Penetration and effects of glyphosate in isolated potato mitochondria

Phytochemistry, Vol.32,No. 1,pp. 9-14, 1993 Printedin Great Britain.

003l-9422/93$5.00+ 0.00 PergamonPressLtd

PENETRATION AND EFFECTS OF GLYPHOSATE IN ISOLATED POTATO MITOCHONDRIA L. ARNAUD, P. RAVANEL,*~C. ANDINGS and M. TISSUT

Lahoratoire de Physiologie Cellulaire V&&ale et *Laboratoire de Pharmacognosie, CNRS, URA 576, Universitt Joseph Fourier, BP 53X,38041Grenoble cedex, France; SLaboratoire de Biochimie,Centre de Recherchede La Dargoire, Rhdne-Poulenc Agro, rue Baizet 6!%00Lyon, France (Received in revised form22 June 1992) Key Word Index-Solanutn

tuberosum; Solanaceae; glyphosate; penetration;

plant mitochondria.

Abstract-The

penetration and effects of the sodium salt of glyphosate, N-(phosphonomethyl) glycine, in isolated potato tuber mitochondria were investigated. The penetration study required the measurement of the volume of mitochondrial water space and this measurement was repeatedly carried out using [14C]dextran giving a value of 3.53 $ mg-’ protein. After a 15 minute incubation period with medium containing 1 /.LMor 1 mM glyphosate, without respiratory substrate, almost no product was found in the membrane pellet, after disruption of the organelles in distilled water. In contrast, glyphosate was found in the mitochondrial water in a range of concentrations close to the external medium concentration. These results show that, at least the diffusion equilibrium between the medium, the intermembrane space and the matrix area was readily reached for glyphosate. The greater part of the product penetrated during the first five minutes of the incubation period. The glyphosate content, in mitochondria operating at 25”, was not changed by adding either substrate (state IV), or substrate + ADP (state III). The probability of an active glyphosate transport by the mitochondrial inner membrane was therefore very unlikely. Glyphosate, at a concentration as high as 50 mM, was unable to change the respiratory activities of isolated potato tuber mitochondria (oxygen consumption rate with different substrates, ADP/O, respiratory control values, etc.).

INTRODUCTION

questions remain largely unanswered, we have chosen to study them.

N-(phosphonomethyl) Glycine, (glyphosate) is the active ingredient of well-known herbicide preparations, such as ‘Round-up’, that are widely used in agriculture for their broad spectrum and low toxicity in animals. The main target of this herbicide is the EPSP synthase, an enzyme catalysing the formation of enolpyruvyl shikimate phosphate from phosphoenolpyruvate and 3-phosphoshikimate [ 11. Through this mode of biochemical action, glyphosate is supposed to control the flux of synthesis of aromatic amino acids and of phenylpropanoids [Z]. Furthermore, its inhibitory action on EPSP synthase seems to induce a powerful accumulation of shibimate inside the treated cells, which might be responsible for the cell necrosis observed in several plant species [S]. In whole plants, several reports have shown that glyphosate can severely affect respiration [4, 51 and Olorunsogo ez al. [6] demonstrated an uncoupling effect on isolated rat liver mitochondria, and an inhibition of the oxygen uptake (state IV and state III respiration) on isolated corn mitochondria was reported [7]. Are the effects of glyphosate on the mitochondrial activities negligible side-effects or not? Is glyphosate able to penetrate inside mitochondria? At present, these

TAuthor to whom correspondence

and this is why

RESULTS Effects of glyphosate on the activities tuber mitockondria (Fig. 1)

of potato

In a first set of experiments, electron transfer between exogenous NADH as electron donor and 0, as electron acceptor was studied in potato mitochondria in the presence. of an uncoupler, CCCP, at 5 FM. Under these conditions, the rate of transfer remained unchanged in the presence of glyphosate, even at a concentration as high as 50 mM. Bovine serum albumin (BSA) added to the incubation medium plays the role of a membrane protectant, able to bind ionizable lipophilic components [8, 93. When BSA is omitted from the medium, mitochondria are more easily a.fTected by chemicals. Even under these conditions, the electron transfer from NADH to O2 remained unaffected by glyphosate up to 50 mM. Glyphosate from 0 to 50 mM was tested on mitochondria oxidizing different substrates in the presence of CCCP (uncoupling state) and also in the presence of ADP (state III). Either with succinate, cl-ketoglutarate, malate or citrate as substrate, the 0, consumption rate remained unchanged by glyphosate. Furthermore, ADP/O and respiratory control were unaffected. These experiments

should be addressed. 9

L. ARNAUD et al

10

Fig.

