Plant Physiol. (1966) 41, 1505-1512
Increased Disease Resistance and Enzyme Activity Induced by Ethylene and Ethylene Production by Black Rot Infected Sweet Potato Tissue' M. A. Stahmann, B. G. Clare,2 and W. Woodbury Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
Received May 2, 1966.
Summiary. Exposuire of root tissuie from a susceptible varietj- of sweet potato to low concentrations of ethylene induiced a resistance to infection by Ceraitocvstis fimlbriaita and an increase in the activity of peroxidase and polyphenoloxidase in the tissuie. Susceptible tissuie that was inocLulated with a pathogenlic strain of C. fimzbri(ata or a nonpathogenic strain that can indtuce resistance liberated more ethylene into closed chambers than tissuie inocuilated wvith strains that di(d not induice resistance. It is stuggested that ethylene may be a stimuluis that diffulses from infected areas into adjoining tissue to initiate metabolic changes which may lead to disease resistance. Polyphenol oxidase bult not peroxidase activity was increased in slices of potato ttubers and parsnip roots treated with ethylene. The activity of these enzymes in root tissule of carrot, radish or tuirnip was not altered by ethylene treatment. the enzymatic activity of roots of sweet potato, The disease of sweet potato roots known as parsnip, carrot, tuirnip, radish and white potato black rot is cauised by certain isolates of the ftungus tuibers. The produiction of ethylene by sweet potato Ceratocystis fimitbriaita Ell. and Halst. The suirface root tissuie inoctulated with pathogenic and nonof root tissue which is normally suisceptible to black pathogeniic isolates of Cer(atocystis is also reported. rot can be made resistant to infection by prior inocuilation with nonpathogenic isolates of C. fitttbriata (16, 17). Root tissuie with this induicedl reMaterials and Methods sistance has increased peroxidase and polvphenol oxidase activitv (16, 17) similar to that fouindl in Preparatioi of Plait Tissiue. Tissuies uised were inoculated natuirally resistant tissuie. V'olatile mawhite potato tuLbers (Solanumm tuberosuim) and roots terials from infected root tissute increased the of sweet potato [Iptiloeoa b(at(itas (L) Lam.], carrot activity of these enzvmes and increased the resist[Dattucus cairotai (L) var. sativa], parsnip [Paistinalca ance of uninfected suisceptible root tissuie (5). sativa (L)], tuirnip [Brassica rapa (L)] and radish Preliminary experiments showed that volatile ma[Ra phlinu(s satizvis (L)]. The sweet potato varieties terials from apples are able to induice similar inwere jtulian (sutsceptible to black rot) and( Suinnycreases in enzymic activity and resistance in norside (resistant to black rot). WVhole roots or tubers mally stusceptible tissue (5). Since apples are were sutrface sterilized with sodiutm hypochlorite known to produice ethylene (2) these resuilts gave soluttion and ctut into slices 2 cm thick. These slices suipport to the view that ethylene may be involved were inocuilated or treated with ethylene. in the changes noted in sweet potato tissuie. Inoculation of Szweet Potaito Tissute. Ctlltuires Ethylene is produced b.' a variety of injtured and of a pathogenic isolate of C. fimiibriat(a Ell. and infected plant tissuies (12) and it was consi(lered Halst. (isolate U-6), another isolate of C. fimibriata that it may be the active volatile material produticed (F-38) which is nonpathogenic and induLices resistby diseased sweet potato tissule. ance and a nonpathogeni, C. miliinor (Hedg.) Huint This paper reports the effect of ethylene on the which does not indul1ce resistance, were grown oIn resistance of sweet potato roots anid its effect oIn potato dextrose agar suipplemented with thiamine (14) at 2 50 for 1 week. Spores from these cultures were suispen(le(l in sterile distilled water aindl uisedl I Sup)orted by funds from the Research Comiimittee as inoculuim. The cuit suirfaces of sweet potato of the Graduate School, University of Wisconsin from slices were inocuilated with 1 ml of spore suispelnsion. funds supplied by the Wisconsin Alumni Research Foundation and by funds from the Herman Frasch Foundation Control slices were treated with sterile distilled for Agricultural Research. Second author in receipt of water. The slices were placed on stainless steel Fulbright Travel Grant. screens above w-et filter paper at the bottom of - Present address: Waite Agricultural Research Incrystallizing dlishes (15 cm X /7.5 cm). Another stitute, University of Adelaide, Ade!aide, South Auistralia. Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org 1505 Copyright © 1966 American Society of Plant Biologists. All rights reserved.
