrequire an increase in the concentration of the enzyme in cellwalls. The activity ofIPs can be limited not by their concentration,but rather by the availability of substrates in situ Thus, evenevidence of no increase in the concentration of wail IPs, assedby measunng the activity in vitro, does not necssarily negate theirpossible role in the enhanced lignin formation. Indeed, the in-creases in activity of wall-bound IPs during the first 16 to 22 hrafter inoculation were relatively smail in our experiments witbcorn as well as in those with reed canarygrass (23, 24). Theincreases should be expected to be even smaller during the firstapproximate 10 hr crucial for the fungal penetration through thecell wall. No data are available for the activity of free IPs in thewalls, including intercellular spaces, in corn. However, this enzymefraction did not significantly increase in infected, wounded, orethylene-treated tissues of other species (5, 6).

Differences between the N and Tms corn inbreds in theirsusceptibility to H. maydis race T may be due to differences in thepermeability of mitochondrial membranes to the T toxin or inplasma membrane receptors (17). However, neither the N inbrednor the Tms one were fully incompatible to the race 0, indicatingthat both races exhibit an additional pathogenic potential notrelated to the T toxin.The fungal populations used in this study were more virulent

than those used in the previous one (14). Although the sporesuspension density was decreased 10-fold, the relatively highvirulency of the fungus could be responsible for differences be-tween experiments in the peroxidase reaction during the 2nd dayafter inoculation.

Seevers and Daly (21) reported a similar peroxidase enhance-ment in both resistant and susceptible wheat lines during the 1stday after the leaves were inoculated with stem rust. Subsequently,the enzyme activity continued to increase in the resistant type,whereas in the susceptible one, it increased only slightly if at all.In this study, the decrease of peroxidase activity in the race T-infected Tms corn suggests even an enhanced degradation ofpreexisting peroxidase molecules due to the pathogen-inducedmetabolic disorders.

Cycloheximide, even at concentrations used normally as aninhibitor of RER-related protein synthesis, is known to be non-specific and to affect various processes including interconversionof pyrimidine nucleotides (18). The loss of turgescence in nonin-oculated corn discs pretreated with cycloheximide at 8 to 10 ,xg/mlindicates an indirect effect on the integrity of membranes; thus,the inhibition of injury-induced peroxidase enhancement in thesetissues did not necessarily result directly from an inhibited de novosynthesis of the enzyme glycoproteins. Cycloheximide could haveinterfered with processes triggering the cell response to injury,which normally leads to activation or synthesis de novo of distinc-tive IPs. By affecting membrane integrity it could have led to arelease of lyases and a significant degradation of IPs even ifperoxidase enhancement took place simultaneously.

In inoculated intact or mechanically injured tissues, the effectof cycloheximide could be even more complex. It has been knownthat an increased synthesis of a large variety of proteins 6 to 18 hrafter inoculation is one ofthe first expressions ofthe host resistanceresponse induced by microbial metabolites (8, 13). Some of theseproteins may contribute to resistance functioning as carbohydrate-recognizing receptors located in the host plasma membrane (1).Cycloheximide as a protein synthesis inhibitor can increase thecompatibility between the host and the pathogen, thus acceleratingand/or increasing metabolic disorders in the host cells and inconsequence interfering with the nonspecific cell response toinjury. This could explain why cycloheximide at concentrationswhich had no effect on the enhancement of distinctive cathodicIPs in noninoculated corn tissues did inhibit this process indieased ones. This could also explain the sharp decrease in thecathodic as well as anodic IPs in infected leaf discs pretreated withthe inhibitor at 8 to 10 i&g/ml.

Increases in IP activity can be induced by a large range of stressfactors. No clear relation has been found between radiosensitivityof corn varieties and irradiation-induced changes in peroxidaseactivity (22). Herbicides or wounding cause not only an enhance-ment of peroxidase activity, but also an increased lignin deposition(14, 15), thus confirming that the latter process also is not specificfor resistance responses to pathogens.One can assume that chemical interactions between a pathogen

and an incompatible host during attempted penetration maytrigger specific resistance responses and some of the not wellunderstood nonspecific responses to injury, such as peroxidase

565Plant Physiol. Vol. 61, 1978

566 BIRECKA AND GARRAWAY Plant Physi6l. Vol. 61, 1978

enhancement. However, the use ofcycloheximide even in this casecannot supply reliable evidence of a specific role of any singlekind of proteins, including peroxidase, in the resistance mecha-nism. The cycloheximide-induced,destruction of the natural re-sistance of potato tuber discs to fluorescent Pseudomonas sp. (25)can serve as a good example.The local peroxidase enhancement in walls of epidermal cells,

observed by Vance et al. (23) during attempted penetration, couldhave been related to a renewed, cycloheximide-sensitive walldeposition (papillae). Young expanding cells, at least in tobaccoand sweet potato tissues (6, 7), reveal a high activity of wall-boundIPs, amounting to over 80%o of the total cell peroxidase activity. Itseems that the methods used by Vance et al. did not allow themto establish a cause-effect relation between the inhibition of IPs inthe wall around the penetration sites and the susceptibility tononpathogenic fungi. Their biochemical data concerning peroxi-dase activity indicate that cycloheximide at concentrations usedhad a toxic effect in the host tissue if, as reported by the authors,cutting injury by itself did not enhance the enzyme. One may takeinto account many factors, besides peroxidase, which could havebroken the reed canarygrass resistance to the fungus.

In most cases ethylene at the concentrations used had no effecton peroxidase activity either in healthy or diseased corn tissues,thus resembling in this respect previously investigated tobaccoleaves and carrot roots (5, 7). Within Graminaceae, C2H4 has beenshown to enhance the enzyme in wheat (10) and to have no effecton it in barley leaves (16). In previous studies on pea varieties (9),the increase in peroxidase activity induced by C2H4 at 80 ,/l/seemed to be due to its toxic action.

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