Plant Cell Reports
Plant Cell Reports (1993) 12:230-232
9 Springer-Verlag 1993
Paraquat tolerance in a photomixotrophic culture of Chenopodium rubrum Sujata Bhargava Botany Department, University of Poona, Pune - 411 007, India Received March 5, 1992/Revised version received November 12, 1992 - Communicated by J. M. Widholm
Abstract :
A paraquat tolerant line of C h e n o p o d i u m rubrum has been compared with paraquat susceptible cultures, in terms of growth, chlorophyll content, photosystem I partial reactions, and the activities of some enzymes involved in detoxificstion of harmful oxygen radicals. Results indicate that paraquat tolerance is manifested through increased activity of superoxide dismulase, peroxidase and catalase, in the tolerant line, only in the presence of paraquai. The behaviour of the paraquat tolerant and susceptible cultures in the absence of paraquat is quite similar. Introduction:
Paraquat is a broad s p e c t r u m contact herbicide. Resistance to paraquat has been observed in several plants like Conyza bona riensis (Shaaltiel and Gressel, 1986), Lolium perenne (Harper and Harvey, 1978), Hordeum $1aucum (Powles and Carnie, 1987) as well as in cell cultures of tobacco (Miller and Hughes, 1980) and tomato ( T h o m a s and Pratt, 1982). The mechanism of paraquat action involves its photoreduction by accepting electrons from photosystem I in chloroplasts. The reduced paraquat reacts with molecular oxygen to form the superoxide radical a n d its subsequent peroxide products (Halliwell, 1984). Paraquat resistance has been attributed to: (a) Decreased paraquat uptake (Kao and Ha ssan, 1985 ) ; (b) Exclusion of paraquat from the symplast (Powles and Carnie, 1987); and (c) Sequestration of paraquat b y elevated activities of superoxide detoxif ying enzymes (Shaaltiel and Gressel, 1986; Furusawa et al., 1984). In this paper, a paraquat tolerant line has been isolated from photomixotrophic cell cultures of C henopodium rubrum. The nature of paraquat tolerance in this line has been investigated using parameters like growth chlorophyll retention, PSI partial reactions, and the activities of some enzymes involved in detoxification of activated oxygen species, namely superoxide dismutase, catalase and peroxidase. Paraquat tolerance has been attributed to a differential activation of these enzymes, in the presence of paraquat.
Materials
and
Methods:
Photomixotrophic cell cultures of C henopodium rubrum L. were raised from a sample provided by Dr. Huesemann, Muenster, Germany, on Murashige and Skoog's (MS) medium supplemented w i t h 10-7M 2,4-D and 2% sucrose (Huesemann et a I., 1979 ). Cultures were illuminated with continuous light of 5000 Lux at a temperature of 25+2~ Paraquat tolerant line was selected by plating suspension cultures of C henopodium rubrum on the same MS medium containing luM paraquat, followed by selection of the green calli which appeared on this medium after a week. The selection procedure was repeated thrice, after w h i c h the green calli w e r e transferred to paraquat free solid medium and maintained as a paraquat tolerant line. A paraquat tolerant line had been isolated earlier by Rana de and Feierabend, 1991) , and the same procedure w a s followed for isolating this line. The paraquat tolerant culture showed an LD 50 value (in terms of chlorophyll retention) of 0.8uM paraquat while the susceptible cultures showed ~an LD 50 value of 0.08uM paraquat. For further experiments, about 30rag fresh weight of calli from the paraquat susceptible and tolerant cultures were inoculated in 20ml of liquid MS m e d i u m w i t h or without paraquat. The concentrations of paraquat used for the susceptible cultures were 0.0SuM (corresponding to its L D 50 value), 0.ZuM and 2uM. Concentrations of paraquat used for the tolerant cultures were 0.8uM (corresponding to its L D 50 value ), and 2uM. Culture conditions were identical to those mentioned earlier. Growth was measured in terms of fresh weight, after filtering the suspensions through a Buchner funnel having Whatman paper No. 1 filter paper. Three replicates w e r e taken for each treatment. Chlorophyll estimation was carried out after 7Z hours of paraquat treatment. Four ml. of 80% acetone w a s added to 50rag cells to extract chlorophyll, which was then determined by the method of Arnon ( 1949 ). Three replicates w e r e taken for each treatment. Chloroplast thylakoid membranes were isolated from the paraquat susceptible and tolerant cultures using a m g t h o d modified from Feiera bend et al. ( 1980 ). The cells were homogenised