l1506
PLANT PHYSIOLOGY
crystallizing dish of the same size was inverted ( 11 ). Polyphenol oxidlase was detected in gels over each (lish containinig root tissute. The jtunction which ha(l been washed for 30 mintutes in 0.1 MN lhetween each set of dishes was covered with a strip phosphate buiffer pH 6.5 utsing a soltution of catechol of alutminutm foil and seale(d with adhesive tape. (1 mg/ml) and proline (I mg/ml) in the same Approximately 200 g of tissute was place(d in each buffer (7). chamber which ha(l a volume of 2.6 liters. The Enzyiic Assays. Peroxidlase activity in extracts tisstie was incutbated at room temperatuire for tip was measutred spectrophotometrically by following to 6 days. the oxidation of pyrogallol at 420 mju or catechol Gas Anattlysis. Samples wvere withdrawnv from at 390 mn (8) at 15 second intervals in the presence the chambers described above usling a Hamilton gas of H..O.,. Polyphenol oxidase was meastured specsyringe. Analyses were carried outt in an Aerotrophotometrically or manlometrically utsing catechol graph 1520 gas chromatography apparatuts eqltipped as substrate (1). with a hydrogen flame ionization (letector. .A stainless steel colutmn (152 cm X 3 mm) packe(d with Results 15 % diethylene glycol sutccinate (D)EGS) on hexamethyldisosilazaine (HMDS) treatedI Chromosorb W\ (80-100 mesh) was tused at 240. Nitrogen carSweet potato tissute treatedl with ethylene showed rier gas was sutpplied at 15 ml per minutte. Hydroa markedl increase in resistance to black rot (fig 1). getn flow wvas 32 ml per minulte. Extracts of treated tissute showed a marked increase Treatmnent of Tissues with Etlhylentc. Slices of in peroxidase (fig 2) and polyphenol oxi(lase (fig tissute aboutt 2 cm thick were placed in 20 1 con3) activity only after electrophoresis in polyacr)yltainers. Ethylene mixtuires of from 8 ppm to 220 amide gels. Polyphenol oxidlase activity couldl not ppm were introdLuced into these chambers lsling the be detected when criu(le extracts were assayed manoevacuation methodl (13). The contaiiners were metrically or spectrophotometrically. This apparent lack of activity was foutnd to be duIte to the presence sealed anid inculbatedl at 250 for either 2 or 3 days. Tisstues incubated in similar contaiiners in the abof inhibitory stl)stances in the extracts. The inseince of added ethylene serveed as controls. The hibitory stubstances colkd lbe remove(l 1h electroslices were then removed and were either tusedl for phoresis or precipitate(d from extracts b)y a(ljulsting the preparatioin of extracts or were inoctilate(l with them to 1.5 M with ammoniitm suilfate. Polyphenol spore suispensionis of C. fimntbri(ata and incubated at oxidase activity was then detectedl in the fraction 250 for 3 wveeks. Resistance to infectionl wtas which was precipitate(d with satuirate(d amniuomili11i assessed after this perio(l. stlfate. After fractionation with ammonlilum sillPreparation of Extr(cts. Cylinlders ('18 mm fate, it was foutnd( that increases in polvphenol oxidase activity in extracts from ethylene treate(d (iam) were cutt from treated root ant(I tul)er slices. D)iscs I mm thick were cutt transversely from these tissute cotuldI be detected spectrophotometricallv cylinders anid( extracte(l under nitrogen in an equtal (table I) and manometrically. The restults obtained 1w these 2 methods were similar. volume of 0.1 zs Tris-hydrochloride buffer, pH 7.4, containiing 0.36 xm sutcrose (6), aind 0.06 Mf ascorbic Peroxidlase activity in crll(le extracts ani(l in exacid. Extractions were (loie at 40 in a mo(lifie(l tracts fractionate(l with ammoniiuim sutlfate w ere sodiutm press. The extracts were centrifulge(d at essentially the same when assayed spectrophoto100,000 X ql for 15; minuLtes aindI the suiperinatanit metrically. There was an approximate 10-foldc infractioins were collecte(d and storedI at -15 . crease in peroxidase activity in sw,eet potato tissuies treated with ethylene (table I). Gel Electrophoresis. Extracts were subjected to electrophoresis in polyacrylami(le gels (10). Gels Extracts from ethylene treated tissiue wheni suIbobtained with extracts of sweet potato were staine(d jectedI to gel electrophoresis showveed increase(l anmto cletect amylase, phosphatase, peroxidase anid( polyylase activity but no appreciable alterationi in aci( phenol oxidase activity. Acid anid alkaliine phosor alkaline phosphatase activity. phatase activity was detected by incubating the gels In extracts from white potato treated with in filtered soltutions of a-naphthyl phosphate (1 ethylene, increased polyphenol oxidase activity was mg/ml) diazo blute B (I mg/ml) and 'MgC., (1 fotund whereas peroxidase activity decrease(l (table mg/ml) in either 0.1 M acetate buiffer pH 4.6 or I). 