Planta (Berl.) 104, 210-219 (1972) 9 by Springer-Verlag 1972
The Effect of Paraquat on Flax Cotyledon Leaves: Physiological and Biochemical Changes N. Harris and A. D. Dodge School of Biological Sciences, University of Bath, Bath, Somerset, U.K. Received December 22, 1971; January 24, 1972 Summary. Some of the physiological and biochemical changes which were found to occur during paraquat (1,1'-dimethyl-4,41-bipyridylium ion) treatment of cotyledon leaves of Linium usitatissimum, are reported. Results showed an inhibition of photosynthetic CO2 uptake and electron flow of isolated chloroplasts. An increase in membrane permeability, changes in the level of the major lipid components and of malondiald~hyde. These are correlated with ultrastructural changes and the discussion includes a proposed mode of action for the herbicides.
Introduction I t is generally accepted that the primary toxic action of the bipyridylium herbicides, paraquat and diquat is a result of the deviation of photosynthetic electron transport within the chloroplast (Akhavein and Linscott, 1968; Calderbank, 1968). In place of a reduction of the natural oxidant, NADP +, bipyridylium ions are reduced to free radicals by a one electron process. Immediate reoxidation follows in the presence of oxygen, to yield hydrogen peroxide (Davenport, 1963). The continued production of the hydrogen peroxide is dependent upon the maintenance of photosynthetic electron transport. Thus, Mees (1960) noted a considerable retardation of herbicidal kill in the presence of the photosynthetic electron transport inhibitor, CMU. However it was reported (Baldwin, Dodge and Harris, 1968) that much of the structural and component breakdown of plant tissue occurred after the cessation of photosynthetic electron transport. The results below concern some of the sequential changes which were found to occur following the paraquat treatment of flax cotyledon leaves. The changes are correlated with ultrastructural changes reported in the previous paper (Harris and Dodge, 1972) and the discussion is concerned with a proposed mode of action for the bipyridylium herbicides. Materials and Methods Flax (Linium usitatissimum var. Redwing) seeds were sown on vermucilite, and maintained under constant illumination. Cotyledon leaf pairs with a few rams of hypocotyl were cut from 8 to 10 days old seedlings and floated on paraquat
Effect of Paraquat on Cotyledon
211
(1 • 10 -4 M) or distilled water, as required. Illumination (2250 lnx) was provided by " d a y glow" neon tubes, and treatments were carried out under constant temperature (24 ~ C • 1~ Leaf pigments were estimated spectrophotometrically after extraction into 80% acetone. Chlorophylls a and b by the method of Arnon (1949) phaeophytins by the method of Vernon (1960) and earotenoids by the method of Kirk and Allen (1965). Total lipids were extracted in chloroform:methanol following the method of Bligh and Dyer (1959). The lipids were separated by thin layer chromatography with silica-gel G (Merk) with plates routinely activated at l l 0 ~ for 20min. Di-isobutyl ketone; acetic acid: water at 80:50:7 was used as solvent (Marinetti, Erbland and Stotz, 1958). The plates were sprayed with phosphomolybdie acid and charred to show total lipid, molybdate-perchloric acid to show phospholipids (Skidmore and Entenman, 1962) and periodate-Schiff reagent, for ~ glycols. After drying the plates in vacuo, further analysis of components was achieved after elution by chloroform:methanol, 1:1. Analysis of the glycolipid followed the method of Brundish, Shaw and Baddiley (1967) with fatty acid methyl esters examined by gas liquid chromatography. Malondialdehyde, a breakdown product of unsaturated fatty acid hydroperoxides, was estimated by the use of thiobarbituric acid (TBA) as described by Heath and Packer (1968). 0.2 gm of cotyledon leaves were ground in 10 ml of distilled water in a mortar, and 3 ml of this preparation was incubated with 5 ml of 0.5% TBA in 20% TCA for 30 min at 95 ~ C. After cooling and eentrifugation to give a clear supernatant the extinction (E) of the solution was measured at 532 and 600 nm. Following E 532 n m - - E 600 nm, to correct for nonspecific absorption, the level of malondialdehyde was estimated by using the mM extinction coefficient of 155 m ~ -~ cm -1 (Heath and Packer, 1968). The rate of potassium efflux from tissue slices was used as an indicator of membrane permeability (Eilam, 1965). 1 mm slices of cotyledon leaves were washed and incubated in distilled water at 20 ~ C. Aliquots of the water were taken at intervals, and the potassium levels estimated by flame photometry. The carbon dioxide exchange of flax cotyledon leaves was measured by using an infra-red gas analyser (Grubb Parsons Ltd.). The plant material was contained within a small perspex chamber (5 • 15 • 100 ram) maintained at constant temperature and humidity. Illumination was provided by a 500 W lamp. Chloroplasts were isolated by the method of Hill and Walker (1959) and photosynthetic electron flow activity was assayed by following the reduction of ferrycyanide as a decrease in E at 420 nm. (Jagendorf and Margulies, 1960). A standard reaction mixture contained, in addition to the chloroplast preparation, 90 ~moles Tris-chloride buffer pH 7.7; 10 ~moles Sodium chloride; 2 i~moles potassium ferricyanide. Illumination was provided by white light of 50 Klux at 20 ~ C.