231 in a medium consisting of 0.45M sucrose, 0.15M TRIS-HCI buffer, pH 7.5, ImM disodium E D T A , 10ram KCI, I m M M g C I 2 . 6 H 2 0 0.01% bovine serum albumin and 4 u M dithioerythreitol. Homogenate w a s filtered through 6 layers of gauze cloth and centrifuged at Z000xg for 20s. Supernatant was again centrifuged at 20O0xg for 2rain. The sediment w a s then w a s h e d with resuspension medium which consisted of 50tnM TRIS-HCI buffer, pH 7.5, i m M M g C I 2 . 6 H 2 0 and I m M disodium EDTA, and suspended in a small volume of the resuspension medium. PSI partial reactions were studied according to the method of Koenig et ai.(1972). Reduced 2,6-DCPIP was used as the electron donor and paraquat as the electron acceptor. PSII reactions were arrested by adding D C M U . A volume of chloroplast membranes corresponding to a chlorophyll concentration of 50ug m l - l w a s used in the assay. Rates of electron transport were measured in terms of m M oxygen evolved h-ling-1 chlorophyll using an Oxygen electrode (Hansatech, D~f 1, England ), in the paraquat tolerant and susceptible cultures grown in the absence of paraquat. Estimation of the activities of enzymes was carried out with an extract made from 70mg cells in iml of 50ram K-phosphate buffer, ph 7.5, using an ultra sonicator (Braun). An initial pulse of 30s, followed by three pulses of 10s at intervals of 10s comprised the sonication cycle. The crude extract w a s centrifuged at 4 ~ C for 10min at 7000xg, and the supernatant used for the enzyme assays. Catalase and peroxidase activity was determined according to the method used by Volk and Feiera bend ( 1989 ). Superoxide dismutase activity was determined by the method of Beauchamp and Fridovich ( 1971 ), in terms of inhibition of the rate of NBT reduction. One unit of SOD activity was the amount which inhibited 50% of the reduction rate of N B T . T h e experiments w e r e carried out twice. Results a n d Discussion:
The paraquat tolerant line s h o w e d less inhibition of growth and better chlorophyll retention as compared to the susceptible cultures after treatment with 2uM paraquat (Table 1 ). Ashton and Ziegler (1987) have s h o w n that 2 u M paraquat inhibits g r o w t h of photoautotrophic and photomixotrophic cultures of C henopodium rubrum, unlike other photosynthetic herbicides eg. Diuron, which only inhibits growth of photoautotrophic cultures. Ho~cever the PSI electron transport activity in chloroplasts isolated from paraquat tolerant and susceptible cultures was similar (Tablel) when grown in s medium without paraquat. Hence paraquat tolerance was probably not associated with changes in the chloroplasts of the tolerant cultures. This observation was also made in paraquat resistant biotypes of Conyza (Fuerst et al., 1985 ) and in paraquat resistant Hordeum glaucum (Powles and Cornic, 1987). Studies on the activities of enzymes involved in detoxifica tion of activated oxygen species, namely, superoxide dismuta se, catalase and peroxidase revealed no major changes in the susceptlble and tolerant cultures (Fig. 1 ). The paraquat tolerant cultures showed slightly decreased activity of superoxide dismutase and peroxidase and slightly increased activity
I.
Table
.
.
.
.
.
.
.
.
.
.
.
.
Growth, chlorophyll content, and PSI partial reactions of paraquat susceptible and tolerant cultures in the presen ce or absence of 2uM paraquat. Results are a mean of three replicates. .
.
.
.
Parameter
.
.
.
.
.
.
.
.
.
.
-paraquat .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Growth(fr. wt.g/2Oml afterl5days .
.
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.
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.
.
+pc raquat
.
.
.
.
.
.
.
.
.
.
.
0.052 + 0.001 .
.
.
1.145 + 0.095
.
.
.
.
.
.
.
.
.
PSI partial 0.391+reaction( m M o l 9,037 02 h - l m g -I chl. ) .
