Physiological Plant Pathology (1975) 7, 99- 111

Malate dehydrogenase in the Fusarial wilt disease of peas

M. N. REDDY~ and M. A. STAHMANN

Department of Biochemistry, College of Agricultural and L$e Sciences, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

(Acceptedfor publication March 1975)

During development of wilt disease in peas caused by Fu.sarium oxysporum f. sp. pisi race 1, the specific activity of malate dehydrogenase (MDH) of stem tissue, at 12 to 14 days after inocula- tion, was increased 280% above that of noninfected plants. This increased specific activity in infected plants was accompanied by an appearance of one, new electrophoretically fast moving isozyme which could not be found in the noninfected plants or in the fungus. The new isozyme in infected plants was a cytoplasmic protein and contained about 21% of the total activity. Wilt development in plants was accompanied by a decrease in mitochondrial malate dehydrogenase and an increase in soluble malate dehydrogenase. Partially purified malate dehydrogenase from plant and fungal tissue had an optimum pH at 7.5 for the reduction of oxaloacetate, and for the oxidation of malate the optimum pH was at 9. The binding affinity of the enzyme was increased upon infection; the Michaelis constants for oxaloacetate and NADH of the enzyme from infected plants was about half that of noninfected plants. The MDH had a maximum velocity for the oxaloacetate at 12 and 50 P*M from noninfected and infected plants, respectively; above these concentrations, oxaloacetate became inhibitory. On heating crude extracts at 55 “C, all fungal MDH was destroyed within 5 min, that from noninfected tissue within 22 min, while 40 min were required for the total loss of activity from infected tissue. Heating acrylamide gels after electrophoresis at 50 “C for 180 min inactivated ail the isozymes from noninfected plants and fungus, whereas heating up to 320 min did not destroy all the isozymes from the infected plants. Aspartate and glutamate increased the activity of the MDH from plants and fungus. Citrate, isocitrate, succinate, a-ketoglutarate, fumarate, maleate and asparagine caused a partial inhibition of the enzyme from noninfected and infected plants; the same compounds except for succinate and asparagine also showed an inhibitory effect on the fungal enzyme. The alteration of physical and chemical properties of malate dehydrogenase following infection and the possible role of this altered enzyme in pathogenesis are discussed.

INTRODUCTION

In animal and plant cells, NAD-dependent malate dehydrogenase (MDH) (EC 1.1.1.37) has been found in two forms, soluble-malate dehydrogenase (s-MDH) and mitochondrial-malate dehydrogenase (m-MDH) [4, 5, 1.2, 30, 32, 381. Recently, other forms of MDH were observed associated with chloroplast fractions of Opuntiu [16] and microbody fractions of spinach [19]. These two forms of MDH differ in several physical, immunological and catalytic properties [5, II, 12, IS, 30, 381. Since these molecular forms are compartmentalized in the cell, they are assumed to have different biological properties, thereby enabling the organism to carry out different functions in different sites [4, II, 19, 31, 321, including an involvement of s-MDH in autotrophic CO, fixation [4, 31, 321.

t Present address: Department of Biochemistry, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, U.S.A.

7

100 M. N. Reddy and M. A. Stahmann

The influence of diseases on multiple molecular forms of enzymes in plants has gained much attention in recent years [24, 26, 27, 371. Among several enzymes, isozyme patterns of MDH were found to be altered both qualitatively and quanti- tatively in several host-parasite combinations [O, 9, 17, 21, 28, 29, 331. In bean leaves, rust development was accompanied by the appearance of one new isozyme of MDH and the continuation of another isozyme that was otherwise lost during the development of healthy leaves [28]. This new isozyme was a soluble, cytoplasmic protein contributed by the plant and the rust disease caused the loss of a mito- chondrial isozyme in bean leaves. A new isozyme of MDH and NADP-dependent malic enzyme (EC 1.1.1.40) was detected durin g the development of bacterial leaf blight in beans [,?I]. In barley infected with powdery mildew, a new MDH isozyme was observed by Johnson et al. [8]. However, in leaf rust of wheat, only the intensity of the isozyme bands was increased but not the number of bands [9]. Using histo- chemical techniques, Viswanathan & Pelletier [33] observed a greater MDH activity around the nonnecrotic areas of the infection sites in potato leaves infected with late blight disease.

