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Inhibition of the photosynthetic system in spinach chloroplasts by the organophosphate insecticide parathion
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
(1977)
1.263-269
Inhibition of the Photosynthetic Chloroplasts by the Organophosphate TAKASHI Pharmaceutical
Institute,
Tohoku
System in Spinach Insecticide Parathion
SUZUKI AND MITSURU UCHIYAMA
University,
Sendai, and National Japan
Received
December
Institute
of Hygienic
Sciences,
Tokyo,
30.1975
Pesticide chemicals are generally categorized as herbicides, insecticides, or fungicides, depending upon the group of pestsagainst which someselectivity of action has been demonstrated. However, since most chemicalsusually have a somewhatbroad specificity, it could not be determined that insecticides are necessarily harmless to plants. Actually, a number of organophosphorousinsecticideshave been shown to have a phytotoxic effect. Our previous studies demonstrated that parathion (O,O-diethyl-O-p-nitrophenyl phosphorothioate) could be photoreduced, dependent on ferredoxin, by spinach chloroplasts (Suzukj and Uchiyama, 1975a) and, independentof ferredoxin, by heated chloroplasts with an artificial electron donor system (Suzukj and Uchiyama, 1975b). In the latter report, the possibility that parathion inhibits photosynthetic electron transfer was suggested. Since the lives of humans and other creatures directly or indirectly depend on photosynthesisin plants, it is necessaryto investigate the possibility mentioned above. It is the purpose of this report to point out that parathion blocks electron transfer from photosystem II to photosystem I in spinach chloroplasts. It is well known that various substancesor treatments inhibit photosynthetic electron transport (Losada and Arnon, 1963; Izawa, 1972). The sitesof action of them may be divided grossly into three as indicated below: Ed I I
:~ I
H,O --I+ Region
-
Y I
P700 -
Q -I+
Quinone A Region
X -+--+ L
2
Fd ---+-+ / v
CYV plastocyanin
NADP+
Region 3
The following inhibitors and treatments are effective in selectively blocking or destroying the water oxidation step (Region 1): ammonia, methylamine, and hydroxylamine (Izawa et al., 1969), Tris washing (Yamashita and Butler, 1968, 1969), chloride removal (Hind et al., 1969), heat treatment (Homann, 1968; Katoh and San Pietro, 1968), manganesedeficiency (Kessler, 19554,and uv irradiation (Yamashita and Butler, 1968b). Chloroplasts thus treated are still capable of transferring electrons from various artificial electron donors to the standard electron acceptors. Copyright @ 1977 by Academic Press, Inc. AU rights of reproduction in any form reserved. Printed in Great Britain
263
264
SUZUKI
AND
UCHIYAMA
The following substances show inhibitory effects on the reducing side of photosystem II (Region 2) and block the electron transport from photosystem II to photosystem I: phenylureas (Wessels and Van der Veen, 1956; Duysens and Amesz, 1962), symmetrical aminotriazines (Moreland et al., 1959; Zweig et al., 1963), 2-alkyl-4hydroxyquinoline oxides (Avron, 196 l), and o-phenanthroline (Nishimura, 1966). These substances inhibit the Hill reaction with NADP+ as electron acceptor but their inhibitory effects are overcome by adding artificial electron donors for photosystem I, such as DCPIP-ascorbate and high concentration of PD-ascorbate.’ Disalicylidenepropanediamine-1,Zdisulfonic acid blocks the electron transfer from X to ferredoxin. Furthermore, ferredoxin-NADP+ reductase antibodies inactivate the reductase and inhibit NADP+ photoreduction (Berzborn et al., 1966). These inhibitions on the reducing side of photosystem I are included in Region 3.
