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New Findings about the Inhibitory Action of Phenylcarbamates and Phenylthiocarbamates on Photosynthetic Apparatus
Pesticide Biochemistry and Physiology 68, 113–118 (2000) doi:10.1006/pest.2000.2499, available online at http://www.idealibrary.com on
New Findings about the Inhibitory Action of Phenylcarbamates and Phenylthiocarbamates on Photosynthetic Apparatus Frantisˇek Sˇersˇenˇ,*,1 Katarı´na Kra´l’ova´,* and Vendelı´n Macho† *Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska´ dolina, 842 15 Bratislava, Slovakia; and †Faculty of Industrial Technologies, University of Trencˇ´ın, T. Vansovej 1054/45, 020 32 Pu´chov, Slovakia Received February 18, 2000, accepted June 3, 2000 The inhibitory effect of alkyl-N-phenylcarbamates (alkyl 5 methyl–octyl) and alkyl-N-phenylthiocarbamates (alkyl 5 methyl–butyl) on the photosynthetic electron transport in spinach chloroplasts was studied. It was found that the site of action of the studied compounds is the intermediate D, i.e., the tyrosine radical located on site 161 in the D2 protein on the donor side of photosystem 2. The efficiency of the studied compounds was inversely proportional to the hydrophobicity of molecules, i.e., the effectiveness showed a decrease with increasing their hydrophobicity. It was found that alkyl-N-phenylcarbamates and alkyl-Nphenylthiocarbamates have the same site of action. q 2000 Academic Press
INTRODUCTION
Derivatives of phenylcarbamic acid (Scheme 1) are biologically active compounds applied mainly as pesticides (1,2). Compounds possessing the biologically active group –NH– CO–X, namely phenylureas, anilides, phenylcarbamates, and phenylthiocarbamates, belong to this group of pesticides (3). The best-known commercially applied herbicides from the above-mentioned group are Diuron or DCMU2 (3-(3,4-dichlorophenyl)-1,1dimethylurea), Pradone (carbetamide (R)-1ethylcarbamoylethyl-N-phenylcarbamate), and Betanex or Desmedipham (3-(ethoxycarbamoylanilino)-N-phenylcarbamate) (4). Camper and Moreland (5) and Wessels and Van der Veen (6) 1 To whom correspondence should be addressed. Fax: 11421-7-65428882. E-mail: [email protected]. 2 Abbreviations used: APC, alkyl-N-phenylcarbamates and alkyl-N-phenylthiocarbamates; Chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP, 2, 6-dichlorophenolindophenol; DMSO, dimethyl sulfoxide; DPC, 1,5-diphenylcarbazide; EPR, electron paramagnetic resonance; PAR, photosynthetically active radiation; PET, photosynthetic electron transport; PQ, plastoquinone pool; PS, photosystem; P680, primary donor of PS 2; P700, primary donor of PS 1, QA and QB, the first and the second quinone acceptor on the oxidized side of PS 2.
studied the effect of isopropyl and ethyl derivatives of N-phenylcarbamates with various substituents at positions 3 and 4 of the benzene ring on the Hill reaction activity of chloroplasts. It was suggested that the site of action is QB, i.e., the second quinone acceptor in herbicide binding protein D1 of photosystem (PS) 2 (7, 8). Before, the relationship between the inhibitory efficiency and the acidity (5) or the molecular structure (9) of the above-mentioned carbamates was studied. In our previous work the effects of ammonium salts of various derivatives of phenylcarbamic acid on photosynthesis were studied (10–14). Carbamates were found to be mitotic poisons that killed roots by inhibiting cell division. They are able to disturb the orientation of the spindle microtubular assembly of dividing cells (15, 16). In contrast to the carbamates, the thiocarbamates affect mainly shoots of plants. They decrease the content of long-chain fatty acid residues in plant lipids and inhibit wax formation (15, 16). This study is aimed: (i) to investigate the effect of alkyl-N-phenylcarbamates and alkyl-Nphenylthiocarbamates (APCs) on photosynthetic electron transport (PET) in spinach chloroplasts, (ii) to determine their site of action in the photosynthetic apparatus of spinach chloroplasts, and
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(iii) to find any correlation between their structure and biological activity. MATERIAL AND METHODS