Results P h o t o s y n t h e t i c c a r b o n d i o x i d e u p t a k e b y f l a x c o t y l e d o n l e a v e s was c o m p l e t e l y i n h i b i t e d w i t h i n 5 h p a r a q u a t t r e a t m e n t , i r r e s p e c t i v e of w h e t h e r t h e l e a v e s w e r e i n c u b a t e d in l i g h t or d a r k n e s s . A t a b o u t t h i s t i m e t h e r e was a g r a d u a l rise in c a r b o n d i o x i d e e v o l u t i o n w h i c h w a s also i n d e p e n d e n t of l i g h t c o n d i t i o n s (Fig. 1). T h e i n h i b i t i o n of p h o t o s y s t e m I I a c t i v i t y , m e a s u r e d as f e r r i c y a n i d e r e d u c t i o n b y c h l o r o p l a s t s i s o l a t e d f r o m p a r a q u a t t r e a t e d leaves, was
212
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Fig. 1. Carbon dioxide uptake and evolution by paraquat treated flax cotyledon leaves. The leaves were incubated with paraquat (1 • 10 -t) in either light o or darkness., and the uptake and evolution of these leaves estimated after treatment
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Treatment
Fig. 2. Ferricyanide reduction by chloroplasts isolated from paraquat treated flax cotyledon leaves, o Leaves treated under light; 9 leaves in darkness
originally shown to be temperature dependent (Baldwin, Dodge and Harris, 1968). With treatment at 24 ~ C, there was no further activity after 19 h eontinuous illumination (Fig. 2). When the illumination
Effect of Paraquat on Cotyledon
213
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Fig. 3. Changes in the level of leaf pigments during the paraquat treatment of flax cotyledon leaves under constant illumination
period was shortened, the loss of activity of isolated chloroplasts was retarded. When a 15 h light incubation period was followed by darkness, the subsequent decline in activity was similar to incubation under continuous illumination. The effect of paraquat treatment on the chlorophyll, carotenoid and phaeophytin levels of flax cotyledon leaves, treated under constant illumination, is shown in Fig. 3. I t is apparent that there was no major breakdown of chlorophylls during the first 20 h treatment. This is an interesting contrast to Fig. 2, and indicates that the major breakdown of the chlorophylls occurred after the cessation of photosynthetic electron transport. Fig. 4 shows that the further breakdown of the
214
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Fig. 4. The effect of light and dark periods on the chlorophyll a and b levels of paraquat treated flax cotyledon leaves, o light treatment;, dark treatment
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Hours Paraquat Treatment Fig. 5. Membrane permeability measured as potassium leakage from slices of paraquat treated flax cotyledon leaves chlorophyll pigments was light dependent, and followed the breakdown of much of the carotenoid pigments. The increased potassium efflux from tissue slices of paraquat treated flax cotyledon leaves, was one of the early manifestations of the applica-
Effect of Paraquat on Cotyledon
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1'8 2'7 3'6 Paraquc~t Treatment
Fig. 6. Malondialdehyde production, as a measure of lipid peroxidation in paraquat treated flax cotyledon leaves tion of paraquat. There was a gradual leakage after 3 h treatment, but the period between 5 and 7 h was marked by a rapid and sudden rise. There was no further liberation after this point (Fig. 5). At about the same time as the increase in potassium efflux, there was a rise in the level of malondialdehyde present (Fig. 6) which continued for the next 30 to 40 h. This was the first indication of an effect of paraquat treatment on the lipid composition of the leaves. Thin layer chromatography of the total lipids extracted from treated leaves showed that the major components were mono- and di-galaetosyl diglyeerides, the phospholipids-phosphatidylethanolamine and phosphatidylcholine, neutral lipids and pigments. Major changes were shown after 36 h paraquat treatment (Fig. 7) when a breakdown of the digalactosyl diglyeeride, phosplipids and pigments was evident. Similarities in f a t t y acid composition, as shown by gas liquid chromatography, indicated t h a t a monogalaetosyl diglyceride was derived from digalaetosyl diglyceride at this time. Further breakdown of both galaetosyl and phospholipids to mono- and diglyeerides occurred within the next 24 h. Discussion
The inhibition of photosynthetic carbon dioxide uptake within 5 h of the application of paraquat was thought to be a direct result of the interaction of these molecules with photosynthetic electron transport
216
N. Harris and A. D. Dodge