0.1 -i Tris-HCI btuffer pH1 9.0 (4). The gels were Polyphenol oxidase activity w,as higher in eximmersed in the appropriate buffer for 30 minuites tracts from parsnip roots treated with ethylene than before being inctlibated in the staining soluitions. in extracts from uintreated roots (fig 3). PolvAmylase was detectedl by incorporating 1 % starch phenol oxidase activity was (letected in extracts in the gels before electrophoresis. After electroonly after electrophoresis in polyacrylamide gels. phoresis the gels were washed with 0.2 M acetate No activity cotuld he dletected spectrophotometrically btl)ffer pH 4.6 for 30 minuites and(I theni iniciubated in either crudcle parsnip extracts or in extracts fracin a soltutionl of iodinle (0.04 mg/ml ), KI ( 10 tionate(l with ammoniutm suilfate. No changes inl mg/ml) in 0.2 M\ acetate buffer pH 4.6. Peroxidlase peroxidlase activity of parsniip extracts followinlg was tletecte(l tusing 20 m_u guaiacol anid 0.3 %onH,2, were dletecte(l. Downloaded from www.plantphysiol.org Septemberethylene 29, 2015 treatment - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE
1507
Resistance Increased by Ethylene Treatment Sweet potato slices treated with ethylene for two days and then inoculated with C. fimbriata. Julian
0 ppm
8 ppm
150 ppm
FIG. 1. Resistance increased by ethylene treatment. Sweet potato slices were exposed to ethylene-air mixtures for 2 days at the concentrations indicated and then inoculated with C. fimbriata.
Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
1509
STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE
r
8; i
a_
1.
I f
I
ti
,i s.
I
40.
__aimNc .t
/ .
I
/*4 FIG. 2. Peroxidase activity of discs cut 2 mm below the surface of susceptible sweet potato tissue treated with 0 (2), 24 (5), and 220 (8) ppm ethylene. Extracts of discs were subjected to electrophoresis on acrylamide gels and stained for enzyme activity.
4 K
l
9 FIG. 3. Polyphenol oxidase activity of discs cut 2 mm below the surface of susceptible sweet potato tissue treated with 0 (4), 8 (5) and 150 (6) ppm ethylene. Extracts of discs were subjected to electrophoresis gels and stained for enzyme activity.
onl acrylamide
Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
1511 I
STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE
Table I. Peroxidase and Polvplicnol Oxidase Activity in Extracts of Swcet Potato Root Tissue and White Potato Tuber Tissue Treated with Ethylene Enzyme activity is expressed as change in optical density at 390 mu/ml of extract/minute. Ethvlene (ppm) 0 8 150
Swseet potato (Julian) Peroxidase Polyphenol oxidase 1.8 0.06 20.5 0.13 26.8 0.13
White potato Peroxidase 0.20 0.16 0.08
Table IT. E!hylene Productioni bl,y Szceet Potato Tissue Inoculated with Fungal Spores Incubated for Three Days
Polyphenol oxidase 0.36
1.50 0.78 Or
Left Uninoculated and
Ethylene produced (ppm) Julian (susceptible) Sunnyside (resistant) None (unwinoculated) 1.5 1.5 Ceratocvstis rtinor* 2.0 4.0 C. fimbriata F38** 13.0 9.0 C. fimbriata U6*** 75.0 15.0 * Nonpathogenic to sweet potato, does not induce resistance to black rot. ** Nonpatlogenic to sweet potato, induces resistance to black rot. *** Produces black rot in susceptible sweet potato. Ethylene treatment had no apparent effect uipon Discussion the peroxidase or polyphenol oxidase activity of carrot, radish or tturnip root tisstue. Very little The results of these experiments indicate that polyphenol oxidase activity was detected in unethylene can increase resistance of sweet potato treated and ethylene treated radish and tturnip extissute to infection by C. fimlbriat(o. They also intracts. dicate that such indtuced resistance is associated with A volatile compouindl with the same retentioni an increase in the peroxidase and polyphenol oxi(lase time as ethylene on the gas chromatographic coltumn activity of ethylene treated tissuies. Ethylene was used in these experiments was demonstrated in the not detected above cultures of C. fimtbriatia grown atmosphere sturrotunding sweet potato tissue inctuon potato dextrose agar btut was detected above bated in closed chambers after being treated with infected sweet potato tisstues at concentrations apwater or being inoculated with C. finmbriata or C. proximating those used in ou r experimental treatm inor (table II). The retention time for both ment of utninoctulated tissue. It is therefore probethylene and the material from sweet potato was able that ethylene is an active principle