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l -2-Aminooxy-3-phenylpropionic acid inhibits phytoalexin accumulation in soybean with concomitant loss of resistance against Phytophthora megasperma f. sp. glycinea
Physiological
Plant Pathology
(1982)
21,65-70
L-;!-Aminooxy-3-phenylpropionic acid inhibits phytoalexin accumulation in soybean with concomitant loss of resistance against Ph ytophthora megasperma f. sp. glycinea P. MoEsT.4 and H. Dejartnumt Schcikkstr.
of B&&w&y,
(Accepted
fw publication
1, D-7800
GRISEBACH
Biologisches Inrtitut F&burg, West Germany March
II der Universit&
1982)
Application of a 1 mu solution of L-2-aminooxy-3-phenylpropionic acid (L-AOPP) to the cut ends of soybean seedlings (cultivar Harosoy 63) 4 h before infection of the hypocotyls with the incompatible race 1 of Phytophthora megasperma f. sp. g&Gea resulted in an inhibition of glyceollin accumulation of about 90%. Experiments on the dependence of the inhibition of glyceollin accumulation on concentration of L-AOPP and on the spread of infection showed a close correlation between decrease in glyceollin content and increase in infected tissue. At an L-AOPP concentration of greater than 500 ELM, the interaction between Harosoy 63 seedlings and race 1 of P. megasjetmo was converted completely from a resistant to a susceptible one. The results are strong support for the assumption that glyceollm plays an important role in the defense reaction of soybean.
INTRODUCTION The question to what extent phytoalexins contribute to specific resistance of plants is still a matter of debate [S, S]. An ideal system to test the importance of phytoalexins for resistance would be a mutant in which phytoalexin synthesis is blocked at a single step. However, at present such mutants are not available. We have therefore tried to inhibit phytoalexin synthesis specifically and to correlate the degree of inhibition with resistance. Towards this goal we used L-2-aminooxy-3-phenylpropion:ic acid [I] (L-AOPP), an inhibitor of phenylalanine ammonia-lyase, to inhibit glyceollin synthesis in soybean seedlings triggered by infection with Phytophthora megas@rma f. sp. glycinea. Here we report results which show a close correlation between inhibition by L-AOPP of glyceollin accumulation in tissue at the infection site and the size of the infected area. MATERIALS AND METHODS Chfzmicals
L-2-Aminooxy-3-phenylpropionic acid was synthesized by the method described by Bri,ggs & Morley [4]. The purity of the compound was checked by high pressure liquid chromatography (HPLC), “H-NMR, and by its optical rotation. Soybean seedlings
Seeds of soybean (Glycine max L. cv. Harosoy 63) were obtained from R. I. Buzzel, 00484059/82/04065+06
$03.00/O
@ 1982 Academic
Press Inc.
(London)
Limited
66 Agriculture Canada, Research Station, Harrow, Ontario. vermiculite and potting soil as described previously [Z].
P. Moesta and H. Grisebach Seedlings were grown in
Fungal cultures Phytophthora megasterma f. sp. glycinea A. A. Hildb. races 1 and 3 were obtained from E. Ziegler, University of Aachen, and were grown as described elsewhere [2]. Inoculation proceduresand analysis of glyceollin The roots of 5-day-old seedlings of soybean were cut off with a razor blade so that a uniform hypocotyl length of 7 cm was obtained. Each seedling was placed in a test tube with 1 ml of distilled water or a solution of L-AOPP in distilled water. The seedlings were then incubated at 25 “C and 60% humidity under illumination with 9500 lx (Osram L40 W/25 fluorescent tubes) for 4 h. Afterwards the seedlings were inoculated with race 1 or 3 of P. megaspermaf. sp. glycinea by one of the following methods. Method A [II] : a slash wound, approximately 1 cm long, was cut with a razor blade into each hypocotyl about 1 cm below the cotyledonary node and a small piece of mycelium was placed in the wound. Method B: an area of epidermis approximately 2 x3 mm was scraped off with a razor blade 1 cm below the cotyledonary node. A small piece of mycelium was placed on top of the wound. After inoculation, the seedlings were transferred to a moist chamber and kept under illumination at 25 “C and lOOo/o humidity for 24 h. For isolation of glyceollin the infected hypocotyl segment plus 5 mm of healthy tissue at each end was