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Action of some herbicides on growth, respiration, plasmalemma integrity, and proton extrusion of Acer pseudoplatanus cells
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
17, 156-161 (1982)
Action of Some Herbicides on Growth, Respiration, Plasmalemma Integrity, and Proton Extrusion of Acer pseudoplatanus Cells II. Amides, Diphenyl Ethers, Nitriles, Phenols, Triazines, JEAN-PIERRE Laboratoire
and Uracils
BLEIN
des Herbicides, INRA, BV 1540, 21034 Dijon Cedex, France Received April 9, 1981; accepted November 27, 1981
The activity of 15 herbicides from different families was assayed on nonphotosynthetic Acer pseudoplatanus ceils, in batch suspension culture. They inhibited growth, stimulated the oxygen consumption by cells, and changed the pH evolution of the culture medium. Amides did not seem to disturb the membrane properties. Some diphenyl ethers and phenols directly modified the proton compartmentation of cells whereas some other diphenyl ethers, nitriies, triazines, and uracils altered permeabilities to potassium and leucine. Changes in membrane permeabilities to protons and medium constituents were discussed.
INTRODUCTION
Many herbicides currently used today (substituted ureas and triazines) act on the energetic metabolism of plants. This was shown by periods of research on inhibitors of photosynthesis (since 1950-1955) or on uncouplers of oxidative phosphorylations (1960- 1965, chemioosmotic theory). In this case the chemicals can be studied with isolated and purified organelles (chloroplasts and mitochondria). Nevertheless, beside the action of herbicides on cellular energetic metabolism, other essential functions of cells could be disturbed with dramatic effects. A perturbation of plasmalemma would modify the cellular exchanges which contribute to maintenance of the pH and ionic equilibria of the cell. Some inhibitors of photosynthesis, such as triazines, change the ionic permeability of nonphotosynthetic callus (1) and of roots (2). Watson et al. (3) indicate that one of the primary sites of action of several herbicides may be on the cell membrane, and Bucholtz and Lavy (4) correlate the effects of alachlor and trifluralin on the root and shoot growth to the plant ability to absorb phosphate and sulfate ions. Consequently we have sought herbicides which act at the plasmalemma level.
The movement of most metabolites and inorganic ions across membranes involves specific carriers linked to the activity of a membrane ATP-dependent proton pump (5). The change in extracellular pH can provide information on the activity of the pump and the consumption of protons linked to the cotransport process between protons and solutes. We also studied the action of herbicides on the oxygen consumption by cells, as mitochondria supply ATP required for proton pump activity. A previous paper (6) reported results obtained with substituted ureas and in the present work, we studied one nitrile, two amides and diphenyl ethers, three phenols and uracils, and four triazines to determine if their inhibition of the cellular growth could be explained by modifications in membrane properties. MATERIALS
Copyrieht Ail rights
0 1982 by Academic Press, 1nc. Of reproduction in my form reserved.
METHODS
Acer pseudoplatanus cells were grown in batch suspension culture and used during the exponential phase of growth. Culture conditions and oxygen uptake determination were described previously. (7). Growth inhibition was determined from the increase in the doubling time of cell dry weight dur156
0048-3575/82/020156-06$02.00/O
AND
ACTION
OF
SOME
HERBICIDES
ing the exponential phase of growth. Results are expressed as percentage of control. One hundred percent was the control level, zero percent was the lethal effect. The cell dry weight was determined from a 5-ml aliquot of suspension, filtered and desiccated overnight at 75°C. Each determination was repeated five times. During the cell growth, the pH of the culture medium changes (8) and its buffering capacity was modified as shown in Fig. 1. The neq H+, appearing or disappearing, were calculated from the pH variation and the buffering capacity (Fig. 1) of the culture medium. In order to improve the extent of the pH variation, fusicoccin was added to the culture medium in some experiments (see legends and (6)). Membrane integrity was determined from the amount of fluorescein released by cells in the culture medium, according to the method of Persidsky and Baillie (9), modified as in (6). For the measurement of leucine uptake, a cell suspension (3-8 mg dry wt/ml) was incubated with 100 PM [14C]leucine (10 &iimmoI, CEA, Fr.). After 10 min, 2-ml aliquots of the suspension were rapidly filtered on Whatman glass-fiber filter (GFIA) and washed twice by 5 ml of fresh culture medium. Cells were scraped from the filter, placed in a scintillation vial, and counted in
of the buffering capacity of the medium during the cell growth. At differenr times, the cell dry weight of the suspension was determined and the culture medium- was titrated, between pH 4.0 and 7.0, to determine its bufering capacity. Cells were removed by centrifugation (lO,OOOg, 10 min). FIG. culture
1. Evolution
ON
Acer
pseudoplazunus
CELLS
157
