Enzyme induction in soybean infected by Phytophthora megasperma f.sp. glycinea

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 217, No. 1, August, pp. 65-71, 1982

Enzyme Induction in Soybean Infected by Phytophthora f .sp. glycinea’ HELGA Bi)RNER

AND

megasperma

HANS GRISEBACH2

Lehrstuhl fiir Bioch.emie der Panzen, Institut fiir Biobgie II der Universitiit Sctinzlestr. 1, O-7800 F&burg iBr., Germany Received December

15, 1981, and in revised form March

F&burg,

15, 1982

Soybean seedlings (Glycine max, cv. Am-soy ‘71) were inoculated in the hypocotyls with mycelium from either race 1 (incompatible) or race 3 (compatible) of Phytophthora megaeperma f. sp. gl@nea, and the activities of phenylalanine ammonia-lyase (PAL), chalcone synthase, glucose-&phosphate dehydrogenase, and glutamate dehydrogenase determined at various times after inoculation. A drastic increase in activity with a subsequent decrease occurred with all enzymes, whereas wound controls showed no or very little change in activity. A higher activity of PAL was reached in the incompatible than in the compatible interaction. Rates of PAL synthesis were determined by pulselabeling with r.,-[35S]methionine and immunoprecipitation of the enzyme. These experiments prove that increase in PAL activity is due to de nova synthesis of the enzyme.

By pulse and pulse-chase experiments with 14C02we had recently shown that in soybean seedlings inoculated with either an incompatible or a compatible race of Phytophthwa mgasperma f.sp. glycinea the level of the phytoalexin glyceollin3 is determined predominantly by its rate of synthesis (1). Hypocotyls that had only been wounded contained only very small amounts of glyceollin. In agreement with these results is the observation that large increases and subsequent decreases of the enzymes phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone synthase, which are involved in glyceollin biosynthesis, occurred in elicitor-treated soybean cotyledons (2). The enzyme dimethylallylpyro-

phosphate:trihydroxypterocarpan dimethylallyl transferase, which catalyzes a late biosynthetic step, was again present only in elicitor-treated cotyledons (3). It can therefore be assumed, in contrast to the claim by earlier investigators (4), that accumulation of soybean phytoalexins after infection is caused by enzyme induction. We have now investigated the changes in activity of a number of enzymes in soybean hypocotyls infected with compatible or incompatible races of Phytophthora megaspermu f.sp. glycinea. Furthermore we have explored the question whether the rise of PAL4 acticity is due to de novo synthesis of this enzyme. MATERIALS

‘This work was supported by Deutsche Forschungsgemeinschaft (SFB 46) and by Fonds der Chemischen Industrie. We thank Professor K. Hahlbrock for the PAL antiserum. a Author to whom all correspondence should be sent. e The term glyceollin is used in this paper for the three isomers which accumulate in infected soybean hypocotyls (1).

AND METHODS

Chemicals [2-‘%]Malonyl-CoA Buchler (Frankfurt/M.)

(59 Ci/mol) from Amersham was diluted with the unla-

* Abbreviations used: PAL, phenylalanine nia-lyase; EDTA, ethylenediaminetetraacetic SDS, sodium dodecylsulfate. 65

ammoacid;

0003-9861/82/090065-07$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

66

BtiRNER

AND GRISEBACH

beled compound to 21 Ci/mol. L-l?S]Methionine (lOO200 Ci/mmol) from Amersham Buchler was dissolved in bidistilled water containing 20 mM mercaptoethanol to an activity of 1 mCi/ml and was stored at -20°C. 4-Coumaroyl-CoA was supplied by Dr. W. Heller. The antiserum against PAL was a gift from Professor K. Hahlbrock. All chemicals were of analytical grade.

BU”ferS The following buffers were used: (A) 10 mM sodium-phosphate, pH 7.2; (B) 100 m?d potassium-phosphate, pH 8, containing 2.8 mM mercaptoethanol and 10% (by vol.) glycerol; (C) the same as B but with 14 mM mercaptoethanol; (D) 100 mM Tris-HCl, pH 7.5, containing 14 mM mercaptoethanol and 10% (by vol) glycerol; (E) 100 mM sodium borate, pH 8.8; (F) 50 mM triethanolamine, pH 7.5, containing 500 mM EDTA NasHa.