1. Polarographic

isolated

potato

glyphosate;

traces

tuber

CCCP,

showing

mitochondria.

the meffectiveness

m-Cl phenylhydrazone;

5 mM; rot., rotenone.

of glyphosate

m, 0.3 mg Mitochondrial

Numbers

(SO mM) on the respiratory

protein;

succinate,

KCN, 30 FM; NADH,

1 mM; citrate,

on traces refer to nmol 0,

consumed

show that glyphosate from 0 to 50 mM was neither an uncoupler nor an inhibitor of the phosphorylation. With malate as substrate, the comparison of the oxidation rate at pH 6.5 and 7.5 gives an accurate measurement of the electron transfer through complex I [lo, 11J. Glyphosate up to 50 mM did not change this process. All these results show that, under our conditions, none of the respiratory activities of isolated potato mitochondria could be changed by glyphosate up to 50 mM (electron transfer, phosphorylation process, substrate, Pi and ADP/ATP transport, Krebs’ cycle enzyme activities). In several herbicidal preparations, glyphosate is used as an isopropylamine salt. Isopropylamine was studied alone up to 50 mM on the same mitochondrial activities and was shown to be without effect. Furthermore, mitochondrial activities were measured after a preincubation period for the organelles, in the presence of either glyphosate or isopropylamine (50 mM): no inhibitory effect was measured. These results raise the question of the penetration yield of glyphosate inside mitochondria.

Penetration of glyphosate into potato tuber mitochondria Principle of the experimental procedure. Using [‘4C]glyphosate, the purpose was to establish penetration kinetics, for the whole period of time during which the isolated mitochondria of the untreated sample showed no change in biological activities (respiratory control and ADP/O values, oxidation rates, etc.). These kinetics had to be compared with those of reference compounds, known to be unable to penetrate inside mitochondria. For this purpose, [‘4CJdextran was chosen as a probe, being unable to diffuse inside the intermembrane space [lo, 123. Furthermore, these kinetics were established

activities

of

6 mM; ATP, 0.3 mM; gly. 10 mM; NAD,

min -’ mg

1 mM; malate,

* protein.

under two different conditions: firstly, kinetic experiments were carried out at 4’, in isolated mitochondria, without respiratory substrate or ADP; secondly, they were established at 25’, in the presence of a substrate (succinate+ATP), either at state IV or at state III, in order to investigate a possible active penetration process and the role of the formation of a proton gradient. Distribution of [14C]dextran between medium and mitochondria. Under our experimental conditions,

[ ‘“Cldextran was distributed between medium and mitochondria as shown in Table 1. Between 5 and 120 min, the equilibrium remained unchanged. The dextran content in S2 + S3 + C represents the amount associated with water between the organelles in the pellets. In the experiments performed with the first method, the amounts of dextran associated with S2 + S3 + C (see Experimental) corresponded, as an average, to 3.00 ~1k 1.57 medium mg-’ protein in the pellet. The total amount of water in the same pellet was 6.36 /II mg- 1 protein. The inner mitochondrial water space (matrix + intermembrane space) was therefore 3.53 ~1 mg-’ protein. Penetration of [14C]glyphosate into mitochondria at 4” without substrate. Under the same experimental condi-

tions as for dextran, [14C]glyphosate was added in the medium at 1 PM and 1 mM. At 4”, without substrate or cofactors, the amounts of glyphosate associated with mitochondria were estimated, after correction of the amounts of medium remaining in the pellets (obtained from the use of dextran). The amount of glyphosate found in S2 + S3 + C (after dextran correction) reached 4% of the total amount present in the medium after 5 min incubation (Fig. 2). This value remained almost constant for the following two hours. (a) After a 2-hr period, all the results obtained showed, repetitively, that in mitochondria maintained at 4” or 25”