taken from each of 10 seedlings and the combined material was placed in a test tube with 5 ml of ethanol and heated to boiling point for 2 min. The ethanol was decanted, the extraction repeated and the tissue washed once more with 5 ml cold ethanol. The collected extracts were evaporated to dryness under reduced pressure and the residue was taken up in 2 x 1 ml of trichloromethane. The solution was then applied to a SepPak silica cartridge (Waters, Kbnigstein), and non-polar components were washed out with a total of 5 ml of CHCl,. The isoflavonoid fraction was eluted with 3 ml of CHCls-3-propanol (1 : 1, v : v). The solvent was evaporated and the residue was taken up in 400 ~1 of 2-propanol. Separation and quantification of the isoflavonoids were achieved by HPLC as described earlier [II]. The dry weight of infected tissue was determined as described by Lazarovits et al. [9] except that the infected tissue was excised from the hypocotyl prior to the extraction of the phytoalexins. RESULTS Inhibition of glyceollin accumulation by L-AOPP Three different methods for the application of the inhibitor were tested. (a) Application ofan L-AOPP solution to the wound. After inoculation of the Harosoy 63 seedlings in a slit wound of the hypocotyl (method A) or in an epidermal wound (method B) with mycelium of the incompatible race I of P. megarlerma f. sp. glycinea, the wound area was covered with strips of cellucotton that had been soaked in the inhibitor solution. In the concentration range examined (1 lt~ to 1 rnM L-AOPP) no reduction of glyceollin content was found at 24 h after inoculation. (b) Application of th-e inhibitor via the roots. When seedlings were placed with their roots in a 300 FM L-AOPP solution 24 h before inoculation, approximately 45%
Inhibition
of glyceollin
67
accumulation
inhibition of glyceollin accumulation was found. At lower inhibitor concentration no effect was observed. Since very large amounts of L-AOPP were needed with this method, higher concentrations of L-AOPP were not tested. At 300 PM L-AOPP a ret.ardation in hypocotyl elongation was observed. (c) A#j&cation of tb inhibitor through the cut base ofhyjoco@s. Removal of the root had no measurable effect on glyceollin accumulation and resistance against race 1 of P. megasprma f. sp. glycinea. Inhibition of glyceollin accumulation was about 65% when a 1 mM L-AOPP solution was applied at the time of inoculation and increased
5
IO Retention
I5 time
20
25
(min)
FIO. 1. HPLC analysisof soybean hypocotyls 24 h after infection with race 1 of Phytc#dwra WM@$@~WJ f. sp. glgcinc. Upper curve: control in absence of L-AOPP. 1 to 3, Glyceollin isomersI-III; 4, daidaein (7, 4’.diiydroxy-isoflavone). Lower curve: pre-incubated for 4 h with 1 mn L-AOPP. Experimental conditions see Methods. Seedlings were infected by method
A.
P. Moesta
66
and H. Grisebach
to about 90% when the seedlings were pre-incubated for 4 h with the inhibitor. Figure 1 shows the drastic decrease in the amount of the 3 glyceollin isomers [IO] and of daidzein caused by 1 mM L-AOPP. Furthermore, it is evident that inhibition of glyceollin synthesis does not lead to accumulation of other compounds that are eluted from the SepPak column by chloroform/propanol (see Methods). The glyceollin concentration in infected tissue is listed in Table 1. Inhibition by L-AOPP resulted in an even lower level of glyceollin than was reached after infection with the compatible race 3. Correlation between inhibition of glyceollin accumulation and resistance The dependence of the inhibition of glyceollin accumulation on concentration of L-AOPP and the accompanying increase in infected tissue are shown in Fig. 2. A TABLE
Inhibition of gbceollin
1
accumulationby L-AOPP Glyceollin@ concentration after 24 h incubation (pg mg-a dry wt of infected tissue)
Race
of P. ?negas@nna f. sp. gl*a Race 1 Race1 Race 3 Race 3
Addition
1 mM L-AOPPC 1 rnM L-AOPP
Infection
method
Infection
A
20.9f2.3’ 2.3*0*4 2.6kO.2 1.5
a The term glyceollin is used in this paper for the structural accumulate in a constant ratio of 8 : 1 : 1 [II]. * Standard deviation as determined from at least 3 independent c The seedlings were pre-incubated for 4 h with the inhibitor.
method
22.6h2.0 2.8kO.3 6.2f1.2 2.7 isomers
I to III,
which
experiments.