Soybean Seedlirqp Seeds of soybean (Glycine mux L. cv. Amsoy 71) were obtained from Agricultural Seed Improvement Association, Indiana 4798, Illinois. Seedlings were grown under sterile conditions in vermiculite and potting soil as described previously (5).

Fungal Cultures Phytophthora me~asper?nu f. sp. g&inea (formerly P. m.egasperma var. sojae) A.A. Hildb. races 1 and 3 were obtained from E. Ziegler, University of Aachen, and were grown for 7-8 days as described elsewhere (5).

Test of Virulence The test was carried out as described previously (1) following a procedure described by Wade and Albersheim (6).

Inoculation of Soybean Seedlings All operations were carried out in a laminar flow hood with sterile solutions. A set of 10 5-day-old seedlings was placed on a 20 X 20-cm glass plate and the roots were covered with a layer of cellucotton soaked in distilled water. A second strip of cellucotton was placed over the cotyledons to keep the seedlings in place. A slash wound, approx 1 cm long, was cut with a razor blade into each hypocotyl about 0.5 cm below the cotyledonary node and a small piece (approx 0.3 cm) of mycelium was placed into each wound. For controls two to three drops of buffer A were put into the wounds. The inoculated seedlings were incubated in the dark at 25°C and 100% humidity.

Preparation of Cell Free Extracts from Hwo@h All operations were carried out at 4°C. The inoculated portions of the hypocotyls were cut out 0.5 cm above and below the wound. For each enzyme assay 10 hypocotyl segments were frozen in a mortar with liquid nitrogen and ground to a fine powder. After addition of 2 ml buffer B, C, or D (see enzyme assays) per gram fresh weight the slurry was slowly stirred for 1 h. After centrifugation at SO,0009 for 20 min the cell-free extract was stirred with 10% (w/w) Dowex 1X2 (Cl- form equilibrated with the respective buffer) for 0.5 h. The extract was cleared by centrifugation and used for enzyme assays.

Enzyme Assays Phenylalanine ammonia-lyase. Extracts were prepared with buffer B. Enzyme activity was determined in buffer E by the absorbance change at 290 nm in presence of 20 mM L-phenylalanine (7, 8). Chalwne synthase. Extracts were prepared with buffer C. Assay method 3 according to Schrbder et al. (9) was used. The incubation time was 15 min.

Glucose-b-phosphate dehydrogenase (EC 1.1.1.49). Extracts were prepared with buffer C or D. Enzyme activity was determined in buffer F by the absorbance change at 365 nm (10). The test volume was reduced to 0.75 ml. Glutamate dehydrogenme (EC l..&l.$). Extracts were prepared with buffer C. A published procedure was used for measurement of enzyme activity (11).

Protein Determination Protein was determined by a modified Lowry procedure in presence of 0.5% sodium dodecyl sulfate with serum albumin as standard (12).

Labeling of PAL with [35SJWethionine Three seedlings were used for one determination. After inoculation of soybean hypocotyls with races 1 or 3 of P. nzegasperma for various lengths of time, the cellucotton over the cotyledons was removed. L[%S]Methionine (2 j&i) in a volume of 2 ~1 water was injected into each hypocotyl about 2 mm below the wound. After 1 h incubation e-cm-long hypocotyl seg? ments were cut out and frozen in liquid nitrogen. The cell-free extract was prepared with buffer E containing 2.8 mM mercaptoethanol as described above.