Synthesis of APC Eleven alkyl-N-phenylcarbamates (alkyl 5 methyl–octyl) and three alkyl-N-phenylthiocarbamates (alkyl 5 methyl–butyl) were prepared by catalyzed reductive carbonylation of nitrobenzene with carbon oxide in presence of alcohols (17,18) or alkanethiols (19–21). Study of Photoreduction and Photooxidation of 2,6-Dichlorophenolindophenol in Spinach Chloroplasts Chloroplasts were prepared from fresh market spinach according to the method of Walker (22), partially modified by Sˇersˇenˇ et al. (23). The rate of electron transport through PS 2 in spinach chloroplasts was monitored by investigation of the photoreduction rate of 2,6-dichlorophenolindophenol (DCPIP) according to Kra´l’ova´ et al. (11). Briefly: DCPIP (40 mmol/ dm3) was added to a chloroplast suspension with 30 mg chlorophyll (Chl)/dm3 in phosphate buffer (pH 7.2). During irradiation (900 mmol m22 s21 PAR) of the above-mentioned suspension plus an appropriate amount of APC, with light from a halogen lamp (250 W) through a 5-cm water filter, a decrease in the DCPIP absorption band at 600 nm was monitored by a spectrophotometer (Specord UV VIS, Zeiss Jena, Germany). The rate of electron transport in spinach chloroplasts through photosystem 1 was monitored by investigation of the photooxidation rate of DCPIPH2 according to Xiao et al. (24). Briefly, the chloroplast suspension (30 mg Chl dm23) in phosphate buffer (pH 7.2) contained 40 mmol dm23 DCPIP, 40 mmol dm23 sodium ascorbate,
and 0.1 mmol dm23 of methylviologen, which was used as a final electron acceptor of PS 1. DCMU (0.02 mmol dm23) was added to each sample to interrupt the supply of electrons into PS 1. After irradiation of the samples prepared in this manner, which contained an appropriate amount of APC, an increase in the DCPIP absorption band was monitored as described above. In all above-mentioned experiments the APCs were added to chloroplast suspensions in the form of dimethyl sulfoxide (DMSO) solution. The maximal DMSO content in the samples (ca. 5%) had no noticeable effect on the photoreduction and photooxidation rate of DCPIP. All measurements were carried out at 258C. EPR Study of Chloroplast Suspensions EPR spectra were recorded by the instrument ERS 230 (WG, Akademie der Wissenschaften, Berlin, Germany) operating in X band at 5 mW microwave power and 0.5 mT modulation amplitude. EPR spectra of the untreated suspensions of spinach chloroplasts in the presence of the studied compounds were recorded in the dark and in the light. The chlorophyll content in samples was 3 g dm23; APCs were added in DMSO solution. The added DMSO (up to 10% vol) had no observable effect on EPR spectra of chloroplasts. The irradiation was carried out with a 250-W halogen lamp fitted with a 5-cm water filter and the intensity was 300 mmol m22 s21 PAR. All EPR experiments were carried out at 258C. RESULTS AND DISCUSSION
DCPIP photoreduction was inhibited in chloroplasts treated with APCs. The IC50 values of APCs (molar concentrations of inhibitors causing a 50% decrease of biological activity with respect to the untreated control sample) varied in the range from 8.5 (for methyl-N-phenylthiocarbamate) to 208 mmol dm23 (for isobutyl-Nphenylcarbamate). The corresponding IC50 values are presented in Table 1. By comparing the observed IC50 values with those previously published for isopropyl (1259 mmol dm23) (9) and
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EFFECT OF PHENYLCARBAMATES ON PHOTOSYNTHESIS TABLE 1 Inhibition of DCPIP Photoreduction in Chloroplasts Treated with APCs Expressed by the IC50 Values Alkyl
Log P
IC50 (mmol dm23)
Methyl Isopropyl n-Butyl sec-Butyl Isobutyl tert-Butyl n-Pentyl
1.52 2.28 2.73 2.74 2.73 2.35 3.12
10 65 30 35 208 136 74
allyl (500 mmol dm23) (6) derivatives of phenylcarbamates, it is evident that our values are lower. We suggest that this discrepancy can be caused by the higher purity of our APC samples or by the different quality of chloroplasts used. With respect to the finding that the site of DCPIP action in the photosynthetic chain of electron transport is the plastoquinone pool PQ on the acceptor side of PS 2 (25), the decreased rates of DCPIP photoreduction in the presence of APCs suggest that APCs inhibit PET through PS 2. The rate of PET through PS 1 was studied by DCPIPH2 photooxidation using its ability to supply electrons to PS 1 (25). It was found that, in chloroplasts treated with APC so that the electron transport through PS 2 was inhibited up to 90%, the rate of photooxidation of DCPIPH2 did not decrease but did the opposite. A small increase in photooxidation of