in Vitro Mode of Action N.MITo, Agricultural
R. SATO, M. MIYAKADO, Reseurch
Laboratory, Takarazuka Research Center, Takarazuka. Hyogo 665. Japan
Received November 22, 1990; accepted February 14, 1991 The effect of N-Phenylimide photobleaching herbicides on in vitro synthesis of protoporphyrin was examined. The N-phenylimide photobleaching herbicide S-23142 [N-(4-chloro-2-fluoro-5propargyloxyphenyl)-3,4.5,6-tetrahydrophthalimide] inhibited the Mg-protoporphyrin IX synthesis of intact plastids by 50% at 10-i’ M. Protoporphyrin IX synthesizing activity was solubilized from plastid membranes by n-dodecyl B-D-maltoside, and the solubilized activity was inhibited by several photobleaching herbicides. In vitro binding of N-phenylimide photobleaching herbicide, S23121 [N-[4-chloro-2-fluoro-5-[( I-methyl-2-propynyl)oxyl]phenyl]-3,4,5.6-tetrahydrophthalimide], to a solubilized plastid fraction was examined. A good correlation between protoporphyrin IX synthesizing activity and [“‘C]S-23121 specific binding was observed. Binding site afftnity (K,) was 8.9-9.8 nM. The binding was displaced by a diphenylether photobleaching herbicide, acifluorfenethyl, and another N-phenylimide. S-23142, but not by a photosystem II electron transport inhibitor, DCMU. Bound [“C]S-23121 was dissociated by an excess of cold S-23121, which indicated the reversible binding of S-23 12 I. !c 1991 Academic Press. Inc.
There are varieties of herbicides called photobleaching herbicides, the mode of action of which has been studied extensively. Photobleaching herbicides require light and oxygen for the expression of their herbitidal activities and induce photooxidation of polyunsaturated fatty acids, leading to the destruction of membrane functions (1, 2). Recently, Matringe ef ul. and Witkowski and Halling reported that photobleaching herbicides inhibit protoporphyrinogen oxidase (PPO).’ which is responsible for the biosynthesis of protoporphyrin IX (3-5). Although they propose that the inhibition of PPO leads to the accumulation of a strong phytotoxic photosensitizer, protoporphyrin IX (Proto) (3-9, some experimental evi-
’ Abbreviations used: PPO, protopophyrinogen oxidase; Proto, protoporphyrin IX; Mg-Proto, Mgprotoporphyrin IX: Tes. N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid: Hepes. N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; DM, ndodecyl B-D-maltoside; BSA. bovine serum albumin; Copro, coproporphyrin III.
dence suggests that the inhibition of PPO is not the sole factor for the accumulation of Proto (6). Thus, the in vitro mode of action of photobleaching herbicides, such as the mechanism of Proto accumulation and the herbicide binding site, is yet to be conclusive. One of the effective methods to further understand the in vitro mode of action of the herbicide is to study the binding of the herbicide to its target site, but to date, only a few studies have been made in this respect (7). S-23 121 is an experimental photobleaching herbicide that has a N-phenylimide moiety in the molecule (Fig. 1). The mode of action of N-phenylimide herbicides has been studied using an S-23 121 derivative, S-23142, and has been shown to be similar to that of diphenylether herbicides, another type of photobleaching herbicide, in cucumber cotyledons Q-10). Although Sandmann et al. reported the inhibition of PPO by a N-phenylimide herbicide, chlorophthalim (1 I), the in vitro mode of action of the N-phenylimide herbicide is not fully understood. Thus for further understanding of the mode of action of photobleaching 128
00483575191 $3.00 Copyright 0 1991 by Academic Press, Inc. Au rights of reproduction in any form reserved.