in volatile 20 seconds. In table II the approximate amotunts materials from infected tisstues which cauise the of this compouind produtced by the variouisly treated increased resistance and peroxidase activity in tintisstues are expressed as ppm ethylene. The values inocuilated sweet potato tissule (5)). were obtained bv comparing areas tin(ler peaks on Higher levels of ethylene were observed above the chromatograms at a retention time of 20 seconds sweet potato tissue inoculated Nvith a nQnpathogenic with areas uinder peaks produiced by known constrain of C. fimlbriatat (induicer, F-38) which incentrations of ethylene at the Fame retention time. duices resistance to black rot than above uninocuilated However, these values and the identification of the tissuie or tissue inocuilated with nonpathogenic C. compouind as ethylene mutst be regarded as tentative, mtinor (noninducer) which does not indtuce resistfor oinly 1 type of column was used. Uninocuilated ance to pathogenic strains of C. fimitbriata. This resistant or susceptible tissue produiced very little suiggests that ethylene may be involved in the induicdetectable ethylene. Produiction of ethylene intion of resistance to black rot in suisceptible tissuie creased following inoculation with all 3 fuingi but inocuilated with inducers (16,17). Peroxidase and the increase was larger when the nonpathogenic polyphenol oxidase activity were also increase(d in strain of C. finbriata (F-38) which induice(d retissuie with indtuced resistance to black rot (16, 17). sistance w-as used than when the nonpathogenic It is possible that ethylene is one of the stimuili strain of C. imlitnor which did not induice resistance which move from areas of C. fimibriaita infection in was uised. It was largest when the pathogenic strain sweet potato into adjoining tissue to initiate the was tused. Under the experimental conditions tused, metabolic changes which lead to resistance to fuirethylene produiction decreased after 4 days inclubather penetration by the pothogen. Since ethylene tion. This decrease may have been duie to decreased can increase peroxidase and polyphenol oxidase O., and increased CO., levels withiin the seale(d activity in sweet potato tissule it appears that these conttitiners. enzymes may be involved in the resistance mecha-
Fungus
Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
1512
PLANT IPHYSIOLOGY
However, as was previou sly noted (17), increases in the activity of these enlzymes alone cainniot lie solely responsible for resistanice. Other factors are involved an(l these may inclul(le the rate of synthesis or transport of substrates to the areas of increase(d polyphenol oxidlase activity. It is not known whether the inhibitor of polyphenol oxidase foulnd( in sweet potato extracts is effective in vivo and hence howN mulch of the increase in activity of this en7zyme is effective in the tissule. However, there is a growving body of ev%i-idence that peroxi(lases act in oxi(latioin reactions requiiring only catalytic amotunits of hy-drogen peroxide (18). Increase(l activity of these 2 enzymes in sweet potato tissuie stimulate(d by ethylene couldk therefore very likely give rise to abnormal or increase(d aromatic biosynthesis resulltinig in the formation of 1)oth physical andcl chemical barriers to infectioln (9, 15). Little change in peroxidase or polyphenol oxidlase activity was note(l in eth, Aene treate(d carrot, tulrInip or radlish tissule. Polyphenol oxidase buit not peroxi(lase activity was inlcrease(l in ethy lenie treated potato and(I parsnip tissuie. It is possible that the changes note( iin ethylene tr.-ated sweet potato tissule are peculliar to that species. Ho\w ev er, it seems probable that ethylene has a more genieral role in the (lefense mechanisms of plailts. It may act as a stimu1lLls of localized metabolic changes lea(dinig to necrotic and(1 hypersensitive reactions in p'anits following infectioln. Since ethYlene is capable of produicinig chlor -phyll degra(lationi in fruiits (2), it is also possible that ethylene pro(duiction in infecte(d greeni planit tissuie may caulse the chlorosis so commonly observed inl sulchi tissu1es.
t,c,
nism.
Literature Cited 1. .\ALBiEG1INA, F. A. NI. 1964. Clilorogenic aci(d oxidases fromil ip)tato tuber slices p)artial piurificationl anid prop)erties. Phy tochenilstry 3: 65-72. _. ButV i
Increased Disease Resistance and Enzyme Activity Induced by Ethylene and Ethylene Production by Black Rot Infected Sweet Potato Tissue' M. A. Stahmann, B. G. Clare,2 and W. Woodbury Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
Received May 2, 1966.
