biosynthesis and metabolism of glyceollin i in soybean hypocotyls following wounding or inoculation...

Physiological and Molecular Plant Pathology (1987) 31, 387--405

Biosynthesis and metabolism of glyceollin I in soybeanhypocotyls following wounding or inoculation withPhytophthora megasperma f . sp . glycinea

M. K . BHATTACHARYYA * and E. W. B . WARDt* Department of Plant Sciences, University of Western Ontario, and tResearch Centre, Agriculture Canada University Sub PostOffice, London, Ontario, .N6A 5B7, Canada

(Acceptedfor publication April 1987)

In unwounded soybean hypocotyls, pulse labelled with [ 14 C]phenylalanine and inoculated withPhytophthora megasperma Esp . glycinea, rates of [ 14 C]-incorporation and glyceollin I accumulationwere higher in resistant than in susceptible responses throughout the time-course of the experiment .This distinction was masked in hypocotyls that were wounded and inoculated . In such hypocotyls,high rates of [ 14C]-incorporation developed that were similar for the first 11 h in resistant andsusceptible responses, although much more glyceollin I accumulated in the former. High rates of[ 14 C]-incorporation also developed in uninoculated wounded hypocotyls but only small amounts ofglyceollin I of high specific radioactivity were detected . Estimates of phenylalanine ammonia-lyaseactivity indicated that the metabolic flux through phenylalanine was limited in wounded controlsbut potentially very high in resistant responses . Differences in rates of[14C] -incorporation and inspecific radioactivity of accumulated glyceollin I presumably indicate differences in the relativecontributions of mobile internal pools and externally applied phenylalanine, in addition to rates ofbiosynthesis . Rapid decline in [ 14 C]-glyceollin I was demonstrated in wounded controls in pulse-chase experiments with phenylalanine as chase, but not in inoculated hypocotyls, due to continued[ 14C1-incorporation during the chase period . Rapid metabolism was demonstrated in all interac-tions and in wounds when cinnamic acid was used as the chase, but there was no evidence thatdifferences in glyceollin I accumulation were due to differential rates of metabolism . Additionalevidence for metabolic activity was provided by pulse feeding with [ 14 C] glyceollin I . It is concludedthat the stimulus of wounding or infection induces a metabolic pathway in which glyceollin I is notan end product . The accumulation of higher levels of glyceollin I in resistant than in susceptibleresponses appears to be due to earlier initiation and subsequently higher rates of biosynthesis in theformer .

INTRODUCTION

The possibility that phytoalexins may be intermediates in constitutive secondarymetabolic pathways and that their accumulation may be regulated as much by rates ofmetabolism as by rates of biosynthesis has been considered by several authors [14, 23, 27,2.9, 33, 36] . Inhibition of metabolism rather than elicitation of biosynthesis, then, wouldbe the key mechanism controlling phytoalexin accumulation in incompatible interac-tions . Evidence for phytoalexin metabolism by plants is provided by time-course studiesof phytoalexin concentrations in infected or stressed tissue in which phytoalexin levelsdecline after reaching a maximum (for example, see references [3, 15, 21, 22, 24]) and

tTo whom correspondence should be addressed .

388

M. K. Bhattacharyya and E . W . B . Ward

particularly by the disappearance of phytoalexins supplied to or radiolabelled withinplant tissues [14, 23, 27, 361 .

In the interaction of soybean hypocotyls (Glycine max L . Merr.) with Phytophthoramegasperma Drechs . f. sp . glycinea (Hildeb.) Kuan and Erwin, Yoshikawa et al . [36]concluded, from pulse-labelling experiments using L-[U 14C]phenylalanine as precur-sor, that rates of biosynthesis of glyceollin in compatible and incompatible interactionswere similar, even though much higher concentrations of glyceollin accumulated in thelatter. They also concluded from pulse-chase experiments and from feeding glyceollin tohypocotyl tissues that glyceollin was metabolized in uninoculated tissue, but that thisactivity was inhibited following infection, especially in the incompatible interactions .These results suggested that the differential accumulation of glyceollin in the two inter-actions primarily reflected differences in the rate of metabolism of glyceollin . Quitedifferent conclusions were reached for the same host-pathogen system by Moesta &Grisebach [21] . These authors used [ 14C]carbon dioxide as the precursor . They foundthat although rates of biosynthesis were the same in both compatible and incompatibleinteractions for the first 12 h following inoculation, biosynthesis subsequently becamemore rapid in the incompatible interaction . It was only at this stage that differences inglyceollin accumulation between the two interactions were detected . No evidence forsignificant glyceollin metabolism was obtained by these authors, and it was concludedthat the differential in glyceollin accumulation was due to the differences in biosyntheticrates that ultimately developed . A difficulty with the conclusions of both groups ofinvestigators is that they imply that elicitation of glyceollin biosynthesis is the same forcompatible and incompatible races and that specificity is a secondary and laterphenomenon . A further inconsistency between the two studies is that differences in thedegree of restriction of the pathogen in compatible and incompatible interactions weredetected by Yoshikawa et al. [35] within 8 h of inoculation, a time at which according toMoesta & Grisebach [21], but not according to Yoshikawa et al. [35, 36], glyceollinconcentrations in both types of interactions were the same .

In this paper biosynthesis and metabolism of glyceollin I (the major isomer accumu-lating in hypocotyls [3]) were examined in both wounded and intact hypocotyls . Theresults indicate that (1) glyceollin I biosynthesis and metabolism are induced followingwounding and/or infection, (2) the time-course of both biosynthesis and glyceollin Iaccumulation in compatible and incompatible interactions are distinctly different, (3)differences in the timing and rate ofbiosynthesis rather than metabolism appear to be themajor factors leading to differential glyceollin I accumulation, and that (4), in wound-inoculated hypocotyls, wound responses initially mask responses to infection . Through-out the paper the term glyceollin is used with reference to undefined mixtures of theglyceollin isomers .

MATERIALS AND METHODS

PathogenThe culture used was a single zoospore isolate (R1 .19) derived from Phytophthoramegasperma f. sp . glycinea race 1 [30] . It was grown routinely on V8 juice agar medium at25 °C . Zoospore suspensions were prepared from 5-day-old cultures on the same mediumin Petri dishes by repeatedly flooding with sterile distilled water until sporangia

Glyceollin I in soybean hypocotyls

389developed and then incubating the cultures overnight under a layer of sterile distilledwater (3-4 mm) to permit release of the zoospores . Inoculum in all cases consisted of a10 tl drop of a standardized zoospore suspension (10 5 ml') .