I. EFFECT
OF PARATHION
ON HILL
REACTIONS
Parathion inhibited the Hill reactions with NADP+ and ferricyanide as electron acceptors as indicated in Table 1. At a concentration of 2.3 x 10e5 M, parathion caused an inhibition of approximately 45% on both photoreductions. Table 1 also shows that photoreducing activity of chloroplasts, to which are added othe NADP+ phenanthroline for blocking electron transfer and which are supplemented with DCPIP-ascorbate, was only slightly inhibited by parathion. This result indicates that parathion does not inhibit on the side of photosystem I but on the side of photosystem II, since it is believed that DCPIP-ascorbate is an electron donor system for photosystem I. TABLE INHIBITORY
EFFECTS
OF PARATHION
1
ON THE PHOTOREDUCTION FERRICYANIDE
OF NADP+
OR POTASSIUM
Hill reaction activity NADP+
(umollmg
Additions
Inhibition w>
Chl. hr)
Inhibition (%)
-
34.4 18.6
45.9
84.4 47.1
44.2
20.2
-
19.2
4.9
Control Parathion” Control
K-Fe-CN
reduced (umollmg Chl . hr)
DCPIP,
ascorbate,
reduced
and o-phenanthrolineb Parathiona
” Parathion was added at a concentration of 2.3 x 10e5 M. b 67 ,UM DCPIP and 6.7 mM ascorbate were added as an artificial electron donor system: 0.1 mM o-phenanthroline was also added as an inhibitor of electron flow from photosystem II to photosystem I. I Abbreviations used: Fd, ferredoxin; hydroquinone; DCP, diphenylcarbazide.
DCPIP,
2,6-dichloroindophenol;
PD, p-phenylenediamine;
HQ,
INHIBITION
OF
CHLOROPLAST
0
PHOTOSYNTHESIS
1
2
Illumination
BY
265
PARATHION
3
time
(min)
FIG. 1. Inhibitory effect of parathion on DCPIP photoreduction. Figure 1 showsthe time course of DCPIP photoreduction in the presenceor absence of parathion. The decreasein optical density at 610 nm, which is an index of DCPIP reduction, was distinctly suppressedby the addition of parathion. At 2 min of illumination, parathion causedan inhibition of approximately 30%. II.
SITE
OF INHIBITION
In order to clarify the site of action of parathion on the photosynthetic electron transport chain, the following experiment was carried out. NADP+ photoreducing activities of chloroplasts and various added electron donor systemswere assayedin the absenceof parathion or in its presenceat a concentration of 2.7 x 1O-5 M. The results are summarized in Table 2. TABLE EFFECTS
OF PARATHION
2
ACTIVITIESOFCHLOROPLASTS IN THE PRESENCEOFVARIOUSELECTRONDONORSYSTEMS ON
NADP+
PHOTOREDUCTION
NADP+
Electron donors
67 ,u~ DCPIP + 6.7 mM ascorbate 670 ,u~ PD + 67 mM ascorbate 33 PMPD f 330 ,~UM ascorbate 200 PM HQ + 330 ,UM ascorbate 0.5 mM DPC
reducedbmol/mg Chl . hr)
Control 29.9 20.3 60.4 33.1 30.2 19.7
a Parathion was added at a concentration of 2.7 x 10m5M.
Parathion” 11.6 14.7 57.1 15.5 10.1 6.0
Inhibition (%I 61.2 27.6 5.5 53.2 66.6 69.5
266
SUZUKI
AND
UCHIYAMA
Although each electron donor system more or less affected the Hill activity of chloroplasts, such an effect does not interfere with the discussion for estimating the inhibition site of parathion. When any artificial electron donor system was not added, parathion caused an inhibition of 6 1%. The inhibitory effect was partially overcome by adding DCPIP-ascorbate and almost completely by adding a higher concentration of PD-ascorbate. The restoration rate of activity by DCPIP-ascorbate was smaller in the present experiment than in the foregoing one (Table 1). It may be due to the different reaction systems in the two experiements, namely the presence or absence of ophenanthroline in the reaction medium. DCPIP-ascorbate and a higher concentration of PD-ascorbate are known to be electron donor systems for photosystem I. On the other hand, a lower concentration of PD-ascorbate, HQ-ascorbate, and DPC (Vernon and Shaw, 1969) are known to be electron donor systems for photosystem II. These electron donor systems could scarcely overcome the inhibitory effect of parathion. From the experimental results described above, it is concluded that parathion inhibition takes palce between photosystem II and photosystem I, similar to the phenylurea and triazine inhibition in Region 2 mentioned before. III. EFFECT OF
VARIOUS
CONCENTRATIONS
OF PARATHION
Figure 2 illustrates the ability of chloroplasts to reduce NADP+ in the presence of various concentrations of parathion. In this experiment, parathion at a concentration of
0
FIG. 2. Effect
of various
5
1 Parathion
concentration
concentrations
of parathion
10 (x
on NADP+
10-‘M)
photoreduction.