Fig. 7. Silica-gel G plates of lipid extracts from control (C) and 36 h paraquat treated (P) flax cotyledon leaves. (i) Sprayed with phosphomolybdie acid. (ii) Sprayed with periodate--Schiff reagent. (iii) Sprayedlwith molybdate-- perchlorie acid with the consequent inhibition of NADP § reduction (Zweig, Shavit and Avron, 1965). The time to total inhibition was probably based on that taken for complete penetration of the herbicide into the cotyledon leaves, a process which was, in these experiments, independent of light. The uptake of the bipyridylinm herbicides by whole plants, on the other hand, may be increased in darkness (Brian, 1967). The increase in potassium efflux and the rise in the level of malondialdehyde occurred next during the course of herbicidal action. The rise in potassium efflux probably represented a change in permeability of the tonoplast and plasmalemma which resulted in its release from the treated tissue. Malondialdehyde is a breakdown product of tri-unsaturated fatty acid hydroperoxides (Patton and Kurtz, 1951; Kohn and Liversedge, 1944) and its rise at this time may also be related to the membrane permeability changes. Electron mierographs of flax cotyledon leaves, treated with paraquat under identical conditions, showed after 6 h the rupture of the tonoplast in many cells of the upper spongy mesophyll (Harris and Dodge, 1972). I t is considered that the products of the interaction of the herbicide with photosynthetic electron transport, which include hydrogen peroxide and peroxy and hydroxyl free radicals derived from the hydrogen peroxide, attacked the lipids of the cell membranes, and in particular the tonoplast where it was located in
Effect of P a r a q u a t on Cotyledon
217
close proximity to the chloroplasts. The free radicals probably instigated a deteriorative chain reaction in the membrane, which led to its rapid destruction with malondialdehyde being an eventual product. Neish (1939) reported that catalase was associated with the chloroplast and Black and Myers (1966) suggested that the hydrogen peroxide formed by interaction of the bipyridylinm herbicides with the photosynthetic electron transport system would be rapidly broken down. More recent evidence however, points to this enzyme being associated with extra chloroplastic bodies, the peroxisomes (Tolbert, Oeser, Kisaki, Hageman and Yamazaki, 1968) (Yamazaki and Tolbert, 1970) and being almost totally absent from the chloroplasts (Gregory, 1968). A result of the rupture of the tonoplast would be a loss of the normal cell compartmentalisation and the cell organelles would presumably be subjected to severe osmotic changes. Although mitochondrial activity was not assayed in these experiments, electron micrographs showed swelling of the mitochondria and other cytoplasmic organelles soon after tonoplast rupture, (Harris and Dodge, 1972) and by about 10 h they were apparently no longer present in the disorganised cytoplasm. The complete loss of photosynthetic electron transport from water occurred after 19 h paraquat treatment under continuous illumination although photosystem I alone was potentially operative for a few h longer (Baldwin et al., 1968). It is assumed that after 19 h treatment there would be no further production of toxic products by the normally accepted interaction of paraquat with photosynthetic electron transport. It is significant that considerable structural breakdown occurred after 19 h treatment (Harris and Dodge, 1972) and also the light promoted breakdown of the chlorophylls occurred after this point when the level of carotenoid had dropped considerably. It is possible that the photooxidation of the chlorophylls occurred due to the loss of the normal carotenoid protective mechanism (Koski and Smith, 1951; Anderson and Robertson, 1970). Heath and Packer (1968) demonstrated that isolated chloroplasts could undergo a cyclic peroxidation initiated by light, and suggested that this was a result of an overloading of the excited chlorophyll energy trapping system. In their system chlorophyll was bleached, there was a consumption of oxygen and malondialdehyde was produced. It is possible that after the cessation of electron transport, and thus without the mediation of paraquat, this type of photoinduced pcroxidation was responsible for considerable cellular damage. However, the rapidity of the structural breakdown would suggest that other destructive processes were in operation. Matile (1968) reported that the plant cell vacuole contained a number of hydrolytic enzymes, including proteases, hue]eases, phosphatases and esterases. The continued and rapid breakdown of celhflar components could therefore be partly due 15 Planta (Berl.), Bd. 104
218
N. Harris and A. D. Dodge:
t o t h e release of these enzymes after t h e r u p t u r e of t h e t o n o p l a s t . A l t e r n a t i v e l y t h e osmotic u p s e t following t o n o p l a s t b r e a k d o w n w o u l d cause t h e r u p t u r e of l y s o s o m e - l i k e organelles w i t h i n t h e c y t o p l a s m a n d l e a d to a similar effect. I n either r e s p e c t t h e r u p t u r e of t h e t o n o p l a s t is a significant event, a n d i t is considered t h a t i t is this e v e n t a t a n e a r l y stage of t h e herbicide t r e a t m e n t , t h a t leads to t h e i r r e v e r s i b i l i t y of t h e action. Acknowledgement is made to the S.R.C. for a research studentship to N.H. and to Plant Protection Ltd., Jealott's Hill who were associated with this work under the C.A.P.S. scheme. References Akhavein, A.A., Linscott, D . L . : The dipyridylium herbicides, paraquat and diquat. Residue Rev. 23, 97-i45 (1968). Anderson, I. C., Robertson, D. S.: Role of carotenoids in protecting chlorophyll from photodestruction. Plant Physiol. 35, 531-534 (1960). Arnon, D . I . : Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). Baddiley, J., Buchanan, J. G., Handsehumaker, R. E., Prescott, J . F . : Chemical studies in the biosynthesis of purine nucleotides I. The preparation of Nglycylglycosylamines. J. chem. Soc. 2818-2823 (1956). Baldwin, B. C., Dodge, A. D., Harris, N. : Recent advances in studies of the mode of action of the bipyridylium herbicides. Proc. 9th Br. Weed Control Conf. 639-644 (1968). Black, C. C., Myers, L. : Some biochemical aspects of the mechanisms of herbicidal activity. Weeds 14, 331-338 (1966). Bligh, E. G., Dyer, W. J. : A rapid method of total lipid extraction and purification. Canad. J. Biochem. Physiol. 37, 911-917 (1959). Brian, R. C. : Darkness and the activity of diquat and paraquat on tomato, broad bean and sugar beet. Ann. appl. Biol. 60, 77-85 (1967). Brundish, D. E., Shaw, N., Baddiley, J.: The structure and possible function of the glycolipid from Staphylococcus lactis 13. Biochem. J. 105, 885-889 (1967). Calderbank, A. : The bibyridylium herbicides. Advanc. Pest Control Res. 8, 127235 (1968). Davenport, H . E . : The mechanism of cyclic phosphorylation by illuminated chloroplasts. Proc. roy. Soc. B 157, 332-345 {1963). Eilam, Y. : Permeability changes in senescing tissue. J. exp. Bot. 16, 614-627 (1965). Gregory, R. P . F . : An improved preparative method for spinach catalase and evaluation of some of its properties. Bioehim. biophys. Acta (Amst.) 159, 429439 (1968). Harris, N., Dodge, A. D. : The effect of paraquat on flax cotyledon leaves: Changes in fine structure. Planta (Berl.)104, 201-209 (1972). Heath, R. L., Packer, L. : Photoperoxidation in isolated chloroplasts. I. Kinetics and stochiometry of fatty acid peroxidation. Arch. Biochem. 125, 189-198 (1968). Hill, R , Walker, D.A.: Pyocyaninc and phosphorylation with chloroplasts. Plant Physiol. 34, 240-245 (1959). Jagendorf, A.T., Margulies, M.M.: Inhibition of spinach chloroplast photochemical reactions by p-chlorophenyl 1-1, dimethyl urea. Arch. Biochem. 90, 184-195 (1960).
Effect of Paraquat on Cotyledon
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Kirk, J. T. 0., Allen, 1~. L.: Dependence of chloroplast pigment synthesis on protein synthesis: effect of actidione. Biochem. biophys. Res. Commun. 21, 523-530 (1965). Kohn, H., Liversedge, M.: On a new aerobic metabolite whose production by brain is inhibited by amorphine, emetrine, ergotamine, epinephrine and menadione. J. Pharmacol. exp. Ther. 82, 292-300 (1944). Koski, V. M., Smith, J. H. C. : Chlorophyll formation in a mutant, white seeding. Arch. Biochem. 84, 189-195 (1951). Marinetti, G. V., Erbland, J., Stotz, E.: Phosphatides of pig heart cell fractions. J. biol. Chem. 288, 562-565 (1958). Matile, Ph.: Enzyme der Vakuolen und Wurzelzellen yon Maiskeimlingen. Ein Beitrag zur funktionellen Bedeutung der Vakuole bei der intrazellularen Verdaunng. Z. Naturforsch. 21b, 871-878 (1966). Mees, G. C. : Experiments on the herbicidal action of 1,1-ethylene-2,2-dipyridylium dibromide. Ann. appl. Biol. 48, 601-612 (1960). Xmeish,A. C. : Studies on chloroplasts II. Their chemical composition and distribution of certain metabolites between the chloroplasts and the remainder of the leaf. Biochem. J. 88, 300-308 (1939). Patton, S., Knrtz, G. : 2-Thiobarbituric acid as a reagent for detecting milk fat oxidation. J. Dairy Sci. 84, 669-674 (1951). Skidmore, :N. D., Entenman, C. : Two dimensional thin layer chromatography of rat liver phosphatides. J. Lipid Res. 3, 471-475 (1962). Tolbert, LN.E., Oeser, A., Kisaki, T., Hageman, 1%.H., Yamazaki, R. K. : Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. biol. Chem. 248, 5179-5184 (1968). Vernon, L. P. : Spectrophotometric determination of chlorophylls and phaeophytins in plant extracts. Analyt. Chem. 82, 1144-1150 (t960). Yamazaki, R. K., Tolbert, hT. E.: Enzymic characterization of leaf peroxisomes. J. biol. Chem. 245, 5137-5144 (1970). Zweig, G., Shavit, N., Avron, M. : Diquat (1,1-ethylene-2,2-dipyridyliumdibromide) in photoreactions of isolated chloroplasts. Biochim. biophys. Acta (Amst.) 109, 332-346 (1965). A. D. Dodge School of Biological Sciences University of Bath Bath, Somerset, U.K.
15"
.N. Harris Present address Botany School University of Cambridge Cambridge, U.K.