HostSeeds of soybeans, cultivar Harosoy (susceptible to Phytophthora megasperma f. sp . glycinearace 1) and its isoline Harosoy 63 (resistant) were supplied by R . I . Buzzell, ResearchStation, Agriculture Canada, Harrow, Ontario . Seedlings were grown in trays ofvermiculite in the dark for 6 days as described previously [32] .

Inoculation and incubationThe seedlings were arranged horizontally on moist cellucotton in glass trays [32] .Hypocotyls were inoculated by placing a 10-µl drop of zoospore suspension, or steriledistilled water in the case of controls, onto the surface of the intact hypocotyl about 2 cmbelow the cotyledons or into a wound at the same position. Wounds were made with ascalpel by removing a strip of tissue from the hypocotyl surface similar in area to thatcovered by a drop of inoculum (3 mm long x 0 .5 mm deep) . Wounds were inoculatedimmediately after wounding except where indicated otherwise . Trays of inoculatedseedlings were closed with plastic film to maintain high humidity and incubated in thedark at 25 "C .

Radioactive compoundsL-[U14C]Phenylalanine (50 µCi ml- ' ; 522 mCi mmol - ') was obtained fromAmersham Corporation . [ 14C]Glyceollin I (0 .05 µCi ml -1 ; 0 . 1mM) was prepared byapplying t.-[U 14C]phenylalanine for 1 h to water-inoculated wounds 25h afterwounding, to obtain glyceollin I of high specific activity . Extraction and purification ofglyceollin I is described below .

Feeding ofradioactive compoundsRoutinely, L-[U 14C]phenylalanine was supplied to inoculated sites and wounds in 5-pldrops after removing the inoculum drops or water . In an alternative procedure usedin one experiment, hypocotyls were cut 2 cm below the inoculated site and 5 .tl ofL-[U 14C]phenylalanine solution was applied to the cut end. A minimum of 10hypocotyls were used for each treatment and each hypocotyl received a 5-pl drop of0 . 1 MM L-[U14C]phenylalanine (50 µCi ml - ') . Except where indicated otherwise, pulseperiods were 1 h, after which glyceollin I was isolated and [ 14C] -incorporationmeasured. Decline of radioactivity in glyceollin I following pulse feeding was determinedfor various chase periods . Inoculated sites were washed to remove L-[U 14C]phenylala-nine and either [ 12C] phenylalanine (10 pl, 1 mm), as used by Yoshikawa et al . [36], ortrans-cinnamic acid (15 µl, 1 mm in 5 mm phosphate buffer, pH 5 . 5) to inhibit phenylala-nine ammonia lyase activity [26], was applied to the inoculated sites . In the cinnamicacid chase experiment, hypocotyls were kept moist under wet cellucotton and severedabout 6 .5 cm below the cotyledons . The cotyledons were removed immediately beforefeeding L-[U 14C]phenylalanine (12 . 5 µCi ml - ', 0-025 mm to wounded hypocotyls;25 µCi ml - ' 0.05 mm to unwounded hypocotyls) to 20 sites per treatment . The pulseperiod was from 8 . 5 to 9 . 5 h after inoculation, except in wounded control hypocotyls

390


where it was delayed to permit detectible accumulation of glyceollin I and was from14-15 h after wounding . In addition to chasing with cinnamic acid at the sites of wound-ing and inoculation, 2 mm cinnamic acid in 10 mm phosphate buffer (pH 5 .5) was addedalso to the wet cellucotton covering the lower cut end of the hypocotyl .

To determine the rate of metabolism of exogenously supplied glyceollin I, 5-µl dropscontaining [14C] glyceollin I were applied to infection sites at various times after inocu-lation and/or wounding and residual glyceollin I in surface fluids and tissue extracts wasdetermined after incubation for 30 min .

Extraction and determination ofglyceollin IFor each determination, sections (approx . 2 cm long) containing infected and woundedtissues were excised from 10 or more hypocotyls and the glyceollin isomers were extractedby boiling in 95% ethanol for 2 min . The extract was decanted and together with twoethanol rinses of the tissues evaporated to dryness under reduced pressure . The residuewas dissolved in 1 ml of water and extracted three times with 2 ml ethyl acetate. Thecombined ethyl acetate fractions were reduced to dryness and redissolved in 100 µl ofethyl acetate and together with two 100-µ1 rinses subjected to TLC (Whatman LK6DFsilica gel, benzene : methanol, 95 : 8) . The glyceollin isomers were detected as a singleband by fluorescence quenching under UV light . The corresponding silica gel wasscraped from the plate and the glyceollin isomers were eluted with ethyl acetate .Glyceollin I was separated from isomers II and III by HPLC [2] using an analyticalcolumn (silica, Whatman Partisil 5, 250 x 4 .6 mm i.d .) and 4 .5% isopropanol in hexane(flow rate 1 . 5 ml min -1 ) as the mobile phase . Glyceollin I was detected by its absorptionat 286 nm . The retention time was 13 .58-14 .33 min, and this afforded complete separ-ation from isomers I I and III . The U V spectrum of the isolated glyceollin I conformed tothat of a purified standard and of published reports [2, 12] . It was quantified from itsabsorbance at 286 nm by reference to a standard curve prepared from purified glyceollinI and the extinction coefficient, 10 800 [2] .

Measurement of/ 14 C]-incorporation into glyceollin IThe fraction corresponding to the glyceollin peak detected by HPLC was collected in ascintillation vial, mixed with 10 ml of scintillation fluid (4 g of Omnifluor in 1 litre oftoluene) and radioactivity (in d min_ 1) measured in a scintillation counter (BeckmanLS 9000) .

Purity of [ 14C]glyceollin IThis was determined for the glyceollin I preparations obtained in the experiment illus-trated in Fig. 1 . Following the first TLC separation, samples from all the pulse timeswithin each treatment were combined giving bulked samples for wounded inoculatedand intact inoculated cv . Harosoy and Harosoy 63 . For the wounded water controls theHarosoy and Harosoy 63 samples were combined also and spiked with purified[12C] glyceollin I to provided sufficient material for further analysis . The bulked sampleswere subjected to HPLC, the glyceollin I fractions were collected and samples were takenfor measurement of radioactivity as described . The remaining portions were subjected to


391TLC in three solvent systems (Table 1), again followed by HPLC and measurement ofradioactivity .