In an earlier paper [17], we reported that the vascular infection of peas with Fusarium wilt resulted in the appearance of a new, fast moving isozyme of MDH and this isozyme was not seen either from the healthy plants or in the fungal pathogen. The alteration of isozyme patterns of MDH in several host-parasite combinations prompted us to further study the behavior of this enzyme under the influence of wilt disease in peas. In the present work, we report new data showing that the changes in the physical and catalytic properties of MDH are due to the formation of a new MDH isozyme.

MATERIALS AND METHODS

Preparation of plant and fungal material

Peas, Pisum sat&m “Davis perfection”, and race I of Fusarium oxysporum f. sp. pisi were used in this study. The procedures for growing pea seedlings and the fungus, and the method. of inoculating seedlings were given earlier [17]. At 12 to 14 days after inoculation, the noninfected and infected plants were harvested by cutting the whole plants at ground level, all leaves were removed and acetone powders were prepared from the stem tissues. Acetone powders also were prepared from freshly harvested 6- to ‘i-day-old fungal mycelium. The fungus was grown in potato dextrose broth (Difco) on a shaker for 6 to 7 days at room temperature (22 to 24 “Cj. At the end of the growth period, the mycelium was collected, washed once with water and used for preparing the acetone powders. The procedure for making acetone powders was given earlier [17, 181. Acetone powders from plant and fungal tissues were stored at -20 “C in airtight containers over a desiccant, until used. All powders were used within 2 to 3 months after their preparation.

Preparation of enyme extracts

The acetone powders from noninfected and infected plants, and from the fungus were extracted with a buffer, pH 7, containing 50 mM-Bis-Tris, and O-1 O/o each of cysteine and ascorbic acid. About 50 to 100 mg acetone powder was mixed with

Malate dehydrogenase in pea wilt 101

1 ml extraction buffer maintained for 20 min at 2 to 4 “C, centrifuged at 100 000 g for 10 min and the resulting supernatants, after appropriate dilutions, were used in most of the experiments. These supernatants were termed “crude extracts”.

In some experiments, a partially purified preparation was employed. Solid ammonium sulphate was added to the crude extracts from acetone powders to a required concentration, the precipitate was dissolved in the Bis-Tris extraction buffer and dialyzed quickly against large quantities of the above buffer for 2 h with 2 changes of buffer. During the preliminary experiments it was found that the maximum activity was precipitated at an ammonium sulphate concentration range of 60 to 80% saturation from the extracts from noninfected plants, at 45 to 80% from the extracts from infected plants and at 45 to 70% from the fungal extracts. By this procedure, a two- to threefold increase in specific activity was obtained. These preparations were termed “partially purified preparations”, and were used for the determination of the Michaelis constants (Ic,), substrate inhibition studies and for the determination of the effects of organic and amino acids.