The Effect of Paraquat on Flax Cotyledon Leaves: Physiological and Biochemical Changes N. Harris and A. D. Dodge School of Biological Sciences, University of Bath, Bath, Somerset, U.K. Received December 22, 1971; January 24, 1972 Summary. Some of the physiological and biochemical changes which were found to occur during paraquat (1,1'-dimethyl-4,41-bipyridylium ion) treatment of cotyledon leaves of Linium usitatissimum, are reported. Results showed an inhibition of photosynthetic CO2 uptake and electron flow of isolated chloroplasts. An increase in membrane permeability, changes in the level of the major lipid components and of malondiald~hyde. These are correlated with ultrastructural changes and the discussion includes a proposed mode of action for the herbicides.
Introduction I t is generally accepted that the primary toxic action of the bipyridylium herbicides, paraquat and diquat is a result of the deviation of photosynthetic electron transport within the chloroplast (Akhavein and Linscott, 1968; Calderbank, 1968). In place of a reduction of the natural oxidant, NADP +, bipyridylium ions are reduced to free radicals by a one electron process. Immediate reoxidation follows in the presence of oxygen, to yield hydrogen peroxide (Davenport, 1963). The continued production of the hydrogen peroxide is dependent upon the maintenance of photosynthetic electron transport. Thus, Mees (1960) noted a considerable retardation of herbicidal kill in the presence of the photosynthetic electron transport inhibitor, CMU. However it was reported (Baldwin, Dodge and Harris, 1968) that much of the structural and component breakdown of plant tissue occurred after the cessation of photosynthetic electron transport. The results below concern some of the sequential changes which were found to occur following the paraquat treatment of flax cotyledon leaves. The changes are correlated with ultrastructural changes reported in the previous paper (Harris and Dodge, 1972) and the discussion is concerned with a proposed mode of action for the bipyridylium herbicides. Materials and Methods Flax (Linium usitatissimum var. Redwing) seeds were sown on vermucilite, and maintained under constant illumination. Cotyledon leaf pairs with a few rams of hypocotyl were cut from 8 to 10 days old seedlings and floated on paraquat
Effect of Paraquat on Cotyledon
211
(1 • 10 -4 M) or distilled water, as required. Illumination (2250 lnx) was provided by " d a y glow" neon tubes, and treatments were carried out under constant temperature (24 ~ C • 1~ Leaf pigments were estimated spectrophotometrically after extraction into 80% acetone. Chlorophylls a and b by the method of Arnon (1949) phaeophytins by the method of Vernon (1960) and earotenoids by the method of Kirk and Allen (1965). Total lipids were extracted in chloroform:methanol following the method of Bligh and Dyer (1959). The lipids were separated by thin layer chromatography with silica-gel G (Merk) with plates routinely activated at l l 0 ~ for 20min. Di-isobutyl ketone; acetic acid: water at 80:50:7 was used as solvent (Marinetti, Erbland and Stotz, 1958). The plates were sprayed with phosphomolybdie acid and charred to show total lipid, molybdate-perchloric acid to show phospholipids (Skidmore and Entenman, 1962) and periodate-Schiff reagent, for ~ glycols. After drying the plates in vacuo, further analysis of components was achieved after elution by chloroform:methanol, 1:1. Analysis of the glycolipid followed the method of Brundish, Shaw and Baddiley (1967) with fatty acid methyl esters examined by gas liquid chromatography. Malondialdehyde, a breakdown product of unsaturated fatty acid hydroperoxides, was estimated by the use of thiobarbituric acid (TBA) as described by Heath and Packer (1968). 0.2 gm of cotyledon leaves were ground in 10 ml of distilled water in a mortar, and 3 ml of this preparation was incubated with 5 ml of 0.5% TBA in 20% TCA for 30 min at 95 ~ C. After cooling and eentrifugation to give a clear supernatant the extinction (E) of the solution was measured at 532 and 600 nm. Following E 532 n m - - E 600 nm, to correct for nonspecific absorption, the level of malondialdehyde was estimated by using the mM extinction coefficient of 155 m ~ -~ cm -1 (Heath and Packer, 1968). The rate of potassium efflux from tissue slices was used as an indicator of membrane permeability (Eilam, 1965). 1 mm slices of cotyledon leaves were washed and incubated in distilled water at 20 ~ C. Aliquots of the water were taken at intervals, and the potassium levels estimated by flame photometry. The carbon dioxide exchange of flax cotyledon leaves was measured by using an infra-red gas analyser (Grubb Parsons Ltd.). The plant material was contained within a small perspex chamber (5 • 15 • 100 ram) maintained at constant temperature and humidity. Illumination was provided by a 500 W lamp. Chloroplasts were isolated by the method of Hill and Walker (1959) and photosynthetic electron flow activity was assayed by following the reduction of ferrycyanide as a decrease in E at 420 nm. (Jagendorf and Margulies, 1960). A standard reaction mixture contained, in addition to the chloroplast preparation, 90 ~moles Tris-chloride buffer pH 7.7; 10 ~moles Sodium chloride; 2 i~moles potassium ferricyanide. Illumination was provided by white light of 50 Klux at 20 ~ C.