Determination offree L-phenylalanineTissues of 20 lesions and wounds in 10 hypocotyls were excised and ground in methanolwith a mortar and pestle. The slurry was centrifuged (microcentrifuge, 15 000 r min- 1 )and the pellet was dried (65 °C) and weighed . The methanol was evaporated from thesupernatant and the residue dissolved in 3 ml high purity water (Milli-Q system,Millipore) . The pH of the solution was adjusted to 2 . 0 with 1 M HCl and extracted threetimes with ethyl ether . The aqueous phase was applied to a cation exchange column(Rexyn 101, H + form, 0 . 9 x 7 . 0 cm) . The column was washed with water, and aminoacids were eluted with 1N NH4OH (12 ml) . The eluate was lyophilized and used for thedetermination of L-phenylalanine by HPLC, in general following procedures describedby Bidlingmeyer et al. [4] . The lyophilized sample was dissolved in 0 .5 ml of water and50 tl was taken for analysis . The water was removed under nitrogen at low pressure andthe residue was dried a second time after redissolving in ethanol : water : triethylamine(2 : 2 : 1, v/v) . The dried residue was derivatized with 20 pl of freshly prepared derivatiz-ing reagent (ethanol : triethylamine : water : phenylisothiocyanate; 7 : 1 : 1 : 1) at roomtemperature for 20 min . The reagent was removed by evaporation and the residueredissolved in 100 µl sample diluent (710 mg of Na 2HPO4 mixed with I litre of water,titrated to pH 7 . 4 with 10% phosphoric acid and mixed with acetonitrile to give a finalconcentration of5% by volume) . It was injected (15 pl) on to a Waters Pico-Tag column(column temperature 38'C, Waters model 710B WISP injector, model 510 pumps,model 480 variable wave length detector and model 840 data system) and L-phenylala-nine was determined by the Pico-Tag (Waters) method following instructions providedby the manufacturer . L-Phenylalanine concentrations were expressed as nmol mg -1 dryweight of excised tissue . Data are based on two replications .

Measurement ofphenylalanine ammonia-lyase activityTissues were excised from 20 lesions or wounds in 10 hypocotyls per treatment, immersedimmediately in liquid nitrogen and stored at - 70 °C. They were ground in ice coldbuffer (0 . 1 M sodium borate, pH 8 .8, in 2 mm mercaptoethanol) using a mortar andpestle . The slurry was centrifuged at 15 000 r min- 1 for 4 min and the supernatant storedat - 70 °C until assayed for enzyme activity [17] . The assay mixture consisted of 300 µMsodium borate (pH 8 .8), 3011M L-phenylalanine and 1 ml of supernatant in a totalvolume of 3 ml . After incubation at 40 °C for 1 h, activity was determinedspectrophotometrically at 290 nm against a mixture identical except for substitutionof n-phenylalanine for the L-isomer . Activity was expressed as nmol cinnamic acidmin-1 mg -1 dry weight of tissues .

RESULTS

Analyses for glyceollin I only are reported here . Glyceollins II and III, which areproduced in hypocotyls in much smaller amounts than glyceollin I, were frequentlyinsufficient to provide reliable estimates of [14C]-incorporation, and could not be puri-fied to constant specific radioactivity . The combination of TLC and HPLC separations

392

M. K . Bhattacharyya and E. W . B. WardTABLE I

Effect of purification on the specific radioactivity (d min - ~ pg - ') of î 14 C/glvceollin I recovered fromsoybeans hypocotyls after wounding or inoculation with Phytophthora megaspermaf sp . glycinea race Iin pulse-feeding experiments with L-f U' 4 C j phenylalanine s . Data are means and standard errors from threereplications . Specific radioactivity after the second TLC is expressed as a percentage (in parentheses) of that

after HPLC

acv . Harosoy is susceptible and cv . Harosoy 63 is resistant to Phytophthora megasperma f. sp .glycinea race 1 .

b Wounded and inoculated sites were pulsed with L-[U 14C]phenylalanine (5 pd, 50 µmCiml - ', 0 1 mm) for I h prior to analysis at the times indicated in Fig . I (a) . Samples for eachcultivar and treatment were bulked, see Materials and Methods .

`In wounded controls, glyceollin from both cultivars was combined after the first TLC andspiked with purified glyceollin I .

(see Methods) yielded glyceollin I sufficiently pure for interpretation of incorporationand metabolic experiments with confidence (Table 1) .

Estimations of glyceollin I rate of biosynthesisIn uninoculated wounded hypocotyls, [ 14C]-incorporation into glyceollin I following1-h pulses with L-[U 14C]phenylalanine was demonstrated I1 h following wounding[Fig . 1(a)] . The rate of incorporation increased very rapidly at subsequent pulse times .However, only trace amounts of glyceollin I (< 1 pg) were detected until 15 h followingwounding and only small quantities were demonstrated after longer periods [Fig . 1(b)] .Trace amounts ofglyceollin I were also detected in unwounded controls, however [14C]-incorporation from L-[U 14C]phenylalanine on the intact surface was only 5-10% of thatin wounded hypocotyls.

In inoculated wounds, [ 14C]-incorporation into glyceollin I was demonstrated 7 hfollowing inoculation . It increased rapidly and was similar in both cultivars until 11 h[Fig . I (a)] . Incorporation then declined in the compatible interaction (Harosoy) but

Cultivar and treatment

After initialTLC (SO) and

HPLC

Second TLC

S1 S2 S3

Harosoy, Harosoy 63, wounded only' 1908±21 2028±22 1858± 17 1987±32(106) (97) (104)

Harosoy, wounded, inoculated 346±6 345±3 361+2 335±4(100) (104) (97)

Harosoy 63, wounded, inoculated 480±2 467±4 480±14 467± 13(97) (100) (97)

Harosoy, unwounded, inoculated 116±2 104±2 106±3 110+1(90) (91) (95)

Harosoy 63, unwounded, inoculated 275±3 278±4 290 ± 4 280+10(101) (105) (102)

Solvent system Rr glyceollin ISO Benzene : methanol (95 :8) 0 . 35Sl Diethylether : n-hexane (60 :40) 0 . 48S2 Chloroform : methanol (100 : 5) 0 . 37S3 n-Hexane : diethylether : glacial acetic acid (65 : 30 : 3) 0 . 26


393

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180

ô 150N

CW

. 30

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0

4 7

1I

15

19

26

4 7

Incubation period (h)

FIG . 1 . Îa) Incorporation of [ 14 C] into glyceollin I from t,-[U 14 C]phenylalanine in l h pulseperiods, and (b) accumulation of glyceollin I in soybean hypocotyls . Intact hypocotyls inoculatedwith Phyto(hthora megasperma f. sp. glycinea race L •, cv . Harosoy (susceptible) ; O, cv . Harosoy 63(resistant) . Wounded inoculated hypocotyls : ∎, cv . Harosoy ; D, cv . Harosoy 63 . Wounded controlhypocotyls : A, cv . Harosoy ; 0, cv . Harosoy 63. Incubation period indicates the time followinginoculation .