Summiary. Exposuire of root tissuie from a susceptible varietj- of sweet potato to low concentrations of ethylene induiced a resistance to infection by Ceraitocvstis fimlbriaita and an increase in the activity of peroxidase and polyphenoloxidase in the tissuie. Susceptible tissuie that was inocLulated with a pathogenlic strain of C. fimzbri(ata or a nonpathogenic strain that can indtuce resistance liberated more ethylene into closed chambers than tissuie inocuilated wvith strains that di(d not induice resistance. It is stuggested that ethylene may be a stimuluis that diffulses from infected areas into adjoining tissue to initiate metabolic changes which may lead to disease resistance. Polyphenol oxidase bult not peroxidase activity was increased in slices of potato ttubers and parsnip roots treated with ethylene. The activity of these enzymes in root tissule of carrot, radish or tuirnip was not altered by ethylene treatment. the enzymatic activity of roots of sweet potato, The disease of sweet potato roots known as parsnip, carrot, tuirnip, radish and white potato black rot is cauised by certain isolates of the ftungus tuibers. The produiction of ethylene by sweet potato Ceratocystis fimitbriaita Ell. and Halst. The suirface root tissuie inoctulated with pathogenic and nonof root tissue which is normally suisceptible to black pathogeniic isolates of Cer(atocystis is also reported. rot can be made resistant to infection by prior inocuilation with nonpathogenic isolates of C. fitttbriata (16, 17). Root tissuie with this induicedl reMaterials and Methods sistance has increased peroxidase and polvphenol oxidase activitv (16, 17) similar to that fouindl in Preparatioi of Plait Tissiue. Tissuies uised were inoculated natuirally resistant tissuie. V'olatile mawhite potato tuLbers (Solanumm tuberosuim) and roots terials from infected root tissute increased the of sweet potato [Iptiloeoa b(at(itas (L) Lam.], carrot activity of these enzvmes and increased the resist[Dattucus cairotai (L) var. sativa], parsnip [Paistinalca ance of uninfected suisceptible root tissuie (5). sativa (L)], tuirnip [Brassica rapa (L)] and radish Preliminary experiments showed that volatile ma[Ra phlinu(s satizvis (L)]. The sweet potato varieties terials from apples are able to induice similar inwere jtulian (sutsceptible to black rot) and( Suinnycreases in enzymic activity and resistance in norside (resistant to black rot). WVhole roots or tubers mally stusceptible tissue (5). Since apples are were sutrface sterilized with sodiutm hypochlorite known to produice ethylene (2) these resuilts gave soluttion and ctut into slices 2 cm thick. These slices suipport to the view that ethylene may be involved were inocuilated or treated with ethylene. in the changes noted in sweet potato tissuie. Inoculation of Szweet Potaito Tissute. Ctlltuires Ethylene is produced b.' a variety of injtured and of a pathogenic isolate of C. fimiibriat(a Ell. and infected plant tissuies (12) and it was consi(lered Halst. (isolate U-6), another isolate of C. fimibriata that it may be the active volatile material produticed (F-38) which is nonpathogenic and induLices resistby diseased sweet potato tissule. ance and a nonpathogeni, C. miliinor (Hedg.) Huint This paper reports the effect of ethylene on the which does not indul1ce resistance, were grown oIn resistance of sweet potato roots anid its effect oIn potato dextrose agar suipplemented with thiamine (14) at 2 50 for 1 week. Spores from these cultures were suispen(le(l in sterile distilled water aindl uisedl I Sup)orted by funds from the Research Comiimittee as inoculuim. The cuit suirfaces of sweet potato of the Graduate School, University of Wisconsin from slices were inocuilated with 1 ml of spore suispelnsion. funds supplied by the Wisconsin Alumni Research Foundation and by funds from the Herman Frasch Foundation Control slices were treated with sterile distilled for Agricultural Research. Second author in receipt of water. The slices were placed on stainless steel Fulbright Travel Grant. screens above w-et filter paper at the bottom of - Present address: Waite Agricultural Research Incrystallizing dlishes (15 cm X /7.5 cm). Another stitute, University of Adelaide, Ade!aide, South Auistralia. Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org 1505 Copyright © 1966 American Society of Plant Biologists. All rights reserved.