L- A0 PP (pi) FIO. 2. Dependence of glyceollin content (0) of soybean seedlings and amount tissue (A) on concentration of L-AOPP. For experimental conditions see Methods. were infected for 24 h with race 1 of Phytophthora megaspsrmaf. sp. g&ma by surface (method B).
of infected Seedlings inoculation
B
lnhiibition
of glyceollin
accumulation
69
clear correlation can be seen between decrease in glyceollin content and increase in infected tissue. At L-AOPP concentrations higher than approximately 500 pi, the interaction between the Harosoy 63 seedlings and race 1 of P. megaspermaf. sp. glyt%zeawas converted completely from a resistant to a susceptible interaction. The hypocotyls appeared dark green and water-soaked and eventually collapsed. As shown in Table 2 L-AOPP caused a 6-fold increase in the amount of infected tissue after 24 h. The size of infected area reached in presence of the inhibitor was the same as that found in the compatible interaction. In contrast to its effect on the incompatible reaction, L-AOPP did not influence the size of the infected area in the compatible interaction. TABLE
Quantitative
effect of L-AOPP
2
on @read of infection
in soybean hJpoco@
Dry weight (mg) after Race
of P. megasperma f. sp. glycirm Race 1 Race 1 Race 3 Race3 a Standard deviation b The seedlings were
Addition
1 rnM L-AOPPb 1 mu
L-AOPP
as determined pre-incubated
Infection
method
A
of infected tissue 24 h incubation Infection
19.&l-&2*1” 38.6f1.7 37.2 f2.0 43.2 frrom at least 3 independent for 4 h with the inhibitor.
method
B
5.1 j, 1.0 29*9*0*9 30.553.9 30.9 experiments.
The observations on the loss of resistance in the presence of L-AOPP were confirmed by microscopical studies. Sections of seedlings (1 cm long) infected by surface inoculation (method B) and incubated for 21 h were fixed, embedded in parafhn, cut on a microtome (15 urn) and stained with safranin/fast green [7]. While hyphal growth in the resistant interaction was confined to the first 4 to 6 cell layers of the hypocotyl, fungal hyphae were found in all cell layers of seedlings treated with 1 mu L-AOPP. No morphological changes were observed in the hypocotyl tissue of uninfected seedlings which had been treated with the same concentration of the inhibitor. DISCUSSION
It was shown previously that accumulation of glyceollin after infection of soybean see:dlings with P. megaspermaf. sp. glyciwa is regulated by its rate of synthesis [ll]. An increase in the activity of phenylalanine ammonia-lyase (PAL), an enzyme involved in biosynthesis of glyceollin, was observed after infection [IZ]. This increase was considerably higher in the incompatible (resistant) than in the compatible reaction [a. According to these results it was expected that inhibition of PAL by L-AOPP would prevent the accumulation of glyceollin after infection, and this sulpposition has now been confirmed. To assess the significance of the inhibition experiments, other possible effects of L-AOPP must be considered. L-AOPP would be expected to inhibit lignification, and if induced lignification at the infection site plays a role in the defense reaction of soybean, such an inhibition could contribute to the loss of resistance. So far induced lignification has not been reported in soybean.