Preparation of particulate and soluble fractions

Particulate and soluble fractions were obtained by following the procedures of Axelrod [I] and Danner & Ting [4]. About 10 to 20 g of freshly cut stem tissues, from noninfected and infected plants at different days after inoculation, were homogenized at 2 to 4 “C for 2 min, with a Virtis homogenizer at high speed, in 20 to 40 ml of 50 mM-phosphate buffer, pH 7.1, containing 0.4 M-sucrose, and 0.1 y. each of cysteine and ascorbic acid. The homogenate was filtered through two layers of cheesecloth and centrifuged for 5 min at 500 g to remove cellular debris. The supernatant was further centrifuged at 10 000 g for 15 min to obtain a supernatant (soluble) and pellet (particulate) fraction. The particulate fraction was washed two times with fresh sucrose-phosphate buffer, the resulting supernatant was discarded, and finally the pellet was resuspended in 2 ml of fresh 50 mMa-phosphate buffer, pH 7.1, containing O*02°h Triton X- 100 and O-1 y. each of cysteine and ascorbic acid, and kept in ice for 30 min. Buffer with 0.02% Triton X-100 and without sucrose was used to facilitate solubilizing of the pellet fraction. After solubilization, the particulate fraction was centrifuged for 10 min at 100 000 g, the supernatant of this fraction was termed “solubilized particulate fraction”, and was used for assaying the MDH activity. For determining the isozyme patterns on acrylamide gels, the “soluble” and “solubilized particulate fractions” were mixed with sufficient sucrose to attain 20% final concentration of sucrose, and about 150 to 200 ~1 of these samples containing about 600 to 800 pg of protein were used.

Protein determination

The protein in all the samples was precipitated with 10% trichloroacetic acid (TCA), washed once with fresh 10% TCA, dissolved in 3% NaOH and determined by the microbiuret method [Z] using bovine serum albumin as a standard. Precipitation with TCA was used to avoid interference from sucrose and phenolic compounds.

Electrophoresis

Polyacrylamide disk gel electrophoresis was carried out as described by Davis [S] with modifications that were published earlier [17]. A sample of 100 to 150 yl

102 M. N. Reddy and M. A. Stahmann

containing about 600 pg protein was used per gel. Crude extracts, and soluble and solubilized particulate fractions from noninfected and infected plants and from the fungus were used for electrophoresis. The isozyme patterns of MDH were visualized by staining the gels as described by Honold et al. [IO]. The stained gels were trans- ferred to 7% acetic acid, scanned with a Joyce chromoscan and the relative peak areas were obtained as integral values. The isozyme patterns were recorded by photographing the gels on Kodak Plus-X panchromatic film. Isozymes in each gel were numbered with the isozyme having the greatest mobility towards the anode being numbered one [34]. The plant and fungal enzymes were separately numbered.

Enzyme assays

The enzyme preparations from noninfected and infected plants and from the fungus were assayed spectrophotometrically, with a Zei.ss spectrophotometer equipped with an automatic recorder and sample change, at 340 nm essentially as described by Danner & Ting [4]. For the forward reaction (oxaloacetate + NADH-tL-malate + NAD), the standard reaction mixture contained O-2 ml of 6 mmoxaloacetate (neutralized with KOH), O-3 ml of 1.2 ma-NADH, 2.4 ml of 100 mM-Bis-Tris buffer (pH 7.5) containing 0.1 o/0 each of cysteine and ascorbic acid, and 0.1 ml appro- priately diluted enzyme preparations. In the preliminary experiments, pH 7.5 was found to be optimum for the forward reaction and this pH was used in all the experi- ments, unless otherwise specified. Suitable precautions as described by Weimberg [35, 361 were taken for the preparation and u.sage of oxaloaceta.te and NADH solutions. The reaction was started by the addition of enzyme and the rate of oxidation of NADH was followed. The enzyme was appropriately diluted with the extraction buffer to get an initial straight line for at least 1 to 2 min. The rate of reaction was linear with respect to the enzyme concentration up to a. A/& &min = O-025. Initial reaction rates were used for calculating the enzyme activities. The activity was expressed in units and one unit was equivalent to a change of 0.01 A/min under the above conditions and the specific activity was expressed as uni.ts per mg protein.

In a few experiments, the enzyme was assayed in the reverse direction also (L-malate + NAD+oxaloacetate + NADH) where the reaction mixture contained 0.2 ml of 1 M-L-malic acid (neutralized with KOH); 0.2 ml of 50 mM-NAD, 1 to 2 ml of appropriately diluted enzyme and 100 mM-Tris-HCl buffer (pH 9), to make a final volume of 3 ml. In the preliminary experiments, pH 9 was found to be optimum for the oxidation of L-malate. The activity was calculated in units and each unit was equivalent to a change of 0.01 A/ min, and the specific activity was expressed as the units per mg protein.