Beckman “ready solv. HP.” Results are expressed as percentage of control. For the determination of potassium uptake, a cell suspension was incubated 60 min with 10 mM KC1 and 86Rb (125 nCi/ml cell suspension, Radiochemical Centre, Amersham, U.K.). Cells were filtered as mentioned for leucine uptake, but not washed, and counted in water by the Cerenkov effect. Results are expressed as percentage of control. Analytical grade herbicides were dissolved in dimethyl sulfoxide, and the final concentration of solvent in each assay was 1%. Controls were carried out with 1% dimethyl sulfoxide. Fifteen herbicides were assayed: ametryne, atrazine, bromacil. chlorthiamid, desmetryne, dichlobenil, dinoseb, DNOC, fluorodifen, ioxynil, lenacil, metribuzin, nitrofen, propanil, and terbacil. They were provided by Chem-service and Pestanal, or were graciously supplied by the Bayer, Ciba-Geigy, DuPont de Nemours, RhBne-Poulenc, and RousselUclaf Companies. RESULTS
In a first step, we assayed the 15 herbicides (100 @4) to screen the molecules acting on proton extrusion and without any action on oxygen consumption. Results are summarized in Fig. 2. Chlorthiamid, propanil, fluorodifen, nitrofen, dichlobenil, ioxynil, dinoseb, and DNOC had a lethal effect. Ametryne, atrazine, desmetryne, bromacil, lenacil, and terbacil inhibited cell growth to some extent, whereas metribuzin stimulated it. Ioxynil, dinoseb, and DNOC strongly stimulated oxygen consumption by cells. To a lesser extent, so did propanil, fluorodifen, dichlobenil, and atrazine; the other herbicides had no effect. Proton extrusion was not changed by metribuzin and was just slightly decreased by chlorthiamid and propanil during the first 60 min (Fig. 3). The other herbicides decreased the proton extrusion and nitrofen, dichlobenil, ioxynil, dinoseb, and
158
JEAN-PIERRE
BLEIN
n .qH?/mln 100
pM of
mg
dry
ld weight
i- " h t r 0 1 chlorthiamid propanil fluorodifen nitrofen dichlobenil ioxynil dinoseb DNOC ametryne atrazine desmetryne metribuzin bromacil lenacil terbacil
FIG. 2. Action of herbicides (100 pM) on cell growth, oxygen consumption, and proton extrusion by ceils, and on plasma/emma integrity. The growth of cell suspension was measured as the doubting time of cell dry weight during the exponential phase of growth. Results are expressed as percentage of control. 100% was the control level, 0% was a lethal effect. The total oxygen consumption by cells was determined by Clark electrode measurements and expressed as percentage of control. 100% was the control level. The neq H+ were determined from the measurement of the pH variation of a fresh culture medium in the presence of fusicoccin (0.03 phi), and calculated during the first 60 min with I pH unit = 42.9 peq H+ (Fig. i). Results were an average of three repetitions. Control value was 9.6 f 1.3. Cell concentration was 8 mglml. The plasmalemma integrity was expressed as the percentage of total fluorescein contained in loaded cells released in 60 min, and determined by OD (485 nm) of the culture medium. Results are an average ofjive repetitions. Control was 22 * 2%.
DNOC were the most active, but over the first 60 min, propanil, desmetryne, and atrazine had a stronger effect (Fig. 3a and b). Dichlobenil, ioxynil, fluorodifen, and nitrofen changed the initial pH (about 0.03 to 0.04 pH unit), measured 10 min after herbicidal treatment but just after the addition of fusicoccin (see the legend of Fig. 3). Only fluorodifen, ioxynil, dinoseb, and DNOC seemed to disturb the membrane integrity. DISCUSSION
1. Herbicides Which Killed Cells (a) With a strong effect on proton extrusion and oxygen consumption. Among the chemicals which killed cells were ioxynil, DNOC, and dinoseb, which have been shown to uncouple oxidative phosphorylation and destroy the membrane structures
(10, 11). For these three herbicides, a good relationship exists between their effects on proton extrusion and membrane integrity. Ioxynil was the most active and DNOC was the least potent. The change in membrane integrity (membrane permeability to fluorescein) might result from variations in intracellular concentration of protons which increased the amount of neutral form of fluorescein (more permeant) according to a previous discussion (6). (6) With effect on proton extrusion but a slight effect on oxygen consumption. Dichlobenil which inhibited proton extrusion like ioxynil, did not stimulate oxygen consumption to the same extent. It did not change the membrane integrity and contrary to ioxynil, it has no hydroxyl group. The variations in the pH of the culture medium might not result from a migration of protons across plasmalemma, but from a
ACTION
OF
SOME
HERBICIDES
ON
Acer
pseudoplutunus
(c) With a slight effect on proton extrusion and oxygen consumption. The two amides, chlorthiamid and propanil, had a lethal effect. They did not act on membrane integrity and they slightly changed the proton extrusion, but propanil stimulated oxygen consumption and it was more active on proton extrusion after the first 60 min. Chlorthiamid did not seem to disturb membrane properties whereas the stimulatory action of propanil on respiration could be interpreted as an action on mitochondria. This is substantiated by Hofstra and Switzer who showed that propanil is an uncoupler of oxidative phosphorylation ( 13). These chemicals are reported to act on several cellular sites, such as proteins and RNA synthesis (14) which could explain the lethal effect on cell suspensions.