Immunoprecipitatic and Determination of Radioactivity in PAL Subunit To a 400-~1 extract from three hypocotyl segments 25 ~1 of PAL-antiserum was added. In a duplicate experiment 30 pl antiserum was used. The incubation

ENZYME

INDUCTION

IN Phytophthora-INFECTED

was kept at room temperature for 30 min and for another 12 h in the refrigerator. For separation of the immunoprecipitate 400 pl of sucrose buffer (1 M sucrose, 50 mM Tris-acetate, pH 7.7,30 mM NaCl, 2% Triton X-100.4 PM methionine) was added. After centrifugation for 20 min at 10,OOOgthe supernatant was removed with a pipet. The pellet was resuspended in 500 ~1 of the Tris-acetate buffer (see above) without sucrose. After centrifugation the washing procedure was repeated. The pellet was then treated for 5 min at 95°C with 20 pl 125 mM Tris-HCl (pH 6.8) containing 4.2% SDS, 20% glycerol, 1.4 mM mercaptoethanol, and 0.001% bromophenol blue. This solution was analyzed by slab gel electrophoresis in presence of dodecyl sulfate with a discontinuous gel system (14). Radioactive zones were detected by a fluorographic method (15). For determination of radioactivity a 2 X 4-mm gel piece of the region which corresponded in the fluorogram to the PAL-subunit (M, 83,000) was cut out and treated with 400 ~130% H202 for 2 h at 50°C in a scintillation vial. After another 1 h at room temperature 4 ml of a Triton scintillation fluid (toluene with 6 g/liter 2,5-diphenyloxazole and Triton X-100 2:l (v/v)) was added. Radioactivity was counted approx 24 h after addition of Triton. For controls gel pieces from regions of the slab gel without protein bands were used.

Total Incorporation of [35SJMethionine into Extractable Proteins To 5 ~1 of crude extract were added 500 ~1 bidistilled water and 500 111of a solution containing 1 N NaOH, 0.5 M HcOc, and 1 mM methionine. After shaking the mixture for 1 min protein was precipitated with 60% trichloroacetic acid. Protein was collected on a filter (Whatman GF/CJ and rinsed with 3% trichloroacetic acid. After drying the filters were counted in the Triton scintillation fluid. RESULTS

Assay for Infectivity of Phytophthwa Races on Soybean Seedlings When we started the experiments, seeds of the soybean cultivar Harosoy 63, which had been used by us in earlier investigations (1) were not available. We therefore used the cultivar Amsoy 71, which shows the same response to races 1 and 3 of l? megasperma as Harosoy 63. No significant differences in the accumulation patterns of glyceollin were found between Harosoy 63 (1) and Amsoy 71 seedlings. In the hypocotyl inoculation assay (1, 6) over 95%

0

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5 Time

15 after

inoculation

25 (h)

FIG. 1. Changes in phenylalanine ammonia-lyase activity in soybean hypocotyls after inoculation with P. wz.egoqermo race l(0) or race 3 (A) and in wound controls (0). Duplicate assays with different enzyme concentrations were carried out. The mean values of three consecutive enzyme activities were plotted against the mean time span (three moving point calculation (13)). Maximal deviation was + 10 pkat/kg protein.

of the seedlings inoculated with the compatible race 3 toppled between 25 and 45 h after infection. In contrast, all seedlings inoculated with the incompatible race 1 remained healthy. Induction of Phenylulanine AmmoniaLyase Five-day-old whole Amsoy ‘71 seedlings were inoculated with race 1 or 3 of P. megasperma by placing a piece of mycelium into a slash wound in the hypocotyl 0.5 cm below the cotyledonary node. The mycelium used for the inoculations was 7 days old in the case of race 1 and 8 days old in the case of race 3 since the latter grows somewhat more slowly on the sitosterol medium (21). PAL activity was determined between 0 and 25 h after inoculation in a 1.5-cm-long hypocotyl section of the infection site. For controls the wound was treated with buffer. The crude extract could be stored for 8 days at -70°C without loss of enzyme activity. Figure 1 shows the changes in PAL activity with races 1 and 3. Enzyme activity increases

68

BtiRNER AND GRISEBACH

Time after inoculation

lh)

FIG. 2. Changes in chalcone eynthase activity in soybean hypocotyls after inoculation with P. megasperma race l(0) or race 3 (A) and in wound controls (0). Duplicate assays with 10 and 20 pl of crude extracts were carried out. Statistical analysis of the values was as in Fig. 1. Maximal deviation was + 0.2 Nkat/kg protein.

