metabolic regulation in diseased leaves. i. the ... · metabolic regulation in diseased leaves. i....

Plaiit Plhysiol. ( 1966) 41, 289-297

Metabolic Regulation in Diseased Leaves.I. The Respiratory Rise in Barley Leaves Infected with Powdery Mildew'

K. J. Scott2 and Robert M. Smillie3Biology Department. Brookhaven National Laboratory, Upton, New York

andBiochemistry Department, University of Sydney, Australia

Received April 12, 1965.

Siiiiniarv Photosynthetic anld resl)iratory activities hlave lbeeni ni eature(l in leaves ofHor(Icitinl zildgarc L. var. Manchuria (barlev) after infection wvith IErysiplic graininisvar. hlordci (powdery miiildew). Two isogeniic linies, onie resistalnt to inlfectioln anld theotlher highly suscep)tible, were examinied.

'T'lhese isogeniic linies shoNwed very different physiological responses followving inifec-tionl. Photosvinthesis anid the chlorophyll colntelnt of resistanlt leavesx as unlaffected I)vinifectioni. Respirationi inlcreased slightly anld this 'was accopll)aniedl by smiall inicreasesin activities of enzymes of glycolysis, the pelitose-P pathway anld the tricarboxviic acidcvcle.

The inlfectioln of susceptible leaves resulted in a slight inicrease in photosynthesis 48lhoturs after iniocuilatioln, hutt sub)sequelltlv there v-as a progressive (lecrease in the photo-synlthlesi.s of these leaves comlpared with that of niolninifected leaves. The capacity ofinifecte(d leaves fol- partial reactionls of photosynthesis suclh as the Hill reactioni and(I thephotoredtlctioll of niicotinianiiide adeniiie linuitcleotide 1phosphate (NADP ) decreasedlduring the later stages of inifectioni. The lcvels of chlorophyll. N.I)HPH-dlial)horase andaldolase Also (leclille(l. h'l'ere xwas no detectaile (lifferenice in the respiration of inlfecteandi noninifected leaves ulnitil 48 hoturs after inlocuilatioln. After this tilmie, the infectedleaves shoxved a higher respiration, the n7aximum differenlce occtirrinig about 14-lholorsafter iniocuilationl. The resl)iratorv incirease was nlot acconmpalie(l bx significallnt changesin the levels of enzymies of glvcolvsis anid( the tricarboxvlic acid cycle \vith the excel)tionof walate dehvdrogenase vhiclh -vas lower in inifected leaves. In conitrast, the activitiesof glutcose-6-P dlehldrogenase anid 6-P-gluconate (lehvdroge-nase shox\vedl chalnl-es similarto that observed for respirationi.

Tlhe respiration anid the activities of glIcose-6-P dehvdrogeniase and( 6-0P-gluconlatedelhvdrog-enase did niot ilncrease in inifected leaves of etiolate(lplants, even xvheni excellelntgrowvth of the funguis wxas established by growing the planits in \White's basal imediilum su)-plementedl with suicrose. TI'he respirationi of a susceptible nmuitant barley (tthe vellow-greeni virescelnt miiutanit of the variety HinmalaIya) vhen growvn in the light at 11 xvas niotchanged bV inifectioni altlhouigh the characteristic respiratory ri_ e occuirre(l in plaiits ogroNvinat 150. At the lower teml)erature chloroplasts fail to develop in this muititalt, althouighdevelopimelnt is normial at 150.

It is suggested that the pathogeni is niot directly resl)ollsible for the ilncrease in respira-tion in greeni leaves, rather that this is a response in the host cells to a loss of lphotosyn-thetic capacitv.

The miietabolislm of susceptible cereal leaves is al-1 The at tered after ilnfectioni vN-ith obligate fungCal pathogens.The researcli at Brookhaven\National Laboratory;was carriedl out uni(ler the auspices of the United States erate of llotosnthesisr i

Atomic Energy Commnission. The researclh at the Uni- early stage of inifectioni anid later declinies (1. 129 13).versity of Sydney w as sul)p)orte(l by a granit from tbe Respirationl is increased in inifected leaves (2) anidRural Cre(lit Development Funid of Australia. the available experimental evidelnce int(licates that the

- Presenit adc Iress: Bioclhemistry Departmenit, Univer- hexose mionophosphate shlulnt is hyperactive (26).sity of Svydley, \ rtraiP.

Presenit adldress>: Plant Plhvsiology Ullit, C.S.I.R.O. Icreased leels of RNa (17) free aminifo acids (27)and(I Sclbool of Bih)li-cal Scienlces, Univ,ersity of Sydlney, an(l auxins (26) have been relortetI in infected cerealAuustralia. leaves. 'I'he basic factors responsible for these

289 www.plantphysiol.orgon November 28, 2018 - Published by Downloaded from

Copyright © 1966 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

PLANT PHYSIOLOGY

chalnges are unknown as is thle physiological signific-anlce of tllese changes in the developmiient of the lhost-p)athogeni combinationl.