.
0.185 + 0.018
0.585+ 0.039
.
.
-paraquat
.
0.01_+ 0.001
1.33+ 0.064
.
.
Paraquat tolerant cultures
+paraquat .
0.37 + _ 0.03
Chlorophyll content(rag g-lfr.wt. ) after3days .
.
Paraqust susceptible cultures
.
.
.
.
.
.
0.92 + 0.085
.
.
.
.
0.338_+ 0.035
.
.
.
.
.
.
.
.
.
.
.
~
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
of c a t a l a s e . A lack of correlation between the activities of these enzymes and paraquat resistance has been reported by Powles and Cornic (1987) and Carroll, et a!. (1988). Shaaltiel and Gressel, (1986) have showed a positive correlation between the activities of the oxygen radical detexifica tion system in the chloroplasts and paraqust resistance. An ability of this o x y g e n radical detoxification system in providing cross tolerance to photoinhibitory light has been reported by Jansen et al. ( 1989 ), and a lack of cross tolerance to photoinhitory light has been shown by Ranade and Feierabend (1991). In the presence of paraquat, the susceptible and tolerant cultures of Chenopodium rubrum behaved very differently. While there was a decrease in the activities of all three enzymes in the susceptible cultures, an increase in superoxide dismula se and peroxidase activity w a s seen in the tolerant cultures. Also, catalase activity was only slightly reduced in the tolerant cultures. This trend was apparent even when the activities of these enzymes were compared at their respective LD 50 values for paraquat (Fig. 1 ). Differential induction or activation of these enzymes by paraquat in the tolerant and susceptible cultures could be an explanation for their higher activities in tolerant cultures, and this will be investigated through further experiments. Studies on the oxygen radical detexif ying system from paraquat tolerant and susceptible plants, in the presence of paraquat have not yet been reported,
Acknowledgements: Financial support by the German Academic Exchange Service, and the Department of Science and Technology, India, is gratefully acknowledged. The author is also thankful to Dr. J. Feiera bend, Frankfurt, for providing facilities to carry out a part of this work, and to Dr. W. Huesemann for a sample of Chenopodium r u b r u m cultures. Technical assistance
232 of Ms. Varsha Vartak in a s s a y s is a l s o a c k n o w l e d g e d .
0"2 c
T o~
0.1
m
'T
o~
%
4"
=..
200 -
% u~
100-
e-
0 0 " 0 8 0.2
2
0
0"8
2
paraquat ]j M
Fig.
i.
enzyme
References:
0"3
%
the
Activity of s u p e r o x i d e dismutase (A), c a t a l a s e (B) a n d p e r o x i d a s e (C) in paraquat susceptib]e (lanes 1,2,3 & 4) a n d tolerant cultures (lanes 5,6 & 7) in the absence (lanes 1 & 5) a n d p r e s e n c e of d i f f e r e n t concentrations of paraquat, after 3 days of treatment. Readings are a mean of two experiments.
Arnon D I (1949) Plant Physiol. 24:115. A s h t o n A R, Z i e g l e r P (1987) P l a n t S c i e n c e 51: 2 6 9 - 2 7 6 . Beauchamp C O, Fridovich I (1971) Anal. B i o c h e m . 44: 2 7 6 - 2 8 7 . Carroll E, Schwarz O, Hickok 1 (1988) Plant Physiol. 87: 6 5 1 - 6 5 4 . Feierabend J, Meschede D, Vogel K -D (1980) Z. Pflanzenphysiol. 98: 61-78. Fuerst E P, Nakatani H Y, Dodge A D, Penner D, Arntzen C J (1985) Plant Physiol. 77: 9 8 4 - 9 8 9 . F u r u s a w a I, K u n i s u k e T, P e r n t i p T h a n u t o n g , Mizuguchi A, Yazaki M, Kosi Asada (1984) Plant and Cell Physiol. 25(7): 1247-1254. H a l l i w e l l B (1984) C h l o r o p l a s t Metabolism: T h e s t r u c t u r e a n d f u n c t i o n of c h l o r o p l a s t s in green leaf cells. Clarendon Press, Oxford. Ilarper D B, Harvey B M R (1978) Plant Cell E n v i r o n m . I: 2 1 1 - 2 1 5 . Huesemann W, Plohr A, Barz W (1979) Protoplasma 100: 1 0 1 - 1 1 2 . Jansen M A K, Shaaltiel Y, Kazzes D, Canaani O, Malkin S, Gresse] J (1989) P l a n t P h y s i o l . 91: 1 1 7 4 - 1 1 7 8 . Kao S M, Hassan H M (1985) J. Biol. Chem. 26: 10478-10481. Koenig F, Menke W, C r a u b n e r H, Schmidt G If, Radunz A (1972) Z. Naturforsch. 27(b)(I0): 1225. Miller O K, Hughes K W (1980) In V i t r o 16(12): 1085-1091. Powles S B, Cornic G (1987) Aust. J. P l a n t P h y s i o l . 14: 81-89. Ranade S, Feierabend J (1991) J. Plant Physiol. 137: 7 4 9 - 7 5 2 . Shaaltiel Y, Gressel J (1986) Pestic. Biochem. Physiol. 26: 2 2 - 2 8 . Thomas B R, Pratt D (1982) Theor. Appl. G e n e t . , 63: 1 6 9 - 1 7 6 . Volk S, Feierabend J (1989) Plant Cell Environm., 12: 7 0 1 - 7 1 2 .