FIG. 1. Chemical tobleaching herbicides.
herbicides, we examined the effect of Nphenylimide herbicides on the in vitro accumulation of protoporphyrin and studied the binding of the herbicide to its target site. MATERIALS
Plant materials. Corn seeds (Zea mays cv. Golden Cross Bantam) were soaked in water for 1 day and germinated on wet vermiculite for 7 days in darkness. Enclosed leaves were detached from the coleoptiles and allowed to green for 4 hr under fluorescent light (1.8 W/m’, 25°C) just before plastid isolation. Preparation and solubilization of plastids. Intact corn plastids were isolated following a procedure of Prado et al. (12). The
plastid pellet isolated from 40 g of corn leaves was suspended in 5 ml of buffer A (20 mM Tes, 10 mM Hepes, 1mM MgCl,, 0.5 mM EDTA, pH 7.7). and n-dodecyl P-D-maltoside (DM) was added to give a final concentration of 1%. The plastid suspension was kept at 4°C in darkness for 30 min, then centrifuged at 100,OOOg for 1 hr to get a solubilized plastid fraction. Measurement of protoporphyrin sizing activities. Mg-protoporphyrin
fuged, and the resulting pellet was extracted by 1.5 ml of cold acetone and 0.5 ml of 0.12 N NH,OH. The extracts were combined and analyzed by HPLC. For the quantitative determination of Mg-Proto, the porphyrin was extracted into ether and the amount was determined according to Ref.
(MgProto) synthesis by intact plastids was measured basically following a procedure of Prado et al. (12). Briefly, plastids were incubated in 1 ml of reaction mixture (500 pmol sucrose, 4 kmol ALA, 1 Fmol MgC12, 1 pmol EDTA, 20 pmol Tes, 10 Fmol Hepes, 1.5 p.mol ATP, 4 pmol glutathione, 0.6 krnol NAD, 2 mg BSA) for 2 hr in darkness, and the reaction was stopped by freezing. The reaction mixture was extracted by 3 ml of cold acetone, centri-
Proto synthesizing activities of solubilized plastids were measured using &aminolevulinic acid (ALA) as the substrate. Fifty microliters of substrate solution (1 kmol ALA, 1 kmol glutathione, 0.15 p,mol NAD, 2.5 p,mol ATP) was added to 200 p.1of solubilized plastid fraction (equivalent to 0.4 to 0.5 mg protein) to start the reaction. The mixture was incubated for 2 hr in darkness, and the reaction was stopped by adding 750 ~1 of 90% acetone containing 0.1 N NH,OH. After centrifugation at 10,OOOg for 5 min, the Proto synthesized was measured using HPLC. HPLC system. A 5-pm C,, reversedphase column (Nucleosil 5C,,, 250 x 4.6 mm) was used with a solvent system of methanol:O. I M ammonium phosphate (9: 1, v/v), and a flow rate of 1 ml/min was maintained. Porphyrin detection was performed with a fluorescence monitor (Shimadzu RF535) with excitation and emission wavelength settings of 410 and 594 nm, respectively, for Mg-Proto and Mg-Proto methyl ester; 420 and 630 nm, respectively, for Proto. The amount of protoporphyrins was determined by the fluorescence monitor calibrated by authentic samples. Binding assays. Equilibrium assays for S-23121 binding were done by incubating 1 ml of a mixture containing a solubilized plastid fraction, 120 nM [phenyl“C]S-23121 (7.21 GBq/mmol), and either with 10 PM unlabeled S-23121 or without an unlabeled compound. The binding mixtures in duplicate tubes were incubated at 30°C for 1 hr, placed on ice, and filtered through a glass fiber filter coated with polyethyleneimine as described by Bruns et al. (13). After filtration, the filters were washed three times with 5 ml of ice-cold 10
mM Tris-HCl buffer (pH 7.6). Specific bindings were defined as the radioactivity difference between the incubation with and without unlabeled S-23 12 1. The binding data were analyzed as described by Scatchard, and the K, value was determined (14). Disruption of plusrids. Plastids isolated by the method described above were suspended in buffer A. For lysis, the suspension was kept at 4°C for 30 min. For sonication, the suspension was sonicated for 15 set twice at 20 kHz by a Tomy Model UD201 sonicator. For detergent solubilization, DM was added to the suspension to give a final concentration of 1%. Then each suspension was centrifuged at 100,OOOg and Proto synthesizing activity and S-23121 binding activity in the supernatant were assayed. Chrmicals. [phenyl-‘4C]S-23 12 I and photobleaching herbicides were synthesized at Takarazuka Research Center, Sumitomo Chemical Co., Ltd. Proto was from Sigma Chemicals. Coproporphyrin III (Copro), Mg-Proto, and Mg-Proto methyl ester were provided by Toyo Hakka Co.. Ltd.