Glyphosate penetration in mitochondria

11

Table 1. Dextran space in pellets of potato tuber mitochondria. Distribution of dextran (expressed as % of the applied quantities) between the different compartments of a mitochondrial pellet after several incubation periods, and under two sets of experimental conditions First method Time Fraction Sl s2 s3 C

5 min

120 min

89.37 +6.02% 9.75 f 5.40% 0.65 f0.46% 0.23 kO.19%

88.25 &-5.55% 10.39 &4.96% 1.03 kO.55% 0.33 *0.22%

Second method Dextran space in pellets (~1)

3.00 /41 f 1.57

15 min 98.37 + 0.49% 1.29 kO.49% 0.15 *0.08% 0.18 *0.08%

Dextran space in pellets (~1)

6.10 ~1 f 1.17

The volume V of the dextran space in a pellet (corresponding to 1 mg mitochondrial protein) is calculated as follows: if A represents the radioactivity of 1 ~1of the [‘*C]dextran solution, V is obtained with the use of the equation: V = S, + S, + C/A. Compartments are: Sl, medium after centrifugation; S2, fresh medium having washed the mitochondria pellet; S3, distilled water in which the washed mitochondria were burst; C, final pellet of disrupted mitochondria. First method: 5 min: mean of six replicates; 120 min: mean of 13 replicates; second method: 15 min, mean of six replicates. Values are indicated as % of the total amount + U.

Time (min)

Fig. 2. [‘*C]Glyphosate absorption kinetics for isolated potato tuber mitochondria. The percentage of [ ‘*Cjglyphosate was obtained from S2 + S3 +C values after subtraction of the amount of product corresponding to the dextran space.

without substrate or ADP, the concentration of glyphosate in the mitochondrial water space seemed to reach a value close to the external concentration. (b) The shape of the curve, showing a rapid penetration phase during the first five minutes, suggests that this was probably partly the result of diffusion inside the intermembrane space (42% of the total mitochondrial water space), which is known to be freely accessible to small hydrophilic compounds [la]. (c) Diffusion inside the matrix seemed to be obtained for the most part over the same time scale (no diffusion change between 15 and 120 min). (d) The amounts of glyphosate corresponding to the C fraction were very low (below 10% of S2+ S3 + C on average). C probably has two origins: firstly a contamination by the glyphosate content of the matrix and of the

rinsed intermembrane space, and, secondly, the true content of the membranes. The latter one is certainly very low. Log P for glyphosate is ca -4 [ 133. Log P represents the logarithm of the partition coefficient between octanol and water and routinely expresses the lipophilic character of a molecule. Here, that means 10000 molecules of glyphosate remain in the water space for one molecule in a lipidic compartment represented by N-octanol. In potato mitochondria, the lipidic phase represents 0.47 mg (0.42 fi) per mg protein [14], and the mitochondrial water space is 3.53 ~1 (see below). According to the log P value, 0.14 molecule of glyphosate may be associated with the lipophilic membrane space for 10000 in the water space. This ratio 14/106 explains why the C/S2 + S3 value was very low, being almost impossible to measure. When

12

L.

et al.