More miiust be knoxwn abl)out the fundaiiienltalmetablolic chanlgres that occur in greenl leaves follox-inw inifection before a comp)rehensive l)ittulre of thephlyvsiolog-ai(y hiocheyiiistry of host-pathogen rela-tiolis can l)e ohtained. Tlhe aimii of the preseLt studyis to learni more al)out the changes in the respiratoryan(l plhotosyntlhetic mletabolismii of infecte(d leaves am',what initiates and controls these changes. Two as-lects of this stu(lv that are of special l)iochenii1caliniterest slhoul( l)e emphasized. Firstly. it is relevantto the genleral problem of metabolic regutlationi inplanit cells sul)jected to coniditionis of stress. In thisinstance the stress is the invadling p)athogei. Tlshepreseince of the pathogen mlav result in tlle formlationof phytotoxins and, where successfuil l)arasitismii hasbeen established, to a depletionl of imllportanit nietabo-lites in the leaf and( premature senescenlce. Thus, in-formation onl the mletabolic chaniges occurring in in-fected leaf tissue woould contribute to our understand-ing of metabolic regulation in )lant cells (during sene-scenice or exposure to adverse en-vironmental condi-tionls. Secondllv, it is of interest to (leterminie xxwhetherthese nmetabolic changes in the leaf are necessary forsuiccessfuil parasitismn or whether thex only reflectthe resl)onse of the leaf to the presellce of the patll-ogenl an(l as suich (lo niot (lirectlv affect -rowth of thepatlog-eii

TI'he best (loctlmlenite(l imetabolic chang-e occtirriniiliplalit cells infected by one of a wide rani-e ofpathogens is that of increase(l respiratioll (18). In-creased activities of glutcose-- l' delevdro-enase anl6l- glticoonate dlehlvdrogenase (14, 25) as w-ell as (le-creased C6/C, ratios (9, 28) accomlpany the rise inresl)iration in cereal leaves after infectioln by rusts orowd(lery miildews. These observations shoxx that

resl)iratorv eniergy-prodtucing mechanism]s are alteredafter- inifectioni. Less is kniowin about the photosvn-tlhesis of infected leaves. Siince photosynthesis isaniotlher il)portant sotirce of energy andcl carboni forcelluilar syintheses in greeni leaxves, any imlpairmiientof plhotosyntlhesis following inifection is likely to af-feet other cellula- l)rocesses includinig respiration.The inl)ortalnce of colisi(leriing both photosynthesisali(l resl)iration in inlfecte(l leaves wxas highlightedl bythe unexl)ecte(l observation that the respirationi ofetiolate(d leaves wxas not increased by infection (1/7).

Measurements of photosynthetic anid respiratoryactivities in barley leaves infecte(d wN-ith I2rvsiph¢graolloilis var. 1ho dcli are described in this paper.Tsogenic lines, susceptible and resistalnt to infection,xere tise(l. Tle restilts are coml)paredl with mieasure-miienits of resl)iratorv activity in infected etiolate(dleaves anid a chlorophvll-less mnutant. Ini a subse-qtient paper these results wxill be cor-relatedI with theprodluction of photosynithetic and respiratorx initer-miediates in itnfecte(d alnd noninlfecte(d leaveS.

Preliiiinary accouints o)f plart of thi.s w ork havebeeni piresenited ( 24, 25).

Materials and Methods

Pll/o;t Mlaterials. toiiless othervise stated 2 iso-genlic linies of Hordcuo vzdgarc I. var. Manchuriabarley xx-ere uisedI as the experimental plant miiate-

r-ial. ( )ne linle, designatedl as M 1622, xlas highly sus-ceptible to in fectioi by IF'rysiplie grumlmiiis var. Ii ordli,Alarcial, ratce 3 ( 19) (powdery imildew-) and tlleother, designated as i\l 1(23, was resistalnt to thispathogen. Seeds were so0xvn in pots of soil eitlher ina coiitrolle(d grow th roomii or in an air-conlditioie(lgreenhouse. 'I'lTe temiperature of the roomil was imiaini-tained at 21 + 1° at a white light intenisitx of 20,00(0lux for 16 hiouirs per d(ay. h'l'e temperattire of thegreennouse was 21 + 3'.

Etiolated l)lants of the stlscel)tible strainxweregroxvn in eitlher of 2 wxays. Firstly, )lants xxeregroxx1 lln lpots of soil that xvere kept in continuousdarkness. SecondIx-, stirface sterilized seeds xveregerminated iii(ler aseptic con(litionson fine-mieslh xxiregaYuze resti1g ala)oxe at sterile soluitioni of White'sbasal sallt ilme(liumii ( 33) ) and(I 2 % sticrose. Lighlt-plroof stainless steel conitaineri-s xere used. Sterileair xas coitintiouolx bubbled througih the imie(litum toaerate the roots.

(ireeci or etiolated l)lants x ere inoculated 5 to 7(lays a fter sow ing xx lien the fil-st leavesx rerc about

cml high. Thle plants xvere inocuIltted x itlh PF. (I1ralmi,,iis var. lor(dci by (dlistil,g tlle leaxves wx itli spI)oresfrm bl avlx lv inetedte(I susceptible pl)aI-nts. 1.tioldatedlanlts xwer'- explose(l to dim (dlaligllt fol- I to 2 mill

lltse (luriig- inioctilationi. \t selected times aifterinioculalttion the top s cmii of both nolninifected anl in-fecte(d leaves xx ere harvested for experimental tise.