Results P h o t o s y n t h e t i c c a r b o n d i o x i d e u p t a k e b y f l a x c o t y l e d o n l e a v e s was c o m p l e t e l y i n h i b i t e d w i t h i n 5 h p a r a q u a t t r e a t m e n t , i r r e s p e c t i v e of w h e t h e r t h e l e a v e s w e r e i n c u b a t e d in l i g h t or d a r k n e s s . A t a b o u t t h i s t i m e t h e r e was a g r a d u a l rise in c a r b o n d i o x i d e e v o l u t i o n w h i c h w a s also i n d e p e n d e n t of l i g h t c o n d i t i o n s (Fig. 1). T h e i n h i b i t i o n of p h o t o s y s t e m I I a c t i v i t y , m e a s u r e d as f e r r i c y a n i d e r e d u c t i o n b y c h l o r o p l a s t s i s o l a t e d f r o m p a r a q u a t t r e a t e d leaves, was
212
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\o
O
9
9
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Ev0tuti0n
/
i
}
o
\ /
Hours
~
Z
Paraquat
Treatment
Fig. 1. Carbon dioxide uptake and evolution by paraquat treated flax cotyledon leaves. The leaves were incubated with paraquat (1 • 10 -t) in either light o or darkness., and the uptake and evolution of these leaves estimated after treatment
Eloo
~ac .2 6C
\
2C kl-
;
~;
1'5
Hours Paraquat
\,
20
I
2s
Treatment
Fig. 2. Ferricyanide reduction by chloroplasts isolated from paraquat treated flax cotyledon leaves, o Leaves treated under light; 9 leaves in darkness
originally shown to be temperature dependent (Baldwin, Dodge and Harris, 1968). With treatment at 24 ~ C, there was no further activity after 19 h eontinuous illumination (Fig. 2). When the illumination
Effect of Paraquat on Cotyledon
213
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,~ z,C c
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1o Paraquat
do
~'o
Treatment
Fig. 3. Changes in the level of leaf pigments during the paraquat treatment of flax cotyledon leaves under constant illumination
period was shortened, the loss of activity of isolated chloroplasts was retarded. When a 15 h light incubation period was followed by darkness, the subsequent decline in activity was similar to incubation under continuous illumination. The effect of paraquat treatment on the chlorophyll, carotenoid and phaeophytin levels of flax cotyledon leaves, treated under constant illumination, is shown in Fig. 3. I t is apparent that there was no major breakdown of chlorophylls during the first 20 h treatment. This is an interesting contrast to Fig. 2, and indicates that the major breakdown of the chlorophylls occurred after the cessation of photosynthetic electron transport. Fig. 4 shows that the further breakdown of the
214
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5
1'8
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Hours Paraquat Treatment
Fig. 4. The effect of light and dark periods on the chlorophyll a and b levels of paraquat treated flax cotyledon leaves, o light treatment;, dark treatment
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Hours Paraquat Treatment Fig. 5. Membrane permeability measured as potassium leakage from slices of paraquat treated flax cotyledon leaves chlorophyll pigments was light dependent, and followed the breakdown of much of the carotenoid pigments. The increased potassium efflux from tissue slices of paraquat treated flax cotyledon leaves, was one of the early manifestations of the applica-
Effect of Paraquat on Cotyledon
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Fig. 6. Malondialdehyde production, as a measure of lipid peroxidation in paraquat treated flax cotyledon leaves tion of paraquat. There was a gradual leakage after 3 h treatment, but the period between 5 and 7 h was marked by a rapid and sudden rise. There was no further liberation after this point (Fig. 5). At about the same time as the increase in potassium efflux, there was a rise in the level of malondialdehyde present (Fig. 6) which continued for the next 30 to 40 h. This was the first indication of an effect of paraquat treatment on the lipid composition of the leaves. Thin layer chromatography of the total lipids extracted from treated leaves showed that the major components were mono- and di-galaetosyl diglyeerides, the phospholipids-phosphatidylethanolamine and phosphatidylcholine, neutral lipids and pigments. Major changes were shown after 36 h paraquat treatment (Fig. 7) when a breakdown of the digalactosyl diglyeeride, phosplipids and pigments was evident. Similarities in f a t t y acid composition, as shown by gas liquid chromatography, indicated t h a t a monogalaetosyl diglyceride was derived from digalaetosyl diglyceride at this time. Further breakdown of both galaetosyl and phospholipids to mono- and diglyeerides occurred within the next 24 h. Discussion
The inhibition of photosynthetic carbon dioxide uptake within 5 h of the application of paraquat was thought to be a direct result of the interaction of these molecules with photosynthetic electron transport
216
N. Harris and A. D. Dodge