Ojtimum pH

The effect of pH was examined for both oxidation of L-malate as well as for reduction of oxaloacetate. The buffers used were: 100 mm-Bis-Tris buffer, for pH 6.6 to 7.4, 100 mM-Tris-HCl buffer for pH 7.5 to 9.2 and 100 mM-glycine-NaOH buffer for pH beyond 9.2.

Malate dehydrogenase in pea wilt

E$ect of substrate concentration

103

The rate of enzymic reaction as a function of oxaloacetate concentration was examined in the forward reaction by using varying amounts (0 to 240 pmol) of oxaloacetate in the reaction mixture.

Michaelis constants

The Michaelis constants (X,) were determined using partially purified enzyme preparations. The X, values for oxaloacetate and NADH were determined at pH 7.5 and for L-malate and NAD, pH 9 was used. These constants were obtained from extrapolation of double reciprocal plots of Lineweaver & Burk [13] using initial rates versus substrate concentrations over a range which did not show apparent inhibition.

Thermal stabilip

The partially purified preparation of MDH from plants and fungus were used for the quantitative determinations and crude extracts were used for the acrylamide gel electrophoresis. The suitably diluted enzyme preparations having the same activity were heated at 45 or 55 “C+O*l in a constant temperature water bath. Samples were withdrawn at specified time intervals, cooled immediately in an ice bath, centrifuged for 10 min at 100 000 g to remove the precipitate and the supernatant was assayed.

For determining the heat stability of isozymes, about 150 to 200 ~1 of crude extracts containing 600 pg protein were used for electrophoresis on acrylamide gels. The gels, after electrophoresis, were transferred to test tubes containing 200 mM- Tris-HCl buffer, pH 7.5, and kept in a water bath at 50 “C. At appropriate time intervals each gel was taken out from the hot water bath, cooled immediately in ice cold buffer and stained for MDH isozymes.

Efect of dzyerent organic and amino acidr

The effect of various organic and amino acids on the activity of MDH was tested by using partially purified plant and fungal preparations. All the compounds were adjusted to pH 7.5 with KOH or HCl and used at a final concentration of 10 mM for the forward reaction (OAA-tL-malate). A control consisted of water alone and the results were expressed as percentage of the control.

RESULTS

In crude extracts from acetone powders of stem tissue, 12 to 14 days after inoculation, the specific activity of malate dehydrogenase (MDH) was increased 280% above that in the extracts from infected plants (Table 1). When the crude extracts were used for electrophoresis on polyacrylamide gels, a single broad isozyme was seen in the gels containing extracts from noninfected plants, four (or five) isozymes in the extracts from infected plants, and two in extracts from the fungal tissue (Plate 1). On the basis of relative mobility and intensity of MDH isozymes from the host and from the pathogen, the bulk of the enzyme activity in the extracts from infected plants appears to be from the host rather than from the pathogen. In gels containing

104 M. N. Reddy and M. A. Stahmann

TABLE 1

Malate dehydrogenase activity from noninfected and infected pea plan&

Sample Specific

activity5 Increase in infected over noninfected (%)

Noninfected 6.2 -

Infected 17.4 280

a Specific activity (AAatO nm 0.01 /min/mg protein) in crude extracts from acetone powders of stem tissue at 12 to 14 days after inoculation. The enzyme was assayed for the reduction of oxaloacetate essentially as described by Danner 6; Ting [4].

the extracts from the infected plants, even though the same protein concentra- tion was used, a new isozyme with a faster electrophoretic mobility was present, in addition to the correspondin g isozymes of noninfected plants and fungus. This new isozyme was not obtained when a mixture of extracts from noninfected plants and fungal tissues were used for electrophoresis. The relative intensities of the isozymes in the gel from extracts of infected plants: as revealed by the Chromoscan, indicated that about 21% of the total activity was contributed by this new isozyme (Table 2).