Inhibition ofprotoporphyrin synthesis by photobleaching herbicides. The effect of S-
23 142 on Mg-Proto synthesis by intact plastids was examined. First, porphyrins synthesized from ALA by intact plastids were analyzed by HPLC. Porphyrins were separated by reversed-phase column and detected by a fluorescence monitor at 594 and 630 nm. Two peaks (peaks A and B) were detected at 594 nm, and one peak (peak Cl was detected at 630 nm (Fig. 2). Peaks A, B, and C were estimated to be Mg-Proto, its methyl ester, and Proto, respectively, because of the coincidence of their retention times and fluorescence spectra with those of authentic samples. The specific activity of Mg-Proto, its methyl ester, and Proto synthesis was 0.54-0.69, 0.12-0.2 1, and 0.21-0.37 nmol/mg protein, respectively. Synthesis of these three kinds of porphyrins was completely inhibited by 1 @4 S23142 (Fig. 2). Figure 3 shows the dosedependent inhibition of Mg-Proto synthesis by S-23 142. S-23 142 inhibited Mg-Proto
% 2 L-.-
FIG. 2. HPLC reaction mixture detection was by done three times
profiles of porphyrines .synthesked by intact plastids. Porphyrins extracted from the were separated by reverse-phase HPLC as described ander Materials and Methods; jluorescence (excitation. 4/O nm; emission, 594 or 630 nm). This experiment M’(IS with similar results.
$ IZOL E
0” IOO6 c
L zo,.“u’l.-‘-’ -11
FIG. 3. Effect of S-23142 on Mg-Proto synthesis by intut plustids from either ALA (0) or Proto (0). Amounts of Mg-Proio were expressed as percentage qf ~,ontrol value. Spec~ific activity of control was 0.4Y0.67 nmol hr ’ . mg protein ‘. The result represents the average of t\c’o independent measurements of duplic ate samples.
synthesis by 50% at IO-” M when ALA was given as the substrate. By contrast, no inhibition was observed when Proto was given as the direct substrate as were other photobleaching herbicides (4).
Solubilization of Proto synthesizing activiry. Corn plastids were solubilized by 1%
’ min Retention
DM and the activity for synthesizing protoporphyrin was examined by giving ALA as the substrate. About 40-42% of the plastid proteins were solubilized by DM. Figure 4 shows the HPLC profile of porphyrins synthesized by solubilized plastids. Two peaks (peaks D and E) were detected at 630 nm. No peak was detected at 594 nm (data not shown). Peaks D and E were estimated to be Copro and Proto, respectively, because of the coincidence of their retention time and fluorescence spectra with those of authentic samples. The specific activity of Proto synthesizing activity was 0.279.36 nmol/mg protein. About 13% of “protoporphyrin” (Mg-Proto, Mg-Proto methyl ester, and Proto) synthesizing activity was solubilized by the detergent when the Mg-Proto. Mg-Proto methyl ester, and Proto synthesizing activities of intact plastids were compared with the Proto synthesizing activity of solubilized plastids. The synthesis of Proto was inhibited by I PM S-23142 (Fig. 4). Figure 5 shows the dosedependent inhibition of Proto synthesizing
FIG. 4. HPLC profiles of porphyrins synthesized by solubilized plastid fraction. Porphyrins accumuloted in the reaction mixture were separated by HPLC as described under Materials and Methods; detection was b.vffuorescence (excitation, 410 nm; emission. 630 nmf. This experiment was done three times with similar results.