2.2 x lo-’ M gives approximately 50% inhibition. More than 5 x 10e5 M parathion caused only a slight increase in inhibition. It may be due to the very low solubility of parathion in water. In effect, more than 10d4 M parathion led to a noticeable error in the measurement of optical density at 340 nm because of the turbidity of the reaction medium.
INHIBITION
OF
CHLOROPLAST
PHOTOSYNTHESIS
BY
PARATHION
267
IV. INHIBITORY EFFECT OF OTHER ORGANOPHOSPHOROUS INSECTICIDES Basedon the results obtained so far, it becameof interest to determine whether or not other organophosphorous insecticides inhibit the photosynthetic electron transport in the same manner as parathion. As described in Table 3, all four insecticides tested, paraoxon, sumithion, diazinon, and disyston, at a concentration of 5 x 1O-5M, caused 20% inhibition, which was lessthan half of the parathion inhibition. Within the limits of this experiment, we can conclude that the inhibitory effect of parathion on the photosynthetic electron transport system is common to other organophosphorous insecticides. TABLE
3
INHIBITORY EFFECT OF SOME ORGANOPHOSPHOROUS INSECTICIDES ON NADP+ PHOTOREDUCTION
Insecticides”
NADP+ reduced &mol/mg Chl . hr)
Inhibition (%I
33.4
55.1
Parathion Paraoxon Sumithion Diazinon Disyston
15.0 25.8
22.8
26.1
21.9
25.7 24.5
23.1 26.1
a Each insecticide was added as 5 ,uI of an ethanolic solution at a final concentration of 5 x 10m5 M; 5 ~1 of ethanol was added to the control reaction medium.
V.
DISCUSSION
The results described here reveal that parathion blocks electron transfer from photosystem II to photosystem I and consequently inhibits the Hill activities of chloroplasts. Although a conclusion cannot be drawn due to an insufficient number of organophosphorous insecticides tested, it is likely that the inhibitory effect of parathion is a general one, of the same nature as that of other organophosphorous insecticides. Furthermore it is probable that most of the inhibitory effects on spinach chloroplasts are of a general nature affecting chloroplasts from all plants in a similar way (Izawa and Good, 1972). The inhibitory effect of organophosphorousinsecticidestested on the photosynthetic electron transport system is considerably weak as compared with that of DCMU (3(3,4-dichlorophenyl)l,l-dimethylurea) or Atrazine (2-chloro-4-(2-propylamino)-6ethylamino-S-triazine) which are potent and specific inhibitors of the Hill reaction and are used as herbicides. The concentrations of theseherbicides giving 50% inhibition of electron transport in isolated chloroplasts are: DCMU, 5 x lo-* to lo-’ M; Atrazine, 5 x 1O-7M. However, it must be rememberedthat parathion is still a fairly active inhibitor as compared with other Hill reaction inhibitors: for example, o-phenanthroline gives a 50% inhibition at about 10-j M. In a review concerning the interaction of pesticides with aquatic microorganisms, Ware and Roan (1970) described the effect of pesticides on photosynthesis in
268
SUZUKI
AND
UCHIYAMA