l1506
PLANT PHYSIOLOGY
crystallizing dish of the same size was inverted ( 11 ). Polyphenol oxidlase was detected in gels over each (lish containinig root tissute. The jtunction which ha(l been washed for 30 mintutes in 0.1 MN lhetween each set of dishes was covered with a strip phosphate buiffer pH 6.5 utsing a soltution of catechol of alutminutm foil and seale(d with adhesive tape. (1 mg/ml) and proline (I mg/ml) in the same Approximately 200 g of tissute was place(d in each buffer (7). chamber which ha(l a volume of 2.6 liters. The Enzyiic Assays. Peroxidlase activity in extracts tisstie was incutbated at room temperatuire for tip was measutred spectrophotometrically by following to 6 days. the oxidation of pyrogallol at 420 mju or catechol Gas Anattlysis. Samples wvere withdrawnv from at 390 mn (8) at 15 second intervals in the presence the chambers described above usling a Hamilton gas of H..O.,. Polyphenol oxidase was meastured specsyringe. Analyses were carried outt in an Aerotrophotometrically or manlometrically utsing catechol graph 1520 gas chromatography apparatuts eqltipped as substrate (1). with a hydrogen flame ionization (letector. .A stainless steel colutmn (152 cm X 3 mm) packe(d with Results 15 % diethylene glycol sutccinate (D)EGS) on hexamethyldisosilazaine (HMDS) treatedI Chromosorb W\ (80-100 mesh) was tused at 240. Nitrogen carSweet potato tissute treatedl with ethylene showed rier gas was sutpplied at 15 ml per minutte. Hydroa markedl increase in resistance to black rot (fig 1). getn flow wvas 32 ml per minulte. Extracts of treated tissute showed a marked increase Treatmnent of Tissues with Etlhylentc. Slices of in peroxidase (fig 2) and polyphenol oxi(lase (fig tissute aboutt 2 cm thick were placed in 20 1 con3) activity only after electrophoresis in polyacr)yltainers. Ethylene mixtuires of from 8 ppm to 220 amide gels. Polyphenol oxidlase activity couldl not ppm were introdLuced into these chambers lsling the be detected when criu(le extracts were assayed manoevacuation methodl (13). The contaiiners were metrically or spectrophotometrically. This apparent lack of activity was foutnd to be duIte to the presence sealed anid inculbatedl at 250 for either 2 or 3 days. Tisstues incubated in similar contaiiners in the abof inhibitory stl)stances in the extracts. The inseince of added ethylene serveed as controls. The hibitory stubstances colkd lbe remove(l 1h electroslices were then removed and were either tusedl for phoresis or precipitate(d from extracts b)y a(ljulsting the preparatioin of extracts or were inoctilate(l with them to 1.5 M with ammoniitm suilfate. Polyphenol spore suispensionis of C. fimntbri(ata and incubated at oxidase activity was then detectedl in the fraction 250 for 3 wveeks. Resistance to infectionl wtas which was precipitate(d with satuirate(d amniuomili11i assessed after this perio(l. stlfate. After fractionation with ammonlilum sillPreparation of Extr(cts. Cylinlders ('18 mm fate, it was foutnd( that increases in polvphenol oxidase activity in extracts from ethylene treate(d (iam) were cutt from treated root ant(I tul)er slices. D)iscs I mm thick were cutt transversely from these tissute cotuldI be detected spectrophotometricallv cylinders anid( extracte(l under nitrogen in an equtal (table I) and manometrically. The restults obtained 1w these 2 methods were similar. volume of 0.1 zs Tris-hydrochloride buffer, pH 7.4, containiing 0.36 xm sutcrose (6), aind 0.06 Mf ascorbic Peroxidlase activity in crll(le extracts ani(l in exacid. Extractions were (loie at 40 in a mo(lifie(l tracts fractionate(l with ammoniiuim sutlfate w ere sodiutm press. The extracts were centrifulge(d at essentially the same when assayed spectrophoto100,000 X ql for 15; minuLtes aindI the suiperinatanit metrically. There was an approximate 10-foldc infractioins were collecte(d and storedI at -15 . crease in peroxidase activity in sw,eet potato tissuies treated with ethylene (table I). Gel Electrophoresis. Extracts were subjected to electrophoresis in polyacrylami(le gels (10). Gels Extracts from ethylene treated tissiue wheni suIbobtained with extracts of sweet potato were staine(d jectedI to gel electrophoresis showveed increase(l anmto cletect amylase, phosphatase, peroxidase anid( polyylase activity but no appreciable alterationi in aci( phenol oxidase activity. Acid anid alkaliine phosor alkaline phosphatase activity. phatase activity was detected by incubating the gels In extracts from white potato treated with in filtered soltutions of a-naphthyl phosphate (1 ethylene, increased polyphenol oxidase activity was mg/ml) diazo blute B (I mg/ml) and 'MgC., (1 fotund whereas peroxidase activity decrease(l (table mg/ml) in either 0.1 M acetate buiffer pH 4.6 or I). 