J. Biosci., Vol. 3 Number 1, March 1981, pp. 23-27. © Printed in India. The inhibition of photosynthetic electron transportby methyl parathion

P. R. ANBUDURAI, R. MANNAR MANNAN and SALIL BOSEDepartment 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. Thepesticide was found to inhibit the Hill reaction with all the Hill oxidants tested, namelypotassium ferricyanide,2,6-dichlorophenol indophenol and para-benzoquinone. The concen-tration of the pesticide required to inhibit 50% of the control Hill activity (I50value) was foundto vary with the different Hill oxidants.

Keywords. Chloroplast electron transport; methyl parathion; Hill reaction.

Introduction

Insecticides are being increasingly used to improve agricultural production. Theseinsecticides, besides effectively controlling pest attack, may affect plant photo-synthesis and thereby decrease photosynthetic productivity to a considerableextent. In fact, the once widely used organochlorine insecticides have been shownto exhibit inhibitory effects on photosynthetic processes (Bowes and Gee, 1971;Bowes, 1972). Organochlorine insecticides are now being replaced mostly byorganophosphorous insecticides (Fest, 1977) and so far no study has been under-taken to find out whether this new group of pesticides has any effect on the photo-synthetic process. The present report shows that methyl parathion, one of themost widely used organophosphorous insecticides inhibits photosynthetic electrontransport 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 theoxidation of hydroquinone (BDH, Bombay) with potassium bromide. Abbreviations used: DCP1P,Dichiorophenol indophenol; DPC, Diphenyl Carbazide.

23

24 Anbudurai et al

Isolation of chloroplasts

Sorghum vulgare CV20 was grown in the University botanical garden undernatural conditions. The chloroplasts were isolated by grinding 10-day oldSorghum leaves in 50 mM phosphate buffer, pH 6.5, containing 400 mM sorbitol, 2mM EDTA; 10 mM NaCl, 5 mM MgCl2 and 1 mM MnCl2 in a Sorvall Omnimixer at50% line voltage for 5 s thrice with 5 s gaps in between. The homogenate wasfiltered through two layers of nylon cloth of 100 μ mesh size. The filtrate wascentrifuged at 2000 g for 2 min, the chloroplasts pellet washed once with thegrinding buffer and resuspended in the same buffer.

Assay for Hill reaction

The reaction mixture for the Hill reaction contained 50 mM phosphate buffer pH7.5, 100 mM sorbitol, 2 mM EDTA. 5 mM MgCl2 chloroplasts equivalent to 20-25µg of chlorophyllper ml of reaction mixture and any one of the Hill oxidants namelypotassium ferricyanide 1 mM, dichlorophenolindophenol 50 μΜ, or benzoquinone2.5 μΜ. The light-dependent oxygen evolution with a Hill oxidant was taken as ameasure 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 InstrumentsCo., Yellow Spring, Ohio, USA) hooked to a Heath (Model EU 20B) servorecorder. The chloroplasts prepared in the above manner were found to be un-coupled 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 diphenylcarbazide) after inactivating the water splitting system by heating the chloroplastsfor 2 min at 50°C. Dichlorophenolindophenol was used as an electron acceptor(Yamashita and Butler, 1968).

Light-dependent reduction of dichlorophenolindophenol was assayed photo-metrically by monitoring the decrease in the absorbance at 610 nm using aSpectronic 70 (Bausch and Lomb) spectrophotometer (Trebst, 1972).

The reaction mixture was exposed to actinic light for 30s and the actual amountof dichlorophenolindophenol reduced was estimated using an extinction coeffi- cient 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-nitrophenylthiophosphate) and also the active ingredient of methyl parathion were used in allthese experiments.

Inhibition of photosynthesis by methyl parathion 25 Results and discussion Figure 1 shows the inhibition of Hill activity of isolated, broken Sorghum vulgaremesophyll chloroplasts by methyl parathion with the three different Hill oxidants.

Figure 1. Effect of methyl parathion on the Hill reaction in isolated chloroplasts, withdifferent Hill oxidants.

Methyl parathion was found to inhibit the Hill activity with all the Hill oxidantstested, 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) wasconsiderably weaker than that of 3-(3,5-dichlorophenyl)-l, 1-dimethylurea (I50:0.05μΜ) or atrazine (I50: 0.5 μΜ) which are potent inhibitors of the Hill reaction (Izawaand Good, 1972). However methyl parathion is comparable with other Hillreaction inhibitors such as O-phenathroline which has an I 50 at 10 μΜ (Izawa andGood, 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 thewater splitting system. The observation that the I50 value for the inhibition of theHill reaction by methyl parathion varies with the different oxidants indicates thatmethyl parathion inhibits the electron transport between PS II and PS I without

26 Anbudurai et al affecting the water splitting system. This possibility was verified with diphenyl-carbazide as an artificial electron donor to the PS II reaction centre afterinactivating the water splitting system by heat treatment (table 1). The effect of

Table 1. Inhibition of the PS II reaction by methyl parathion.

a 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 isolatedchloroplasts is entirely due to methyl parathion and not because of some othersubstances present in it. This has been confirmed using the active ingredient ofmethyl parathion where the same effect as that of the commercial product wasnoted (results not shown).

The inhibition of photosynthetic electron transport in isolated chloroplasts bymethyl parathion leads to the logical question of whether methyl parathion at thelevel present in the environment has any effect on the photosynthetic processunder 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 translocatedinside 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 sprayedat the concentrations of 250-400 ppm the residue level in the plant drops down to 2-5 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 methylparathion required to inhibit 50% of the Hill reactions of isolated chloroplasts is inthe range of 25 to 47 μΜ (16-29 ppm). Thus, under field conditions, as a result ofthe quick degradation of methyl parathion, such a concentration may be notedonly for one or two days after spraying. However, a short term effect immediatelyafter spraying may still be considerable.

Inhibition of photosynthesis by methyl parathion 27

This circumstantial evidence suggests that under the field conditions, methylparathion may not inhibit the photosynthetic process as long as it is sprayed at theoptimal concentration. However when this pesticide is sprayed in higher concen-trations and/or non-uniformly, the photosynthetic process may be inhibited to aconsiderable extent. These results highlight the danger of indiscriminate sprayingand 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.