To assess the extenit of ftingal (lexvelol)mellt,leaxvesx ere staine(l accordini- to the lprocedure (le-scrihed )v \\hit'e and Raker ( 32) 1and examiine(d lli-croscol)icallv. LRefore heiaxvilx inifected( leaves xrrctisedl ill exl)erimelnts, niycelia and coill(lia were i-e-moxed 1)v gentlyvbruslhing the leaves xitl (lltll) cot-toil sxx-a:bs.

llcasiircm'ciiots o/ Jliotosy,licsi('Sis mid RCsPI;ra!imoPhotosynthetic raLtes of leaif segmllenits ( 2.5; cm illleigth ) xvere determiine(d m"Maliometricall- b)y meastlr-ilg ( ). prodtiction at 250 (luring illmiiiiinatioln. ThleC) >, conicenitrationi xx as ll.aintained ait 1 % by thle useof a CO().. buffer ( 11 ) RPespiratorxy rates of leaf,-cements xvel-e (letel-mliell(I imaomietricallly bx ineas-

tiiiilig ( ) constiimption il (dlililesk(es l)rexiolslx dc-scribe(l (16).

Tiv\Ylmic Assa y.y. A; x-ariosi tlimCes after- 11iocuilation1, lionliiifected(Iad in fecte(l leaves rel-c harx-e.tedfor dietermillations of enzsmic alctivity. Samples oflcaves (500 n- fl- xxt) xver- lhoiiio-eniizd in -lass'T'eni Broeck lband( honio-elli 'ers contaillig- 0.05; xi'rixs aIdjtiste(l to pH 7.8 xx itb HCCl. Th'l homiogen--tes xvere cliltutedI to 1 5.0 ml wxith the ''ris and x erec-uitriftge(d (it 144,000 X !j for- 31) uilitites. Thlesul)erllnltant fluiids x erc assaxvci for (.-nzx mic activities.

'11' lireciipiltate troiii tbc 144.1)1 , prdClltipitctc

2'90

www.plantphysiol.orgon November 28, 2018 - Published by Downloaded from Copyright © 1966 American Society of Plant Biologists. All rights reserved.


SCOTT ANI) SA I LIA1 -METABOLIC RElGULATION IN DISEASED LEAVES

was niot routinely assayed for enzymiiic activitv sinicefor most enzymes it was not possible to obtain reliablespectrophotometric assays because of excessive ab-

sorption and scattering of the mleasurinig beam.However, attempts were made to demonstrate gltu-cose-6-P dlehydrogenase activity in the 144,000 X g

precipitate since this enzyme showed striking changesin infected leaves. No significant activity was foundin the 144,000 X g precipitate from either noninfectedor infected leaves.

All preparations were carried out at 40 anid assays

at 230. Activities are expressed as Mmoles of sub-strate utilized per mlinute per g fresh weiglht of theoriginal leaf sample.

Enzymic activities were mleasured by continuouslyrecording the progress of the reaction usilng a Caryrecording spectrophotometer, model 14. Reactionimlixttures for assays of allolase, phosplhohexoisoiler-

ase, 3-P-glyceronmtitase, enolase, pyruvate kinase.NADP+-isocitrate (lehydrogenase, aconitase, mialatedehvdrogeniase, glucose-6-P dehydrogenaso an(l 6-P-glutconate dehydrogenase have been described else-where (23). NADH- and NADPH-cytochrome c

reduictases were assayed using a reaction iimixtureconsisting of leaf extract. Tris (50 m11M). cytochroniec (40 /,cM), and NADH (100 Mm) or NADPH (100,AM). The increase in absorbancy at 550 Mm was

recordled. NADPH-diaphorase was assayed by theabsorbancy decrease at 600 nmpt in a reaction mixturecontaining leaf extract, Tris (50 mM), 2,6-dichloro-phenol indophenol (50 Mm) and NADPH (100 mM).

Chloroplast fragments from either noninfected or

infected barley leaves were isolated following theprocedure used for spinach by San Pietro and Lang

(21). The capacity of these fragnments to photore-duce NADP+ was measured spectrophotometricallyas previously described (29). The reaction mixture(0.8 ml) contained barley chloroplast fragments (12Mug chlorophyll), Tris (25 mM), MgCl9 (3 mM),NADP+ (125 UM) and excess photosynthetic py-

ridine nucleotide reductase (30 MAg) prepared fromspinach (21). The photoreduction of 2,6-dichloro-phenol indophenol by chloroplast fragments was meas-

ured similarly in a reaction mixture (0.8 ml) contain-ing chloroplast fragments (2 Mg chlorophyll), Tris (20niM) and 2,6-dichlorophenol indophenol (50 um).

Chlorophyll was estimated according to the pro-

ce(lolre of MacKiniiey (15 ).

Results

Photosynthetic and Respiratory Activities of Siis-ceptible Plants. Changes in photosynthesis and res-

piration as a function of the stage of fungal develop-ment were made by measuring photosynthetic andrespiratory.activities at various times after inoculation(fig 1). The rate of photosynthesis 48 hours afterinoculation is slightly higher in infected leaves thanin noninfected leaves. After this time the rate ofphotosynthesis of the infected leaves is lower than

that of the noninfected leaves. This difference be-comes more pronounced as the infection progresses.There is little difference in respiration during theearly stages of infection, but after 48 hours the res-piration is higher in infected leaves. The respira-tion of the infected leaves beginis to rise sharply about96 hours after inoculation, reaches a peak at about

:E

.120039

o 800

0

-

"' 400

PHOTOSYNTHESIS RESPIRATION

) \ ---1'/c

-31\

O 48 96 144 192 48 6 144 192HOURS AFTER INOCULATION

E0

1.r

ro

,

1v 0

FIG. 1. Rates of photosynthesis and respiration innoninfected and infected0 ----- 0 infected.

6.

4.

2.

(.9

cn 0,1

LI

w

m 0.

E

"I

w

O.

m

CI)

0)E

0.