Detergent (1% DM) 2 ;j ‘51 2 z 27
2 ? P
I I 50 100 Pro10 synthesizing
\ .,.,.,.,.I.. -10
I I , 150 200 250 actlvlty (pnWhrimg protein) I I 20 40
5. Dose-dependent inhibition of solubilized Proto synthesizing activity by several photobleaching herbicides. Proto synthesizing activity nws mensured as described under Materials und Methods. Specific activity of control wn.s 0.29-0.34 nmol . hr ’ mg protein-‘. The result represents the average of tuv independent measurements of duplicate samples. FIG.
activity of solubilized plastids by several photobleaching herbicides. The S-23 142 and S-23 121 concentrations needed to inhibit Proto synthesizing activity by 50% (I,,) was 7 and 3.2 r&I, respectively (Fig. 5). In vitro binding of [‘4c]S-23121 to the solubilized plastid fruction. Binding of [‘4C]S-23121 to the solubilized plastid fraction was diminished by the simultaneous addition of excess amounts of unlabeled S23142, and the specific binding of [‘4C]S23121 was completely abolished by heating the solubilized plastids at 90°C for 10 min (data not shown). Plastids were disrupted as described under Materials and Methods and the Proto synthesizing activity and the [‘4C]S-23121 binding in the lOO.OOOg supernatant were measured. A good correlation between Proto synthesizing activity and [ 14C]S-23 121 binding was observed. Both activities were solubilized most effectively by the detergent treatment (Fig. 6). Figure 7 shows the displacement of [r4C]S-23 12 1 binding by the IV-phenylimide herbicide, S-23142, and the diphenylether herbicide, acifluorfen-ethyl (AFE). The [14C]S-23 12 1 binding was displaced both by
FIG. 6. Solubilization of Proto synthesizing activity and [‘4C]S-23121 binding activity. Plastids were disrupted as described under Muterials and Methods and centrifuged at lOO,OOOg for I hr, and both activities in the supernutant were assayed. This experiment HUS done three times wjith similar results.
S-23 142 and AFE. But DCMU, a photosynthetic electron transport inhibitor, did not displace the [r4C]S-23121 binding. The binding affinity of [r4C]S-23121 was determined by the slope of the Scatchard plot. Scatchard plot analysis of [‘4C]S23121 binding data at 30°C gave a dissocia-
FIG. 7. Displacement of [“‘CIS-23121 binding to a solubilized plastid fraction by photobleaching herbicides. The amount of [“‘C]S-23121 bound wus corrected for nonspecific binding in the presence of IO WM unlabeled S-23121 and plotted as a percentage of the amount obtained without competitors. Total and specific binding of control were 7.6-8.2 and 3.6-4.1 pmol.mg protein I. respectively. The result represents the average of two independent measurements of duplicate samples.
FIG. 8. Scutchardplot analysis of S-23121 binding data. by Scatchard (12) and the data corrected for nonspecific Results represent one of two independent measurements. the Scatchard plot (Experiment I. 8.9 nM; Experiment 2,
tion constant of K, = 8.9-9.8 nibI (Fig. 8). The S-23121 concentration needed to inhibit Proto synthesizing activity by 50% (I,,) was 3.2 n&I (Fig. 5). Figure 9 shows the reversible binding of S-23 121. After incubating the solubilized plastids with [14C]S-23121, an excess of unlabeled S-23121 was added. About 60% of the labeled S-23 121 was dissociated from the solubilized plastid fraction after the addition of unlabeled S-23121. DISCUSSION
Although several studies have been made on the inhibition of protoporphyrin synthesis by photobleaching herbicides (3-5), the in vitro mode of action of photobleaching herbicides is not fully understood. For instance, it is not known whether the inhibition of PPO is the sole factor for Proto accumulation in vivo (6), and there is little information of the binding site of photobleaching herbicide. Thus, it is of interest to examine the effect of photobleaching herbicides on the in vitro accumulation of porphyrin and to study the binding of the herbicide to its target site.
protein) S-23121 binding was analyzed as described binding were plotted. Inset: binding curve. K, value was determined from the slope of 9.8 nM).