II 15

19 26

increased in the incompatible interaction (Harosoy 63) until 15 h and then also declined .Accumulation of glyceollin I, however, was distinctly different in the two interactions[Fig . 1(b) ] . In the incompatible interaction it accumulated rapidly from 7 h after inocu-lation, but was not detected until 11 h in the compatible interaction, and subsequentlyaccumulated relatively slowly .

In contrast to the similarities in early rates of[14C]-incorporation in both interactiontypes in inoculated wounded hypocotyls, rates of incorporation in the incompatibleinteraction in unwounded hypocotyls differed from those in the compatible interactionwhen first demonstrated at 7 h following inoculation [Fig . 1(a)] . The patterns ofglyceollin I accumulation in the two interactions in unwounded hypocotyls wereconsistent with the differences in incorporation rates .

Specific radioactivity of[14C]glyceollin I differed widely in the different treatmentsand also varied with time after inoculation and wounding (Table 2) . Thus, specificactivities were very high in wounded control hypocotyls and also in the compatibleinteraction in wounded hypocotyls, although they declined rapidly in the latter . Thisdiffered from the incompatible interaction in wounded hypocotyls in which specificactivities were low initially and remained lower than in the compatible interaction . In

394

M. K . Bhattacharyya and E . W . B . WardTABLE 2

Specific radioactivity (d min -1 pg - ') of i 14 C/glyceolhn I recovered following pulse feeding with i;U14 C,'phenylalanine . from soybean hypocotyls wounded or inoculated with Phytophthora megasperma

f. sp . glycinea race l'

'Wounded and inoculated sites were pulsed with L-[U 14C] phenylalanine (5 µl, 50 µCi ml - '0 . 1 mm) for 1 h prior to analysis at the times indicated .

b cv. Harosoy is susceptible and cv . Harosoy 63 is resistant to Phytophthora megasperma f. sp .glycinea race 1 .

`Specific radioactivities were calculated from the incorporation of [ 1O C] into glyceollin Iduring the pulse period and the mean hourly accumulation of glyceollin I .

contrast, in the unwounded hypocotyls specific activities in both interactions weresimilar and remained low at all pulse times .

The possibility that wounding or type of interaction might affect access of surfaceapplications of L-[U 14C]phenylalanine to sites of biosynthesis and hence influenceincorporation rates was examined in the following experiment . Comparisons were madeOf[14C] -incorporation from L-[U 14C]phenylalanine applied either to infection sites orthrough the cut ends ofhypocotyls . For the same interaction type much more [ 14C] wasincorporated in wounded than in unwounded hypocotyls following application to infec-tion sites but there were no differences in incorporation following feeding through cutends ofhypocotyls (Table 3) . Therefore, quantitative comparisons ofincorporation ratesbetween inoculated wounded and unwounded hypocotyls cannot be made when precur-sor is applied to the inoculated site . On the other hand the data also demonstrate that thedifferential in [14C] -incorporation between compatible and incompatible interactionswas not affected by the feeding method . Hence comparisons of incorporation rates in thetwo interaction types are valid when precursor is applied to the inoculated site .

Estimation ofglyceollin I metabolismIn pulse-chase experiments to determine apparent rates of [ 1QC]glyceollin I metab-olism, a rapid decline in labelled glyceollin I was demonstrated only in the woundedcontrols (Fig . 2) . In inoculated hypocotyls, radiolabel incorporation continued duringthe chase period in incompatible interactions and the amount of [ 14C]glyceollin Iremained more or less constant in the compatible interactions . When cinnamic acid wasused as the chase instead of phenylalanine there was a rapid decline in [14C]glyceollin Iin both control and inoculated hypocotyls (Fig . 3) . Rates of metabolism were similar incompatible and incompatible interactions both in wounded and in intact hypocotyls .

Additional evidence for high rates of glyceollin metabolism were provided bymeasuring the recovery of [ 14C] glyceollin I supplied for 30 min periods at various timesafter wounding or after inoculation of wounded or intact hypocotyls (Fig. 4) .

Time frominoculation

(h)

Unwoundedinoculated

Woundedinoculated

Woundedonly

Harosoyb Harosoy 63 Harosoy Harosoy 63 Harosoy Harosoy 63

11 1927' 1755 72 032 4934 65 281 65 482

15 1509 1875 16 977 5011 93 146 43 35619 934 1272 4508 2067 62 112 78 563

Glyceollin I in soybean hypocotylsTABLE 3

Influence of method of feeding L-1 UI °C]phenylalanine on incorporation of/ "CJ into glyceollin I followinginoculation of wounded or intact soybean hypocotyls with Phytophthora megaspermaf. sp . glycinea race 1 .

Data are means and standard errors of (14C]glyceollin 1(d min - 1) for three replicated experiments

a All plants were inoculated, cv Harosoy, is susceptible and cv . Harosoy 63 is resistant . L-[Ut4C]Phenylalanine (50 pCi ml_ 1, 0 .1 mm) was applied (5 pl) to each of 10 hypocotyls .b In method 1, inoculum drops were removed from inoculated sites 10 h after inoculation andreplaced with L-[U14C]phenylalanine for I or 2 h before analysis . In method 2, at 10 h afterinoculation hypocotyls were severed c . 2 .5 cm below inoculation sites and the cotyledonsremoved . A 5 pl drop of L-[U14C]phenylalanine was applied to the lower cut surface of theinoculated section of each hypocotyl and incubated for l or 2 h before analysis .

Metabolism was negligible shortly following wounding and inoculation but by 4 .5-5 .0 hit was very rapid in wounded hypocotyls (inoculated and controls) . Thereafter, the rateof metabolism continued to increase in the wounded controls but apparently declined inthe inoculated wounded hypocotyls, especially in the incompatible interaction . Rates ofmetabolism were much less in intact hypocotyls than in wounded hypocotyls especiallyin the incompatible interaction .