0.:

0.

leaves. noninfected,

NAOPH- DIAPHORASE CHLOROPHYLL

o0 3.1

o / ~~~~~~~~~~~~~~~~~~~~2.1

O I O

.2DHDOENLASE ALHDROLEASE

o I 0-

3

2

NADPH- CYTOCHROME cREDUCTASE

NADH-CYTOCHROME c

REDUCTASE

N%

.0.1

0.

0.

0

I

w.0

cr

.0

E

sOH

'._

0

w

1.0 E:

LL

C')cn

1.6 C7

.E

"

48 96 144 192 48 96 144 192

HOURS AFTER INOCULATION

FIG. 2. Activities of photosynthetic and respiratoryenzymes and the chlorophyll content of noninfected andinfected leaves. O noninfected, 0 -----0 in-fected.

291

V V



PLAN-T PHYSIOLOGY

144 loumrs and tlhen (leclilnes. Th'lle miiaxinluimm respira-torv rate occturs about 24 houirs after the colmmilenice-menit of sl)oruilation.

Changes in chlorophyll conitenit aill enzymic activ-

ities following inifectioni are show n in figuire 2 an(l

table I. Chlorophyll and the activities of aldolase

AFi(;. 3. Fungal development on leaves of barley plants

gro\wn un(ler (liffereint enviroinmenital conditions. A,

greeni leaves; B, etiolate(l leaves fromii plants groxvxnin soil; C, etiolate(d leaves fromii plants grown on

\Vhite's basic mediumii with 2 % sucrose. The leaveswere fixedl 144 hours after inoculationi. MagnificationX8(1.

ancl NAA)PHI-Idiaphorase (chloroplast transhydrogen-ase) are decrease(I in inifecte(d leaves. A similir (lif-ference is exideint in the measurewents of the litht-dle)ell(lepnt re(dnictioln of N ADP)1' or 2,6-dichlorophenollolindophenol. Ini p)hotosvnthetic tissnes 10os5t of thealdolase is localize(l in the chloroplasts (3(h).

Inicreases in the activities of gluicose-6-' (lellh(lro-geniase, 6-P-gluconate (lehv(lrogenase and NA\I)IIIcvtochlrome c reducta,'e occIII in inifecte(d leaves(fig 2). Tlhe onset of these increases occurs le-tween 48 and(1 96 hotur-s after inoculation coincdi(lilnwith the oniset of the higher respliratorv rates all(llower l)photosynthetic rates ill inlfecte(d leax es. lI(itllthe rate of respiration alndI the activities of the 2eizvim es of the pentose-l' pJathw\av in inifecte(d leavesreach a maximuiilm at 144 houris after inoculationI.By wax of conitrast thel-e is little chanlge in the ac-

Table 1. Photosviitlictic (1m(d RxcSpiratOr, Activitics ot

SuscOptible A mOninfectedtmid In fccted (,tr('en LcavesMaterial was assaye(l 144 loturs after inoculation witlh

E. gramiinis var. hordei. The mcveliumii was remove(l bvbrushing.

Activitvumoles/min per g fr wvtNon-infected Iinfected

PhotosynithesisRespirationPhotosynithetic NAI)P reductionHill reactionP-hexoisomerase3-P-glyceromutasePyruvate kinaseIsocitrate dehycirogenaseAconitaseMalate dehydrogenase

8.20.265.704.802.102.70

1261.02

0.4(014.0

3.60.542.052.281.141.07

9(1.01.010.437.00

292



SCOTT AND SMILLIE- METABOLIC REGULATION IN DISEASED LEAVES

tivity of NADH-cytochrome c reductase between the2 tyl)es of leaves.

The activities of enzyn- es of the glycolytic path-way between 3-P-glycerate and )pyrtlvate anid severaltricarboxylic acid enzymes are niot higher in infectedleaves. Usually sliglht decreases in activity occuirespecially of malate dehydrogenlase which is con-sistently lower in infected tissle.

Rcspiration of Infected Etiolated Leaves. Theresults already given indlicate that the respiratoryrise in infected tissue mnay be related to the decreasedphotosy.lthetic activity since both of these changesoccurred about the sanme timne. Accordingly, experi-ments were conducte(d to determine the effect of in-fectioni oni the resl)iration of etiolated plants. Frungalgrowth was not as good in etiolated leaves as ingreen leaves but imacroscopically-visible colonies wereobserved at the timle of sporulationi (fig 3). Asshown in figture 4, these infected leaves do not showsignificant increases in respirationi or the activities ofglucose-6-P dehydrogienase and 6-P-gluconate de-hydrogenase. Activities of aldolase, enolase andNADH-cytochromiie c reductase are similar in in-fectedl and n-oninfectedl leaves.

'The possibility remained that the lack of a suitablegrowth substrate was limiting both the rate of res-piration in etiolated leaves and the growth of thefungus. Further experiments were performed usingetiolated plants grown on White's basal medium con-taining 2 % sucrose. Leaves froml etiolated plantsgrowin under these conditions supported better ftiigalgrowth than leaves from etiolated lplants grown insoil; in fact fungal development was comparable tothat obtained oln green leaves (fig 3). However,

these etiolated leaves exhibited little or nio increasesin respiration, glucose-6- P dehydrogenase, 6-P-glucon-ate dehydrogeniase or isocitrate dehydrogeniase at 144hours after illoculationi (table II).