We first examined the effect of Nphenylimide photobleaching herbicides on the in vitro porphyrin synthesis in intact plastids. The Mg-Proto synthesis was strongly inhibited by S-23142 only when ALA was given as the substrate. No inhibition was observed when Proto was given as the direct substrate, which suggests that the target site of S-23142 is the enzyme which is responsible for the Proto synthesis (3-5). When protoporphyrins synthesized from ALA by intact plastids were analyzed by HPLC, it was found that no Proto accumulation was observed. Illumination of the reaction mixture (3 W/m2) after the reaction did not result in the increase of the Proto level (data not shown). Recently, the effect of acifluorfen-Na on the in vitro synthesis of Proto by isolated plastids from cucumber was investigated, and it was reported that no Proto accumulation was observed in vitro (6). This result, together with our results, may indicate that the mechanism of Proto accumulation by photobleaching herbicides is a complicated phenomenon that involves more than a single inhibition step at the PPO level.
9. A time course of dissociution of S-23121 the soluhilkd plustid fracrion. The solubilized plusrid fraciion nws incubated with [“‘C]S-23121 (120 nmfor I hr und IO FM unlabeled S-23121 M’CIS udded lo the mixture. The rudiouctivity hound to the solubiItedplustidfruction MUS meusured and expressed us u percentage qf fhe radioartivity just byfive the uddition of unlabeled S-23121 (0). The radioactivities bound to the sohtbilized plustid fruction without the addition of unlabeled S-23121 M’ere plotled (0). The result repwsents the average of IMW independent meusrtrrments of duplicate samples.
We next attempted to study the binding of the photobleaching herbicide to its target site. But one of the problems in conducting binding studies was the lipophilic nature of the compounds. When plastid membranes were used for binding experiments, large amounts of nonspecific binding to membrane lipids make the interpretation of the data difficult. In order to avoid this problem, we attempted to solubilize protoporphyrin synthesizing activity which is sensitive to photobleaching herbicides. Although Proto synthesizing activity was solubilized from plastid membranes by 1% DM, Mg-Proto synthesizing activity was abolished by detergent treatment. This result is consistent with the report that the Mg-Proto synthesis in vitro requires intact plastid (15). Solubilized Proto synthesizing activity was less sensitive to S-23142 than Mg-Proto synthesizing activity of isolated intact plastids. Although the significance of this observation is unclear, the site(s) for S-23142 may be subject to proteolytic de-
gradation which makes the Proto synthesizing activity insensitive to S-23142 as described by Jacobs ef al. (16). Next we examined the in vitro binding of the N-phenylimide photobleaching herbicide, S-23121, to the solubilized plastid fraction. The binding of [14C]S-23 121 was studied by the glass fiber filter filtration method described by Bruns et al. (13). We first demonstrated the specific and reversible binding of the N-phenylimide photobleaching herbicide to solubilized plastid fractions. Both Proto synthesizing and S-23 12 1 binding activities were solubilized most effectively by detergent treatment and not by sonication or lysis of intact plastid. This result indicates that the target site of S-23 121 binds to the plastid membrane . The binding site affinity (K,) was 8.9-9.8 nM which was around the same as the I,,, value for the Proto synthesizing activity of the solubilized plastid fraction. The small difference between these two values may be explained by the use of different batches of solubilized plastid fraction. Tisher and Strotmann studies the binding of photosynthetic electron transport inhibitors to thylakoid membranes and showed that there is a good correlation between I,, values for photosynthetic electron transport and K,, of the electron transport inhibitors, which leads to the conclusion that the sites of binding and inhibition of the electron transport inhibitors are identical (17). The satisfactory correspondence of the K, of S23 12 I to the I,,, for Proto synthesizing activity suggests that the sites of binding and inhibition of S-23 12 I are identical. [‘4C]S-23121 binding was displaced by the diphenylether herbicide AFE. This indicated that both N-phenylimide and diphenylether herbicides share the same binding site. Jacobs et al. studied the mode of PPO inhibition by a diphenylether herbicide and suggested the irreversible binding of the herbicide (16), but in the case of Nphenylimide herbicides, reversible binding was suggested in this study. These facts suggest that the mode of PPO inhibition by