/ I k-In+ 0
+I0
+20
MqH+
0
+10
2. Herbicides Which Inhibited Growth to Some Extent
+20
159
CELLS
Cell
All other chemicals, except atrazine, did not change the uptake of oxygen by cells. They decreased to some extent the proton extrusion without change in membrane integrity. Their action on cell growth could result from slight modifications in membrane permeability.
FIG. 3. Time course of pH modifications of the culture medium. Fusicoccin (0.3 PM) was added at zero time. Herbicides (100 pM) were added 10 min before. Cell concentration was 8 mg dry wtlml. (a) A, Control; 0, nitrofen; q , chlorthiamid; 0, j7uorodifen; n , propanil. (b) A, Control; l , desmetryne; 0, ioxynil; 0. atrazine; n , dichlobenil.
3. Herbicides Which Stimulated Cell Growth
change in metabolites and/or inorganic ions membranes permeability. Glen et al. reported that dichlobenil greatly reduces the transport of assimilates from leaves (12). Fluorodifen and nitrofen effects seemed, respectively, like ioxynil and dichlobenil. Thus, their action on membrane structures must be equally comparable. The action of these four herbicides on proton extrusion by cells can be of two kinds. The first action was very fast ( < 10 min) and could correspond to the solubilization of the herbicides into the membranes. The second one lasted for a longer time (150 min) and could result from the activity of the modified membranes by herbicides.
Metribuzin was not active on oxygen consumption, membrane integrity, and proton extrusion by cells. Cell growth was stimulated by about 35%. Freney reported simular results with simazine (5 to 10 mA4) which increase the dry weight and growth of maize tops by 27% (15). The activity of ametryne, bromacil, desmetryne, dichlobenil, lenacil, nitrofen, and terbacil at a lower concentration (lO@V) on cell growth and proton extrusion by cells was determined (Fig. 4). Bromacil, desmetryne, and terbacil had no action on proton extrusion by cells; whereas bromacil did not affect cell growth, desmetryne and terbacil increased it. Ametryne and dichlobenil equally increased the amount of H+
160
JEAN-PIERRE
dichlobenil
FIG. 4. Action of herbicides (IO t&l) on cell growth and proton extrusion. For experimental conditions see Fig. 2. Fusicoccin concentration was 1 uM. Cells were not resuspended in fresh medium and the buffering capacity of the culture medium required for the calculation of proton equivalents was issued from Fig. 1,
which appeared in the culture medium but only dichlobenil had a lethal effect while ametryne had no action on cell growth. It means this same change in protons extrusion observed resulted from different actions. Lenacil and nitrofen were the most active on proton extrusion. Nitrofen had a lethal effect and the smaller action of lenacil(33% inhibition) could result from a metabolization of this herbicide. At 10 pM, nitrofen, dichlobenil, and lenacil were the most active. Proton extrusion establishes an electrochemical gradient of protons across the plasmalemma of cells, and thus provides the driving force for the proton coupled uptake of ions of organic solutes (16, 17). Therefore we assayed several herbicides
BLEIN
(100 pM) for proton extrusion, without fusicoccin, and effects on leucine and potassium uptake (Fig. 5). Bromacil was slightly active and some other herbicides such as ametryne, desmetryne, or terbacil induced an acidification of the culture medium without considerable change in leucine and potassium uptake. Nitrofen and dichlobenil also increased acidification of the medium, but they inhibited leucine uptake; while nitrofen inhibited potassium uptake, dichlobenil stimulated it. As shown in Fig. 4, ametryne and dichlobenil had the same action on the amount of proton equivalents which appeared but only dichlobenil modified the cell growth and the leucine and potassium uptake. Lenacil induced an alkalinization of the culture medium. It strongly inhibited the leucine uptake and increased the potassium uptake (25fold of control). CONCLUSION
Use of easy techniques such as pH determination of the culture medium or oxygen consumption by cells allowed us to detect molecules which modify the cellular exchanges across the plasmalemma without effect on mitochondria, i.e., without change in direct proton compartmentation of the cell as it would be with uncouplers of oxidative phosphorylation. Results with phenols (ioxynil, dinoseb, and DNOC)
lenacil terbacil dichlobenil nitroien
FIG. 5. Action of herbicides (100 pM) on proton extrusion (without fusicoccin) and on leucine and potassium uptake. For experimental conditions to determine the amount of neq H+ see Fig. 4. Leucine and potassium “Rb uptake were measured after an incubation of IO and 60 mitt, respectively. Herbicide and labeled tracer were added at zero time. Experiments were repeated three times.