Fig. 7. Silica-gel G plates of lipid extracts from control (C) and 36 h paraquat treated (P) flax cotyledon leaves. (i) Sprayed with phosphomolybdie acid. (ii) Sprayed with periodate--Schiff reagent. (iii) Sprayedlwith molybdate-- perchlorie acid with the consequent inhibition of NADP § reduction (Zweig, Shavit and Avron, 1965). The time to total inhibition was probably based on that taken for complete penetration of the herbicide into the cotyledon leaves, a process which was, in these experiments, independent of light. The uptake of the bipyridylinm herbicides by whole plants, on the other hand, may be increased in darkness (Brian, 1967). The increase in potassium efflux and the rise in the level of malondialdehyde occurred next during the course of herbicidal action. The rise in potassium efflux probably represented a change in permeability of the tonoplast and plasmalemma which resulted in its release from the treated tissue. Malondialdehyde is a breakdown product of tri-unsaturated fatty acid hydroperoxides (Patton and Kurtz, 1951; Kohn and Liversedge, 1944) and its rise at this time may also be related to the membrane permeability changes. Electron mierographs of flax cotyledon leaves, treated with paraquat under identical conditions, showed after 6 h the rupture of the tonoplast in many cells of the upper spongy mesophyll (Harris and Dodge, 1972). I t is considered that the products of the interaction of the herbicide with photosynthetic electron transport, which include hydrogen peroxide and peroxy and hydroxyl free radicals derived from the hydrogen peroxide, attacked the lipids of the cell membranes, and in particular the tonoplast where it was located in
Effect of P a r a q u a t on Cotyledon
217
close proximity to the chloroplasts. The free radicals probably instigated a deteriorative chain reaction in the membrane, which led to its rapid destruction with malondialdehyde being an eventual product. Neish (1939) reported that catalase was associated with the chloroplast and Black and Myers (1966) suggested that the hydrogen peroxide formed by interaction of the bipyridylinm herbicides with the photosynthetic electron transport system would be rapidly broken down. More recent evidence however, points to this enzyme being associated with extra chloroplastic bodies, the peroxisomes (Tolbert, Oeser, Kisaki, Hageman and Yamazaki, 1968) (Yamazaki and Tolbert, 1970) and being almost totally absent from the chloroplasts (Gregory, 1968). A result of the rupture of the tonoplast would be a loss of the normal cell compartmentalisation and the cell organelles would presumably be subjected to severe osmotic changes. Although mitochondrial activity was not assayed in these experiments, electron micrographs showed swelling of the mitochondria and other cytoplasmic organelles soon after tonoplast rupture, (Harris and Dodge, 1972) and by about 10 h they were apparently no longer present in the disorganised cytoplasm. The complete loss of photosynthetic electron transport from water occurred after 19 h paraquat treatment under continuous illumination although photosystem I alone was potentially operative for a few h longer (Baldwin et al., 1968). It is assumed that after 19 h treatment there would be no further production of toxic products by the normally accepted interaction of paraquat with photosynthetic electron transport. It is significant that considerable structural breakdown occurred after 19 h treatment (Harris and Dodge, 1972) and also the light promoted breakdown of the chlorophylls occurred after this point when the level of carotenoid had dropped considerably. It is possible that the photooxidation of the chlorophylls occurred due to the loss of the normal carotenoid protective mechanism (Koski and Smith, 1951; Anderson and Robertson, 1970). Heath and Packer (1968) demonstrated that isolated chloroplasts could undergo a cyclic peroxidation initiated by light, and suggested that this was a result of an overloading of the excited chlorophyll energy trapping system. In their system chlorophyll was bleached, there was a consumption of oxygen and malondialdehyde was produced. It is possible that after the cessation of electron transport, and thus without the mediation of paraquat, this type of photoinduced pcroxidation was responsible for considerable cellular damage. However, the rapidity of the structural breakdown would suggest that other destructive processes were in operation. Matile (1968) reported that the plant cell vacuole contained a number of hydrolytic enzymes, including proteases, hue]eases, phosphatases and esterases. The continued and rapid breakdown of celhflar components could therefore be partly due 15 Planta (Berl.), Bd. 104
218
N. Harris and A. D. Dodge:
t o t h e release of these enzymes after t h e r u p t u r e of t h e t o n o p l a s t . A l t e r n a t i v e l y t h e osmotic u p s e t following t o n o p l a s t b r e a k d o w n w o u l d cause t h e r u p t u r e of l y s o s o m e - l i k e organelles w i t h i n t h e c y t o p l a s m a n d l e a d to a similar effect. I n either r e s p e c t t h e r u p t u r e of t h e t o n o p l a s t is a significant event, a n d i t is considered t h a t i t is this e v e n t a t a n e a r l y stage of t h e herbicide t r e a t m e n t , t h a t leads to t h e i r r e v e r s i b i l i t y of t h e action. Acknowledgement is made to the S.R.C. for a research studentship to N.H. and to Plant Protection Ltd., Jealott's Hill who were associated with this work under the C.A.P.S. scheme. References Akhavein, A.A., Linscott, D . L . : The dipyridylium herbicides, paraquat and diquat. Residue Rev. 23, 97-i45 (1968). Anderson, I. C., Robertson, D. S.: Role of carotenoids in protecting chlorophyll from photodestruction. Plant Physiol. 35, 531-534 (1960). Arnon, D . I . : Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). Baddiley, J., Buchanan, J. G., Handsehumaker, R. E., Prescott, J . F . : Chemical studies in the biosynthesis of purine nucleotides I. The preparation of Nglycylglycosylamines. J. chem. Soc. 2818-2823 (1956). Baldwin, B. C., Dodge, A. D., Harris, N. : Recent advances in studies of the mode of action of the bipyridylium herbicides. Proc. 9th Br. Weed Control Conf. 639-644 (1968). Black, C. C., Myers, L. : Some biochemical aspects of the mechanisms of herbicidal activity. Weeds 14, 331-338 (1966). Bligh, E. G., Dyer, W. J. : A rapid method of total lipid extraction and purification. Canad. J. Biochem. Physiol. 37, 911-917 (1959). Brian, R. C. : Darkness and the activity of diquat and paraquat on tomato, broad bean and sugar beet. Ann. appl. Biol. 60, 77-85 (1967). Brundish, D. E., Shaw, N., Baddiley, J.: The structure and possible function of the glycolipid from Staphylococcus lactis 13. Biochem. J. 105, 885-889 (1967). Calderbank, A. : The bibyridylium herbicides. Advanc. Pest Control Res. 8, 127235 (1968). Davenport, H . E . : The mechanism of cyclic phosphorylation by illuminated chloroplasts. Proc. roy. Soc. B 157, 332-345 {1963). Eilam, Y. : Permeability changes in senescing tissue. J. exp. Bot. 16, 614-627 (1965). Gregory, R. P . F . : An improved preparative method for spinach catalase and evaluation of some of its properties. Bioehim. biophys. Acta (Amst.) 159, 429439 (1968). Harris, N., Dodge, A. D. : The effect of paraquat on flax cotyledon leaves: Changes in fine structure. Planta (Berl.)104, 201-209 (1972). Heath, R. L., Packer, L. : Photoperoxidation in isolated chloroplasts. I. Kinetics and stochiometry of fatty acid peroxidation. Arch. Biochem. 125, 189-198 (1968). Hill, R , Walker, D.A.: Pyocyaninc and phosphorylation with chloroplasts. Plant Physiol. 34, 240-245 (1959). Jagendorf, A.T., Margulies, M.M.: Inhibition of spinach chloroplast photochemical reactions by p-chlorophenyl 1-1, dimethyl urea. Arch. Biochem. 90, 184-195 (1960).
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Kirk, J. T. 0., Allen, 1~. L.: Dependence of chloroplast pigment synthesis on protein synthesis: effect of actidione. Biochem. biophys. Res. Commun. 21, 523-530 (1965). Kohn, H., Liversedge, M.: On a new aerobic metabolite whose production by brain is inhibited by amorphine, emetrine, ergotamine, epinephrine and menadione. J. Pharmacol. exp. Ther. 82, 292-300 (1944). Koski, V. M., Smith, J. H. C. : Chlorophyll formation in a mutant, white seeding. Arch. Biochem. 84, 189-195 (1951). Marinetti, G. V., Erbland, J., Stotz, E.: Phosphatides of pig heart cell fractions. J. biol. Chem. 288, 562-565 (1958). Matile, Ph.: Enzyme der Vakuolen und Wurzelzellen yon Maiskeimlingen. Ein Beitrag zur funktionellen Bedeutung der Vakuole bei der intrazellularen Verdaunng. Z. Naturforsch. 21b, 871-878 (1966). Mees, G. C. : Experiments on the herbicidal action of 1,1-ethylene-2,2-dipyridylium dibromide. Ann. appl. Biol. 48, 601-612 (1960). Xmeish,A. C. : Studies on chloroplasts II. Their chemical composition and distribution of certain metabolites between the chloroplasts and the remainder of the leaf. Biochem. J. 88, 300-308 (1939). Patton, S., Knrtz, G. : 2-Thiobarbituric acid as a reagent for detecting milk fat oxidation. J. Dairy Sci. 84, 669-674 (1951). Skidmore, :N. D., Entenman, C. : Two dimensional thin layer chromatography of rat liver phosphatides. J. Lipid Res. 3, 471-475 (1962). Tolbert, LN.E., Oeser, A., Kisaki, T., Hageman, 1%.H., Yamazaki, R. K. : Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. biol. Chem. 248, 5179-5184 (1968). Vernon, L. P. : Spectrophotometric determination of chlorophylls and phaeophytins in plant extracts. Analyt. Chem. 82, 1144-1150 (t960). Yamazaki, R. K., Tolbert, hT. E.: Enzymic characterization of leaf peroxisomes. J. biol. Chem. 245, 5137-5144 (1970). Zweig, G., Shavit, N., Avron, M. : Diquat (1,1-ethylene-2,2-dipyridyliumdibromide) in photoreactions of isolated chloroplasts. Biochim. biophys. Acta (Amst.) 109, 332-346 (1965). A. D. Dodge School of Biological Sciences University of Bath Bath, Somerset, U.K.
15"
.N. Harris Present address Botany School University of Cambridge Cambridge, U.K.