In a time course study, it was found that about two times more MDH activity was present in the soluble fractions of the noninfected plants than in the particulate fractions (Fig. 1). Further, these total activitie s di.d not alter appreciably over a 20-day period. However, in the infected plants, the total activities associated with the particulate and soluble fractions varied to a large extent following inoculation. The enzyme activity associated with the particulate fraction increased up to 9 days, but

Days after inoculation

FIG. 1. Distribution of malate dehydrogenase in soluble (cytoplasmic) and particulate (mitochondrial) fractions of noninfected and infected pea plants at different days abler inocula- tion.

Malate dehydrogenase in pea wilt 105

after 9 days the activity in the particulate fractions decreased rapidly. During this period, enzyme activity in soluble fractions increased rapidly up to 9 to 12 days after inoculation and maintained, thereafter, at about the same level with a slight decrease.

TABLE 2

Relative intensities of isozymes of malate dehydrogenase in crude extracts from infected pea plants

ISXyme Relative number intensity’” O/o of total

1 62 21.4 2 144 49.6 3 51 17.6 4 33 11.4

a The relative intensity of each isozyme was expressed as integral values obtained in densitometer tracings of electropherograms. The isozymes in the gel (cf. gel no. 2 in Plate 1) were numbered with the one having the greatest mobility toward the anode being numbered one [34]. Crude extracts ( 100 to 150 pl containing 600 yg protein) from acetone powders of pea stem tissue, obtained at 12 to 14 days after inoculation, were used for the acrylamide disk gel electrophoresis. The gels were nm as per Davis [6] with few modifications [17] and stained for the MDH as per Honold et al. [IO]

Only one isozyme with the same mobility as the band from noninfected plants was obtained from the particulate fractions (gel 6, Plate 2), whereas three or four isozymes were seen with soluble fractions (gel 5, Plate 2). The new isozyme was detected only in the soluble fractions but not in the particulate fractions. Further, all the fungal isozymes also seemed to be present in the soluble fractions.

The effect of varying the pH on MDH activity was examined for both the oxidation of L-malate and for the reduction of oxaloacetate. The optimum pH, for both plant and fungal enzymes, was at 7.5 for the reduction of oxaloacetate, and 9 for the oxidation of L-malate (Fig. 2).

l ‘

0 t 111111111 6.8 7.2 7.6 P 8.4 8.4 8.8 9.2 9.6 IO IO.4

rH FIG. 2. Effect of pH on malate dehydrogenase activity in the extracts from noninfected and

infected pea plants and culture grown mycelium of F. oxysporum f. sp. pisi race 1. (- - - -) non- infected; (. * . . .) infected; (-) fungus.

106 M. N. Reddy and M. A. Stahmann

For the reduction of oxaloacetate, the rate of enzyme reaction was examined as a function of oxaloacetate concentration. The general shapes of the rate versus oxaloacetate concentration curves at pH 7.5 were different for the enzymes from non- infected and infected plants (Fig. 3). The enzyme has a maximum velocity at an oxaloacetate concentration of 12 pM from infected plants. Higher levels of oxalo- acetate were inhibitory for the enzyme from both noninfected and infected plants where 50:/o of the activity was inhibited at or a.bove 125 pM concentration.

TABLE 3

Apparent Michaelis constants (17,) of malate dehydrogenasefor oxaloacetat.e9 L-malate and 3Jz4D in the partially puriJied preparations from noninfected and iqfectcd pea plants and from cuitwe growz

mycelium of F. oxysporum f. sj. pisi me 1

Michaelis constants (M)a o/O Drcrease in

Pea plants infected over F. oxysporum Substrate Noninfected Infected noninfected f. sp. p;si race 1

Oxaloaceta.te 3.0 x IO-5 1.5 x 10-j 50 1.9 x 10-s NADH 3.1 x IO-4 1.4x 10-t 55 1.0 x 10-4 L-Malate 1.8 x 1O-2 1.0 x 10-Z 44 3.9 x 10-Z NAD 3.3 x 10-4 3.0 x 10-4 9 2.7 x iO-”

n The Michaelis constants (K,,) were obtained by extrapolation of double reciprocal plots of Lineweaver & Burk [13] using initial rate versus substrate concentrations.