(fresh wt) Dowex 1. With 10 or 20 ~1 crude extract the reaction was linear for at least 30 min. The product of the reaction was identified as naringenin by thin-layer chromatography on cellulose with 15% acetic acid (R’= 0.43). The formation of the flavanone is due to the presence of chalcone isomerase (EC 5.5.1.6) in the crude extract. Besides naringenin a minor radioactive compound with Rf 0.52 was detected. Figure 2 demonstrates the changes in chalcone synthase activity after inoculation with races 1 and 3. In both cases a drastic increase in enzyme activity was observed, with maximal activity being reached about 18 h after inoculation. No significant difference in maximal enzyme activity between hypocotyls infected with the two different races was found, but there seems to be a faster decline with race 3. Chalcone synthase activity in wounded plants without infection was the same as in intact seedlings and did not change with time. Mycelium of races 1 and 3 did not contain the synthase activity. Glucose-b-Phosphate Dehgdrogenase

with an apparent lag phase of about 2.5 h. Significant differences in PAL activity between seedlings infected with race 1 and those infected with race 3 did not occur until about 14 h after inoculation. Subsequently a considerably higher PAL activity was reached in the incompatible interaction (race 1). Maximal PAL activity was, respectively, about 10 and 6 times higher with races 1 and 3 than in the wound controls. Controls showed no significant increase in enzyme activity during 21 h. PAL activity with reference to fresh weight showed the same course of enzyme activity. No PAL activity was present in extracts from mycelium of races 1 or 3 which had been prepared in the same way as the hypocotyl extracts. Chalcone Synthase Highest chalcone synthase activity was obtained when the hypocotyl segments were extracted in buffer C containing 10%

Enzyme activity was about twice as high in buffer D than in buffer C. As shown in Figs. 3A and B a rapid rise in enzyme activity after inoculation with races 1 and 3 took place. With both races maximal enzyme activity with a value ‘7 to 8 times higher than the basal level was reached at about 17 h after infection. The wound controls showed a small increase in activity with time. Mycelium of races 1 and 3 had glucose-&phosphate dehydrogenase activity of 360 pkat/kg. Glutamate Dehydrogenase Figure 4 shows the changes in enzyme activity after inoculation with races 1 and 3. After 16 h incubation a considerably higher dehydrogenase activity was reached with race 3. Wounded controls had only very low enzyme activity. Mycelium of races 1 and 3 showed a glutamate dehydrogenase activity of 245 Nkat/kg in buffer C.

ENZYME

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INDUCTION

IN Ph@pWzoru-INFECTED

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5000

4000

0. 0

L 0 5 Time after

10

15 inoculation

20

25

(h)

0

1 5

I 10

, 15

Time after inoculation

I 20

25

(h)

FIG. 3. (A) Changes in glucose-6-phosphate dehydrogenase activity in soybean hypocotyls after inoculation with P. mc~ospermo race 1 (0, 0) and in wound controls (0). Assay in buffer C (open symbols) or buffer D (solid symbols). (B) Inoculation with race 3 (A, A). In contrast to Figs. 1, 2, and 4 the symbols represent the measured values.

Incorporation of [S5SjMethionine into PAL Subunit To investigate whether the rise in PAL activity after inoculation with P. megasperma was due to de nova synthesis of the enzyme, PAL was pulse-labeled by incorporation of L-[35S]methionine at various times after inoculation. In the first experiments a solution of the labeled compound was supplied directly to the inoculation site. With this method 35S incorporation into PAL was low. A much higher incorporation was achieved when the precursor solution was injected into the hypocotyls about 2 mm below the slit wound. PAL was then isolated from the crude extract by immunoprecipitation of the enzyme with a specific antiserum. After slab gel electrophoresis the radioactive PAL subunit (M, 83,000) was detected by fluorog-

raphy (15) and its radioactivity determined by scintillation counting. The rate of PAL synthesis after inoculation with races 1 and 3 is shown in Fig. 5. In both cases a drastic increase in the rate of synthesis is observed, but the increase is significantly higher after infection with the incompatible race. After reaching a maximum at about 7-8 h after inoculation, the synthetic rate rapidly declines. In wound controls there was only low 35S incorporation and no significant change was observed up to 15 h after infection. DISCUSSION