ARNAUD

changing the concentration of glyphosate from 1 PM to 1 mM, the results were similar (results not shown). Penetration of [‘4C]glyphosate inta mitochondria at 25” in the presence of succinate as substrate. At 25”, under

aerobic conditions, mitochondria were maintained for 15 min either in the presence of succinate (+ATP) or without respiratory substrate. In each case [14C]glyphosate was added. From the beginning to the end of the experiment, small samples of the mitochondrial suspension were taken to verify the stability of the mitochondrial activities (respiratory rates, respiratory control, ADP/O) and if there was enough succinate to maintain, in the sample with substrate, an oxidative rate corresponding to a state IV situation during the experiment. The amounts of glyphosate present in S2 + S3 + C, at 25”, at state IV or without substrate are shown in Table 2. The results were similar to the corresponding values measured at 4 (results not shown). Furthermore, substrate oxidation and the maintenance of state IV for the whole experiment, which is responsible for a strong acidification of the intermembrane space, did not induce any important

change in glyphosate distribution equilibrium after 15 min. The results obtained either at 1 PM or at 1 mM were nearly identical. Znjkence of ATP synthesis on glyphosate penetration into potato mitochondria. ATP genesis and excretion by

the mitochondria had no stimulative effect on glyphosate penetration (Table 3). Furthermore, ATP accumulation inside the intermembrane space seemed to lower glyphosate diffusion in this compartment.

DISCUSSION

Glyphosate is a herbicide primarily acting on EPSP synthase, an enzyme located inside the stroma of plastids [15]. Furthermore, a powerful loading of this compound into the phloem is known to occur [16]. As a whole, at least under field conditions, this herbicide is able to pass through several biological membranes such as plasmalemma and plastid envelopes. The question arises therefore of the penetration of this compound through other

Table 2. Presence of glyphosate in the different mitochondrial fractions, after incubation at 25” with 1 PM and 1 mM glyphosate, either in the presence or the absence of succinate Percentage of the diffusion equilibrium

Concentration Without substrate

1.8lkO.69 0.36kO.17 0 2.17kO.62

Glyphosate (1 mM) s2 s3 C S2+S3+C

Without substrate

State IV

pm01 mg-i protein

Glyphosate (1 PM) s2 s3 C S2+S3+C

State IV

0.87 kO.43 0.31 kO.12 0 1.18kO.79 nmol mg-’

3.02_+0.63 0.49 kO.01 0 3.5lkO.54

24+ 12% 9+3% 0 33 +22%

51&19% 10+5% 0 61+ 17%

protein

3.70f0.40 0.41*0.19 0 4.11 kO.37

85& 18% 14*3% 0 101* 15%

105~11% 11+5% 0 116*10%

Without substrate: mean of 10 values, state IV: mean of six values ( + 0).

Table 3. Distribution of glyphosate in the S2, S3, C fractions, at 25”, either under phosphorylating conditions, or not Percentage of the diffusion equilibrium

pm01 mg - 1 protein 1lrM

Without substrate

State III

Without substrate

State III

s2 s3

216kO.44 0.42 + 0.05

2.14kO.26 0.20*0.06

61+12% 12+1%

60+7% 6+2%

C S2+S3+C

0 2.58 f 0.26

0 2.34kO.23

0 73k7%

0 66*7%

Mean of three replicates *a.