P. Moesta
70
and H. Grisebach
The following physiological and biochemical effects of L-AOPP in soybean seedlings were observed 72 h after treatment with inhibitor [6] : inhibition of fresh weight gain, increase of extractable PAL activity, and increase in the amount of phenylalanine. These effects would not be expected to influence the defense reaction. No effect on soluble proteins was observed. Since in our experiments the seedlings were in contact with the inhibitor for only 28 h, long term effects of L-AOPP do not have to be taken into consideration. While unknown side effects of L-AOPP on the resistance reaction cannot be excluded, the close correlation between the decrease in glyceollin content and loss of resistance is strong support for the assumption that this phytoalexin plays an important role in the defense reaction of soybean against P. mega.rperma f. sp. glycinea. This work was supported by Deutsche Forschungsgemeinschaft (SFB 46). P.M. thanks the Studienstiftung des Deutschen Volkes for a stipend. We thank Dr R. I. Buzzel for soybean seeds. REFERENCES 1. mm,
N. & G~DEKE, K. H. (1977). of L-phenylalanine ammonia-lyase in 2. AYERS, A, R., EBEL, J., FINELLI, F., interactions IX. Quantitative assay present in the extracellular medium
a-Aminooxy-8-phenylpropionic acid-a potent inhibitor vitro and in vivo. Plant S&cc U.&S 8,313-317. BERGER, N. & ALBERSHEIM, P. (1976). Host-pathogen of elicitor activity and characterization of the elicitor of cultur*, of Phytophthora mgas@etnta var. sqjae. Plant
PhysMlogV57,751-759. 3. BURNER,
H.
& GRISEBACH,
mega.s@nnaf. sp. glycinca.
H.
(1982).
Enzyme
induction
in soybean
infected
by
Phy@hthoru
Archives Biochemistry and Biabhysics (in press).
4. Bamos, M. T. & MORLEY, J. S. (1979). Aminooxyanalogues of aspartame and gastrin L-terminal tetrapeptide amide. 3oumal of the Chqkal SO&Y I, 2 138-2 143. 5. CRUICIKSHANK, I. A. M. (1980). Defense triggered by the invader: chemical defenses. In P&at Disease,Ed. by J. G. Horsfall & E. B. Cowling, Vol. V, pp. 247-267. Academic Press, New York. 6. DUKE, S. O., HOAOLAND, R. E. & ELMORE, C. D. (1980). Effects of glyphosate on metabolism of phenolic compounds. V. L-a-aminooxy+phenylpropionic acid and glyphosate effects on phenylalanine ammonia-lyase in soybean seedlings. Plant Physiology 65, 17-2 1. 7. FRANK, J. A. & PAXTON, J. D. (1970). Time sequence for phytoalexin production in Harcuoy and Harosoy 63 soybeans. PhytopathologyGO,3 15-3 18. 8. GRISEBACH, H. & EBEL, J. (1978). Phytoalexins, chemical defense substances of higher plants? Angewandh Gemie, International Edition in English 17, 635-647. 9. LZAROVITS, G., ST&EL, R. & WARD, E. W. B. (1981). Age-related changes in specificity and glyceollin production in the hypocotyl reaction of soybeans to Phytophthora megas@nna var. sojae. Phytojathology 71, 94-97. 10. Lvrm, R. L., MULHEIRN, L. J. & LEWORTHY, J. C. (1976). New pterocarpinoid phytoalexins of soybean. 3wnal of the Chemical Society,Chemical Communications497-498. 11. MOESTA. P. & GRBEBACH. H. (19811. Investiaation of the mechanism of rrlvceollm accumulation in soybean infected by khyk$hthok mega.s@&a Esp. glycinea. Archives of &hemistry and Biophysics 215 462-467. 12. Z&RINGER, U., EBEL, J. & GRISEBACH, H. (1978). Induction of phytoalexin synthesis in soybean. Elicitor-induced increase in enzyme activities of flavonoid biosynthesis and incorporation of mevalonate into glyceollin. Archives of Bio&mist~ and Biophysics M&450-455.
Plant Pathology
(1982)
21,65-70
L-;!-Aminooxy-3-phenylpropionic acid inhibits phytoalexin accumulation in soybean with concomitant loss of resistance against Ph ytophthora megasperma f. sp. glycinea P. MoEsT.4 and H. Dejartnumt Schcikkstr.
of B&&w&y,
(Accepted
fw publication
1, D-7800
GRISEBACH
Biologisches Inrtitut F&burg, West Germany March
II der Universit&
1982)
Application of a 1 mu solution of L-2-aminooxy-3-phenylpropionic acid (L-AOPP) to the cut ends of soybean seedlings (cultivar Harosoy 63) 4 h before infection of the hypocotyls with the incompatible race 1 of Phytophthora megasperma f. sp. g&Gea resulted in an inhibition of glyceollin accumulation of about 90%. Experiments on the dependence of the inhibition of glyceollin accumulation on concentration of L-AOPP and on the spread of infection showed a close correlation between decrease in glyceollin content and increase in infected tissue. At an L-AOPP concentration of greater than 500 ELM, the interaction between Harosoy 63 seedlings and race 1 of P. megasjetmo was converted completely from a resistant to a susceptible one. The results are strong support for the assumption that glyceollm plays an important role in the defense reaction of soybean.