0.1 -i Tris-HCI btuffer pH1 9.0 (4). The gels were Polyphenol oxidase activity w,as higher in eximmersed in the appropriate buffer for 30 minuites tracts from parsnip roots treated with ethylene than before being inctlibated in the staining soluitions. in extracts from uintreated roots (fig 3). PolvAmylase was detectedl by incorporating 1 % starch phenol oxidase activity was (letected in extracts in the gels before electrophoresis. After electroonly after electrophoresis in polyacrylamide gels. phoresis the gels were washed with 0.2 M acetate No activity cotuld he dletected spectrophotometrically btl)ffer pH 4.6 for 30 minuites and(I theni iniciubated in either crudcle parsnip extracts or in extracts fracin a soltutionl of iodinle (0.04 mg/ml ), KI ( 10 tionate(l with ammoniutm suilfate. No changes inl mg/ml) in 0.2 M\ acetate buffer pH 4.6. Peroxidlase peroxidlase activity of parsniip extracts followinlg was tletecte(l tusing 20 m_u guaiacol anid 0.3 %onH,2, were dletecte(l. Downloaded from www.plantphysiol.org Septemberethylene 29, 2015 treatment - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE
1507
Resistance Increased by Ethylene Treatment Sweet potato slices treated with ethylene for two days and then inoculated with C. fimbriata. Julian
0 ppm
8 ppm
150 ppm
FIG. 1. Resistance increased by ethylene treatment. Sweet potato slices were exposed to ethylene-air mixtures for 2 days at the concentrations indicated and then inoculated with C. fimbriata.
Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
1509
STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE
r
8; i
a_
1.
I f
I
ti
,i s.
I
40.
__aimNc .t
/ .
I
/*4 FIG. 2. Peroxidase activity of discs cut 2 mm below the surface of susceptible sweet potato tissue treated with 0 (2), 24 (5), and 220 (8) ppm ethylene. Extracts of discs were subjected to electrophoresis on acrylamide gels and stained for enzyme activity.
4 K
l
9 FIG. 3. Polyphenol oxidase activity of discs cut 2 mm below the surface of susceptible sweet potato tissue treated with 0 (4), 8 (5) and 150 (6) ppm ethylene. Extracts of discs were subjected to electrophoresis gels and stained for enzyme activity.
onl acrylamide
Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
1511 I
STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE
Table I. Peroxidase and Polvplicnol Oxidase Activity in Extracts of Swcet Potato Root Tissue and White Potato Tuber Tissue Treated with Ethylene Enzyme activity is expressed as change in optical density at 390 mu/ml of extract/minute. Ethvlene (ppm) 0 8 150
Swseet potato (Julian) Peroxidase Polyphenol oxidase 1.8 0.06 20.5 0.13 26.8 0.13
White potato Peroxidase 0.20 0.16 0.08
Table IT. E!hylene Productioni bl,y Szceet Potato Tissue Inoculated with Fungal Spores Incubated for Three Days
Polyphenol oxidase 0.36
1.50 0.78 Or
Left Uninoculated and
Ethylene produced (ppm) Julian (susceptible) Sunnyside (resistant) None (unwinoculated) 1.5 1.5 Ceratocvstis rtinor* 2.0 4.0 C. fimbriata F38** 13.0 9.0 C. fimbriata U6*** 75.0 15.0 * Nonpathogenic to sweet potato, does not induce resistance to black rot. ** Nonpatlogenic to sweet potato, induces resistance to black rot. *** Produces black rot in susceptible sweet potato. Ethylene treatment had no apparent effect uipon Discussion the peroxidase or polyphenol oxidase activity of carrot, radish or tturnip root tisstue. Very little The results of these experiments indicate that polyphenol oxidase activity was detected in unethylene can increase resistance of sweet potato treated and ethylene treated radish and tturnip extissute to infection by C. fimlbriat(o. They also intracts. dicate that such indtuced resistance is associated with A volatile compouindl with the same retentioni an increase in the peroxidase and polyphenol oxi(lase time as ethylene on the gas chromatographic coltumn activity of ethylene treated tissuies. Ethylene was used in these experiments was demonstrated in the not detected above cultures of C. fimtbriatia grown atmosphere sturrotunding sweet potato tissue inctuon potato dextrose agar btut was detected above bated in closed chambers after being treated with infected sweet potato tisstues at concentrations apwater or being inoculated with C. finmbriata or C. proximating those used in ou r experimental treatm inor (table II). The retention time for both ment of utninoctulated tissue. It is therefore