Plant Cell Reports (1993) 12:230-232
9 Springer-Verlag 1993
Paraquat tolerance in a photomixotrophic culture of Chenopodium rubrum Sujata Bhargava Botany Department, University of Poona, Pune - 411 007, India Received March 5, 1992/Revised version received November 12, 1992 - Communicated by J. M. Widholm
Abstract :
A paraquat tolerant line of C h e n o p o d i u m rubrum has been compared with paraquat susceptible cultures, in terms of growth, chlorophyll content, photosystem I partial reactions, and the activities of some enzymes involved in detoxificstion of harmful oxygen radicals. Results indicate that paraquat tolerance is manifested through increased activity of superoxide dismulase, peroxidase and catalase, in the tolerant line, only in the presence of paraquai. The behaviour of the paraquat tolerant and susceptible cultures in the absence of paraquat is quite similar. Introduction:
Paraquat is a broad s p e c t r u m contact herbicide. Resistance to paraquat has been observed in several plants like Conyza bona riensis (Shaaltiel and Gressel, 1986), Lolium perenne (Harper and Harvey, 1978), Hordeum $1aucum (Powles and Carnie, 1987) as well as in cell cultures of tobacco (Miller and Hughes, 1980) and tomato ( T h o m a s and Pratt, 1982). The mechanism of paraquat action involves its photoreduction by accepting electrons from photosystem I in chloroplasts. The reduced paraquat reacts with molecular oxygen to form the superoxide radical a n d its subsequent peroxide products (Halliwell, 1984). Paraquat resistance has been attributed to: (a) Decreased paraquat uptake (Kao and Ha ssan, 1985 ) ; (b) Exclusion of paraquat from the symplast (Powles and Carnie, 1987); and (c) Sequestration of paraquat b y elevated activities of superoxide detoxif ying enzymes (Shaaltiel and Gressel, 1986; Furusawa et al., 1984). In this paper, a paraquat tolerant line has been isolated from photomixotrophic cell cultures of C henopodium rubrum. The nature of paraquat tolerance in this line has been investigated using parameters like growth chlorophyll retention, PSI partial reactions, and the activities of some enzymes involved in detoxification of activated oxygen species, namely superoxide dismutase, catalase and peroxidase. Paraquat tolerance has been attributed to a differential activation of these enzymes, in the presence of paraquat.