INTRODUCTION The question to what extent phytoalexins contribute to specific resistance of plants is still a matter of debate [S, S]. An ideal system to test the importance of phytoalexins for resistance would be a mutant in which phytoalexin synthesis is blocked at a single step. However, at present such mutants are not available. We have therefore tried to inhibit phytoalexin synthesis specifically and to correlate the degree of inhibition with resistance. Towards this goal we used L-2-aminooxy-3-phenylpropion:ic acid [I] (L-AOPP), an inhibitor of phenylalanine ammonia-lyase, to inhibit glyceollin synthesis in soybean seedlings triggered by infection with Phytophthora megas@rma f. sp. glycinea. Here we report results which show a close correlation between inhibition by L-AOPP of glyceollin accumulation in tissue at the infection site and the size of the infected area. MATERIALS AND METHODS Chfzmicals
L-2-Aminooxy-3-phenylpropionic acid was synthesized by the method described by Bri,ggs & Morley [4]. The purity of the compound was checked by high pressure liquid chromatography (HPLC), “H-NMR, and by its optical rotation. Soybean seedlings
Seeds of soybean (Glycine max L. cv. Harosoy 63) were obtained from R. I. Buzzel, 00484059/82/04065+06
$03.00/O
@ 1982 Academic
Press Inc.
(London)
Limited
66 Agriculture Canada, Research Station, Harrow, Ontario. vermiculite and potting soil as described previously [Z].
P. Moesta and H. Grisebach Seedlings were grown in
Fungal cultures Phytophthora megasterma f. sp. glycinea A. A. Hildb. races 1 and 3 were obtained from E. Ziegler, University of Aachen, and were grown as described elsewhere [2]. Inoculation proceduresand analysis of glyceollin The roots of 5-day-old seedlings of soybean were cut off with a razor blade so that a uniform hypocotyl length of 7 cm was obtained. Each seedling was placed in a test tube with 1 ml of distilled water or a solution of L-AOPP in distilled water. The seedlings were then incubated at 25 “C and 60% humidity under illumination with 9500 lx (Osram L40 W/25 fluorescent tubes) for 4 h. Afterwards the seedlings were inoculated with race 1 or 3 of P. megaspermaf. sp. glycinea by one of the following methods. Method A [II] : a slash wound, approximately 1 cm long, was cut with a razor blade into each hypocotyl about 1 cm below the cotyledonary node and a small piece of mycelium was placed in the wound. Method B: an area of epidermis approximately 2 x3 mm was scraped off with a razor blade 1 cm below the cotyledonary node. A small piece of mycelium was placed on top of the wound. After inoculation, the seedlings were transferred to a moist chamber and kept under illumination at 25 “C and lOOo/o humidity for 24 h. For isolation of glyceollin the infected hypocotyl segment plus 5 mm of healthy tissue at each end was taken from each of 10 seedlings and the combined material was placed in a test tube with 5 ml of ethanol and heated to boiling point for 2 min. The ethanol was decanted, the extraction repeated and the tissue washed once more with 5 ml cold ethanol. The collected extracts were evaporated to dryness under reduced pressure and the residue was taken up in 2 x 1 ml of trichloromethane. The solution was then applied to a SepPak silica cartridge (Waters, Kbnigstein), and non-polar components were washed out with a total of 5 ml of CHCl,. The isoflavonoid fraction was eluted with 3 ml of CHCls-3-propanol (1 : 1, v : v). The solvent was evaporated and the residue was taken up in 400 ~1 of 2-propanol. Separation and quantification of the isoflavonoids were achieved by HPLC as described earlier [II]. The dry weight of infected tissue was determined as described