probethylene and the material from sweet potato was able that ethylene is an active principle in volatile 20 seconds. In table II the approximate amotunts materials from infected tisstues which cauise the of this compouind produtced by the variouisly treated increased resistance and peroxidase activity in tintisstues are expressed as ppm ethylene. The values inocuilated sweet potato tissule (5)). were obtained bv comparing areas tin(ler peaks on Higher levels of ethylene were observed above the chromatograms at a retention time of 20 seconds sweet potato tissue inoculated Nvith a nQnpathogenic with areas uinder peaks produiced by known constrain of C. fimlbriatat (induicer, F-38) which incentrations of ethylene at the Fame retention time. duices resistance to black rot than above uninocuilated However, these values and the identification of the tissuie or tissue inocuilated with nonpathogenic C. compouind as ethylene mutst be regarded as tentative, mtinor (noninducer) which does not indtuce resistfor oinly 1 type of column was used. Uninocuilated ance to pathogenic strains of C. fimitbriata. This resistant or susceptible tissue produiced very little suiggests that ethylene may be involved in the induicdetectable ethylene. Produiction of ethylene intion of resistance to black rot in suisceptible tissuie creased following inoculation with all 3 fuingi but inocuilated with inducers (16,17). Peroxidase and the increase was larger when the nonpathogenic polyphenol oxidase activity were also increase(d in strain of C. finbriata (F-38) which induice(d retissuie with indtuced resistance to black rot (16, 17). sistance w-as used than when the nonpathogenic It is possible that ethylene is one of the stimuili strain of C. imlitnor which did not induice resistance which move from areas of C. fimibriaita infection in was uised. It was largest when the pathogenic strain sweet potato into adjoining tissue to initiate the was tused. Under the experimental conditions tused, metabolic changes which lead to resistance to fuirethylene produiction decreased after 4 days inclubather penetration by the pothogen. Since ethylene tion. This decrease may have been duie to decreased can increase peroxidase and polyphenol oxidase O., and increased CO., levels withiin the seale(d activity in sweet potato tissule it appears that these conttitiners. enzymes may be involved in the resistance mecha-
Fungus
Downloaded from www.plantphysiol.org on September 29, 2015 - Published by www.plant.org Copyright © 1966 American Society of Plant Biologists. All rights reserved.
1512
PLANT IPHYSIOLOGY
However, as was previou sly noted (17), increases in the activity of these enlzymes alone cainniot lie solely responsible for resistanice. Other factors are involved an(l these may inclul(le the rate of synthesis or transport of substrates to the areas of increase(d polyphenol oxidlase activity. It is not known whether the inhibitor of polyphenol oxidase foulnd( in sweet potato extracts is effective in vivo and hence howN mulch of the increase in activity of this en7zyme is effective in the tissule. However, there is a growving body of ev%i-idence that peroxi(lases act in oxi(latioin reactions requiiring only catalytic amotunits of hy-drogen peroxide (18). Increase(l activity of these 2 enzymes in sweet potato tissuie stimulate(d by ethylene couldk therefore very likely give rise to abnormal or increase(d aromatic biosynthesis resulltinig in the formation of 1)oth physical andcl chemical barriers to infectioln (9, 15). Little change in peroxidase or polyphenol oxidlase activity was note(l in eth, Aene treate(d carrot, tulrInip or radlish tissule. Polyphenol oxidase buit not peroxi(lase activity was inlcrease(l in ethy lenie treated potato and(I parsnip tissuie. It is possible that the changes note( iin ethylene tr.-ated sweet potato tissule are peculliar to that species. Ho\w ev er, it seems probable that ethylene has a more genieral role in the (lefense mechanisms of plailts. It may act as a stimu1lLls of localized metabolic changes lea(dinig to necrotic and(1 hypersensitive reactions in p'anits following infectioln. Since ethYlene is capable of produicinig chlor -phyll degra(lationi in fruiits (2), it is also possible that ethylene pro(duiction in infecte(d greeni planit tissuie may caulse the chlorosis so commonly observed inl sulchi tissu1es.
t,c,
nism.
Literature Cited 1. .\ALBiEG1INA, F. A. NI. 1964. Clilorogenic aci(d oxidases fromil ip)tato tuber slices p)artial piurificationl anid prop)erties. Phy tochenilstry 3: 65-72. _. ButV i