13

Glyphosate penetration in mitochondria types of biological membranes, such as the mitochondrial inner membrane. Furthermore, the role played by the cell physiological state on such a possible penetration was another important question. For the time scale used in this study (between 0 and 120 min), it appeared that the estimated value of glyphosate concentration in the inner mitochondrial water space was close to the diffusion equilibrium. This result was established without doubt, although the accuracy of these determinations was low. Considering the value (0.58) of the ratio between the matrix space and the whole mitochondrial water, the diffusion had to occur inside these two compartments. The possibility of an active transfer of glyphosate through the inner mitochondrial membrane seems highly unlikely. It was interesting to investigate whether the relative matrix alkalinization at state IV might lead to a greater concentration inside mitochondria. In fact, experiments showed that this was not the case. Log P for glyphosate, which is close to -4 [13] at neutral pH, explains the distribution of this compound between hydrophilic spaces (matrix, intermembrane space, medium) and membranes. With lipophilic compounds, such as pentachlorophenol or phemnedipham, they would concentrate inside membrane [17, 183 and therefore, greatly increase C. In the present case, the highest concentration was obtained in S2. The first Fick’s law, 4 = - P(C, -C,), expresses a flux/time unit/surface unit, with P being a permeability coetIicient, C, and C, being the concentrations in compartments 1 and 2. As the possibility of an active membrane transport seems very unlikely, Fick’s law has to be used to understand the diffusion from the medium to the matrix. The permeability coefficient P probably has a low value, which is, however, limited by the fact that the membrane is very thin (60 A). As a consequence, one may explain why glyphosate cannot be a potent typical protonophoric uncoupler of the phosphorylation process, although it is able to transport protons at biological pH [19]. This compound could never reach a sufficient concentration inside the membrane, and it is not mobile enough to be able to transport protons at the same rate as they are produced during the electron transfer from endogenous NADH to 0,. In fact, at the highest concentration of glyphosate used here (50 mM), the membrane concentration may reach a maximum of 5 PM. In contrast, to achieve complete uncoupling activity, the weak lipophilic acid pentachlorophenol used at 5 PM in the medium (devoid of BSA) was present at a calculated concentration reaching 13.5 mM in the membrane [17]. It is therefore possible to understand why biochemical processes taking place inside the membrane (vectorial transport of protons, electron transfer) are not likely to be affected by glyphosate. In contrast, at the diffusion equilibrium, glyphosate would reach the same concentration in the matrix as in the medium (highest concentration used in respiratory tests, 50 mM). Under these conditions, either Krebs’ cycle enzymes or transporters of hydrophilic compounds (dicarboxylic acid, phosphate, ADP/ATP, etc.) might have been affected by the herbicide. Our results showed that this was not the case in potato tuber mitochondria. Under the same conditions,

these mitochondrial activities were not affected by the presence of 50 mM isopropylamine. EXPERIMENTAL

Preparation of mitochondria. Mitochondria from potato tubers ( Solanum tuberosum L.) were prepared by methods previously described [20]. 0, exchange measurements. O2 exchange was followed polarographically at 25”, using a Clark-type electrode system. For plant mitochondria, the reaction medium contained 0.3 M mannitol, 5 mM MgCl,, 10 mM KCl, 10 mM, Pi buffer, and in some cases, 0.1% BSA. All incubations were carried out at pH 7.2, except when malate was the respiratory substrate (pH 6.5 and 7.5). Protein measurements. Protein contents were determined according to ref. [21]. Determination of dextran and glyphosate spaces. First method: a tightly coupled mitochondrial suspension, containing up to 25 mg protein ml-’ was divided in 1 ml fractions and put in several 1.8 ml Microfuge tubes. Three different physiological situations were studied: mitochondria without respiratory substrate, mitochondria at state IV (presence of succinate+ ATP) and mitochondria at state III (presence of succinate+ ATP and ADP). Aq. solns of radiolabelled dextran and glyphosate were added. After a 5-, 15- or 120~mln period of incubation at 4” or 25”, under a gentle orbital stirring movement (100 t-pm), the suspension was centrifuged (5 min, 12000 g in a Microfuge 12, Beckman). At the end of the incubation period, an aliquot of mitochondria was taken for polarographic control of respiratory activity. High activities were maintained under all our experimental conditions. After centrifugation, the supernatant (Sl) was prepared for counting and the pellet was gently resuspended in 1 ml of reaction medium. After a second centrifugation, as described above, a supernatant (S2) was collected. The pellet was then washed in H,O and centrifuged, leading to a supernatant (S3) and a pellet (C) corresponding to burst mitochondria. Radioactivity of the different fractions Sl, S2, S3 and C was evaluated using a liquid scintillation spectrophotometer (Intertechnique, model SL 30). Second method: in or&r to improve the accuracy of the procedure, in a second set of experiments, large bottles (250 ml) were used for centrifugation. One ml of mitochondrial pellet was incubated in 11 ml reaction medium. Under these conditions, a centrifugation (12 000 g, 5 min) led to a very good separation of pellets and supernatants, lowering the risks of contamination between these two fractions. In the experiments at state IV and state III, the amounts of succinate and ADP were calculated to maintain the organelles in these states, during the whole incubation time, and to maintain, with mannitol, an osmotic pressure of 0.3 M. To ensure a large amount of O2 during the incubation period, a flux of air was maintained in each bottle constantly submitted to an orbital movement (125 rpm). In experiments with dextran, S2 + S3 + C was used to measure the dextran space in the centrifuged pellet. The amount of penetrated glyphosate was measured as (S2+ S3 +C) glyphosate