I I Oo 50

I I I I I 150 250 350

Oxaloaceiate conc.(pmol)

FIG. 3. Effect of oxaloacetate concentration on ma!a.te dehydrogenase activity in partially purified extracts from noninfected and infected pea plants as measured by the rate of NADH oxidation.

PI.A.L.E I. Isozymc patterns of malatc dehydrogrnasr in the extracts from : 1. noninfvctcd; 2. infected pea plants; 3. culture grown mycelium ofF. oxy.c~orum f. sp. pi& race 1. Crude extracts from acetone powders of pea stem tissue. obtained at 12 to 14 days after inoculation, were used for the acrylamide gel electrophoresis. Note the presence of one dark band in the extracts from noninfected plants, four (two dark and two light) or five (two dark and three light) bands in the extracts from infected plants, and two bands in the extracts from fungus. The band with the highest mobility was present only in the oztracts from infected plants.

(.facing jxqe 1061

PLATE .ticulatt 3. inocl 6).

Is0 mitol lion

mic) j 12 d

.iort 1

Malate dehydrogenase in pea wilt 107

The Michaelis constants (K,) observed for oxaloacetate, NADH L-malate and NAD are given in Table 3. These constants for all four substrates of the MDH from infected plants were lower than that from extracts of noninfected plants. Following infection, the K, values for oxaloacetate and NADH were reduced 50 and 550/, respectively, whereas for L-malate and NAD, the constants were 44 and 9%, respectively, lower.

The thermal stability of MDH varied depending upon the source of the enzyme (Fig. 4). The enzyme from the fungus was more susceptible to heat than the enzyme

Time (min)

FIG. 4. Thermal inactivation of malate dehydrogenase at 45 and 55 “C in extracts from non- infected and infected pea plants and culture grown mycelium of F. oxysgorum f. sp. pisi race 1. Partially purified plant and fungal extracts adjusted to contain the same quantity of protein were heated for the specified times, cooled immediately in an ice bath, centrifuged for 10 min at 100 000 g and the supernatants assayed for the MDH activity. (- - - -) Noninfected; (. . . . .) infected; (-) fungus.

from plants. With the fungal enzyme 50% of the activity was lost in 4 min at 45 “C and in about 1 min at 55 “C. The MDH from infected plants was more stable than from noninfected plants. Fifty per cent of the MDH activity was inactivated at 45 “C in 40 min from the noninfected plants and in 100 min from infected plants; whereas at 55 “C, it took only 3 and 4 min, from noninfected and infected plants, respectively. Further, at 55 “C, total loss in activity of MDH occurred within 5, 22 and 40 min in the extracts from fungus, noninfected and infected plants, respectively.

When the gels after electrophoresis and before staining were heated at 50 “C, the thermal stability of the isozyme bands of MDH were different depending upon the source of the enzyme (Fig. 5). In the gels containing the MDH from noninfected plants and fungus, all the isozymes were inactivated in 180 min. Among the isozymes from fungus, the faster moving two isozymes were inactivated faster within 15 min than the slower moving isozyme. In contrast to the MDH from fungus and non- infected plants, the enzyme from infected plants was comparatively stable to heat; some activity was seen in the gel even after 320 min of heating.

108 M. N. Ready and M. A. Stahmann

The effects of various organic and amino acids on MDH varied depending upon the source of the enzyme (Table 4). Citrate and isocitrate were less inhibitory to the MDH from the fungus than from the plants. Succinate activated the MDH from fungus whereas it inhibited the plant enzyme. Similarly, aspartate and glutamate had a greater activation on the fungal enzyme than on the MDH from plants.