Respiratiou of ani Iiifected Chlorophyll-less Mu-tauit. Froml the experiments cited above it appearsthat the increase in respirationi in the green leaves isrelated to an impairment of chloroplast nmetabolism

I

Ia

I'XU)O uJ0C,jv

0

0aI

8

a:

LLJ

c

E

:1

az

V)

IV0

RESPI RATION

o.8P-I

0.6L

GLUCOSE 6-PHOSPHATE0.9iK DEHYDROGENASE

0.69 ~~~~~I0.3

0

ALDOLASE-"4.0

4 2.0

NADH-CYTOCHROME cREDUCTASE

0.8

1- -. -J: L. 0.2

_ _ 0.26- PHOSPHOGLUCONATE

DEHYDROGENASE- 0.3

- 0.2

4 8 96 144 48 96 144

HOURS AFTER INOCULATION

FIG. 4.zymes in0 0

-L

'I_

V)

H

h)

G

L,c,

E

a,)

E

0.1

Respiration and activities of respiratory en-noninifected and infected etiolated leaves.noninfected, 0 -----0 infected.

Table II. Respiratory ArtivitY in Etiolated Leaves GroCm oan White's BasalSalt Medium SupplentcOod zcith Sufcr-ose

Material was harvested 144 hours after inoculation.

Enzymatic activityTissue Respiration 5Lmoles substrate utilized/min per g fr wt

Al 0/hr per g fr wt Glucose-6-P 6-P-gluconate Isocitratedehydrogenase dehydrogenase dehydrogenase

Infected 220 0.28 0.15 0.43Noninfected 210 0.24 0.14 0.41

Table III. Respiration anid Chlorophyll Con1tcint of Manichiuria Straini M/1622 and aChloroPhyll Afniitaut of the Varicty Himnalaya after Infection

Growth Chlorophylltemperature mg/g fr wt

11° 0110 0150 0.9715° 0.69110 1.5411° 1.0615 ' 1.9315° 1.24

RespirationAl O.,/hr per 100

mg fr wt

2425538045914373

2?(93

State ofleaf

NoninfectedInfectedNoninfectedInfectedNoninfectedInfectedNoninfectedInfected

Variety

HimalayaHimalayaHimalayaHimalayaManchuriaManchuriaManchuriaManchuria



PLANT PITYSIOLOGY

or, alternatively, to somiie light reaction not directly-associated with the chloroplasts. This point was i1-vestigated fuirther usinig a mutant of barlev. Thismiiutanit (the yellow-green virescenit mutant of thevariety Himalaya), susceptible to powdery mildew in-fection. forms chlorophyll if grown in the light above13°. Below this temiiperatture chlorophyll is not syn-thesized. Seedlinigs of this mutant Nwere growni ingrowth chambers at 110 and 15°. The plants wvereilluminated for 16 hours per dav. Plants of thevariety Manchuria were also grown in the samechambers as controls. Some of the first leaves ap-proximately 5 cm high were inoculated with spores ofE'. graniinis var. hordei. W,Vhen the fungus wassporuilating (144 hrs after inioculation at 15° and 240hlrs at 110), leaves were harvested anid after remiovalof the mycelia and spores the rate of respiration andthe chlorophyll content of the leaves were determiiinecl.As shown in table III there is a marked increase inrespiration only in those inifected leaves containingchlorophyll, namely AManchturia grown at 110 and 150and the mutant of Himalaya grown at 150. No in-crease in respiration was observed in the inlfecte(dmutant that was growni at 110. The apparently lowerrespiration of the noninfected mutant of Himalaya

grown at 11 0 can be largely\attributed to a lowerprotein contenit per g fresh weight.

Resistanit Straini. The pattern of photosynthesisaindl respiration in the resistanlt strain after inifectionimaav be colntrasted with that in the susceptible strain.As shown in table IV', infection does not alter the rateof photosynthesis nor the chlorophyll content of theresistant straini for at least 168 hours after inocula-tion. The respiration is increased in this strain butthe onset of the rise is earlier and the magniituide isnot as great as in the susceptible strain.

The results of a typical experiment showing the ac-tivities of respiratory enzymes in the resistant strainare listed in table V. Commliieincing about 48 hoursafter inoculation, infected leaves show slight increasesin the activities of these enzymes. These increasesoccur niot only in enzymes of the pentose-P pathwaybut also in glvcolytic andi tricarhoxylic acid cycleenzymlles. The respiratory response to infectioni inthe resistanit strain apll)ears to bc (lifferelnt from thatin the suisceptible straini.

Discussion

Isogenic lines of i-esistallt alillsusceptil)le straitls

Table IN'. Effec-t of Powdery Mildetc Infcction onl Respiration, Photosynthesis antdChlorophyll Content in Infected Resistant Leaves

Respiration Photosynthesis ChlorophyllHrs after '.l O.,/hr per 100 mg fr wxt ul O.,/hr per 100 mg fr wt mg/g fr wtinoculation Noninfecte(d Infected Noninfected Iinfected Noniinfected Iiifected

0 38 95024 27 31 1100 1080 1.2 1.348 26 33 1210 1200 1.8 1.772 24 35 1170 1150 1.6 1.496 23 32 950 960 1.5 1.4144 21 30 840 870 1. 1.4168 19 29 650 680 1.2 1.3