DCPIPH2 was observed. To exclude any reduction of primary donor of PS 1 (P700) by electrons from PS 2, DCMU was added into the chloroplast suspension. It is known that the site of inhibitory action of DCMU is QB and therefore DCMU is able to interrupt electron transport from the primary donor of PS 2 (P680) to P700 (25). One example of the APC effect on PET through PS 1 is presented in Table 2 (third column) using the very effective compound methyl-N-phenylcarbamate. The behavior of all studied APCs was similar. From the fact that APCs do not decrease the rate of DCPIPH2 photooxidation it can be assumed that the studied compounds do not inhibit PET throug PS 1. For closer identification of the site of APC
Alkyl n-Hexyl n-Heptyl n-Octyl Allyl Methylthio n-Propylthio n-Butylthio
Log P
IC50 (mmol dm23)
3.52 3.92 4.31 2.26 1.86 2.68 3.07
107 120 175 81 8.5 35 91
action in the chain of PET, an artificial electron donor, 1,5-diphenylcarbazide (DPC), acting in the site of Z/D intermediates which are located on the donor side of PS 2, has been used (26). These intermediates secure the electron transport from the oxygen-evolving complex to P680 (27). With respect to the fact that DPC at 0.002 mol dm23 produces complete restoration of PET in chloroplasts inhibited by APC (see Table 2, fourth column), we can assume that the part of the PET chain from the intermediates Z/D through P680, pheophytin, the first and the second quinone acceptor QA and QB up to plastoquinone pool PQ, is not damaged by APCs. This finding is contradictory to that of Tischer and Strotmann (7) and Moreland (8), who suggested QB as the site of action of phenylcarbamates. EPR spectroscopy is a method which enables us to determine the interactions of some photosynthesis-inhibiting compounds with photosynthetic centers. Intact chloroplasts of higher plants exhibit, at room temperature, EPR signals in the region of free radicals (g , 2.0), known as signal I and signal II, which are connected with both photosystems PS 1 and 2 (28). To illustrate the APCs’ effects on the photosynthetic apparatus registered by EPR spectroscopy, methyl-N-phenylcarbamate was selected (Fig. 1). In the presence of APCs the intensity of the slow constituent of signal II (signal IIslow) shows a decrease (Fig. 1B, solid line). With respect to the fact that signal IIslow belongs to the intermediate D, i.e., to the tyrosine radical (TyrD) which is situated on the donor side of PS 2 in the position 161 of the D2 protein (29), it can be
´ L’OVA ´ , AND MACHO ˇ , KRA SˇERSˇEN
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TABLE 2 The Example of Methyl-N-phenylcarbamate Action upon PET through PS 2 and PS 1
Type of chloroplasts Control 155 mmol dm23 of methylN-phenylcarbamate a
Rate of DCPIP photoreduction (mmol dm23 s21 g Chl21)
Rate of DCPIPH2 photooxidation (mmol dm23 s21 g Chl21)
Rate of DCPIP photoreduction in the presence of DPCa (mmol dm23 s21 g Chl21)
15.9
18.5
16.0
1.6
20.1
15.9
23
The DPC concentration was 0.002 mol dm .
assumed that APCs interact with the intermediate D. Whereas for measurable EPR signals a relatively high chlorophyll concentration (3 g dm23) in the chloroplast sample is necessary, the applied inhibitor concentration causing the above-mentioned effect is high too (0.05 mol
dm23). The comparison of the molar ratios of Chl: methyl-N-phenylcarbamate corresponding to the 50% decrease of the investigated parameters (DCPIP photoreduction and decrease of EPR signal IIslow) shows that these ratios are comparable (3:1 for DCPIP photoreduction and 2:1 for the EPR experiment (not documented here)). Due to the interaction between APCs and intermediate D, the inhibition of the electron transport through the photosynthetic transport chain occurs, causing restricted reduction of oxidized P700. This was reflected in the EPR spectra of APC-treated chloroplasts by a large
FIG. 1. EPR spectra of untreated spinach chloroplasts (A) and chloroplasts treated with 0.05 mol dm23 of methylN-phenylcarbamate (B). The solid lines were recorded in the dark and the dashed lines were recorded in the light. Line a was registered at half the sensitivity of the other lines.
FIG. 2. The dependence of IC50 values of APC on their lipophilicity expressed as log P (M—n-alkyl-N-phenylcarbamates, C—n-alkyl-N-phenylthiocarbamates, D— branched alkyl-N-phenylcarbamates, and 1—allyl-Nphenylcarbamate).