N-phenylimide may be different from that of diphenylether herbicides. Recently, Matringe and colleagues reported the in vitro binding of a diphenylether photobleaching herbicide, acifluorfen, to plastid membranes, and reached the same conclusions as ours (7). They used ‘H-labeled acifluorfen with a high specific activity to detect the binding to plastid membranes. But in order to further purify the binding site of the photobleaching herbicide, it is necessary to solubilize it from the plastid membrane and to detect the specific binding of the herbicide to the solubilized fractions. In this study, we first report on the specific binding of N-phenylimide photobleaching herbicides to solubilized plastids. ACKNOWLEDGMENTS
We thank Professor Nicholas J. Jacobs and Professor Judith M. Jacobs for their valuable discussions and advice. We also thank Toyo Hakka Co. Ltd for the kind donation of porphyrins. REFERENCES
I. S. 0. Duke, J. M. Becerril. T. D. Sherman. J. Lydon, and H. Matsumoto, Protoporphyrin IX’ role in the mechanism of action of diphenyl ether herbicides, Pesric. Sci., in press. 7. K. J. Kunert and A. D. Dodge, Herbicide induced radical formation and antioxidative systems, in “Target Site of Herbicide Action” (P. Boger and G. Sandmann. Eds.), p. 45. CRC Press, Boca Raton, FL, 1989. 3. M. Matringe. 3. M. Camadro, P. Labbe, and R. Scalla, Protoporphyrinogen oxidase inhibition by 3 peroxidizing herbicides, FEBS Lett. 245, 35 (1989). 4. M. Matringe, J. M. Camadro, P. Labbe. and R. Scalla. Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides. Biochern. J. 269, 231 (1989). 5. D. A. Witkowski and B. P. Halling, Inhibition of plant Protoporphyrinogen oxidase by the herbi-
tide acifluorfen-menthyl. PIat Physiol. 90, 1239 (1989). 6. C A. Rebeiz. K. N. Reddy. V. B. Nandihalli. and J. Velu. Tetrapyrole-dependent photodynamic herbicides, Plwrorhem. Photobid. 52, 1099 (IWO).
7. R Varsano, M. Matringe, N. Magnin. R. Momet. and R. Scalla. Competitive interaction of three peroxidizing herbicides with the binding of [7H]acifluorfen to corn etioplast membranes, FEES Len. 272, 106 (1990). 8. R Sato. E. Nagano. H. Oshio, and K. Kamoshita, Diphenylether-like physiological and biochemical actions of S-23142, a novel N-phenylimide herbicide, Pesric. Biochem. Physiol. 28, 194 (1987). 9. N. Mito, R. Sato. M. Miyakado. H. Oshio, and S. Tanaka. Mode of action of N-phenylimide photobleaching herbicide, S-23 142. Plant Physiol. Suppl. 93, 8 (1990). IO. R. Sato. Mechanism of action of diphenylethertype herbicides. C/tern. Ragd. P/ants (To/go) 25, 68 (1990). I I. G. Sandmann. B. Nicolaus. and P. Boger, Typical peroxidative parameters verified with mungbean seedling. soy bean cells and duckweed, Z. Nnrurforsch-C 45, 512 (1990). 12. A. D. Prado. B. M. Chereskin, P. A. Casterferanco. V. R. Franceshi. and B. E. Wezelman. ATP requirement for Mg chelatase in developing chloroplasts. Planr Physiol. 65, 956 ( 1980). 13. R. F. Bruns, K. Lawson-Wendling. and T. A. Pugsley. A rapid filtration assay for soluble receptors using polyethylenimine-treated filter, And. Biochem. 132, 74 I 1983). 14. G. Scatchard, The attraction of proteins for small molecule and ions, Ann. N.Y. Acud. Sci. 51, 660 (1949). 15. C. A. Rebeiz, J. C. Crane. and C. Nishijima. The biosynthesis of metal porphyrins by subchloroplastic fractions, Plant Ph,vsiol. 50, I85 (1972). 16. J. M. Jacobs. N. J. Jacobs, S. E. Borotz. and M. L. Guerinot. Effect of the photobleaching herbicide. acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles. soy bean root nodules. and bacteria, Arch. Biochem. Biophys. 280, 369 (1990). 17. W. Tischer and H. Strotmann, Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport, Biochim. Biopftys. Acre 460, I13 (1977).