ACTION
OF
SOME
HERBICIDES
which act like protonophores on plasmalemma and mitochondrial membrane were used as controls and back up the criticism previously made about the method of Persidsky and Baillie to determine membrane integrity (6). The results reported in this work can be interpreted as plasmalemma perturbations only if comparisons are made. They allow us to identify chemicals acting on plasmalemma. Among the 15 herbicides studied, three were very active on A. pseudoplatanus cells. They were dichlobenil, nitrofen, and lenacil. It does not imply the same would occur in plants in which penetration, translocation, and metabolization have to be taken into account. We are presently studying these three herbicides to determine the sensitivity of potential sites in plasmalemma. The small growth inhibition induced by lenacil is due to a quick metabolization of the herbicide by cells (manuscript in preparation). The differences observed in the effect of herbicides on proton extrusion in the presence or in the absence of fusicoccin could be explained by the existence of two different mechanisms for proton extrusion (18). Another possibility would be that, in the presence of fusicoccin, the proton pump activity is no longer influenced by cellular regulation (19). REFERENCES
1. K. Zippel, M. Mader, and M. Bopp, The effect of some herbicides on ion accumulation in tissue culture of Anagallis arvensis, Angew. Bot. 5 1, 77 (1977). 2. F. Renosto, S. Nardi, and G. Ferrari, Atrazine inhibition of ion uptake by plant roots, Pesric. Biachem. Physiof. 11, 243 (1979). 3. M. C. Watson, P. G. Bartels, and K. C. Hamilton, Action of selected herbicides and Tween 20 on oat (Avena sariva) membranes, Weed Sci. 28, 122 (1980).
ON
Acer pseudoplatunus
CELLS
161
4. D. L. Bucholtz and T. L. Lavy, Alachlor and trifluralin effects on nutrient uptake in oats and soybeans, Agron. J. 71, 24 (1979). 5. E. Marre, Fusicoccin-A tool in plant physiology, Annu. Rev. Plant Physiol. 30, 273 (1979). 6. J. P. Blein, Action of some herbicides on growth. respiration, plasmalemma integrity and proton extrusion of Acer pseudoplatanus cells. I. Substituted ureas, Pestic. Biochem. Physiol. 16, 179- 186 (1981). 7. J. P. Blein, Cyanide stimulation of respiration of Acer pseudoplatanus cells in batch suspension culture and activation of the alternative pathway, Planf Sci. Lerr. 19, 65 (1980). 8. A. Kurkdjian, Action de la fusicoccine sur la morphologie et le grandissement de celhtles d’Acer pseudoplatanus cultivees in vitro, Physiol. veg.,
17, 305 (1979).
9. M. D. Persidsky and C. S. Baillie, Fluorometric test of cell membrane integrity, Cryobiology, 14, 322 (1977).
10. T. E. Ferrari and D. E. Moreland, Effects of 3,5dihalogenated-4-hydroxybenzonitriles on the activity of mitochondria from white potato tubers, Plant Physiol.. 44, 429 (1969). 11. F. M. Ashton and A. S. Crafts, “Mode of Action of Herbicides,” p. 256, Wiley, New York, 1973. 12. R. K. Glen, 0. A. Leonard, and D. E. Bayer. Some effects of dichlobenil, Res. Prog. Rep. West. Sac. Weed Sci., 150 (1971). 13. G. Hofstra and C. M. Switzer, The phytotoxicity of propanil, Weed Sci. 16, 23 (1968). 14. R. S. Morrod, Effects on plant cell membrane structure and function, in “Herbicides” (L. J. Audus, Ed.), Vol. 1, p. 281, Academic Press, London/New York/San Francisco, 1976. 15. J. R. Freney, Increased growth and uptake of nutrients by corn plants treated with low levels of simazine, Ausr. J. Agr. Res. 16, 257 (1965). 16. F. W. Ben&up, Cell electrophysiology and membrane transport, Prog. Bat. 40, 84 (1978). 17. R. J. Poole, Energy coupling for membrane transport, Annu. Rev. Plunt Physiol. 29, 437 (1978). 18. R. E. Cleland and T. Lomax, Hormonal control of H+-excretion from oat cells, in “Regulation of Cells Membrane Activities in Plants (E. Marre and 0. Ciferri, Eds.), p. 161, Elsevier. Amsterdam, 1977. 19. R. Colombo, A. Bonetti, R. Cerana, and P. Lado, Effect of plasmalemma ATPase inhibitors diethylstilbestrol and orthovanadate, on fusicoccin-induced H+ extrusion in maize roots. P/ant Sci. Left.. 21, 305 (1981).