EFFECT OF PHENYLCARBAMATES ON PHOTOSYNTHESIS
increase of signal I (Fig. 1B, dashed line). For better identification of signal I it is shown in Fig. 1B as the dotted part of the dashed line. For visualization of signal I, the EPR spectra of APC-treated chloroplasts in the light were recorded at two amplifications. This great increase of signal I is caused by interruption of the electron flow from PS 2 to PS 1 and results in the absence of P700 reduction, which is oxidized in the light to P700+. From this finding it can be assumed that APCs do not impair PS I, which is in good accordance with our finding that APCs do not effect the photooxidation of DCPIPH2. On the other hand, the fast constituent of signal II (signal IIvery fast) belonging to the intermediate Z, i.e., to the tyrosine radical (TyrZ) which is situated in the position 161 of the D1 protein on the donor side of PS 2 (29), was not affected by APCs (Fig. 1B, dashed line, the part with a longer dash). The most active inhibitor was methylthio derivative (IC50 5 8.5 mmol dm23) and, with increasing lipophilicity of the studied compounds, the photosynthesis-inhibiting activity showed a linear decrease (with r 5 0.96) for compounds with linear alkyl substituents (Fig. 2, marked as ▫ and C). This fact is illustrated in Fig. 2, where the lipophilicity is expressed by the partition coefficient octanol/water (P). The theoretical log P values were calculated using the method of Crippen and according to the work of Loos et al. (30). The inhibitory activity of compounds with branched alkyl substituents (R 5 isopropyl, tert butyl, isobutyl; Fig. 2, marked as D) was lower than that of their linear isomers. Their lower effectiveness can be connected with the fact that, for the achievement of the site of action in the photosynthetic apparatus, the branched substituents represent a higher steric hindrance than their linear isomers. Our findings differ from the results of Hansch and Deutsch (9), who found that the inhibition of the Hill reaction in chloroplasts produced by ethyl and isopropyl derivatives of N-phenylcarbamates with different substituents on the benzene ring (in positions 3 and 4) shows an increase with increasing lipophilicity of the compounds. From our results and the finding of Hansch and
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Deutsch (9) it can be concluded that for the inhibitory efficiency of APCs on photosynthesis, not only is the lipophilicity of the molecule a determining factor but also the global molecular structure. Based on our results we suggest a new site of APC action, intermediate D. On the other hand, the previously studied less hydrophobic derivatives of phenylcarbamic acid which have been applied in the form of ammonium salts interacted not only with the intermediate D, but also with the intermediate Z and with the manganese cluster (10–14) situated in the oxygen evolving complex which is located in the polar regions of thylakoid membranes. ACKNOWLEDGMENTS This study was supported by the Scientific Grant Agency of the Ministry of Education, Slovak Republic (Grant 1/ 7262/20). The authors thank Dr. D. Loos from the Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, for the calculation of log P values and Dr. D. Mikula´sˇova´ from the Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, for her assistance in the preparation of chloroplasts.
REFERENCES 1. M. Sitting (Ed.), “Pesticide Manufacturing and Toxic Materials Control Encyclopedia,” pp. 196, 254, 527, 604, 646, 695, Noyes Data corp., Park Ridge, NJ, 1980. 2. N. N. Mel’nikov, “Pesticides,” pp. 273–278, Chimia, Moscow, 1987 [in Russian]. 3. A. Trebst, Inhibitors in electron flow: Tools for the functional and structural localization of carriers and energy conservation sites, Methods Enzymol. 69C, 675 (1980). 4. J. Markovicˇ, “Zoznam povoleny´ch prı´pravkov na ochranu rastlı´n 1995 (The list of allowed preparations to protection of plants 1995),” AT Publishing, Bratislava, 1995 [in Slovak]. 5. E. D. Camper and D. E. Moreland, Correlations between acidity of substituted phenylamides and inhibition of the Hill reaction, Biochim. Biophys. Acta 94, 383 (1965). 6. J. S. C. Wessels and R. Van der Veen, The action of some derivatives of phenylurethane and of 3-phenyl1,1-dimethylurea on Hill reaction, Biochim. Biophys. Acta 19, 548 (1956). 7. W. Tischer and H. Strotmann, Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport, Biochim. Biophys. Acta 460, 113 (1977). 8. D. E. Moreland, Mechanisms of action of herbicides, Ann. Rev. Plant. Physiol. 31, 597 (1980).