phytoplankton. Several workers have reported the effect of pesticides on the growth and survival of photosynthetic microorganisms (Gregory et al., 1969; Moore, 1970; Poorman, 1973). With the exception of herbicides, however, reports of this type of research in higher plants have apparently not been published to date. The ultimate source of all biological energy is solar energy which is obtained via photosynthetic reactions. Furthermore, residues of pesticides, including organophosphorous insecticides, have been widely demonstrated. Thus they occur in almost every part of the environment, for example, in air, rain, water, soil, and plants. In these respects, their effect on photosynthetic reactions should not be overlooked even if it is minor. The results in this report seem to show a new type of action of organophosphates. More detailed experiments are required in order to clarify the effects of them on photosynthetic reactions in photosynthetic microorganisms as well as in higher plants. REFERENCES AVRON, M. (1961). The inhibition of photoreactions of chloroplasts by 2-alkyl-4-hydroxyquinoline Noxides. Biochem. J. l&735-739. BERZBORN, R., MENKE, W., TREBST, A., AND PISTORIUS, E. (1966). Inhibition of photosynthetic reactions of isolated chloroplasts by chloroplast antibodies. Z. Nuturforsch. B21,1057-1059. DUYSENS, L. M. N., AND AMESZ, J. (1962). Function and identification of two photochemical systems in photosynthesis. Biochim. Biophys. Acta b&243-260. GREGORY, W. W., REED, J. K., AND PRIESTER, L. E., JR. (1969). Accumulation of parathion and DDT by some algae and protozoa. J. Protozool. 16,69-7 1. HIND, G., NAKANISHI, H. Y., AND IZAWA, S. (1969). The role of Cl- in photosynthesis. I. The Clrequirement of electron transport. Biochim. Biophys. Acta 172,277-289. HOMANN, P. (1968). Effect of manganese on the fluorescence of chloroplasts. Biochem. Biophys. Res. Commun. 33,229-234. IZAWA, S., AND GOOD, N. E. (1972). Inhibition of photosynthetic electron transport and photophosphorylation. In Methods in Enzymology (A. San Pietro, ed.), pp. 355-377. Academic Press, New York. IZAWA, S., HEATH, R. L., AND HIND, G. (1969). The role of chloride ion in photosynthesis. III. The effect of artificial electron donors upon electron transport. Biochim. Biophys. Acta 180,388-398. KATOH, S., AND SAN PIETRO, A. (1968). A comparative study of the inhibitory action on the oxygenevolution system of various chemical and physical treatments of Euglena chloroplasts. Arch. Biochem. Biophys. 128,378-386. KESSLER, E. (1955). On the role of manganese in the oxygen-evolving system of photosynthesis. Arch. Biochem. Biophys. 59,527-529. LOSADA, M., AND ARNON, D. I. (1963). Selective inhibitors of photosynthesis. In Metabolic Inhibitors (R. M. Hochster and J. H. Quastel, eds.), pp. 558-593. Academic Press, New York. MOORE, R. B. (1970). Effect of pesticides on growth and survival of Euglena gracilis Z. Bull. Environ. Contam. Toxicol. 5,226-230. MORELAND, D. E., GENTNER, W. A., AND HILTON, J. L. (1959). Mechanism of herbicidal action of 2chloro-4,6-bis(ethylamino)-S-triazine. Plant Physiol. 34,432-435. NISHIMURA, M. (1966). Oxidation-reduction reactions of cytochromes in red algae. Brookhaven Symp. Biol. 19,132-142. POORMAN, A. E. (1973). Effects of pesticides on Euglena gracilis. I. Growth studies. Bull. Environ. Contam. Toxicol. 10,25-28. SUZUKI, T., AND UCHIYAMA, M. (1975a). Photoreduction of parathion by spinach chloroplasts. Chem. Pharm. Bull. 23,2 175-2 178. SUZUKI, T., AND UCHIYAMA, M. (1975b). Photoreduction of parathion by spinach chloroplasts. II. Ferredoxin-independent photoreduction of parathon by heated chloroplasts with an artificial electron donor system. Chem. Pharm. Bull. 23,2290-2294.