by Lazarovits et al. [9] except that the infected tissue was excised from the hypocotyl prior to the extraction of the phytoalexins. RESULTS Inhibition of glyceollin accumulation by L-AOPP Three different methods for the application of the inhibitor were tested. (a) Application ofan L-AOPP solution to the wound. After inoculation of the Harosoy 63 seedlings in a slit wound of the hypocotyl (method A) or in an epidermal wound (method B) with mycelium of the incompatible race I of P. megarlerma f. sp. glycinea, the wound area was covered with strips of cellucotton that had been soaked in the inhibitor solution. In the concentration range examined (1 lt~ to 1 rnM L-AOPP) no reduction of glyceollin content was found at 24 h after inoculation. (b) Application of th-e inhibitor via the roots. When seedlings were placed with their roots in a 300 FM L-AOPP solution 24 h before inoculation, approximately 45%
Inhibition
of glyceollin
67
accumulation
inhibition of glyceollin accumulation was found. At lower inhibitor concentration no effect was observed. Since very large amounts of L-AOPP were needed with this method, higher concentrations of L-AOPP were not tested. At 300 PM L-AOPP a ret.ardation in hypocotyl elongation was observed. (c) A#j&cation of tb inhibitor through the cut base ofhyjoco@s. Removal of the root had no measurable effect on glyceollin accumulation and resistance against race 1 of P. megasprma f. sp. glycinea. Inhibition of glyceollin accumulation was about 65% when a 1 mM L-AOPP solution was applied at the time of inoculation and increased
5
IO Retention
I5 time
20
25
(min)
FIO. 1. HPLC analysisof soybean hypocotyls 24 h after infection with race 1 of Phytc#dwra WM@$@~WJ f. sp. glgcinc. Upper curve: control in absence of L-AOPP. 1 to 3, Glyceollin isomersI-III; 4, daidaein (7, 4’.diiydroxy-isoflavone). Lower curve: pre-incubated for 4 h with 1 mn L-AOPP. Experimental conditions see Methods. Seedlings were infected by method
A.
P. Moesta
66
and H. Grisebach
to about 90% when the seedlings were pre-incubated for 4 h with the inhibitor. Figure 1 shows the drastic decrease in the amount of the 3 glyceollin isomers [IO] and of daidzein caused by 1 mM L-AOPP. Furthermore, it is evident that inhibition of glyceollin synthesis does not lead to accumulation of other compounds that are eluted from the SepPak column by chloroform/propanol (see Methods). The glyceollin concentration in infected tissue is listed in Table 1. Inhibition by L-AOPP resulted in an even lower level of glyceollin than was reached after infection with the compatible race 3. Correlation between inhibition of glyceollin accumulation and resistance The dependence of the inhibition of glyceollin accumulation on concentration of L-AOPP and the accompanying increase in infected tissue are shown in Fig. 2. A TABLE
Inhibition of gbceollin
1
accumulationby L-AOPP Glyceollin@ concentration after 24 h incubation (pg mg-a dry wt of infected tissue)
Race
of P. ?negas@nna f. sp. gl*a Race 1 Race1 Race 3 Race 3
Addition
1 mM L-AOPPC 1 rnM L-AOPP
Infection
method
Infection
A
20.9f2.3’ 2.3*0*4 2.6kO.2 1.5
a The term glyceollin is used in this paper for the structural accumulate in a constant ratio of 8 : 1 : 1 [II]. * Standard deviation as determined from at least 3 independent c The seedlings were pre-incubated for 4 h with the inhibitor.
method
22.6h2.0 2.8kO.3 6.2f1.2 2.7 isomers
I to III,
which
experiments.
L- A0 PP (pi) FIO. 2. Dependence of glyceollin content (0) of soybean seedlings and amount tissue (A) on concentration of L-AOPP. For experimental conditions see Methods. were infected for 24 h with race 1 of Phytophthora megaspsrmaf. sp. g&ma by surface (method B).