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9. C. Hansch and E. W. Deutsch, The structure–activity relationship in amides inhibiting photosynthesis, Biochim. Biophys. Acta 112, 381 (1966). 10. L’. Mitterhauszerova´, K. Kra´l’ova´, F. Sˇersˇenˇ, J. Cso¨llei, and E. Krempaska´, Effect of dibasically substituted local anesthetics on photosynthesis, green algae and plants, Cˇekoslov. Farm. 40, 209 (1991) [in Slovak]. 11. K. Kra´l’ova´, F. Sˇersˇenˇ, and J. Cˇizˇma´rik, Dimethylaminoethylesters of alkoxy-substituted phenylcarbamic acids, Chem. Papers 46, 266 (1992). 12. K. Kra´l’ova´, F. Sˇersˇenˇ, and J. Cso¨llei, Inhibitory effects of some esters of 2- and 3-substituted alkoxyphenylcarbamic acids on photosynthesis, Biol. Plant. 34, 253 (1992). 13. K. Kra´l’ova´, F. Sˇersˇenˇ, and J. Cizˇma´rik, Inhibitory effect of piperidinoethylesters of alkoxyphenylcarbamic acids on photosynthesis, Gen. Physiol Biophys. 11, 261 (1992). 14. F. Sˇersˇenˇ and K. Kra´l’ova´, Concentration-dependent inhibitory and stimulating effects of amphiphilic ammonium salts upon photosynthetic activity of spinach chloroplasts. Gen. Physiol. Biophys. 15, 27 (1996). 15. F. M. Ashton and A. S. Crafts, “Mode of Action of Herbicides,” pp. 200, 289, Wiley, New York, 1973. 16. C. Fedtke, “Biochemistry and Physiology of Herbicide Action,” p. 134, Springer-Verlag, Berlin, 1982. 17. V. Macho, L. Vojcˇek, M. Schmidtova´, and Z. Zˇidek, SK Patent No. 277853 1995. 18. V. Macho, D. Mravec, M. Schmidtova´, L. Vojc´ek, and J. Hudec, SK Patent No. 279119 1998. 19. V. Macho, L. Vojcˇek, and M. Schmidtova´, SK Patent No. 277855 1995. 20. V. Macho, Reductive carbonylation and carbonylative
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New Findings about the Inhibitory Action of Phenylcarbamates and Phenylthiocarbamates on Photosynthetic Apparatus Frantisˇek Sˇersˇenˇ,*,1 Katarı´na Kra´l’ova´,* and Vendelı´n Macho† *Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska´ dolina, 842 15 Bratislava, Slovakia; and †Faculty of Industrial Technologies, University of Trencˇ´ın, T. Vansovej 1054/45, 020 32 Pu´chov, Slovakia Received February 18, 2000, accepted June 3, 2000 The inhibitory effect of alkyl-N-phenylcarbamates (alkyl 5 methyl–octyl) and alkyl-N-phenylthiocarbamates (alkyl 5 methyl–butyl) on the photosynthetic electron transport in spinach chloroplasts was studied. It was found that the site of action of the studied compounds is the intermediate D, i.e., the tyrosine radical located on site 161 in the D2 protein on the donor side of photosystem 2. The efficiency of the studied compounds was inversely proportional to the hydrophobicity of molecules, i.e., the effectiveness showed a decrease with increasing their hydrophobicity. It was found that alkyl-N-phenylcarbamates and alkyl-Nphenylthiocarbamates have the same site of action. q 2000 Academic Press
INTRODUCTION
Derivatives of phenylcarbamic acid (Scheme 1) are biologically active compounds applied mainly as pesticides (1,2). Compounds possessing the biologically active group –NH– CO–X, namely phenylureas, anilides, phenylcarbamates, and phenylthiocarbamates, belong to this group of pesticides (3). The best-known commercially applied herbicides from the above-mentioned group are Diuron or DCMU2 (3-(3,4-dichlorophenyl)-1,1dimethylurea), Pradone (carbetamide (R)-1ethylcarbamoylethyl-N-phenylcarbamate), and Betanex or Desmedipham (3-(ethoxycarbamoylanilino)-N-phenylcarbamate) (4). Camper and Moreland (5) and Wessels and Van der Veen (6) 1 To whom correspondence should be addressed. Fax: 11421-7-65428882. E-mail: [email protected]. 2 Abbreviations used: APC, alkyl-N-phenylcarbamates and alkyl-N-phenylthiocarbamates; Chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP, 2, 6-dichlorophenolindophenol; DMSO, dimethyl sulfoxide; DPC, 1,5-diphenylcarbazide; EPR, electron paramagnetic resonance; PAR, photosynthetically active radiation; PET, photosynthetic electron transport; PQ, plastoquinone pool; PS, photosystem; P680, primary donor of PS 2; P700, primary donor of PS 1, QA and QB, the first and the second quinone acceptor on the oxidized side of PS 2.