5 i

5

4 3 2 1

1 1

(bl

FIG. 5. Thermal inactivation of isozymes of maiate dehydrogenase ( MDH)

q

i

nt he extracts from noninfected (a), and infected (b) pea plants, and culture grown my&urn of F. ox~~s~‘~wum f. sp. p& race 1 (c). About 150 to 200 ~1 of crude extracts from acetone powders containing 600 pg protein were placed on each acrylamide gel and the gels after electrophoresis were heated in 200 mwTris-HCl buffer, pH 7.5, at 50 “C for: 0 (1): 15 (2), 30 (3), 60 (4)$ 180 (5) and 320 (6) min. After the specified time, the gels were cooled immediately and stained for MDH isozymes. The bands in the gels containing extracts from infected plants stained darker, even though the extracts from noninfected and infected plants contained the same amount of protein.

TABLE 4

Effect of various organic and amino acids on the activity qf malate dehydrogenase from noninfected and infected pea plants andfrom F. oxysporum~f. rp. pisi race 1

O/ Increase (t-j or decrease (-) over controlb Pea plants

F. oxysporum Infected Compound” f. sp. @isi race 1 Noninfected Infected heated”

Citric acid -47 -81 -61 -80 Isocitric acid -48 -80 -77 -76 a-Ketoglutaric acid -70 -86 -77 -69 Fumaric acid -32 -57 -52 -60 Succinic acid +G -40 -39 -59 Maleic acid -27 --Fl - 56 -64 Aspartic acid +78 4” 12 +28 +46 Glutamic acid $83 +8 +28 +25 Asparagine 0 -t18 -8 0

cI All compounds were adjusted to pH 7.5 and were added at i0 rnM final concentration to the reaction mixtures and the forward reaction (oxaioacetate+r*-maiate) was measured at pH 7.5.

b Control consisted of reaction mixture without the added compounds. c The partially purified MDH from the infected plants was heated at 45 “C for 10 min,

cooled immediately in an ice bath, centrifuged at 100 000 gfor 10 min and the supernatant was assayed for the MDH activity. This procedure was followed to reduce the presence of MDH from fungus, and by this treatment nearly 907/o of the plant enzyme were inactivated.

of the fungal enzyme and 2056

Malate dehydrogenase in pea wilt 109

MDH from infected plants behaved as a mixture of plant and fungal enzymes. To avoid the influence of fungal enzyme, the extracts from infected plants were heated for 10 min at 45 “C, cooled immediately, centrifuged and used. By this treat- ment, nearly 90% of the fungal enzyme was inactivated. When this “heated enzyme” from the infected plants was compared to the enzyme from noninfected plants, some differences in the effects of these inhibitory compounds were observed (last column, Table 4). Succinate inhibited 40 and 60% of MDH activity from noninfected and infected plants, respectively. Similarly, or-ketoglutarate inhibited 77 and 69% of MDH from noninfected and infected plants, respectively. However, in the presence of aspartate, MDH activity was increased 46% for the enzyme from infected plants whereas only 13% activation was obtained for the enzyme from noninfected plants.

DISCUSSION

The evidence presented in this report indicates that during development of Fusarial wilt disease in peas, the specific activity of malate dehydrogenase was increased 280% compared to the activity in the noninfected plants. An increase or decrease of the specific activity following infection does not indicate whether the change in enzyme activity was due to an alteration in the activity of the existing proteins or the appearance or disappearance of electrophoretically different proteins. Further, increased activity could be due to the enzyme from the pathogen. However, in the present work, the increased specific activity appeared to be from the host, since the increased activity was followed by the appearance of a new isozyme band in the host. This isozyme was considered a new one since the corresponding band having the same electrophoretic mobility was not present in the fungus. Increased specific activity and the presence of new isozymes or an increase in intensity of the existing isozymes of MDH following infection were observed in several other host-parasite combinations [S, 9, 17, 20, 21, 28, 291.