BIOCHEMISTRY
AND
PHYSIOLOGY
17, 156-161 (1982)
Action of Some Herbicides on Growth, Respiration, Plasmalemma Integrity, and Proton Extrusion of Acer pseudoplatanus Cells II. Amides, Diphenyl Ethers, Nitriles, Phenols, Triazines, JEAN-PIERRE Laboratoire
and Uracils
BLEIN
des Herbicides, INRA, BV 1540, 21034 Dijon Cedex, France Received April 9, 1981; accepted November 27, 1981
The activity of 15 herbicides from different families was assayed on nonphotosynthetic Acer pseudoplatanus ceils, in batch suspension culture. They inhibited growth, stimulated the oxygen consumption by cells, and changed the pH evolution of the culture medium. Amides did not seem to disturb the membrane properties. Some diphenyl ethers and phenols directly modified the proton compartmentation of cells whereas some other diphenyl ethers, nitriies, triazines, and uracils altered permeabilities to potassium and leucine. Changes in membrane permeabilities to protons and medium constituents were discussed.
INTRODUCTION
Many herbicides currently used today (substituted ureas and triazines) act on the energetic metabolism of plants. This was shown by periods of research on inhibitors of photosynthesis (since 1950-1955) or on uncouplers of oxidative phosphorylations (1960- 1965, chemioosmotic theory). In this case the chemicals can be studied with isolated and purified organelles (chloroplasts and mitochondria). Nevertheless, beside the action of herbicides on cellular energetic metabolism, other essential functions of cells could be disturbed with dramatic effects. A perturbation of plasmalemma would modify the cellular exchanges which contribute to maintenance of the pH and ionic equilibria of the cell. Some inhibitors of photosynthesis, such as triazines, change the ionic permeability of nonphotosynthetic callus (1) and of roots (2). Watson et al. (3) indicate that one of the primary sites of action of several herbicides may be on the cell membrane, and Bucholtz and Lavy (4) correlate the effects of alachlor and trifluralin on the root and shoot growth to the plant ability to absorb phosphate and sulfate ions. Consequently we have sought herbicides which act at the plasmalemma level.
The movement of most metabolites and inorganic ions across membranes involves specific carriers linked to the activity of a membrane ATP-dependent proton pump (5). The change in extracellular pH can provide information on the activity of the pump and the consumption of protons linked to the cotransport process between protons and solutes. We also studied the action of herbicides on the oxygen consumption by cells, as mitochondria supply ATP required for proton pump activity. A previous paper (6) reported results obtained with substituted ureas and in the present work, we studied one nitrile, two amides and diphenyl ethers, three phenols and uracils, and four triazines to determine if their inhibition of the cellular growth could be explained by modifications in membrane properties. MATERIALS
Copyrieht Ail rights
0 1982 by Academic Press, 1nc. Of reproduction in my form reserved.
METHODS
Acer pseudoplatanus cells were grown in batch suspension culture and used during the exponential phase of growth. Culture conditions and oxygen uptake determination were described previously. (7). Growth inhibition was determined from the increase in the doubling time of cell dry weight dur156
0048-3575/82/020156-06$02.00/O
AND
ACTION
OF
SOME
HERBICIDES
ing the exponential phase of growth. Results are expressed as percentage of control. One hundred percent was the control level, zero percent was the lethal effect. The cell dry weight was determined from a 5-ml aliquot of suspension, filtered and desiccated overnight at 75°C. Each determination was repeated five times. During the cell growth, the pH of the culture medium changes (8) and its buffering capacity was modified as shown in Fig. 1. The neq H+, appearing or disappearing, were calculated from the pH variation and the buffering capacity (Fig. 1) of the culture medium. In order to improve the extent of the pH variation, fusicoccin was added to the culture medium in some experiments (see legends and (6)). Membrane integrity was determined from the amount of fluorescein released by cells in the culture medium, according to the method of Persidsky and Baillie (9), modified as in (6). For the measurement of leucine uptake, a cell suspension (3-8 mg dry wt/ml) was incubated with 100 PM [14C]leucine (10 &iimmoI, CEA, Fr.). After 10 min, 2-ml aliquots of the suspension were rapidly filtered on Whatman glass-fiber filter (GFIA) and washed twice by 5 ml of fresh culture medium. Cells were scraped from the filter, placed in a scintillation vial, and counted in
of the buffering capacity of the medium during the cell growth. At differenr times, the cell dry weight of the suspension was determined and the culture medium- was titrated, between pH 4.0 and 7.0, to determine its bufering capacity. Cells were removed by centrifugation (lO,OOOg, 10 min). FIG. culture
1. Evolution
ON
Acer
pseudoplazunus
CELLS
157