studied the effect of isopropyl and ethyl derivatives of N-phenylcarbamates with various substituents at positions 3 and 4 of the benzene ring on the Hill reaction activity of chloroplasts. It was suggested that the site of action is QB, i.e., the second quinone acceptor in herbicide binding protein D1 of photosystem (PS) 2 (7, 8). Before, the relationship between the inhibitory efficiency and the acidity (5) or the molecular structure (9) of the above-mentioned carbamates was studied. In our previous work the effects of ammonium salts of various derivatives of phenylcarbamic acid on photosynthesis were studied (10–14). Carbamates were found to be mitotic poisons that killed roots by inhibiting cell division. They are able to disturb the orientation of the spindle microtubular assembly of dividing cells (15, 16). In contrast to the carbamates, the thiocarbamates affect mainly shoots of plants. They decrease the content of long-chain fatty acid residues in plant lipids and inhibit wax formation (15, 16). This study is aimed: (i) to investigate the effect of alkyl-N-phenylcarbamates and alkyl-Nphenylthiocarbamates (APCs) on photosynthetic electron transport (PET) in spinach chloroplasts, (ii) to determine their site of action in the photosynthetic apparatus of spinach chloroplasts, and
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(iii) to find any correlation between their structure and biological activity. MATERIAL AND METHODS
Synthesis of APC Eleven alkyl-N-phenylcarbamates (alkyl 5 methyl–octyl) and three alkyl-N-phenylthiocarbamates (alkyl 5 methyl–butyl) were prepared by catalyzed reductive carbonylation of nitrobenzene with carbon oxide in presence of alcohols (17,18) or alkanethiols (19–21). Study of Photoreduction and Photooxidation of 2,6-Dichlorophenolindophenol in Spinach Chloroplasts Chloroplasts were prepared from fresh market spinach according to the method of Walker (22), partially modified by Sˇersˇenˇ et al. (23). The rate of electron transport through PS 2 in spinach chloroplasts was monitored by investigation of the photoreduction rate of 2,6-dichlorophenolindophenol (DCPIP) according to Kra´l’ova´ et al. (11). Briefly: DCPIP (40 mmol/ dm3) was added to a chloroplast suspension with 30 mg chlorophyll (Chl)/dm3 in phosphate buffer (pH 7.2). During irradiation (900 mmol m22 s21 PAR) of the above-mentioned suspension plus an appropriate amount of APC, with light from a halogen lamp (250 W) through a 5-cm water filter, a decrease in the DCPIP absorption band at 600 nm was monitored by a spectrophotometer (Specord UV VIS, Zeiss Jena, Germany). The rate of electron transport in spinach chloroplasts through photosystem 1 was monitored by investigation of the photooxidation rate of DCPIPH2 according to Xiao et al. (24). Briefly, the chloroplast suspension (30 mg Chl dm23) in phosphate buffer (pH 7.2) contained 40 mmol dm23 DCPIP, 40 mmol dm23 sodium ascorbate,
and 0.1 mmol dm23 of methylviologen, which was used as a final electron acceptor of PS 1. DCMU (0.02 mmol dm23) was added to each sample to interrupt the supply of electrons into PS 1. After irradiation of the samples prepared in this manner, which contained an appropriate amount of APC, an increase in the DCPIP absorption band was monitored as described above. In all above-mentioned experiments the APCs were added to chloroplast suspensions in the form of dimethyl sulfoxide (DMSO) solution. The maximal DMSO content in the samples (ca. 5%) had no noticeable effect on the photoreduction and photooxidation rate of DCPIP. All measurements were carried out at 258C. EPR Study of Chloroplast Suspensions EPR spectra were recorded by the instrument ERS 230 (WG, Akademie der Wissenschaften, Berlin, Germany) operating in X band at 5 mW microwave power and 0.5 mT modulation amplitude. EPR spectra of the untreated suspensions of spinach chloroplasts in the presence of the studied compounds were recorded in the dark and in the light. The chlorophyll content in samples was 3 g dm23; APCs were added in DMSO solution. The added DMSO (up to 10% vol) had no observable effect on EPR spectra of chloroplasts. The irradiation was carried out with a 250-W halogen lamp fitted with a 5-cm water filter and the intensity was 300 mmol m22 s21 PAR. All EPR experiments were carried out at 258C. RESULTS AND DISCUSSION