The increased activity in the initial stages corresponded to the increased activity associated with both the soluble and particulate fractions. However, in advanced stages of infection, mitochondrial MDH decreased rapidly. Decreased activity in mitochondria was probably due to the destruction of mitochondria during disease development. Destruction of mitochondria and loss of a mitochondrial MDH isozyme also were observed by Staples & Stahmann [28] during rust development in beans.

The presence of a new isozyme in the infected plants indicate that the proteins with MDH activity are more heterogeneous during disease development than in noninfected plants. Increased heterogeneity of the enzyme from the infected plants was further indicated by its greater heat stability, less susceptibility to the increased oxaloacetate concentrations and differential effects of natural substrates. Similarly, during development of rust diseases in flax and wheat, Shaw and his associates observed an increased activity of ribonuclease and substantial changes in properties of this enzyme, such as substrate specificity, thermal stability and sensitivity toward inhibitors [3, 231. They attributed these changes in kinetic and catalytic properties to the formation of new RNase molecules.

A difference of MDH from infected plants compared to the noninfected plants was indicated by their Michaelis constants. For the oxidation of malate and reduction

110 M. N. Reddy and M. A Stahmann

of oxaloacetate, the & values of the enzyme from infected plants were two to three times lower. The decrease in Michaelis constants means an increase in binding to the substrate. Such increases in binding affinity after infection may be one of the causes for the increased accumulation of metabolites (“metabolic sink:‘) in or around infection sites. In plants, accumulation of metabolites at infection sites has been shown to be an active process [25] and it probably aids in meeting the metabolic requirements for the growth of the pathogen. The pathogen may conceivably induce a change in the binding affinity of the enzyme for the substrate by an alteration of the tertiary or quarternary structure of the enzyme as suggested by MacDonald & Strobe1 [I41 with adenosine diphosphate-glucose pyrophosphorylase in wheat lea.ves infected with Puccinia striiformis. Induced synthesis of multiple forms and changes in the catalytic properties of enzymes are some of the important steps in regulation of host metabolism by the pathogen, which favor its development.

It is interesting to note that the differences in X,X values are greater for oxalo- acetate and NADH than for malate and NAD. This indicates that in infected plants, the affinity of the oxaloacetate and NADH for MDH are increased more than for the malate and NAD. Kaplan [II] postulated a shuttle system where mitochondrial- MDH is oriented towards oxidation of malate in mitochondria, whereas soluble- MDH is oriented towards the reduction of oxaloacetate in cytoplasm. On the basis of this hypothesis and on the basis of the present evidence that activity of soluble- MDH increases and a new isozyme is present in the soluble fraction, the pathogen has exerted some control of the conversion of oxaloacetate to malate. This probably results in the acceleration of the conversion from oxaloacetate to malate rather than malate to oxaloacetate, resulting in the accumulation of malate.

This work was supported by the College of Agricultural and Life Sciences: University of Wisconsin; and by grants from the National Institute of Allergy and Infectious Diseases (Al 101) of the National Institutes of Health, and from the Herman Frasch Foundation.

The authors wish to thank Drs E. L. Gritton and D. F. Hagedorn for supplying pea seeds and fungal cultures.

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bean leaves infected by Southern bean mosaic virus. Phytopathology 56, 669-677. 8. JOHNSON, L. B., BRANNAMAN, B. L. & ZSCHEILE, F. P., JR (1966). Protein and enzyme changes in

barley leaves infected with Erysiphe graminis f. sp. hordei. Phytopathoiop 56, 1405-1410. 9. JOHNSON, L. B., BRANNAMAN: B. L. & ZSCHEILE, F. P., JR (1968). Protein and enzyme changes in

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Malate dehyrogenase in pea wilt 111

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