INHIBITION
OF CHLOROPLAST
PHOTOSYNTHESIS
BY PARATHION
269
L. P., AND SHAW, E. R. (1969). Photoreduction of 2,6-dichlorophenohndophenol by diphenylcarbazide: A photosystem 2 reaction catalyzed by Tris-washed chloroplasts and subchloroplast fragments. Plant Physiol. 44, 1645-1649. WARE, G. W., AND ROAN, C. C. (1970). Interaction of pesticides with aquatic microorganisms and plankton. Residue Rev. 33, 15-45. WESSELS, J. S. C., AND VAN DER VEEN, R. (1956). Action of some derivatives of phenylurethan and of 3phenyl-l,l-dimethylurea on the Hill reaction. Biochim. Biophys. Acta 19,548-549. YAMASHITA, T., AND BLJTLER, W. G. (1968a). Photoreduction and photophosphorylation with Tris-washed chloroplasts. Plant Physiol. 43, 1978-1986. YAMASHITA, T., AND BUTLER, W. G. (1968b). Inhibition of chloroplasts by UV-irradiation and heattreatment. Plant Physiol. 43.2037-2040. YAMASHITA, T., AND BUTLER, W. G. (1969). Inhibition of the Hill reaction by Tris and restoration by electron donation to photosystem II. Plant Physiol. 44,435-438. ZWEIG, G., TAMAS, I., AND GREENBERG, E. (1963). The effect of photosynthesis inhibitors on oxygen evolution and fluorescence of illuminated Chlorella. Biochim. Biophys. Acta 66,196-205. VERNON,
AND
ENVIRONMENTAL
SAFETY
(1977)
1.263-269
Inhibition of the Photosynthetic Chloroplasts by the Organophosphate TAKASHI Pharmaceutical
Institute,
Tohoku
System in Spinach Insecticide Parathion
SUZUKI AND MITSURU UCHIYAMA
University,
Sendai, and National Japan
Received
December
Institute
of Hygienic
Sciences,
Tokyo,
30.1975
Pesticide chemicals are generally categorized as herbicides, insecticides, or fungicides, depending upon the group of pestsagainst which someselectivity of action has been demonstrated. However, since most chemicalsusually have a somewhatbroad specificity, it could not be determined that insecticides are necessarily harmless to plants. Actually, a number of organophosphorousinsecticideshave been shown to have a phytotoxic effect. Our previous studies demonstrated that parathion (O,O-diethyl-O-p-nitrophenyl phosphorothioate) could be photoreduced, dependent on ferredoxin, by spinach chloroplasts (Suzukj and Uchiyama, 1975a) and, independentof ferredoxin, by heated chloroplasts with an artificial electron donor system (Suzukj and Uchiyama, 1975b). In the latter report, the possibility that parathion inhibits photosynthetic electron transfer was suggested. Since the lives of humans and other creatures directly or indirectly depend on photosynthesisin plants, it is necessaryto investigate the possibility mentioned above. It is the purpose of this report to point out that parathion blocks electron transfer from photosystem II to photosystem I in spinach chloroplasts. It is well known that various substancesor treatments inhibit photosynthetic electron transport (Losada and Arnon, 1963; Izawa, 1972). The sitesof action of them may be divided grossly into three as indicated below: Ed I I
:~ I
H,O --I+ Region
-
Y I
P700 -
Q -I+
Quinone A Region
X -+--+ L
2
Fd ---+-+ / v
CYV plastocyanin
NADP+
Region 3
The following inhibitors and treatments are effective in selectively blocking or destroying the water oxidation step (Region 1): ammonia, methylamine, and hydroxylamine (Izawa et al., 1969), Tris washing (Yamashita and Butler, 1968, 1969), chloride removal (Hind et al., 1969), heat treatment (Homann, 1968; Katoh and San Pietro, 1968), manganesedeficiency (Kessler, 19554,and uv irradiation (Yamashita and Butler, 1968b). Chloroplasts thus treated are still capable of transferring electrons from various artificial electron donors to the standard electron acceptors. Copyright @ 1977 by Academic Press, Inc. AU rights of reproduction in any form reserved. Printed in Great Britain
263
264
SUZUKI
AND
UCHIYAMA
The following substances show inhibitory effects on the reducing side of photosystem II (Region 2) and block the electron transport from photosystem II to photosystem I: phenylureas (Wessels and Van der Veen, 1956; Duysens and Amesz, 1962), symmetrical aminotriazines (Moreland et al., 1959; Zweig et al., 1963), 2-alkyl-4hydroxyquinoline oxides (Avron, 196 l), and o-phenanthroline (Nishimura, 1966). These substances inhibit the Hill reaction with NADP+ as electron acceptor but their inhibitory effects are overcome by adding artificial electron donors for photosystem I, such as DCPIP-ascorbate and high concentration of PD-ascorbate.’ Disalicylidenepropanediamine-1,Zdisulfonic acid blocks the electron transfer from X to ferredoxin. Furthermore, ferredoxin-NADP+ reductase antibodies inactivate the reductase and inhibit NADP+ photoreduction (Berzborn et al., 1966). These inhibitions on the reducing side of photosystem I are included in Region 3.