Precursor pools and phenylalanine ammonia-lyase activityThe pulse-labelling experiments (Fig . 1) indicated that wounding alone inducedglyceollin I biosynthesis . The effect of wounding prior to inoculation on glyceollin Ibiosynthesis and metabolism was examined, therefore . Inoculation of hypocotyls thathad been wounded 12 h previously (after wound-induced biosynthesis had been estab-lished ; Fig . 1) resulted in a much earlier onset of glyceollin accumulation than inhypocotyls inoculated at the time of wounding [Fig . 5(a)] . However, the pattern of[14C]-incorporation into glyceollin I after pulse feeding with L-[U14C]phenylalanine[(Fig. 5(a)] was entirely different from that in hypocotyls inoculated at the time ofwounding (Fig . 1) . Rapid incorporation occurred in the compatible interaction and nochanges in incorporation rate occurred in the incompatible interaction . A pulse-chaseexperiment (pulsed 12 h following wounding for 1 h, inoculated at 13 h and chased with1 mm phenylalanine from 15 h) indicated that incorporation of radiolabel into glyceollinI continued during the chase period in the incompatible interaction but declined in thecompatible interaction [Fig. 5(b)] . The inconsistencies in patterns of glyceollin I

395

Cultivar andtreatments Method lb Method 2

Pulse period 10-11 hHarosoyWounded 17290+_966 130+22Unwounded 1281+159 94+34

Harosoy 63Wounded 29 379+ 1042 1241+323Unwounded 3800±422 952+107

Pulse period 10-12 hHarosoy, unwounded 1546+53 1169±80Harosoy 63, unwounded 16917±1047 12 311 ± 1109

396

M. K . Bhattacharyya and E. W. B . Ward

60

50

M0 40

EE 30côuc~ 20Ûa

10

1

IIII

4

8

12

16

Chose period (h)

FIG . 2 . Fate of [ 14C]glyceollin I in soybean hypocotyls in a pulse-chase experiment withL-[U 14C]phenylalanine . [ i4 C]Glyceollin I was synthesized during a I h pulse with L-[U 1AC]

phenylalanine and chased with L-phenylalanine (1 mm) . The pulse was applied from 10-11 hfollowing inoculation or from 14-15 h after wounding in the wounded controls . Intact hypocotylsinoculated with Phytophthora megasperma f. sp . glycinea race 1 : •, cv . Harosoy (susceptible) ; O,cv. Harosoy 63 (resistant) . Wounded inoculated hypocotyls : ∎, cv. Harosoy ; D, Harosoy 63 .Wounded control hypocotyls : A, cv . Harosoy ; A, cv. Harosoy 63 . Chase period commenced at0 h (arrow) immediately following the 1 h pulse from - 1 h. Data are from one of two similarexperiments .

accumulation and [14C]-incorporation suggested that the pulse and pulse-chase datamight reflect precursor pool sizes rather than true rates of biosynthesis and metabolism .

This possibility was examined by determining the relative incorporation of [ 14C]into glyceollin I after a 1 h pulse with L-[U 14C]phenylalanine at two specific radio-activities (522 or 5OmCi m mol -1) (Table 4) . The 10-fold difference in specificradioactivity had little influence on [ 14C]-incorporation in the incompatible interac-tions but in the compatible interactions and the wounded controls incorporation fromthe L-[U 14C]phenylalanine of low specific radioactivity was only about 50% of thatfrom the phenylalanine of high specific radioactivity . This reduction in incorpor-ation suggests that internal pools of phenylalanine were limiting in the controls andcompatible interactions .

Phenylalanine pool sizes were measured at 11 h following inoculation or wounding, atime at which rates of[14C]-incorporation had reached significant levels in control andinoculated hypocotyls [Fig . 1(a)] . Measurements in wounded controls were made alsoafter 15 h to coincide with the first detection of glyceollin I accumulation in woundedcontrols . Phenylalanine concentrations were much lower in inoculated than in controlhypocotyls and, in Harosoy 63, lower in the wounded controls than in the intact hypo-cotyls (Table 5) . The phenylalanine pool in the wounded hypocotyls decreased sig-nificantly between 11 and 15 h . Thus there was in general an inverse correlation between

-10


397

8

6

4

2

0-1

0

I

2

3

Chose period (h)Fte . 3 . Metabolism of [ 14C]glyceollin I in soybean hypocotyls in a pulse-chase experiment with

L-[U 14 C]phenylalanine . [14CGlyceollin I was synthesized during a 1 h pulse with L-[U 14 C]phenylalanine and chased with 1 mm cinnamic acid . The pulse was applied from 8. 5-9 . 5 h follow-ing inoculation or 14-15 h following wounding in the wounded control . Intact hypocotyls inocu-lated with Phytophthora megasperma f. sp. glycinea race 1 : •, cv . Harosoy (susceptible) ; O, cv . Harosoy63 (resistant) . Wounded inoculated hypocotyls: ∎, cv . Harosoy ; O, cv . Harosoy 63. Woundedcontrol hypocotyls : A, cv . Harosoy . Chase period commenced at 0 h (arrow) immediately follow-ing the I h pulse from - I h . Data are from one out of three similar experiments .

phenylalanine pool size and PAL activity and, from other experiments, glyceollin Iaccumulation . However, there were striking differences in PAL activity between inocu-lated and control hypocotyls and activity in incompatible interactions was about threetimes that in compatible interactions (Table 5) .

DISCUSSION

L-[U 14C]Phenylalanine was used in the present study as the experimental precursor of

glyceollin I, in general following procedures described by Yoshikawa et al . [36] . Althoughincorporation ofboth [14C]daidzein and [14C]mevalonate has been reported [1, 37] wedid not obtain useful incorporation of these precursors in pulse-feeding experiments .Moesta & Grisebach [21] used [ 14C]carbon dioxide as a precursor of the glyceollins andsuggested [19,20] that phenylalanine might have the disadvantage that uptake might beinfluenced by experimental conditions . Nevertheless, L-[ 14C]phenylalanine has beensuccessfully employed as a precursor in a number ofstudies of phenylpropanoid biosyn-thesis (for example, see references [1, 7,8]) and the data given in Table 3 indicate that itis an acceptable precursor for comparisons of [ 14C] -incorporation into glyceollin I inincompatible and compatible interactions under the experimental conditions used here .In contrast, the drawbacks to the use of[14C] carbon dioxide appear to be more serious .

4

398

M. K. Bhattacharyya and E. W. B . Ward

20

16

N

oX

Ic 12Ela

4

0 5

10

Incubation period (h)FIG. 4 . Metabolism of externally supplied [ 14C]glyceollin I in soybean hypocotyls . [ 14C]

Glyceollin I was applied to wounds or infection sites for pulse periods of 30 min immediately prior toanalysis at the times indicated . Residual [ 14 C]glyceollin I was determined following each 30 minpulse . Efficiency of recovery from treated tissue without incubation was 64% (1600d min - ') .Intact hypocotyls inoculated with Phytophthora megasperma f. sp . glycinea race l : •, cv . Harosoy (sus-ceptible) ; O, cv . Harosoy 63 (resistant) . Wounded inoculated hypocotyls : ∎, cv. Harosoy ; L, cv .Harosoy 63 . Wounded control hypocotyls : A, cv . Harosoy ; L~, cv. Harosoy 63 . Incubation periodrefers to time following wounding and inoculation . Data are the mean and standard errors for tworeplications (five hypocotyls per replicate) .

As will be discussed below, the aims ofpulse-chase experiments [21 ] may be frustrated bythe remoteness of CO 2 from glyceollin and the large pool of metabolic products that itmight be expected to give rise to .