Partridge and Keen had reported that curves of PAL activity in wounded and P. megasperma inoculated hypocotyls of Harosoy and Harosoy 63 soybeans were

70

BGRNER

Time after

inoculation

AND GRISEBACH

(h)

FIG. 4. Changes in glutamate dehydrogenase activity in soybean hypocotyls after inoculation with P. megospermu race l(0) or race 3 (A) and in wound controls (0). Statistical analysis of the values was as in Fig. 1.

similar (4). They concluded that rise in PAL activity is a simple wound response and/or a nonspecific response to the fungus. Since they found that the PAL level returned to the basal level at 30 h after inoculation, a time at which rapid gly-

Time after

inoculation

ceollin synthesis occurred, they suggested that PAL “activation” has no essential role in glyceollin biosynthesis. Our results are in contradiction to these conclusions. They prove that PAL activity is not induced by wounding and that the degree of induction of PAL by infection is higher with the incompatible race 1 than with the compatible race 3 of P. rnegaS~WWLU. The higher level of PAL reached in the incompatible reaction is in agreement with the accumulation curves of glyceollin in Harosoy 63 (1) and Amsoy ‘71 hypocotyls. The integrated curves of PAL activity and the curves for glyceollin accumulation are shown in Fig. 6. The curves for glyceollin accumulation with races 1 and 3 and those for PAL activity diverge about 12 h after infection and a correspondence between the accumulation of glyceollin and PAL activity is evident (compare earlier results in Ref. (2)). In assessing the accuracy of such a comparison, one must keep in mind that in our analysis we are integrating over a large cell population. It can be estimated that a l-cm-long hypocotyl segment contains about 5 X lo5 cells. Race-dependent differences in PAL activity and glyceollin accumulation in cells around hyphae could occur at an earlier time and would be expected to be much larger (23).

[hl Time after inoculation

FIG. 5. Changes in [%]methionine incorporation into PAL subunit after inoculation with P. meguspermo race l(0) or race 3 (A) and in wound controls (0). Incorporation which represents rate of synthesis is expressed as percentage of % incorporation into total protein. Bars represent maxima1 deviation.

(h)

FIG. 6. Integrated curves for the activity of PAL (-) taken from Fig. 1 and accumulation of glyceollin with race 1 (A) or race 3 (0). The ordinate represents percentage of maximal value. For experimental methods for accumulation see (1).

ENZYME

INDUCTION

IN Phgtophthora-INFECTED

While our results permit the conclusion that PAL exerts an important control function in glyceollin biosynthesis, the participation of other enzymes in the regulation of this pathway should be investigated. Our results further show that the rise in PAL activity is due to de nova synthesis of the enzyme. Experiments on the question whether de nova synthesis is caused by an increase in the quantity of PAL mRNA, e.g., at the transcriptional level, must await the availability of PAL cDNA which is at present being isolated in K. Hahlbrock’s laboratory. Hille et al. (22) have demonstrated that the rise in PAL and chalcone synthase activity after inoculation of soybean cell cultures with a glucan elicitor from P. megaspermu is also caused by de nova synthesis of these enzymes. The same conclusion was reached by Loschke et al. (18) with respect to the rise in PAL activity in peas inoculated with Fusarium so&i and by Hahlbrock et al. about coordinated changes in both PAL and 4-coumarate:CoA ligase activities in parsley cell cultures after treatment with the P. megasperma elicitor (19, 20). The drastic increase of chalcone synthase activity after infection demonstrates that an enzyme of the flavonoid pathway proper (16) (group II enzyme) is also induced. Irradiation of cell suspension cultures of parsley causes selective induction of the enzymes of general phenylpropanoid metabolism and of flavonoid glycoside biosynthesis (16, 17). Glutamate dehydrogenase and glucose-&phosphate dehydrogenase activities were not altered by irradiation (16). According to our results infection causes not only induction of the enzymes involved in the flavonoid pathway but also large increases in the activities of glutamate and glucose-&phosphate dehydrogenase. Further investigations must show how extensively infection changes the general metabolism of the host.

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