Materials
and
Methods:
Photomixotrophic cell cultures of C henopodium rubrum L. were raised from a sample provided by Dr. Huesemann, Muenster, Germany, on Murashige and Skoog's (MS) medium supplemented w i t h 10-7M 2,4-D and 2% sucrose (Huesemann et a I., 1979 ). Cultures were illuminated with continuous light of 5000 Lux at a temperature of 25+2~ Paraquat tolerant line was selected by plating suspension cultures of C henopodium rubrum on the same MS medium containing luM paraquat, followed by selection of the green calli which appeared on this medium after a week. The selection procedure was repeated thrice, after w h i c h the green calli w e r e transferred to paraquat free solid medium and maintained as a paraquat tolerant line. A paraquat tolerant line had been isolated earlier by Rana de and Feierabend, 1991) , and the same procedure w a s followed for isolating this line. The paraquat tolerant culture showed an LD 50 value (in terms of chlorophyll retention) of 0.8uM paraquat while the susceptible cultures showed ~an LD 50 value of 0.08uM paraquat. For further experiments, about 30rag fresh weight of calli from the paraquat susceptible and tolerant cultures were inoculated in 20ml of liquid MS m e d i u m w i t h or without paraquat. The concentrations of paraquat used for the susceptible cultures were 0.0SuM (corresponding to its L D 50 value), 0.ZuM and 2uM. Concentrations of paraquat used for the tolerant cultures were 0.8uM (corresponding to its L D 50 value ), and 2uM. Culture conditions were identical to those mentioned earlier. Growth was measured in terms of fresh weight, after filtering the suspensions through a Buchner funnel having Whatman paper No. 1 filter paper. Three replicates w e r e taken for each treatment. Chlorophyll estimation was carried out after 7Z hours of paraquat treatment. Four ml. of 80% acetone w a s added to 50rag cells to extract chlorophyll, which was then determined by the method of Arnon ( 1949 ). Three replicates w e r e taken for each treatment. Chloroplast thylakoid membranes were isolated from the paraquat susceptible and tolerant cultures using a m g t h o d modified from Feiera bend et al. ( 1980 ). The cells were homogenised
231 in a medium consisting of 0.45M sucrose, 0.15M TRIS-HCI buffer, pH 7.5, ImM disodium E D T A , 10ram KCI, I m M M g C I 2 . 6 H 2 0 0.01% bovine serum albumin and 4 u M dithioerythreitol. Homogenate w a s filtered through 6 layers of gauze cloth and centrifuged at Z000xg for 20s. Supernatant was again centrifuged at 20O0xg for 2rain. The sediment w a s then w a s h e d with resuspension medium which consisted of 50tnM TRIS-HCI buffer, pH 7.5, i m M M g C I 2 . 6 H 2 0 and I m M disodium EDTA, and suspended in a small volume of the resuspension medium. PSI partial reactions were studied according to the method of Koenig et ai.(1972). Reduced 2,6-DCPIP was used as the electron donor and paraquat as the electron acceptor. PSII reactions were arrested by adding D C M U . A volume of chloroplast membranes corresponding to a chlorophyll concentration of 50ug m l - l w a s used in the assay. Rates of electron transport were measured in terms of m M oxygen evolved h-ling-1 chlorophyll using an Oxygen electrode (Hansatech, D~f 1, England ), in the paraquat tolerant and susceptible cultures grown in the absence of paraquat. Estimation of the activities of enzymes was carried out with an extract made from 70mg cells in iml of 50ram K-phosphate buffer, ph 7.5, using an ultra sonicator (Braun). An initial pulse of 30s, followed by three pulses of 10s at intervals of 10s comprised the sonication cycle. The crude extract w a s centrifuged at 4 ~ C for 10min at 7000xg, and the supernatant used for the enzyme assays. Catalase and peroxidase activity was determined according to the method used by Volk and Feiera bend ( 1989 ). Superoxide dismutase activity was determined by the method of Beauchamp and Fridovich ( 1971 ), in terms of inhibition of the rate of NBT reduction. One unit of SOD activity was the amount which inhibited 50% of the reduction rate of N B T . T h e experiments w e r e carried out twice. Results a n d Discussion:
The paraquat tolerant line s h o w e d less inhibition of growth and better chlorophyll retention as compared to the susceptible cultures after treatment with 2uM paraquat (Table 1 ). Ashton and Ziegler (1987) have s h o w n that 2 u M paraquat inhibits g r o w t h of photoautotrophic and photomixotrophic cultures of C henopodium rubrum, unlike other photosynthetic herbicides eg. Diuron, which only inhibits growth of photoautotrophic cultures. Ho~cever the PSI electron transport activity in chloroplasts isolated from paraquat tolerant and susceptible cultures was similar (Tablel) when grown in s medium without paraquat. Hence paraquat tolerance was probably not associated with changes in the chloroplasts of the tolerant cultures. This observation was also made in paraquat resistant biotypes of Conyza (Fuerst et al., 1985 ) and in paraquat resistant Hordeum glaucum (Powles and Cornic, 1987). Studies on the activities of enzymes involved in detoxifica tion of activated oxygen species, namely, superoxide dismuta se, catalase and peroxidase revealed no major changes in the susceptlble and tolerant cultures (Fig. 1 ). The paraquat tolerant cultures showed slightly decreased activity of superoxide dismutase and peroxidase and slightly increased activity
I.