Table V. Activities of Respirators Eifynzmes in Resistanlt Leaves

Glucose-6-P(lehydrogenase

Enzvmic activity,umoles substrate utilized/min per g fr t

6-P-gluconatedehydrogeinase Enolase

Nonnifected0.350.270.240.200.19

Aldolase

Noninfected3.55.64.74.14.3

Tn fectecl

0.280.310.30)0.23

Infected

5.85.65.25.1

Noniiifected0.300.240.250.200.14

Infecte(d

I socitratedelivdrogenase

Noninfected0.670.610.530.380.31

0.290.320.250.15

Infected

0.640.710.520.38

Nownifected I nifected1.3

(.89 1.00.60 0.750.44 0.60

Nialatedlehydrogenase

Noninfecte(d Tnfected141413

1313

13 1412 12

Hrs afterinoculation

0244896168

0244896168

294



SCOTT AND SArILLIE--METABOLIC REGULATION IN DISEASED LEAVES

were used in this investigation but their physiologicalresponse to infection is very different. In contrast tosusceptible leaves, photosynthesis and chlorophyll con-tent in resistant leaves is not altered by infection.Previous microscopic studies (32) have revealed acollapse of cells neighbouring infection courts inresistant varieties, but the extent of this cell collapseappears to be too small to effect a significant decreasein the photosynthetic rate of the leaf. Respirationin resistant leaves is only slightly enhanced as are theactivites of some enzymes of the pentose-P pathway,glycolysis and the tricarboxylic acid cycle. It is dif-ficult at present to assess the significance of the dif-ferent physiological reactions of host cells in termsof resistance or susceptibility.

The characteristic rise in respiration observed insusceptible green leaves after infection with fungalpathogens raises several questions. Firstly, what isthe nature of this increase? Is it merely a quantitativechange of existing respiratory pathways or do quali-tative changes also occur? Is the increase accom-panied by an activation of enzymes or the syn-thesis of new leaf protein? Secondly, what is theregulatory mechanism associated with the increase inrespiration? A third important question. namely thesignificance of this change in the successful establish-ment of the host-pathogen complex, will be examinedin a subsequent paper.

Before discussing these points it should be em-phasized that the rise in respiration observed in in-fected green leaves cannot be attributed solely to thegrowing fungus, nor to those plant cells which arepenetrated by fungal haustoria. This also applies tothe enzymes which show an increase in activity in in-fected leaves. Infected leaves harvested during thelater stages of infection were routinely brushedto remove surface fungi before any nmeasurements wereperformed. At the stage of infection showing themaximum increase in respiration, it was possible toremove most of the pathogen by this procedure. Sep-arate experiments (22) have shown that the increasein glucose-6-P dehydrogenase and 6-P-gluconate de-hydrogenase is still found when the remainder of thepathogen is removed by carefully stripping off theepidermal cells of the leaf. Powdery mildew is anectoparasite and the haustoria do not penetrate fur-ther than the epidermal cells (32).

The enzymic assays on extracts of green leaves(table I and fig 2) suggest that the pentose-P path-way plays an important role in the increased respira-tory activity of infected leaves. This view is sup-ported by the similarity of the curves for respiration,glucose-6-P dehydrogenase and 6-P-gluconate dehy-drogenase in infected leaves (fig 1. 2). the absenceof corresponding changes in total protein (17 ), theactivities of glycolytic and tricarboxylic acid cycleenzymes (fig 2 and table I), and the decreasedC6/C1 ratio (9, 28). The definite increase in theactivities of glucose-6-P dehydrogenase and 6-P-glu-conate dehydrogenase relative to total protein showsthat the respiratory rise is accompanied by the con-

version of inactive forms of these enzymlles to activeforms or else by the synthesis of new protein. Theevidence in this paper does not allow a distinctionbetweeni these 2 possibilities.

Presumably the levels of mlitochondrial enzymesin noninfected leaves are sufficient to cope with anyincreased 0, consumption via mitochondrial oxidativepathways following infection. It is interesting thatthe respiration of both noninfected and infected leavesis increased by dinitrophenol (16).

Several possibilities exist when considering factorsresponsible for the rise in respiration of infectedleaves. Regulatory mechanisms controlling leaf res-piration could be directly influenced by toxins orother substances that are released following infection.These substances could originate from the pathogen,or from leaf cells sensitive to the presence of thepathogen. Regulatory mechanisms for respirationthat are based on the concentrations of certainmetabolites would be affected bv a drain of thesemetabolites to sites of fungal synthesis. Alterna-tively, toxic substanices or altered metabolite levelscould indirectly affect respiration by interfering withsome other phase of leaf metabolism which is inte-grated with the respiratory mechanism. The experi-mental evidence points to the last possibility as beingthe most likely since there appears to be a relationshipbetween the decrease in photosynthesis and the in-crease in respiration. The onset of both changes oc-curs about the same time. An increase in respirationand the activities of glucose-6-P dehydrogenase and6-P-gluconate dehydrogenase was not observed ininfected leaves lacking functional chloroplasts (fig4, tables II, III). In this connection the experimentsof Udvardy et al. (31) are pertinent. They ob-served that an increase in respiration and glucose-6-P dehydrogenase activity occurred in barley leavesafter detachment and that these increases were pre-vented by treating the detached leaves with kinetin.Kinetin retards chlorophyll breakdown in detachedleaves (20).

