Spectronic 70 (Bausch and Lomb) spectrophotometer (Trebst, 1972). The reaction mixture was exposed to actinic light for 30s and the actual amount.
J. Biosci., Vol. 3 Number 1, March 1981, pp. 23-27. © Printed in India.
The inhibition of photosynthetic electron transport by methyl parathion P. R. ANBUDURAI, R. MANNAR MANNAN and SALIL BOSE Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021 MS received 5 July 1980; revised 6 December 1980 Abstract. The effect of methyl parathion (metacid-50), an organophosphorous insecticide, on the Hill reactions of isolated mesophyll chloroplasts of Sorghum vulgare was studied. The pesticide was found to inhibit the Hill reaction with all the Hill oxidants tested, namely potassium ferricyanide,2,6-dichlorophenol indophenol and para-benzoquinone. The concentration of the pesticide required to inhibit 50% of the control Hill activity (I50value) was found to vary with the different Hill oxidants. Keywords.
Chloroplast electron transport; methyl parathion; Hill reaction.
Introduction Insecticides are being increasingly used to improve agricultural production. These insecticides, besides effectively controlling pest attack, may affect plant photosynthesis and thereby decrease photosynthetic productivity to a considerable extent. In fact, the once widely used organochlorine insecticides have been shown to exhibit inhibitory effects on photosynthetic processes (Bowes and Gee, 1971; Bowes, 1972). Organochlorine insecticides are now being replaced mostly by organophosphorous insecticides (Fest, 1977) and so far no study has been undertaken to find out whether this new group of pesticides has any effect on the photosynthetic process. The present report shows that methyl parathion, one of the most widely used organophosphorous insecticides inhibits photosynthetic electron transport in isolated chloroplasts. Materials and methods All the chemicals used were of analar grade. They were obtained from BDH, Bombay (sorbitol and NaCl), Sarabhai. Μ Chemicals, Baroda (K3 [Fe(CN) 6 ], MgCl2, MnCl2, sodium phosphate mono- and dibasic) and from BDH, Poole, England (DCPIP and DPC). Benzoquinone was prepared in the laboratory by the oxidation of hydroquinone (BDH, Bombay) with potassium bromide.
Abbreviations used: DCP1P,Dichiorophenol indophenol; DPC, Diphenyl Carbazide.
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Isolation of chloroplasts Sorghum vulgare CV20 was grown in the University botanical garden under natural conditions. The chloroplasts were isolated by grinding 10-day old Sorghum leaves in 50 mM phosphate buffer, pH 6.5, containing 400 mM sorbitol, 2 mM EDTA; 10 mM NaCl, 5 mM MgCl2 and 1 mM MnCl2 in a Sorvall Omnimixer at 50% line voltage for 5 s thrice with 5 s gaps in between. The homogenate was filtered through two layers of nylon cloth of 100 μ mesh size. The filtrate was centrifuged at 2000 g for 2 min, the chloroplasts pellet washed once with the grinding buffer and resuspended in the same buffer. Assay for Hill reaction The reaction mixture for the Hill reaction contained 50 mM phosphate buffer pH 7.5, 100 mM sorbitol, 2 mM EDTA. 5 mM MgCl2 chloroplasts equivalent to 20-25 µg of chlorophyllper ml of reaction mixture and any one of the Hill oxidants namely potassium ferricyanide 1 mM, dichlorophenolindophenol 50 μΜ, or benzoquinone 2.5 μΜ. The light-dependent oxygen evolution with a Hill oxidant was taken as a measure of Hill activity (Trebst, 1972). The oxygen evolution was followed polarographically under saturating light (>620 nm) using a YSl 4004 Clark oxygen electrode (Yellow Spring Instruments Co., Yellow Spring, Ohio, USA) hooked to a Heath (Model EU 20B) servo recorder. The chloroplasts prepared in the above manner were found to be uncoupled as evidenced by the absence of any effect by NH4C1. Assay for PS II reaction with artificial electron donor The PS II reaction was assayed with an artificial electron donor (0.5 mM diphenyl carbazide) after inactivating the water splitting system by heating the chloroplasts for 2 min at 50°C. Dichlorophenolindophenol was used as an electron acceptor (Yamashita and Butler, 1968). Light-dependent reduction of dichlorophenolindophenol was assayed photometrically by monitoring the decrease in the absorbance at 610 nm using a Spectronic 70 (Bausch and Lomb) spectrophotometer (Trebst, 1972). The reaction mixture was exposed to actinic light for 30s and the actual amount of dichlorophenolindophenol reduced was estimated using an extinction coefficient value of 20 mM—1 (Trebst, 19721). Estimation of chlorophyll Chlorophyll was extracted in 80% acetone and estimated according to Arnon (1949). Insecticide used The commercial product Metacid-50 (R) (Bayer India Limited, Thana, Bombay) containing 50% active ingredient of methyl parathion (50% dimethyl p-nitrophenyl thiophosphate) and also the active ingredient of methyl parathion were used in all these experiments.