DCPIP photoreduction was inhibited in chloroplasts treated with APCs. The IC50 values of APCs (molar concentrations of inhibitors causing a 50% decrease of biological activity with respect to the untreated control sample) varied in the range from 8.5 (for methyl-N-phenylthiocarbamate) to 208 mmol dm23 (for isobutyl-Nphenylcarbamate). The corresponding IC50 values are presented in Table 1. By comparing the observed IC50 values with those previously published for isopropyl (1259 mmol dm23) (9) and
115
EFFECT OF PHENYLCARBAMATES ON PHOTOSYNTHESIS TABLE 1 Inhibition of DCPIP Photoreduction in Chloroplasts Treated with APCs Expressed by the IC50 Values Alkyl
Log P
IC50 (mmol dm23)
Methyl Isopropyl n-Butyl sec-Butyl Isobutyl tert-Butyl n-Pentyl
1.52 2.28 2.73 2.74 2.73 2.35 3.12
10 65 30 35 208 136 74
allyl (500 mmol dm23) (6) derivatives of phenylcarbamates, it is evident that our values are lower. We suggest that this discrepancy can be caused by the higher purity of our APC samples or by the different quality of chloroplasts used. With respect to the finding that the site of DCPIP action in the photosynthetic chain of electron transport is the plastoquinone pool PQ on the acceptor side of PS 2 (25), the decreased rates of DCPIP photoreduction in the presence of APCs suggest that APCs inhibit PET through PS 2. The rate of PET through PS 1 was studied by DCPIPH2 photooxidation using its ability to supply electrons to PS 1 (25). It was found that, in chloroplasts treated with APC so that the electron transport through PS 2 was inhibited up to 90%, the rate of photooxidation of DCPIPH2 did not decrease but did the opposite. A small increase in photooxidation of DCPIPH2 was observed. To exclude any reduction of primary donor of PS 1 (P700) by electrons from PS 2, DCMU was added into the chloroplast suspension. It is known that the site of inhibitory action of DCMU is QB and therefore DCMU is able to interrupt electron transport from the primary donor of PS 2 (P680) to P700 (25). One example of the APC effect on PET through PS 1 is presented in Table 2 (third column) using the very effective compound methyl-N-phenylcarbamate. The behavior of all studied APCs was similar. From the fact that APCs do not decrease the rate of DCPIPH2 photooxidation it can be assumed that the studied compounds do not inhibit PET throug PS 1. For closer identification of the site of APC
Alkyl n-Hexyl n-Heptyl n-Octyl Allyl Methylthio n-Propylthio n-Butylthio
Log P
IC50 (mmol dm23)
3.52 3.92 4.31 2.26 1.86 2.68 3.07
107 120 175 81 8.5 35 91
action in the chain of PET, an artificial electron donor, 1,5-diphenylcarbazide (DPC), acting in the site of Z/D intermediates which are located on the donor side of PS 2, has been used (26). These intermediates secure the electron transport from the oxygen-evolving complex to P680 (27). With respect to the fact that DPC at 0.002 mol dm23 produces complete restoration of PET in chloroplasts inhibited by APC (see Table 2, fourth column), we can assume that the part of the PET chain from the intermediates Z/D through P680, pheophytin, the first and the second quinone acceptor QA and QB up to plastoquinone pool PQ, is not damaged by APCs. This finding is contradictory to that of Tischer and Strotmann (7) and Moreland (8), who suggested QB as the site of action of phenylcarbamates. EPR spectroscopy is a method which enables us to determine the interactions of some photosynthesis-inhibiting compounds with photosynthetic centers. Intact chloroplasts of higher plants exhibit, at room temperature, EPR signals in the region of free radicals (g , 2.0), known as signal I and signal II, which are connected with both photosystems PS 1 and 2 (28). To illustrate the APCs’ effects on the photosynthetic apparatus registered by EPR spectroscopy, methyl-N-phenylcarbamate was selected (Fig. 1). In the presence of APCs the intensity of the slow constituent of signal II (signal IIslow) shows a decrease (Fig. 1B, solid line). With respect to the fact that signal IIslow belongs to the intermediate D, i.e., to the tyrosine radical (TyrD) which is situated on the donor side of PS 2 in the position 161 of the D2 protein (29), it can be
´ L’OVA ´ , AND MACHO ˇ , KRA SˇERSˇEN
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TABLE 2 The Example of Methyl-N-phenylcarbamate Action upon PET through PS 2 and PS 1
Type of chloroplasts Control 155 mmol dm23 of methylN-phenylcarbamate a
Rate of DCPIP photoreduction (mmol dm23 s21 g Chl21)
Rate of DCPIPH2 photooxidation (mmol dm23 s21 g Chl21)
Rate of DCPIP photoreduction in the presence of DPCa (mmol dm23 s21 g Chl21)
15.9
18.5
16.0
1.6
20.1
15.9
23
The DPC concentration was 0.002 mol dm .