One similarity between photosyn-thesis and theoxidative pentose-P pathway is that both requireNAD)P+. A dlecrease in photosynthesis could lead toa higher NADP+/NADPH ratio. NADP+ availabil-ity can limit the operation of the pentose-P pathwayin plant and animal tissues. The Cf,/C, ratio is de-creased in such diverse tissues as maize root tips (7),carrot discs (4), liver slices (8) and corneal epithe-lium (10) by treatment with substances such asmethylene bluie and N-methyl plhenazonllilll sulfatewhich increase tissue levels of NADP+. Increasingthe tissue level of reduced pyridine nucleotide inlactating rat mamnmary gland increases the C6/C,ratio (5). If the availability of NADP+ is a con-trolling factor in the activity of the pentose-P path-way in barley leaves, then a shift in the NADP+/NADPH ratio brought about by a progressive de-struction of an important pathway for NADP+ reduc-tion could conceivably lead to increased activity ofthe pentose-P pathway. Direct measureii ent of py-

295



PLANT '11 YSIOLOGY

ridIinie intleotide levels in other planit tisstues has le(dYamamoto (34) to conicluide that the N ADPn/NADPHI ratio inlcreases in cells as the respiratorypattern chaniges froimi onie in which the Embdem-AM1eyerhof-Parnas pathway p)redomlillates to onle inwrhiclh the l)entose-P patllway becomes increasingly

imiiportanit.On1 the basis of the experimental evi(lence anid ar-

gumiienits presented above wve suiggest that l)ow(lery

mildew inifectioni of susceptible barley leaves cauises

a redutictioll of photosynthesis and that the inlcreasein respirationi is a dlirect response to this loss oflhotosynthetic capacity. The respiratory inicrease isassociate( wvith ellhalncedl activity of the pentoe- 'pathway. TI'he ratio of NADP'/NA.I)PIH may heanl imlJportant conltrol factor linikiing photosynlthesxisali(l the l)entose-P lpatllh a

'I'he (lecrease in photosynthesis in ilnfected leavesappears to resuilt from a (lestr-uictioni of chloroplastcomponents. h'l'ere is a loss of enzymic activity as-

sociate(l with both electroni traniisfer and(I CO., fixationand aldecrease in chlorophyll conitenlt. Cy tologicalstudies (3) of wheat leaves have shown that the sizeof chloroplasts is redtuce(d in leaves infected withj

Putccizii(o gr(aIlinis (rtust). 'I'he agents iniitiating thebreakdown of chloroplast metabolism in infecte(dleaves are uinknown, btut the actioln of phytotoxic sul)-

stanices l)ro(luced by either the pathogen or the hostcells seemiis to be the mlost likely explanation. Bush-nell and( Alleni (6) have shown that aqueous extractsof pow(lery mildew spores applied to barley leavesl)roduce chlorotic bands around(l green islalnds. Sev-eral of the toxinls which have been isolated fromii cutl-

tures of phytopathogeniic funigi couild possibly inter-fere with photosynthetic electroni tranisfer- by actingeither as inhibitors or HTill oxidants. In either case

aln inhibition of p)hotophosp)horylation coul(d resultfollowe(d bv the cessation of l)roteini synthesis incilloroplasts.

TIn ilnfecte(d nonigreeni tissues siuclh as tubers. in-creased respiratory activity is commonly foun(d in thecells adjacent to infected areas. Althouglh the argu-

miients advanced above al)ylv specifically to infectedlphotosynthetic tissues, simzilar regulatorvimechanismsmay be involved ini the increased respiratory activityof both green and nlongreeln cells. In nonigreen cellsa ftungal-ini(duced impairnwent of onle respiratorymlechainismii could lead to the enhanicemiient of an-

other. Another consi(lerationi arisinig fromii the samiie

argunments is that the large inicrease in the respiratoryactivity of green leaves might niot be a propertyp)eculiar to fulngal infectioni but might be effected by

aniy one of several agents, stuch as certaini herbicides,which can cauise the proressive (clestructioii ofchiloroplast metabolism.

Acknowledgments

The able technical assistance of AMr. G. Wells is grate-fully acknowledged. The authors \vish to thank Dr. J. G.Mfoseman, U.S.D.A., Beltsville, for kindly supplying seeds

of the isogenic lines of the variety Mfanchuria anid Dr.H. H. Smitlh, Brookhaven National Iahoratory, forkindly sulp1)lying mutanit anid xx-ild-type seeds of the varietyHimalaya.

Literature Cited

1. x, P. J. 1942. Changes in the mnetabolisnm ofheat leaves indluce(d by infection with l)powderymildew. \m. J. Botany 29: 425-35.

2. .\LLFI, P. J. AND) D. R. GoIDARD. 1938. A respira-tory study of p)o\wdery mildew of wheat. Ami1. J.Botany 25: 613-21.

3. ALi}1x, R. 12-. 1923. Cytological studies of iifec-tion of Baart, Kanred and Mindum wheats byPIu'cinii1 qroomimis lforms IlI and XIX. J. Agr.Researcl 21: 571-604.

4. ArP W.S, T1. AND 14. BEXEERS. 1960. Pathway.s of

glucose (lissiimilati))) in carrot slices. Plant

Nwsiol. 35: 830-38.5. IBIA\(ON SFIEL)I), I. AN)) H. \V. REAM i,G. 1964.

Pathways of glucose metabolism and( nucleic aci(dsyntlhesis. 'Nature 202: 464-66.

6). Bt-sH NELL, \W. R. ANi) P. J. Ai.iLEN. 1962. RZespi-ratorY changes iil barley leaves produced by singlecolonies of 1)o\v\lery mil(le\w. Plant Plhvsiol. 37:751-58.

7. , S. \o) H. BFEVEvRS. 1961. The regulatioiiof pathlvays of glucose cat-tbolism in mniaize roots.Biochemii. J. 80 : 21-7.