Table
.
.
.
.
.
.
.
.
.
.
.
.
Growth, chlorophyll content, and PSI partial reactions of paraquat susceptible and tolerant cultures in the presen ce or absence of 2uM paraquat. Results are a mean of three replicates. .
.
.
.
Parameter
.
.
.
.
.
.
.
.
.
.
-paraquat .
.
.
.
.
.
.
.
.
.
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.
.
Growth(fr. wt.g/2Oml afterl5days .
.
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+pc raquat
.
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.
.
.
.
.
.
.
.
.
0.052 + 0.001 .
.
.
1.145 + 0.095
.
.
.
.
.
.
.
.
.
PSI partial 0.391+reaction( m M o l 9,037 02 h - l m g -I chl. ) .
.
0.185 + 0.018
0.585+ 0.039
.
.
-paraquat
.
0.01_+ 0.001
1.33+ 0.064
.
.
Paraquat tolerant cultures
+paraquat .
0.37 + _ 0.03
Chlorophyll content(rag g-lfr.wt. ) after3days .
.
Paraqust susceptible cultures
.
.
.
.
.
.
0.92 + 0.085
.
.
.
.
0.338_+ 0.035
.
.
.
.
.
.
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.
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.
~
.
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.
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.
.
.
.
of c a t a l a s e . A lack of correlation between the activities of these enzymes and paraquat resistance has been reported by Powles and Cornic (1987) and Carroll, et a!. (1988). Shaaltiel and Gressel, (1986) have showed a positive correlation between the activities of the oxygen radical detexifica tion system in the chloroplasts and paraqust resistance. An ability of this o x y g e n radical detoxification system in providing cross tolerance to photoinhibitory light has been reported by Jansen et al. ( 1989 ), and a lack of cross tolerance to photoinhitory light has been shown by Ranade and Feierabend (1991). In the presence of paraquat, the susceptible and tolerant cultures of Chenopodium rubrum behaved very differently. While there was a decrease in the activities of all three enzymes in the susceptible cultures, an increase in superoxide dismula se and peroxidase activity w a s seen in the tolerant cultures. Also, catalase activity was only slightly reduced in the tolerant cultures. This trend was apparent even when the activities of these enzymes were compared at their respective LD 50 values for paraquat (Fig. 1 ). Differential induction or activation of these enzymes by paraquat in the tolerant and susceptible cultures could be an explanation for their higher activities in tolerant cultures, and this will be investigated through further experiments. Studies on the oxygen radical detexif ying system from paraquat tolerant and susceptible plants, in the presence of paraquat have not yet been reported,
Acknowledgements: Financial support by the German Academic Exchange Service, and the Department of Science and Technology, India, is gratefully acknowledged. The author is also thankful to Dr. J. Feiera bend, Frankfurt, for providing facilities to carry out a part of this work, and to Dr. W. Huesemann for a sample of Chenopodium r u b r u m cultures. Technical assistance
232 of Ms. Varsha Vartak in a s s a y s is a l s o a c k n o w l e d g e d .
0"2 c
T o~
0.1
m
'T
o~
%
4"
=..
200 -
% u~
100-
e-
0 0 " 0 8 0.2
2
0
0"8
2
paraquat ]j M
Fig.
i.
enzyme
References:
0"3
%
the
Activity of s u p e r o x i d e dismutase (A), c a t a l a s e (B) a n d p e r o x i d a s e (C) in paraquat susceptib]e (lanes 1,2,3 & 4) a n d tolerant cultures (lanes 5,6 & 7) in the absence (lanes 1 & 5) a n d p r e s e n c e of d i f f e r e n t concentrations of paraquat, after 3 days of treatment. Readings are a mean of two experiments.
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