assumed that APCs interact with the intermediate D. Whereas for measurable EPR signals a relatively high chlorophyll concentration (3 g dm23) in the chloroplast sample is necessary, the applied inhibitor concentration causing the above-mentioned effect is high too (0.05 mol
dm23). The comparison of the molar ratios of Chl: methyl-N-phenylcarbamate corresponding to the 50% decrease of the investigated parameters (DCPIP photoreduction and decrease of EPR signal IIslow) shows that these ratios are comparable (3:1 for DCPIP photoreduction and 2:1 for the EPR experiment (not documented here)). Due to the interaction between APCs and intermediate D, the inhibition of the electron transport through the photosynthetic transport chain occurs, causing restricted reduction of oxidized P700. This was reflected in the EPR spectra of APC-treated chloroplasts by a large
FIG. 1. EPR spectra of untreated spinach chloroplasts (A) and chloroplasts treated with 0.05 mol dm23 of methylN-phenylcarbamate (B). The solid lines were recorded in the dark and the dashed lines were recorded in the light. Line a was registered at half the sensitivity of the other lines.
FIG. 2. The dependence of IC50 values of APC on their lipophilicity expressed as log P (M—n-alkyl-N-phenylcarbamates, C—n-alkyl-N-phenylthiocarbamates, D— branched alkyl-N-phenylcarbamates, and 1—allyl-Nphenylcarbamate).
EFFECT OF PHENYLCARBAMATES ON PHOTOSYNTHESIS
increase of signal I (Fig. 1B, dashed line). For better identification of signal I it is shown in Fig. 1B as the dotted part of the dashed line. For visualization of signal I, the EPR spectra of APC-treated chloroplasts in the light were recorded at two amplifications. This great increase of signal I is caused by interruption of the electron flow from PS 2 to PS 1 and results in the absence of P700 reduction, which is oxidized in the light to P700+. From this finding it can be assumed that APCs do not impair PS I, which is in good accordance with our finding that APCs do not effect the photooxidation of DCPIPH2. On the other hand, the fast constituent of signal II (signal IIvery fast) belonging to the intermediate Z, i.e., to the tyrosine radical (TyrZ) which is situated in the position 161 of the D1 protein on the donor side of PS 2 (29), was not affected by APCs (Fig. 1B, dashed line, the part with a longer dash). The most active inhibitor was methylthio derivative (IC50 5 8.5 mmol dm23) and, with increasing lipophilicity of the studied compounds, the photosynthesis-inhibiting activity showed a linear decrease (with r 5 0.96) for compounds with linear alkyl substituents (Fig. 2, marked as ▫ and C). This fact is illustrated in Fig. 2, where the lipophilicity is expressed by the partition coefficient octanol/water (P). The theoretical log P values were calculated using the method of Crippen and according to the work of Loos et al. (30). The inhibitory activity of compounds with branched alkyl substituents (R 5 isopropyl, tert butyl, isobutyl; Fig. 2, marked as D) was lower than that of their linear isomers. Their lower effectiveness can be connected with the fact that, for the achievement of the site of action in the photosynthetic apparatus, the branched substituents represent a higher steric hindrance than their linear isomers. Our findings differ from the results of Hansch and Deutsch (9), who found that the inhibition of the Hill reaction in chloroplasts produced by ethyl and isopropyl derivatives of N-phenylcarbamates with different substituents on the benzene ring (in positions 3 and 4) shows an increase with increasing lipophilicity of the compounds. From our results and the finding of Hansch and
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Deutsch (9) it can be concluded that for the inhibitory efficiency of APCs on photosynthesis, not only is the lipophilicity of the molecule a determining factor but also the global molecular structure. Based on our results we suggest a new site of APC action, intermediate D. On the other hand, the previously studied less hydrophobic derivatives of phenylcarbamic acid which have been applied in the form of ammonium salts interacted not only with the intermediate D, but also with the intermediate Z and with the manganese cluster (10–14) situated in the oxygen evolving complex which is located in the polar regions of thylakoid membranes. ACKNOWLEDGMENTS This study was supported by the Scientific Grant Agency of the Ministry of Education, Slovak Republic (Grant 1/ 7262/20). The authors thank Dr. D. Loos from the Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, for the calculation of log P values and Dr. D. Mikula´sˇova´ from the Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, for her assistance in the preparation of chloroplasts.
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