I. EFFECT
OF PARATHION
ON HILL
REACTIONS
Parathion inhibited the Hill reactions with NADP+ and ferricyanide as electron acceptors as indicated in Table 1. At a concentration of 2.3 x 10e5 M, parathion caused an inhibition of approximately 45% on both photoreductions. Table 1 also shows that photoreducing activity of chloroplasts, to which are added othe NADP+ phenanthroline for blocking electron transfer and which are supplemented with DCPIP-ascorbate, was only slightly inhibited by parathion. This result indicates that parathion does not inhibit on the side of photosystem I but on the side of photosystem II, since it is believed that DCPIP-ascorbate is an electron donor system for photosystem I. TABLE INHIBITORY
EFFECTS
OF PARATHION
1
ON THE PHOTOREDUCTION FERRICYANIDE
OF NADP+
OR POTASSIUM
Hill reaction activity NADP+
(umollmg
Additions
Inhibition w>
Chl. hr)
Inhibition (%)
-
34.4 18.6
45.9
84.4 47.1
44.2
20.2
-
19.2
4.9
Control Parathion” Control
K-Fe-CN
reduced (umollmg Chl . hr)
DCPIP,
ascorbate,
reduced
and o-phenanthrolineb Parathiona
” Parathion was added at a concentration of 2.3 x 10e5 M. b 67 ,UM DCPIP and 6.7 mM ascorbate were added as an artificial electron donor system: 0.1 mM o-phenanthroline was also added as an inhibitor of electron flow from photosystem II to photosystem I. I Abbreviations used: Fd, ferredoxin; hydroquinone; DCP, diphenylcarbazide.
DCPIP,
2,6-dichloroindophenol;
PD, p-phenylenediamine;
HQ,
INHIBITION
OF
CHLOROPLAST
0
PHOTOSYNTHESIS
1
2
Illumination
BY
265
PARATHION
3
time
(min)
FIG. 1. Inhibitory effect of parathion on DCPIP photoreduction. Figure 1 showsthe time course of DCPIP photoreduction in the presenceor absence of parathion. The decreasein optical density at 610 nm, which is an index of DCPIP reduction, was distinctly suppressedby the addition of parathion. At 2 min of illumination, parathion causedan inhibition of approximately 30%. II.
SITE
OF INHIBITION
In order to clarify the site of action of parathion on the photosynthetic electron transport chain, the following experiment was carried out. NADP+ photoreducing activities of chloroplasts and various added electron donor systemswere assayedin the absenceof parathion or in its presenceat a concentration of 2.7 x 1O-5 M. The results are summarized in Table 2. TABLE EFFECTS
OF PARATHION
2
ACTIVITIESOFCHLOROPLASTS IN THE PRESENCEOFVARIOUSELECTRONDONORSYSTEMS ON
NADP+
PHOTOREDUCTION
NADP+
Electron donors
67 ,u~ DCPIP + 6.7 mM ascorbate 670 ,u~ PD + 67 mM ascorbate 33 PMPD f 330 ,~UM ascorbate 200 PM HQ + 330 ,UM ascorbate 0.5 mM DPC
reducedbmol/mg Chl . hr)
Control 29.9 20.3 60.4 33.1 30.2 19.7
a Parathion was added at a concentration of 2.7 x 10m5M.