Beckman “ready solv. HP.” Results are expressed as percentage of control. For the determination of potassium uptake, a cell suspension was incubated 60 min with 10 mM KC1 and 86Rb (125 nCi/ml cell suspension, Radiochemical Centre, Amersham, U.K.). Cells were filtered as mentioned for leucine uptake, but not washed, and counted in water by the Cerenkov effect. Results are expressed as percentage of control. Analytical grade herbicides were dissolved in dimethyl sulfoxide, and the final concentration of solvent in each assay was 1%. Controls were carried out with 1% dimethyl sulfoxide. Fifteen herbicides were assayed: ametryne, atrazine, bromacil. chlorthiamid, desmetryne, dichlobenil, dinoseb, DNOC, fluorodifen, ioxynil, lenacil, metribuzin, nitrofen, propanil, and terbacil. They were provided by Chem-service and Pestanal, or were graciously supplied by the Bayer, Ciba-Geigy, DuPont de Nemours, RhBne-Poulenc, and RousselUclaf Companies. RESULTS
In a first step, we assayed the 15 herbicides (100 @4) to screen the molecules acting on proton extrusion and without any action on oxygen consumption. Results are summarized in Fig. 2. Chlorthiamid, propanil, fluorodifen, nitrofen, dichlobenil, ioxynil, dinoseb, and DNOC had a lethal effect. Ametryne, atrazine, desmetryne, bromacil, lenacil, and terbacil inhibited cell growth to some extent, whereas metribuzin stimulated it. Ioxynil, dinoseb, and DNOC strongly stimulated oxygen consumption by cells. To a lesser extent, so did propanil, fluorodifen, dichlobenil, and atrazine; the other herbicides had no effect. Proton extrusion was not changed by metribuzin and was just slightly decreased by chlorthiamid and propanil during the first 60 min (Fig. 3). The other herbicides decreased the proton extrusion and nitrofen, dichlobenil, ioxynil, dinoseb, and
158
JEAN-PIERRE
BLEIN
n .qH?/mln 100
pM of
mg
dry
ld weight
i- " h t r 0 1 chlorthiamid propanil fluorodifen nitrofen dichlobenil ioxynil dinoseb DNOC ametryne atrazine desmetryne metribuzin bromacil lenacil terbacil
FIG. 2. Action of herbicides (100 pM) on cell growth, oxygen consumption, and proton extrusion by ceils, and on plasma/emma integrity. The growth of cell suspension was measured as the doubting time of cell dry weight during the exponential phase of growth. Results are expressed as percentage of control. 100% was the control level, 0% was a lethal effect. The total oxygen consumption by cells was determined by Clark electrode measurements and expressed as percentage of control. 100% was the control level. The neq H+ were determined from the measurement of the pH variation of a fresh culture medium in the presence of fusicoccin (0.03 phi), and calculated during the first 60 min with I pH unit = 42.9 peq H+ (Fig. i). Results were an average of three repetitions. Control value was 9.6 f 1.3. Cell concentration was 8 mglml. The plasmalemma integrity was expressed as the percentage of total fluorescein contained in loaded cells released in 60 min, and determined by OD (485 nm) of the culture medium. Results are an average ofjive repetitions. Control was 22 * 2%.
DNOC were the most active, but over the first 60 min, propanil, desmetryne, and atrazine had a stronger effect (Fig. 3a and b). Dichlobenil, ioxynil, fluorodifen, and nitrofen changed the initial pH (about 0.03 to 0.04 pH unit), measured 10 min after herbicidal treatment but just after the addition of fusicoccin (see the legend of Fig. 3). Only fluorodifen, ioxynil, dinoseb, and DNOC seemed to disturb the membrane integrity. DISCUSSION
1. Herbicides Which Killed Cells (a) With a strong effect on proton extrusion and oxygen consumption. Among the chemicals which killed cells were ioxynil, DNOC, and dinoseb, which have been shown to uncouple oxidative phosphorylation and destroy the membrane structures
(10, 11). For these three herbicides, a good relationship exists between their effects on proton extrusion and membrane integrity. Ioxynil was the most active and DNOC was the least potent. The change in membrane integrity (membrane permeability to fluorescein) might result from variations in intracellular concentration of protons which increased the amount of neutral form of fluorescein (more permeant) according to a previous discussion (6). (6) With effect on proton extrusion but a slight effect on oxygen consumption. Dichlobenil which inhibited proton extrusion like ioxynil, did not stimulate oxygen consumption to the same extent. It did not change the membrane integrity and contrary to ioxynil, it has no hydroxyl group. The variations in the pH of the culture medium might not result from a migration of protons across plasmalemma, but from a
ACTION
OF
SOME
HERBICIDES
ON
Acer
pseudoplutunus
(c) With a slight effect on proton extrusion and oxygen consumption. The two amides, chlorthiamid and propanil, had a lethal effect. They did not act on membrane integrity and they slightly changed the proton extrusion, but propanil stimulated oxygen consumption and it was more active on proton extrusion after the first 60 min. Chlorthiamid did not seem to disturb membrane properties whereas the stimulatory action of propanil on respiration could be interpreted as an action on mitochondria. This is substantiated by Hofstra and Switzer who showed that propanil is an uncoupler of oxidative phosphorylation ( 13). These chemicals are reported to act on several cellular sites, such as proteins and RNA synthesis (14) which could explain the lethal effect on cell suspensions.