Yoshikawa et al. [36] concluded from pulse-feeding experiments that high biosynthe-tic rates were induced by inoculation of wounded hypocotyls and by wounding alone .The rates were similar in both compatible and incompatible interactions, althoughaccumulation ofglyceollin differed in the two interactions and was only just detectable inthe wounded controls . They concluded from these and pulse-chase experiments thatdifferences in accumulation were a reflection of different rates of glyceollin metabolism .Our results with wounded hypocotyls were broadly similar . However, in unwoundedinoculated hypocotyls, differences in glyceollin I accumulation appeared to be relateddirectly to differences in biosynthesis and not metabolism, for rates of both [14C]-incorporation and glyceollin I accumulation were distinctly different throughout thetime-course in both interaction types . This distinction evidently was masked bywounding, both in the present study and those of others [21, 36] .

The pulse-chase experiments, with L-phenylalanine as the chase, provided satisfac-tory evidence for rapid glyceollin metabolism in the wounded controls in both thepresent study and that of Yoshikawa et al . [36] . They failed to do so in the inoculated

Glyceollin 1 in soybean hypocotyls

0

Time (h)

Co

w

80 y

399

wrn

oco

0 ôrErU

âFIG . 5 . Effect of wounding soybean hypocotyls 12 h prior to inoculation with Phytophthora

megasperma f. sp . glycinea on (a) glyceollin I accumulation and incorporation of [ 14 C] into glyceollin Iand (b) the fate of [ 14 C]glyceollin I in a pulse-chase experiment . In (a) L-[U 14Clphenylalanine(5 µl, 50 µCi ml -t , 0. 1 mm, per hypocotyl) was supplied in a 30 min pulse immediately prior to thetime of analysis . In (b) L-[U 14 C]phenylalanine (5 µl, 25 pCi ml -t , 0.05 mm, per hypocotyl) wassupplied for 1 h prior to inoculation (0 h) and chased with I mm phenylalanine from 2 h afterinoculation (arrow) . In both (a) and (b) hypocotyls cv . Harosoy (∎, susceptible) and cv . Harosoy63 (E], resistant) were inoculated at 0 h (12 h after wounding) . Solid line indicates [ 14C]glyceollinI . broken line indicates glyceollin I accumulation .

hypocotyls, evidently due to the development of extensive pools of radiolabelled pre-cursors that continued to supply the biosynthetic pathway during the chase period .Presumably, a much wider pool ofprecursors generated from [14C]carbon dioxide in theexperiments of Moesta & Grisebach [21] increased this problem . They found [ 14 (1]-

incorporation continued to increase for 4-6h after the pulse-period, followed by alimited decline in [ 14C]glyceollin in the incompatible interaction only (half life of 28 h) .In the present study, radioactivity detected in glyceollin I declined rapidly in all interac-tions, after a lag of between 1 and 2 h, with half-lives of about 2 h (Fig . 3), when cinnamicacid was used to feed-back inhibit PAL, following the evidence of Shields et al . [26] .Furthermore, the rates of metabolism of[14C]glyceollin I indicated by these data weresimilar for each of the pairs of compatible and incompatible interactions in eitherwounded or unwounded hypocotyls . This was in spite of differences in specific radio-activities of glyceollin I in the two types ofinteraction (Table 2) . Hence, metabolic ratesare related to glyceollin pools rather than directly to interaction-types . On the basis ofthis experiment, it appears that differences in glyceollin I accumulation in the two typesof interaction, either in wounded or unwounded hypocotyls, are not controlled bydifferential metabolic rates .

Additional evidence for glyceollin I metabolism was provided by pulse-feeding with[ 14C]glyceollin I (Fig . 4) . Our results were consistent with those ofYoshikawa et al . [36],obtained with hypocotyl sections in vitro, but demonstrated further that glyceollin Iapplied to wounds and to inoculated wounded and intact hypocotyls was also metabo-lized. This ability evidently was not constitutive but was induced within 4 . 5 h of wound-ing or infection and hence may involve de novo enzyme synthesis comparable to thatrequired for glyceollin biosynthesis [5, 10, 25] . Half-lives for externally supplied

400 M. K . Bhattacharyya and E . W . B . WardTABLE 4

Influence of specific radioactiaity o/ L-! U 14 ;phenylalanine on incorporation o/ ,' 7dC into glyceollin 1 (dmin - ') in pulse feeding experiments in soybean hypocotyls wounded or inoculated with Phytophthora

megaspermaff sp . glycinca race 1 . Data are means and standard errors, front three replications

acv . Harosoy is susceptible and cv . Harosoy 63 is resistant to Phytophthora megasperma f. sp .glycinea race 1 .

'Wounded and inoculated sites (10 per treatment) were pulsed with 5 pl of 50 µCi MI- ' L-[U14 C]phenylalanine at two molar concentrations A and B as above, for I h from 10 to I 1 hafter inoculation or wounding . Thus A and B received the same amount of radioactivity but Breceived about 10 x as much phenylalanine as A .

`Accumulated glyceollin I (pg) in parenthesis .

[ t4C]glyceollin I were of the same order as those determined in pulse-chase experimentswith cinnamic acid. This experiment serves to emphasize that glyceollin I metabolism isinduced following wounding and infection . It also indicates that metabolic rates arehigher in compatible than incompatible interactions . However, this evidence must betreated with caution, especially in view of the results of the pulse-chase experiments(Fig . 3) . Dilution from endogenous glyceollin, especially in incompatible interactions,may have limited [ 14C] glyceollin I turnover, and access to sites of metabolism may havebeen less efficient in incompatible interactions due to the increasing preponderance ofdead cells with time . Access may have been more efficient also in tissues exposed bywounding .