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Results and discussion Figure 1 shows the inhibition of Hill activity of isolated, broken Sorghum vulgare mesophyll chloroplasts by methyl parathion with the three different Hill oxidants.
Figure 1. Effect of methyl parathion on the Hill reaction in isolated chloroplasts, with different Hill oxidants.
Methyl parathion was found to inhibit the Hill activity with all the Hill oxidants tested, although the concentration of the methyl parathion required for 50% inhibition (I50 value) varied with the different Hill oxidants (figure 1). This inhibitory action of the organophosphorous insecticides (I50: 25-47 μΜ) was considerably weaker than that of 3-(3,5-dichlorophenyl)-l, 1-dimethylurea (I50:0.05 μΜ) or atrazine (I50: 0.5 μΜ) which are potent inhibitors of the Hill reaction (Izawa and Good, 1972). However methyl parathion is comparable with other Hill reaction inhibitors such as O-phenathroline which has an I 50 at 10 μΜ (Izawa and Good, 1972). The Hill reaction in chloroplasts can be inhibited in two general ways, either by blocking the electron transport between the two photosystems or by impairing the water splitting system. The observation that the I50 value for the inhibition of the Hill reaction by methyl parathion varies with the different oxidants indicates that methyl parathion inhibits the electron transport between PS II and PS I without
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affecting the water splitting system. This possibility was verified with diphenylcarbazide as an artificial electron donor to the PS II reaction centre after inactivating the water splitting system by heat treatment (table 1). The effect of Table 1.
a
Inhibition of the PS II reaction by methyl parathion.
Dichlorophenol indophenol; b Diphenyl carbazide.
The PS II reaction was assayed with diphenyl carbazide (DPC) as an electron donor to the PS II reaction centre after inactivating the water splitting system by heat treatment.
the commercial methyl parathion on photosynthetic electron transport in isolated chloroplasts is entirely due to methyl parathion and not because of some other substances present in it. This has been confirmed using the active ingredient of methyl parathion where the same effect as that of the commercial product was noted (results not shown). The inhibition of photosynthetic electron transport in isolated chloroplasts by methyl parathion leads to the logical question of whether methyl parathion at the level present in the environment has any effect on the photosynthetic process under field conditions. For methyl parathion to have any direct effect on photosynthesis under field conditions, it should first enter chloroplasts and persist there for quite some time. There are reports that the methyl parathion enters the plant and is translocated inside it (Coeffin, 1964; Attri and Rattan Lal, 1974c; Kannan and Jayaraman, 1980). But these organophosphorous insecticides are known to be degraded very rapidl within the plants (Agnihotrudu and Mithyanantha, 1978). Thus when it is sprayed at the concentrations of 250-400 ppm the residue level in the plant drops down to 25 ppm on the second day and reaches a non-detectable level in about ten days time (Attri and Rattan Lal, 1974a, b). As shown in figure 1 the concentration of methyl parathion required to inhibit 50% of the Hill reactions of isolated chloroplasts is in the range of 25 to 47 μΜ (16-29 ppm). Thus, under field conditions, as a result of the quick degradation of methyl parathion, such a concentration may be noted only for one or two days after spraying. However, a short term effect immediately after spraying may still be considerable.
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This circumstantial evidence suggests that under the field conditions, methyl parathion may not inhibit the photosynthetic process as long as it is sprayed at the optimal concentration. However when this pesticide is sprayed in higher concentrations and/or non-uniformly, the photosynthetic process may be inhibited to a considerable extent. These results highlight the danger of indiscriminate spraying and pinpoint the need for caution in using this insecticide. References Arnnon, D. I. (1949) Plant Physiol., 24, 1. Agnihotrudu, V. and Mithyanantha, M. S. (1978) Pesticide residues—A review of Indian work, (Rallis India Limited, Bangalore, India). Attri, B. S. and Rattan Lal (1974a) Indian J. Agric. Sci., 44, 361. Attri, B. S. and Rattan Lal (1974b) Indian J. Agric. Sci., 44, 481. Attri, B. S. and Rattan Lal (1974c) Indian J. Agric. Sci., 44, 816. Bowes, G. W. (1972) Plant Physiol., 49, 172 Bowes, G. W. and Gee, R. W. (1971) Bioenergetics, 2, 47 Coeffin, D. E. (1964) Residue Rev., 1, 64. Fest, D. C. (1977) Recent developments of organophosphorous pesticides, Paper presented at the I Int. Congress on Phosphorous Compounds, Rabat. Izawa, S. and Good, N. E. (1972) Methods Enzymol., B24, 355. Kannan, N. and Jayaraman, J. (1980) Pesticide residues in the environment in India, Proc. Symp., Bangalore, November 1978, UAS Tech. Series No. 32, Paper No. 52, p. 244. Trebst, A. (1972) Methods Enzymol., B24, 146. Yamashita, T. and Butler, W. L. (1968) Plant Physiol., 43, 2037.