/ I k-In+ 0
+I0
+20
MqH+
0
+10
2. Herbicides Which Inhibited Growth to Some Extent
+20
159
CELLS
Cell
All other chemicals, except atrazine, did not change the uptake of oxygen by cells. They decreased to some extent the proton extrusion without change in membrane integrity. Their action on cell growth could result from slight modifications in membrane permeability.
FIG. 3. Time course of pH modifications of the culture medium. Fusicoccin (0.3 PM) was added at zero time. Herbicides (100 pM) were added 10 min before. Cell concentration was 8 mg dry wtlml. (a) A, Control; 0, nitrofen; q , chlorthiamid; 0, j7uorodifen; n , propanil. (b) A, Control; l , desmetryne; 0, ioxynil; 0. atrazine; n , dichlobenil.
3. Herbicides Which Stimulated Cell Growth
change in metabolites and/or inorganic ions membranes permeability. Glen et al. reported that dichlobenil greatly reduces the transport of assimilates from leaves (12). Fluorodifen and nitrofen effects seemed, respectively, like ioxynil and dichlobenil. Thus, their action on membrane structures must be equally comparable. The action of these four herbicides on proton extrusion by cells can be of two kinds. The first action was very fast ( < 10 min) and could correspond to the solubilization of the herbicides into the membranes. The second one lasted for a longer time (150 min) and could result from the activity of the modified membranes by herbicides.
Metribuzin was not active on oxygen consumption, membrane integrity, and proton extrusion by cells. Cell growth was stimulated by about 35%. Freney reported simular results with simazine (5 to 10 mA4) which increase the dry weight and growth of maize tops by 27% (15). The activity of ametryne, bromacil, desmetryne, dichlobenil, lenacil, nitrofen, and terbacil at a lower concentration (lO@V) on cell growth and proton extrusion by cells was determined (Fig. 4). Bromacil, desmetryne, and terbacil had no action on proton extrusion by cells; whereas bromacil did not affect cell growth, desmetryne and terbacil increased it. Ametryne and dichlobenil equally increased the amount of H+
160
JEAN-PIERRE
dichlobenil
FIG. 4. Action of herbicides (IO t&l) on cell growth and proton extrusion. For experimental conditions see Fig. 2. Fusicoccin concentration was 1 uM. Cells were not resuspended in fresh medium and the buffering capacity of the culture medium required for the calculation of proton equivalents was issued from Fig. 1,
which appeared in the culture medium but only dichlobenil had a lethal effect while ametryne had no action on cell growth. It means this same change in protons extrusion observed resulted from different actions. Lenacil and nitrofen were the most active on proton extrusion. Nitrofen had a lethal effect and the smaller action of lenacil(33% inhibition) could result from a metabolization of this herbicide. At 10 pM, nitrofen, dichlobenil, and lenacil were the most active. Proton extrusion establishes an electrochemical gradient of protons across the plasmalemma of cells, and thus provides the driving force for the proton coupled uptake of ions of organic solutes (16, 17). Therefore we assayed several herbicides
BLEIN
(100 pM) for proton extrusion, without fusicoccin, and effects on leucine and potassium uptake (Fig. 5). Bromacil was slightly active and some other herbicides such as ametryne, desmetryne, or terbacil induced an acidification of the culture medium without considerable change in leucine and potassium uptake. Nitrofen and dichlobenil also increased acidification of the medium, but they inhibited leucine uptake; while nitrofen inhibited potassium uptake, dichlobenil stimulated it. As shown in Fig. 4, ametryne and dichlobenil had the same action on the amount of proton equivalents which appeared but only dichlobenil modified the cell growth and the leucine and potassium uptake. Lenacil induced an alkalinization of the culture medium. It strongly inhibited the leucine uptake and increased the potassium uptake (25fold of control). CONCLUSION
Use of easy techniques such as pH determination of the culture medium or oxygen consumption by cells allowed us to detect molecules which modify the cellular exchanges across the plasmalemma without effect on mitochondria, i.e., without change in direct proton compartmentation of the cell as it would be with uncouplers of oxidative phosphorylation. Results with phenols (ioxynil, dinoseb, and DNOC)
lenacil terbacil dichlobenil nitroien
FIG. 5. Action of herbicides (100 pM) on proton extrusion (without fusicoccin) and on leucine and potassium uptake. For experimental conditions to determine the amount of neq H+ see Fig. 4. Leucine and potassium “Rb uptake were measured after an incubation of IO and 60 mitt, respectively. Herbicide and labeled tracer were added at zero time. Experiments were repeated three times.