The demonstration (Table 4) that [ 14C]-incorporation into glyceollin I was affectedsignificantly by the specific radioactivity of the phenylalanine fed, suggests that pool sizesor metabolic fluxes greatly influence estimates of biosynthesis in pulse-feeding exper-iments . In controls and compatible interactions the decrease in [ 14C]-incorporationwith the higher of the two phenylalanine concentrations (i .e . low specific radioactivity)would be expected if internal pools of phenylalanine were small relative to quantitiesprovided by the pulse or if endogenous rates of carbon-flow through phenylalanine werelow. The much smaller effect of specific radioactivity of phenylalanine on incorporationin the incompatible interactions suggests that internal precursor pools in these inter-actions were large or that metabolic rates were high relative to quantities provided by the

Cultivar' and treatment

I 14 C]Phenylalanine (specificradioactivity / b

B/A x 100(°-~,)(A) 522 mCi mmol - ' (B) 50 mCi mmol - '

Harosoy, wounded only 6539± 1777 3206± 733 49(1 .9 ± 0 . 5)` (1 . 3 ± 0 . 2)

Harosoy 63, wounded only 6147±280 2770± 170 45(1-2+0-1) (1 . 2 ± 0 . 1)

Harosoy, wounded, inoculated 17290+966 8902± 2813 52(2-8+0-03) (4-5+0-1)

Harosoy 63, wounded, inoculated 29379+1042 28395+38 97(10 . 7±0 .4) (11 .3±2 . 5)

Harosoy, unwounded, inoculated 1281+159 786±55 61(3-8+0-3) (4 .5+0 . 8)

Harosoy 63, unwounded, inoculated 3801+42-2 3286+188 86(21 . 8+3-8) (17 . 4 ± 0 . 9)

Glyceollin I in soybean hypocotylsTABLE 5

Concentrations offree L-phenylalanine and activity of phenylalanine ammonia-lyase in soybean hypocotylsfollowing wounding or inoculation with Phytophthora megaspermaf. sp . glycinea race 1 . Data are

means and standard errors for two replications

401

a cv . Harosoy is susceptible and cv. Harosoy 63 is resistant to Phytophthora megasperma f . sp .glycinea race 1 .

'Based on tissue excised from 20 lesions or wounds from 10 hypocotyls .`Incubation period from the time of inoculation or wounding to the time of analysis .'Hypocotyls in this series were wounded 12 h prior to inoculation, 4 h and 11 h incubation

refer to the period after inoculation until analysis .

pulse . No evidence for the expansion of free phenylalanine pools was found, and concen-trations were lowest where demand appeared to be greatest. However, there were majordifferences in PAL activity (Table 5) that were generally correlated with glyceollin Iaccumulation. Presumably, a very high rate of flow of carbon into the glyceollin bio-synthetic pathway is generated in the incompatible interaction resulting in largeaccumulations of glyceollin I of relatively low specific radioactivity (Table 2) . At theother extreme in the wounded controls the external L-[U 14C] phenylalanine pulse pre-sumably makes a major contribution to the very small endogenous flow, resulting inglyceollin I of high specific radioactivity (Table 2) . Observations that rates of [ 14CJ-incorporation in wounded controls approach [36] or equal (present paper) those inincompatible interactions, therefore, reflect differences in endogenous flow rates but notsimilarities in biosynthetic rates .

It might be expected that more uniform labelling of precursors would be achieved bypulse feeding with [ 14C]-carbon dioxide as employed by Moesta & Grisebach [211, thus

Cultivar" and treatmentPhenylalanine'

(nmol mg -t dry wt)

PAL activity'(nmol cinnamic

mint mg-' dry wt

Hypocotyls unwoundedI 1 h incubation`Harosoy inoculated 67±28 0 .88±0 . 23Harosoy 63 inoculated 56+14 2-61+0-12Harosoy uninoculated 162+9 0 . 15+0-04Harosoy 63 uninoculated 164+8 0 . 18+0-06

Hypocotyls wounded at time of inoculationI l h incubationHarosov inoculated 85+18 0 .92±0 . 04Harosov 63 inoculated 54±20 2 .85±0 . 22Harosoy uninoculated 146+17 0-2+0-03Harosov 63 uninoculated 111+19 0-13+0-06

15 h incubationHarosoy uninoculated 66+5 0 .16±0 . 08Harosoy 63 uninoculated 75+6 0-11+0 . 01

Hypocotyls wounded12 h prior to inoculation'

4 h incubationHarosoy inoculated 67 ± 12 0-49+0-17Harosov 63 inoculated 49+1 1 .06+0. 08

1 1 h incubation - _Harosoy inoculated 25±2 0 .88+0 . 05Harosoy 63 inoculated 28+1 3 .98+0 . 35

402


avoiding some of the difficulties encountered with [ 14C]phenylalanine . That this maynot be the case, however, is suggested by evidence that glyceollin biosynthesis in hypo-cotyls draws heavily on reserves in cotyledons [16] . Such reserves, presumably, wouldnot be labelled during [ 14C]carbon dioxide pulses and would be tapped differentially asdifferent rates of metabolic fluxes developed in compatible and incompatible interac-tions . Significantly, incorporation patterns during the early critical hours followinginoculation in wounded hypocotyls were similar both following [ 14C]carbon dioxidefeeding [21] and [14C] phenylalanine feeding [36, and the present paper] . In all threestudies, rates of incorporation during this period were the same in compatible andincompatible interactions in wounded hypocotyls . Moesta & Grisebach [21 ] consideredthat their data for glyceollin accumulation were consistent with this, for they failed tofind significant differences for the two interactions before 14 h after inoculation(although a trend would appear to have been established earlier) . In the previous studyby Yoshikawa et al. [36], and in our present study, differences in glyceollin accumulationdeveloped much earlier, leading to the interpretations discussed above . The latter resultsappear to be the more reasonable . They are consistent with the early differentiation ofresistant and susceptible responses in soybean hypocotyls [28, 35] and the association ofglyceollin with restriction of spread of the pathogen [18, 34, 35] . There is evidence thattranscription of mRNAs for pathway enzymes takes place within 3 h of inoculation inincompatible interactions but not in compatible interactions [10] . The data of Table 5indicate also that PAL activity in incompatible interactions was several-fold that incompatible interactions at 11 h after inoculation . Furthermore, in roots, accumulation ofglyceollin I and increases in activity of pathway enzymes occur much earlier in incom-patible than in compatible interactions [5, 11] . Börner & Grisebach [6], however, hadpreviously failed to find comparable differences in PAL activity in hypocotyls . Possiblytheir procedure for inoculation with mycelium introduced non-specific elicitors ofglyceollin biosynthesis that, together with the complications due to wounding, obscuredany early differences in response between compatible and incompatible interactions .

Although wounding does not result in the accumulation of significant amounts ofglyceollin during the first 12 h, it accelerates the biosynthesis and accumulation ofglyceollin following infection (Fig . 5) with parallel increases in the activity of PAL(Table 5) . This suggests that some wound responses may be readily diverted to glyceollinbiosynthesis, or that the low level of biosynthesis initiated by wounding can be morerapidly enhanced by inoculation . Such a response may be important in the resistance ofwounds to infection . A similar phenomenon was reported for sweet potatoes [13] . Clearlyinoculated wounds present a much more complex situation for biochemical analysis thaninoculated intact hypocotyls .