Parathion” 11.6 14.7 57.1 15.5 10.1 6.0
Inhibition (%I 61.2 27.6 5.5 53.2 66.6 69.5
266
SUZUKI
AND
UCHIYAMA
Although each electron donor system more or less affected the Hill activity of chloroplasts, such an effect does not interfere with the discussion for estimating the inhibition site of parathion. When any artificial electron donor system was not added, parathion caused an inhibition of 6 1%. The inhibitory effect was partially overcome by adding DCPIP-ascorbate and almost completely by adding a higher concentration of PD-ascorbate. The restoration rate of activity by DCPIP-ascorbate was smaller in the present experiment than in the foregoing one (Table 1). It may be due to the different reaction systems in the two experiements, namely the presence or absence of ophenanthroline in the reaction medium. DCPIP-ascorbate and a higher concentration of PD-ascorbate are known to be electron donor systems for photosystem I. On the other hand, a lower concentration of PD-ascorbate, HQ-ascorbate, and DPC (Vernon and Shaw, 1969) are known to be electron donor systems for photosystem II. These electron donor systems could scarcely overcome the inhibitory effect of parathion. From the experimental results described above, it is concluded that parathion inhibition takes palce between photosystem II and photosystem I, similar to the phenylurea and triazine inhibition in Region 2 mentioned before. III. EFFECT OF
VARIOUS
CONCENTRATIONS
OF PARATHION
Figure 2 illustrates the ability of chloroplasts to reduce NADP+ in the presence of various concentrations of parathion. In this experiment, parathion at a concentration of
0
FIG. 2. Effect
of various
5
1 Parathion
concentration
concentrations
of parathion
10 (x
on NADP+
10-‘M)
photoreduction.
2.2 x lo-’ M gives approximately 50% inhibition. More than 5 x 10e5 M parathion caused only a slight increase in inhibition. It may be due to the very low solubility of parathion in water. In effect, more than 10d4 M parathion led to a noticeable error in the measurement of optical density at 340 nm because of the turbidity of the reaction medium.
INHIBITION
OF
CHLOROPLAST
PHOTOSYNTHESIS
BY
PARATHION
267
IV. INHIBITORY EFFECT OF OTHER ORGANOPHOSPHOROUS INSECTICIDES Basedon the results obtained so far, it becameof interest to determine whether or not other organophosphorous insecticides inhibit the photosynthetic electron transport in the same manner as parathion. As described in Table 3, all four insecticides tested, paraoxon, sumithion, diazinon, and disyston, at a concentration of 5 x 1O-5M, caused 20% inhibition, which was lessthan half of the parathion inhibition. Within the limits of this experiment, we can conclude that the inhibitory effect of parathion on the photosynthetic electron transport system is common to other organophosphorous insecticides. TABLE
3
INHIBITORY EFFECT OF SOME ORGANOPHOSPHOROUS INSECTICIDES ON NADP+ PHOTOREDUCTION
Insecticides”
NADP+ reduced &mol/mg Chl . hr)
Inhibition (%I
33.4
55.1
Parathion Paraoxon Sumithion Diazinon Disyston
15.0 25.8
22.8
26.1
21.9
25.7 24.5
23.1 26.1
a Each insecticide was added as 5 ,uI of an ethanolic solution at a final concentration of 5 x 10m5 M; 5 ~1 of ethanol was added to the control reaction medium.
V.
DISCUSSION
The results described here reveal that parathion blocks electron transfer from photosystem II to photosystem I and consequently inhibits the Hill activities of chloroplasts. Although a conclusion cannot be drawn due to an insufficient number of organophosphorous insecticides tested, it is likely that the inhibitory effect of parathion is a general one, of the same nature as that of other organophosphorous insecticides. Furthermore it is probable that most of the inhibitory effects on spinach chloroplasts are of a general nature affecting chloroplasts from all plants in a similar way (Izawa and Good, 1972). The inhibitory effect of organophosphorousinsecticidestested on the photosynthetic electron transport system is considerably weak as compared with that of DCMU (3(3,4-dichlorophenyl)l,l-dimethylurea) or Atrazine (2-chloro-4-(2-propylamino)-6ethylamino-S-triazine) which are potent and specific inhibitors of the Hill reaction and are used as herbicides. The concentrations of theseherbicides giving 50% inhibition of electron transport in isolated chloroplasts are: DCMU, 5 x lo-* to lo-’ M; Atrazine, 5 x 1O-7M. However, it must be rememberedthat parathion is still a fairly active inhibitor as compared with other Hill reaction inhibitors: for example, o-phenanthroline gives a 50% inhibition at about 10-j M. In a review concerning the interaction of pesticides with aquatic microorganisms, Ware and Roan (1970) described the effect of pesticides on photosynthesis in