ACTION
OF
SOME
HERBICIDES
which act like protonophores on plasmalemma and mitochondrial membrane were used as controls and back up the criticism previously made about the method of Persidsky and Baillie to determine membrane integrity (6). The results reported in this work can be interpreted as plasmalemma perturbations only if comparisons are made. They allow us to identify chemicals acting on plasmalemma. Among the 15 herbicides studied, three were very active on A. pseudoplatanus cells. They were dichlobenil, nitrofen, and lenacil. It does not imply the same would occur in plants in which penetration, translocation, and metabolization have to be taken into account. We are presently studying these three herbicides to determine the sensitivity of potential sites in plasmalemma. The small growth inhibition induced by lenacil is due to a quick metabolization of the herbicide by cells (manuscript in preparation). The differences observed in the effect of herbicides on proton extrusion in the presence or in the absence of fusicoccin could be explained by the existence of two different mechanisms for proton extrusion (18). Another possibility would be that, in the presence of fusicoccin, the proton pump activity is no longer influenced by cellular regulation (19). REFERENCES
1. K. Zippel, M. Mader, and M. Bopp, The effect of some herbicides on ion accumulation in tissue culture of Anagallis arvensis, Angew. Bot. 5 1, 77 (1977). 2. F. Renosto, S. Nardi, and G. Ferrari, Atrazine inhibition of ion uptake by plant roots, Pesric. Biachem. Physiof. 11, 243 (1979). 3. M. C. Watson, P. G. Bartels, and K. C. Hamilton, Action of selected herbicides and Tween 20 on oat (Avena sariva) membranes, Weed Sci. 28, 122 (1980).
ON
Acer pseudoplatunus
CELLS
161
4. D. L. Bucholtz and T. L. Lavy, Alachlor and trifluralin effects on nutrient uptake in oats and soybeans, Agron. J. 71, 24 (1979). 5. E. Marre, Fusicoccin-A tool in plant physiology, Annu. Rev. Plant Physiol. 30, 273 (1979). 6. J. P. Blein, Action of some herbicides on growth. respiration, plasmalemma integrity and proton extrusion of Acer pseudoplatanus cells. I. Substituted ureas, Pestic. Biochem. Physiol. 16, 179- 186 (1981). 7. J. P. Blein, Cyanide stimulation of respiration of Acer pseudoplatanus cells in batch suspension culture and activation of the alternative pathway, Planf Sci. Lerr. 19, 65 (1980). 8. A. Kurkdjian, Action de la fusicoccine sur la morphologie et le grandissement de celhtles d’Acer pseudoplatanus cultivees in vitro, Physiol. veg.,
17, 305 (1979).
9. M. D. Persidsky and C. S. Baillie, Fluorometric test of cell membrane integrity, Cryobiology, 14, 322 (1977).
10. T. E. Ferrari and D. E. Moreland, Effects of 3,5dihalogenated-4-hydroxybenzonitriles on the activity of mitochondria from white potato tubers, Plant Physiol.. 44, 429 (1969). 11. F. M. Ashton and A. S. Crafts, “Mode of Action of Herbicides,” p. 256, Wiley, New York, 1973. 12. R. K. Glen, 0. A. Leonard, and D. E. Bayer. Some effects of dichlobenil, Res. Prog. Rep. West. Sac. Weed Sci., 150 (1971). 13. G. Hofstra and C. M. Switzer, The phytotoxicity of propanil, Weed Sci. 16, 23 (1968). 14. R. S. Morrod, Effects on plant cell membrane structure and function, in “Herbicides” (L. J. Audus, Ed.), Vol. 1, p. 281, Academic Press, London/New York/San Francisco, 1976. 15. J. R. Freney, Increased growth and uptake of nutrients by corn plants treated with low levels of simazine, Ausr. J. Agr. Res. 16, 257 (1965). 16. F. W. Ben&up, Cell electrophysiology and membrane transport, Prog. Bat. 40, 84 (1978). 17. R. J. Poole, Energy coupling for membrane transport, Annu. Rev. Plunt Physiol. 29, 437 (1978). 18. R. E. Cleland and T. Lomax, Hormonal control of H+-excretion from oat cells, in “Regulation of Cells Membrane Activities in Plants (E. Marre and 0. Ciferri, Eds.), p. 161, Elsevier. Amsterdam, 1977. 19. R. Colombo, A. Bonetti, R. Cerana, and P. Lado, Effect of plasmalemma ATPase inhibitors diethylstilbestrol and orthovanadate, on fusicoccin-induced H+ extrusion in maize roots. P/ant Sci. Left.. 21, 305 (1981).