It is concluded that differences in rates of glyceollin I accumulation, and presumablyof other isomers, are related to the development of higher rates of biosynthesis in incom-patible than in compatible interactions and not to differences in metabolism . This isbased on the demonstration that (a) rates of [14C] -incorporation into glyceollin I inincompatible interactions either equalled (wounded hypocotyls) or exceeded(unwounded hypocotyls) those in compatible interactions, (b) in incompatible interac-tions specific radioactivity of accumulated glyceollin I was low due to relatively highercontributions from unlabelled internal pools, (c) PAL activity was stimulated to amuch higher level in incompatible than in compatible interactions, consistent with high


403metabolic flow through the biosynthetic pathway, (d) rapid metabolism of glyceollin Ioccurred in both interactions (and in uninoculated wounds) and rates were related to theaccumulation of glyceollin I and not directly governed by the type of interaction .Accumulation must result from differences between rates of biosynthesis and metabolismand possibly from sequestration of glyceollin I in the more extensive dead tissue ofincompatible interactions . Assuming that the products of glyceollin metabolism are notphytotoxic, it is probably essential that the plant maintains a balance between bio-synthesis and metabolism of glyceollin to avoid harmful accumulation. Glyceollin Ishould be regarded as an intermediate and not an end product of a secondary metabolicpathway .

Since biosynthesis is stimulated in both wounds and infected tissues it may be aresponse to cell damage . Major stimulation presumably would require persistentdamage that occurs in infected tissues to varying degrees but not in wounds . Evidencethat continued stimulation is essential for phytoalexin accumulation in cell-suspensioncultures was provided by Dixon ei al. [9] . A requirement for continued stimulationagainst high background rates of glyceollin metabolism may explain earlier observationsof the spread of a compatible race from restricted lesions with high glyceollin levels thathad been induced by unfavourable temperatures [31 ] or co-inoculation with an incom-patible race of P. megasperma Esp . glycinea [28] .

We are grateful to Dr R . I . Buzzell for supplying soybean seeds and to Dr A . N . Starrattfor advice on analytical procedures . M.K.B. was the recipient of a CommonwealthScholarship for post graduate studies .

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4 . BIDLINGMEYER, B . A ., STEVEN, A . C . & THOMAS, L . T . (1984) . Rapid analysis of amino acids using pre-column derivatization . Journal of Chromatography 336, 93-104 .

BONHOFF, A., LOYAL, R ., EBEL, J . & GRISEBACH, H. (1986) . Race : cultivar-specific induction ofenzymesrelated to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f . sp .glycinea . Archives of Biochemistry and Biophysics 246, 149-154 .

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induction ofrishitin-metabolizing activity in potato tuber tissue disks by wounding and identification of

rishitin metabolites . Phytopathology 68, 720-725 .15 . KEEN, N . 'l' ., ZAKI, A. 1 . & Sims, J . J . (1972) . Biosynthesis of hydroxyphaseollin and related iso(lavanoids

in disease-resistant soybean hypocotyls . Phytochemistry 11, 1031-1039 .16 . KIMPEL, J . A . & KOSUGE, 1' . (1985) . Metabolic regulation during glyceollin biosynthesis in green soybean

hypocotyls. Plant Physiology 77, 1-7 .17 . LAMB, C . J ., MERRITT, T . K . & BU Tr, V. S . (1979) . Synthesis and removal of phenylalanine ammonia-

lyase activity in illuminated discs of potato tuber parenchyme . Biochemica et Biophysica Acta 582,196-212 .

18. LAZAROVITS, G ., STÖSSEL, P. & WARD, E. W . B . (1981) . Age related changes in specificity and glyceollinproduction in the hypocotyl reaction of soybeans to Phytophthora megasperma var . sojae . Phytopathology 71,94-97 .

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20. MOESTA, P. & GRISEBACH, H. (1981) . Investigation of the mechanism of phytoalexin accumulation insoybean induced by glucan or mercuric chloride . Archives of Biochemistry and Biophysics 211, 39-43 .

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27 . STOESSL, A ., ROBINSON, J . R ., RocK, G . L . & WARD, E. W . B . (1976) . Metabolism of capsidiol by sweetpepper tissue : some possible implications for phytoalexin studies . Phytopathology 67, 64-66 .

28. WARD, E. W . B . (1983) . Effects of mixed or consecutive inoculations on the interaction of soybeans withraces of Phytophthora megasperma f . sp . glycinea . Physiological Plant Pathology 23, 281-294 .

29. WARD, E . W . B . & BARRIE, S. D . (1982) . Evidence that 13-hydroxycapsidiol is not an intermediate incapsidiol degradation in peppers . Phytopathology 72, 466-468 .

30. WARD, E . W . B. & BuzzELL, R . I . (1983) . Influence of light, temperature and wounding on the expressionof soybean genes for resistance to Phytophthora megasperma f. sp . glycinea . Physiological Plant Pathology 23,401--409 .

31 . WARD, E. W . B . & LAZAROVITS, G . (1982) . 'Temperature-induced changes in specificity in the interactionof soybeans with Phytophthora megasperma f . sp . glycinea . Phytopathology 72, 826-830 .

32. WARD, E. W. B ., LAZAROVITS, G ., UNWIN, C. H . & BUZZELL, R. 1 . (1979) . Hypocotyl reactions andglyceollin in soybeans inoculated with zoospores of Phytophthora megasperma var . sojae . Phytopathology 69,951-955 .

33. WARD, E. W. B ., STOESSL, A . & STOTHERS, J . B . (1977) . Metabolism of sesquiterpenoid phytoalexinscapsidiol and rishitin to their 13-hydroxy derivatives by plant cells . Phytochemistry 16, 2024-2025 .

34. WARD, E . W .B ., STÖSSEL, P. & LAZAROVITS, G . (1981) . Similarities between age-related and race-specificresistance of soybean hypocotyls to Phytophthora megasperma var . sojae . Phytopathology 71, 504-508 .


40535 . YOSHIKAWA, M., YAMAUCHI, K . & MASAGO, H. (1978) . Glyceollin : its rôle in restricting fungal growth in

resistant soybean hypocotyls infected with Phytophthora megasperma var. sojae. Physiological Plant Pathology12,73 --82 .

36 . YOSHIKAWA, M ., YAMAUCHI, K . & MASAGO, H . (1979) . Biosynthesis and biodegradation ofglyceollin bysoybean hypocotyls infected with Phylophthora megasperma var . sojae . Physiological Plant Pathology 14,157-169 .

37 . ZÄHRINGER, U ., L' BEL, J . & GRISEBACH, H. (1978) . Induction of phytoalexin synthesis in soybean . Elicitor-induced increase in enzyme activities of flavonoid biosynthesis and incorporation of mevalonate intoglyceollin . Archives of Biochemistry and Biophysics 188,450-455 .

biosynthesis and metabolism of glyceollin i in soybean hypocotyls following wounding or inoculation...

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