L. ARNAUD et al.

14

-(S2+S3 +C) dextran, the same amount of r4C being added in each type of experiment. Calculation of the glyphosate concn inside the mitochondrial water space. pw: H,O content of the pellet (fr. wt

-dry wt) corresponding to 1 mg mitochondrial protein. V, vol corresponding to dextran space (see legend of Table 1). Miw, corresponding to the mitochondrial water space (matrix and intermembrane spaces) = pw -V. Matrix space, 0.58 Miw [12]. Average glyphosate concn in the Miw (mM), penetrated [14C]glyphosate (nmol)/Miw (~1). These values, compared with the glyphosate concn in the medium, allow the calculation of the percentage of the diffusion equilibrium. Chemicals. [carbonyl-r4C]Dextran was a NEN research product (M, SOOOt-7OooO; s.a. 0.0021 GBq g-l) and [r4C]glyphosate (s.a. 1.89 GBq mmol-‘) was purchased from Amersham France.

Cole, D. J., Dodge, A. D. and Caseley, J. C. (1980) J. Exp. Botany 31, 1665. Olorunsogo, 0. O., Bababunmi, E. A. and Bassir, 0. (1979) Bull. Environ. Contam. Toxicol. 22, 357. Lopez-Brana, I., Delibes, A. and Garcia-Olmedo, F. (1984) J. Exp. Botany 35, 905. 8. Ducet, G. (1978) Physiol. Veg. 16, 753. 9. Ravanel, P. and Tissut, M. (1986) Phytochemistry 25, 577.

R. (1985) in Mitochondria in Higher Plants. Structure, Function and Biogenesis (Am. Sot. Plant

10. Deuce,

Physiol., ed.). Academic Press, Orlando. 11. Ravanel, P., Tissut, M. and Deuce, R. (1986) Plant Physiol. 80, 500.

12. Cohen, N. S., Cheung, C. W. and Raijman, L. (1987) Biochem. J. 245, 375. R. A. and Edgington, L. V. (1981) 13. Martin, Pest. Biochem. Physiol. 16, 87.

Acknowledgement-We

are grateful to the Region Rhone-Alpes and to Rhone-Poulenc Agro for financial support.

J. M. (1991) PbD Thesis. J. Fourier 14. Routaboul, University, Grenoble, France. 15. Della-Cioppa, G., Bauer, S. C., Klein, B. K., Shah, D. M., Fraley, R. T. and Kishore, G. M. (1986) Proc. Natn. Acad. Sci. 83, 6873.

REFERENCES

Amrhein, N., Schab, J. and Steinruecken, H. C. (1980) Naturwiss. 67, 356. Hollaender, H. and Amrhein, N. (1980) Plant Physiol. 66, 823.

Schultz, A., Munder, T., Hollander-Czytko, H. and Amrhein, N. (1990) Z. Naturforsch. 45, 529. Ali, A. and Fletcher, R. A. (1978) Can. J. Botany 56, 2196.

16. Baoui, H. E., Delrot, S., Besson, J. and Bonnemain, J. L. (1986) Physiol. Veg. 24, 431. 17. Albertin, H., Chiron, S., Nurit, F., Ravanel, P. and Tissut, M. (1991) Phytochemistry 30, 3553. 18. Ravanel, P., Tissut, M., Nurit, F. and Mona, S. (1990) Pest. Biochem. Physiol. 38, 101. 19. Terada, H. (1981) Biochim. Biophys. Acta 639, 225. 20. Deuce, R., Christensen, E. L. and Bonner, W. D. (1972) Biochim. Biophys. Acta 275, 148. 21. Bradford, M. M. (1976) Analyt. Biochem. 72, 248.