268
SUZUKI
AND
UCHIYAMA
phytoplankton. Several workers have reported the effect of pesticides on the growth and survival of photosynthetic microorganisms (Gregory et al., 1969; Moore, 1970; Poorman, 1973). With the exception of herbicides, however, reports of this type of research in higher plants have apparently not been published to date. The ultimate source of all biological energy is solar energy which is obtained via photosynthetic reactions. Furthermore, residues of pesticides, including organophosphorous insecticides, have been widely demonstrated. Thus they occur in almost every part of the environment, for example, in air, rain, water, soil, and plants. In these respects, their effect on photosynthetic reactions should not be overlooked even if it is minor. The results in this report seem to show a new type of action of organophosphates. More detailed experiments are required in order to clarify the effects of them on photosynthetic reactions in photosynthetic microorganisms as well as in higher plants. REFERENCES AVRON, M. (1961). The inhibition of photoreactions of chloroplasts by 2-alkyl-4-hydroxyquinoline Noxides. Biochem. J. l&735-739. BERZBORN, R., MENKE, W., TREBST, A., AND PISTORIUS, E. (1966). Inhibition of photosynthetic reactions of isolated chloroplasts by chloroplast antibodies. Z. Nuturforsch. B21,1057-1059. DUYSENS, L. M. N., AND AMESZ, J. (1962). Function and identification of two photochemical systems in photosynthesis. Biochim. Biophys. Acta b&243-260. GREGORY, W. W., REED, J. K., AND PRIESTER, L. E., JR. (1969). Accumulation of parathion and DDT by some algae and protozoa. J. Protozool. 16,69-7 1. HIND, G., NAKANISHI, H. Y., AND IZAWA, S. (1969). The role of Cl- in photosynthesis. I. The Clrequirement of electron transport. Biochim. Biophys. Acta 172,277-289. HOMANN, P. (1968). Effect of manganese on the fluorescence of chloroplasts. Biochem. Biophys. Res. Commun. 33,229-234. IZAWA, S., AND GOOD, N. E. (1972). Inhibition of photosynthetic electron transport and photophosphorylation. In Methods in Enzymology (A. San Pietro, ed.), pp. 355-377. Academic Press, New York. IZAWA, S., HEATH, R. L., AND HIND, G. (1969). The role of chloride ion in photosynthesis. III. The effect of artificial electron donors upon electron transport. Biochim. Biophys. Acta 180,388-398. KATOH, S., AND SAN PIETRO, A. (1968). A comparative study of the inhibitory action on the oxygenevolution system of various chemical and physical treatments of Euglena chloroplasts. Arch. Biochem. Biophys. 128,378-386. KESSLER, E. (1955). On the role of manganese in the oxygen-evolving system of photosynthesis. Arch. Biochem. Biophys. 59,527-529. LOSADA, M., AND ARNON, D. I. (1963). Selective inhibitors of photosynthesis. In Metabolic Inhibitors (R. M. Hochster and J. H. Quastel, eds.), pp. 558-593. Academic Press, New York. MOORE, R. B. (1970). Effect of pesticides on growth and survival of Euglena gracilis Z. Bull. Environ. Contam. Toxicol. 5,226-230. MORELAND, D. E., GENTNER, W. A., AND HILTON, J. L. (1959). Mechanism of herbicidal action of 2chloro-4,6-bis(ethylamino)-S-triazine. Plant Physiol. 34,432-435. NISHIMURA, M. (1966). Oxidation-reduction reactions of cytochromes in red algae. Brookhaven Symp. Biol. 19,132-142. POORMAN, A. E. (1973). Effects of pesticides on Euglena gracilis. I. Growth studies. Bull. Environ. Contam. Toxicol. 10,25-28. SUZUKI, T., AND UCHIYAMA, M. (1975a). Photoreduction of parathion by spinach chloroplasts. Chem. Pharm. Bull. 23,2 175-2 178. SUZUKI, T., AND UCHIYAMA, M. (1975b). Photoreduction of parathion by spinach chloroplasts. II. Ferredoxin-independent photoreduction of parathon by heated chloroplasts with an artificial electron donor system. Chem. Pharm. Bull. 23,2290-2294.
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