8. CAiIIILL, (G. F. JR., A. B. HAsTxINS, J. ASHMORE,and(l S. ZOTIT. 1958. Stucdies oIn carbohyd(ratemetabolismii in rat liver slices. X. Factors in theregulationi of patlhways of glucose metabolism. J.Biol. Chcm. 230: 125-35.

9. DALY, J. M., R. 'M. SAYRE, \ND J. H. PAZUTR. 1957.The hexose imonophosphate slhtunit as a maj or

respiratory PatIm-av (lurinlg sporulation of rust ofsafflower. P'lanit Plhysiol. 32: 44-48.

10. KIN'OSIITA, J. H. 1957. The stitmiulationi of theplhosphogluconate oxi(dation patlhway by pyruvatein bovine cornleal epithelium. J. Biol. Chemii. 228:247-53.

11. KRE11s, H. .A. 1951. The Use of CO., buffers in111anioni1ctric nicasureincilts of cell metahOi ism. Bio-checmi1. J. 48: 349-59.

12. LA..ST, F. T. 1963. Metabolism of barley leaves inloc-

ulated witfl Frvsiplc Yramimis Merat. Anni. Bot.N.S. 27: 68,5-90.

13. LIVNF, A. 1964. Photosynthesis in healthy and

rust-affected l)lants. Planit Physiol. 39: 614-21.14. ILUNDERSTXDT, J., R. HEITEFtSS, A\ND) 'A. H. Flc-HS.

1962. Aktivitat einiger enzynme des kohlenhydrat-stoffwechlsels aus weizenikeimpflainzeni nach infek-tiO)) nuit Pf'ccimao q(7raoiiis tritici. Natuirwisseil-schlafteni 49: 403.

15. A\lAcKINNIY, G. 1941. Absorp)tion of liglht by

chlorop)hllll solutions. J. Biol. Cliemii. 140: 315-22.16. MI LLERD, A. AND K. J. Sc(OTT. 1956. Host patlhogen

relationis in p)owdery mildewx of barley. II.

Clhanges in respiratory p)atter:). .\ustralian J. Biol.Sci. 9: 37-44.

17. IT.FRRI), A. ANDo K. J. SC-OTT. 1963. Host patho-geni relationis in powdery miiiklew of barley. III.

U'tilization of respiratory energy. Australian J.Biol. Sci. 16: 775-83.

18. MM.LERD, .\. .\ND K, J. SCOTT. 1962. Respiration ofthe disease( l)lant. Amin. P\ev. Plant Physiol. 13:559-74.

296



SCOTT AND SMILLIE-METABOLIC REGULATION IN DISEASED LEAVES

19. MOSEMAN, J. G. 1956. Physiological races ofErvsiphe gramninis f. sp. hordei in Nortlh America.Phytopathology 46: 318-22.

20. PERSON. C., D. J. SAMBORSKI, AND F. R. FORSYTH.1957. Effect of benzimidazole on (letaclied wheatleaves. Nature 180: 1294-95.

21. SAN PIETRO, A. AND H. M. LANG. 1958. Photo-synthetic pyridine nucleotide reductase. I. Partialpurification anid properties of the enizyme fromspinach. J. Biol. Chem. 231: 211-29.

22. SCOTT, K. J. 1965. Respiratory enzymic activitiesin the host and pathogen of barley leaves inifectedwith ErYsiphe grami1inis. Phytopathology. 55:438-41.

23. SCOTT, K. J., J. S. CRAIGIE, AND R. M. SMILLIE.1964. Pathways of respiration in plant tumors.Plant Physiol. 39: 323-27.

24. SCOTT, K. J. AND R. M. SMILLIE. 1962. Enzy1mechanges in barley leaves after inifection with pow-(lery mildew. Plant Physiol. 37: lvii.

25. ScoTT, K. J. AND R. M. SMILLIE. 1963. Possiblerelationship between photosynthesis anid the rise inrespiration in diseased leaves. Nature 197: 1319-20.

26. SIIAW, M. 1963. The physiology and host-parasiterelationis of the rusts. Ann. Rev. Phytopathology1: 259-94.

27. SHAW, M. AND N. CoLoTELO. 1961. The physiologyof host-parasite relations. VII. The effect ofstem rust on the nitrogen and amino acids in wheatleaves. Canl. J. Botany 39: 1351-72.

28. SHAW, M. AND D. J. SAMBORSKI. 1957. Thephysiology of host-parasite relations. III. Thepattern of respiration in rusted and mildewed cerealleaves. C(an. J. Botany 35: 389-407.

29. SMILLIE, R. Ml. 1962. Photosynthetic anid respira-tory activities of growing pea leaves. PlantPhysiol. 37: 716-21.

30. SMILLIE, R. M. 1963. Formiiation and function ofsoluble proteins in chloroplasts. Can. J. Botany41: 123-54.

31. UDVARDY, J., M. HORVATH, K. KI-SBAN, L. DiEZSI,AND G. L. FARKAS. 1964. Alteration of enizymeactivities in detached leaves and their counterac-tion by kinetin. Experientia 20: 214-15.

32. WVHITE, N. H. AND E. P. BAKER. 1954. Hostpathogen relations in powdery mildew of barley.I. Histology of tissue reactions. Phytopathology44: 657-62.

33. WHITE, P. R. 1963. The cultivationi of plant andanimal cells. Ronald Press, New York, p 60.

34. YAMAMOTO, Y. 1963. Pyridine nucleotide contentin the higher plant. Effect of age of tissue.Plant Physiol. 38: 45-54.

297



metabolic regulation in diseased leaves. i. the ... · metabolic regulation in diseased leaves. i....

Documents