of infected Seedlings inoculation
B
lnhiibition
of glyceollin
accumulation
69
clear correlation can be seen between decrease in glyceollin content and increase in infected tissue. At L-AOPP concentrations higher than approximately 500 pi, the interaction between the Harosoy 63 seedlings and race 1 of P. megaspermaf. sp. glyt%zeawas converted completely from a resistant to a susceptible interaction. The hypocotyls appeared dark green and water-soaked and eventually collapsed. As shown in Table 2 L-AOPP caused a 6-fold increase in the amount of infected tissue after 24 h. The size of infected area reached in presence of the inhibitor was the same as that found in the compatible interaction. In contrast to its effect on the incompatible reaction, L-AOPP did not influence the size of the infected area in the compatible interaction. TABLE
Quantitative
effect of L-AOPP
2
on @read of infection
in soybean hJpoco@
Dry weight (mg) after Race
of P. megasperma f. sp. glycirm Race 1 Race 1 Race 3 Race3 a Standard deviation b The seedlings were
Addition
1 rnM L-AOPPb 1 mu
L-AOPP
as determined pre-incubated
Infection
method
A
of infected tissue 24 h incubation Infection
19.&l-&2*1” 38.6f1.7 37.2 f2.0 43.2 frrom at least 3 independent for 4 h with the inhibitor.
method
B
5.1 j, 1.0 29*9*0*9 30.553.9 30.9 experiments.
The observations on the loss of resistance in the presence of L-AOPP were confirmed by microscopical studies. Sections of seedlings (1 cm long) infected by surface inoculation (method B) and incubated for 21 h were fixed, embedded in parafhn, cut on a microtome (15 urn) and stained with safranin/fast green [7]. While hyphal growth in the resistant interaction was confined to the first 4 to 6 cell layers of the hypocotyl, fungal hyphae were found in all cell layers of seedlings treated with 1 mu L-AOPP. No morphological changes were observed in the hypocotyl tissue of uninfected seedlings which had been treated with the same concentration of the inhibitor. DISCUSSION
It was shown previously that accumulation of glyceollin after infection of soybean see:dlings with P. megaspermaf. sp. glyciwa is regulated by its rate of synthesis [ll]. An increase in the activity of phenylalanine ammonia-lyase (PAL), an enzyme involved in biosynthesis of glyceollin, was observed after infection [IZ]. This increase was considerably higher in the incompatible (resistant) than in the compatible reaction [a. According to these results it was expected that inhibition of PAL by L-AOPP would prevent the accumulation of glyceollin after infection, and this sulpposition has now been confirmed. To assess the significance of the inhibition experiments, other possible effects of L-AOPP must be considered. L-AOPP would be expected to inhibit lignification, and if induced lignification at the infection site plays a role in the defense reaction of soybean, such an inhibition could contribute to the loss of resistance. So far induced lignification has not been reported in soybean.
P. Moesta
70
and H. Grisebach
The following physiological and biochemical effects of L-AOPP in soybean seedlings were observed 72 h after treatment with inhibitor [6] : inhibition of fresh weight gain, increase of extractable PAL activity, and increase in the amount of phenylalanine. These effects would not be expected to influence the defense reaction. No effect on soluble proteins was observed. Since in our experiments the seedlings were in contact with the inhibitor for only 28 h, long term effects of L-AOPP do not have to be taken into consideration. While unknown side effects of L-AOPP on the resistance reaction cannot be excluded, the close correlation between the decrease in glyceollin content and loss of resistance is strong support for the assumption that this phytoalexin plays an important role in the defense reaction of soybean against P. mega.rperma f. sp. glycinea. This work was supported by Deutsche Forschungsgemeinschaft (SFB 46). P.M. thanks the Studienstiftung des Deutschen Volkes for a stipend. We thank Dr R. I. Buzzel for soybean seeds. REFERENCES 1. mm,
N. & G~DEKE, K. H. (1977). of L-phenylalanine ammonia-lyase in 2. AYERS, A, R., EBEL, J., FINELLI, F., interactions IX. Quantitative assay present in the extracellular medium
a-Aminooxy-8-phenylpropionic acid-a potent inhibitor vitro and in vivo. Plant S&cc U.&S 8,313-317. BERGER, N. & ALBERSHEIM, P. (1976). Host-pathogen of elicitor activity and characterization of the elicitor of cultur*, of Phytophthora mgas@etnta var. sqjae. Plant
PhysMlogV57,751-759. 3. BURNER,
H.
& GRISEBACH,
mega.s@nnaf. sp. glycinca.
H.
(1982).
Enzyme
induction
in soybean
infected
by
Phy@hthoru
Archives Biochemistry and Biabhysics (in press).
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