The Plant Journal (1998) 14(3), 359–364
SHORT COMMUNICATION
Magnesium ions promote assembly of channel-like structures from beticolin 0, a non-peptide fungal toxin purified from Cercospora beticola Cyril Goudet1,2,†, Anne-Alie´nor Ve´ry1,†,‡, Marie-Louise Milat2, Miche`le Ildefonse3, Jean-Baptiste Thibaud1,*, Herve´ Sentenac1 and Jean-Pierre Blein2 1Biochimie et Physiologie Mole ´ culaire des Plantes, URA 2133 CNRS/ENSA-M/INRA, Place Viala, 34060 Montpellier cedex 1, France, 2Phytopharmacie et Biochimie des Interactions Cellulaires, INRA/Universite´ de Bourgogne, BV 1540, 21034 Dijon Cedex, France, and 3DBMS/BMC/URA 520 CNRS, CENG, 17 rue des Martyrs, 38054 Grenoble cedex 9, France
Summary Beticolins are toxins produced by the fungus Cercospora beticola. Using beticolin 0 (B0), we have produced a strong and Mg2F-dependent increase in the membrane conductance of Arabidopsis protoplasts and Xenopus oocytes. In protein-free artificial bilayers, discrete deflexions of current were observed (12 pS unitary conductance in symmetrical 100 mM KCl) in the presence of B0 (approximately 10 µM) and in the presence of nominal Mg2F. Addition of 50 µM Mg2F induced a macroscopic current which could be reversed to single channel current by chelating Mg2F with EDTA. Both unitary and macroscopic currents were ohmic. The increase in conductance of biological membranes triggered by B0 is therefore likely to originate from the ability of this toxin to organize itself into transmembrane pores in the presence of Mg2F. The pore is poorly selective, displaying permeability ratios PCl/PK, PNa/PK and PCa/PK close to 0.3, 0.65 and 0.4, respectively. Such channel-like activity could be involved in the deleterious biological activity of the toxin, by causing the collapse of ionic and electrical gradients through biological membranes together with Ca2F influx and scrambling of cellular signals.
Received 7 October 1997; revised 2 February 1998; accepted 13 February 1998. *For correspondence (fax 133 4 99 52 57 37; e-mail
[email protected]). †Joint first authorship. ‡Present address: Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK.
© 1998 Blackwell Science Ltd
Introduction Beticolins are non-host-specific toxins produced by the phytopathogenic fungus Cercospora beticola, which is responsible for the prevalent leaf spot disease in sugar beet. Fifteen non-peptide compounds sharing the same polycyclic skeleton with a chlorine atom and partially hydrogenated anthraquinone and xanthone moieties have been isolated (Ducrot et al., 1996 and refs therein) and named beticolin 0 to 14. In addition to their phytotoxicity, beticolins also display a bactericidal activity (Schlo¨sser, 1962) and an anti-proliferative effect on tumour cells (Ding et al., 1996). The mechanisms underlying these biological activities are still poorly understood. Beticolins can chelate Mg21 ions and form an electrically neutral dimeric complex composed of two beticolin molecules and two Mg21 ions (Gome`s et al., 1996). This association with Mg21 strongly increases the partitioning of the toxin into the hydrophobic phase (Mike`s et al., 1994). In plant cells, beticolins have been found to induce loss of solutes, to depolarize transmembrane potential and to inhibit H1 extrusion and K1 uptake (Gapillout et al., 1996; Macri and Vianello, 1979). In plant microsomal vesicles and in purified plasma membranes, they have been shown to inhibit the ATP-dependent proton translocation (Macri et al., 1983; Simon-Plas et al., 1996). Here, we present evidence that beticolin 0 (B0) (Figure 1A) can form, upon addition of Mg21, poorly selective ion channels which are permeable not only to K1, Na1 and Cl– but also to Ca21. The deleterious properties of the fungal toxin probably involve these channel-like properties.
Results
Effect of beticolin 0 on the membrane conductance of mesophyll cell protoplasts In protoplasts obtained from Arabidopsis thaliana mesophyll cells, resistance of excised outside-out patches ranged around 100 GΩ (Figure 1B(a)), as endogenous channel activity was inhibited by high Ca21 concentrations and the absence of ATP in the pipette solution. In these control conditions, addition of B0 to the bath solution strongly increased the patch conductance (30-fold, Figure 1B(b)). Further addition of EDTA reversed this effect (Figure 1B(c)). 359
360 Cyril Goudet et al.
Figure 1. Beticolin 0 increases the conductance of biological membranes. (A) Structure of B0. It consists of an heptacyclic skeleton with a chlorine atom and partially hydrogenated anthraquinone and xanthone moieties. (B) Increase in membrane conductance induced by B0 in a mesophyll protoplast of Arabidopsis thaliana and inhibition of this effect by EDTA. Currents were recorded through an excised outside-out patch clamped for 1 sec at potentials from –80 mV to 180 mV (10 mV step; holding potential 0 mV). The pipette solution contained 50 mM KCl, 1 mM CaCl2, 380 mM mannitol, 5 mM Mes–Tris (pH 6.0). The three panels show records obtained successively from the same patch. (a) The bath contained 50 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 380 mM mannitol, 5 mM Mes–Tris pH 6.0 (control conditions). (b) Bath solution of panel (a) plus 6 µM B0. (c) Bath solution of panel (b) plus 25 mM EDTA. Data shown are representative of four experiments. (C) Mg21-dependent increase in membrane conductance induced by B0 in a Xenopus oocyte. Current was recorded in the –160 to 180 mV range (10 mV step; holding potential –40 mV). The control external medium contained 98 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 and 5 mM Hepes–NaOH (pH 7.4). (a) Currents recorded in the control medium. (b) and (c) Currents recorded from the same oocyte as in (a), upon successive addition of 40 µM B0 (1 B0; panel (b)) and of 1 mM MgCl2 (1 B0 and Mg21; panel (c)) into the control medium. (d) The maximal value of the current was sampled for each voltage–clamp episode shown in panels (a), (b) and (c). The control current (panel (a)) was subtracted from the current traces shown in panels (b) and (c), and the resulting current values were plotted against membrane potential for both 1 B0 (j) and 1 Mg21 and B0 (d) conditions. Data shown are representative of 10 experiments.
Effect of beticolin 0 on the membrane conductance of Xenopus oocytes Under our experimental conditions, the endogenous conductance of Xenopus oocytes was low (range –160 to 180 mV) (Figure 1C(a)). Addition of B0 to the perfusing
solution only slightly increased the currents recorded within the same voltage range (Figure 1C(b)). Further addition of 1 mM MgCl2 to the previous solution (40 µM B0 still present) increased the recorded currents significantly (Figure 1C(c)). The current–voltage relationships established in the presence of B0 with or without 1 mM MgCl2 © Blackwell Science Ltd, The Plant Journal, (1998), 14, 359–364
Beticolin channel-like structure 361
Figure 2. Mg21-dependent currents induced by beticolin 0 (B0) through a synthetic lipid membrane. B0 concentration was 15 µM. Dashed line indicates the zero current level. (A) Channel-like activity of B0. Records of current at different imposed voltages, after subtraction of the leak current. (B) Effect of magnesium on B0 activity. Current through the same bilayer as in panel (A). Traces (a), (b) and (c) represent a continuous record. Traces (a) and (b): time course of the increase in current upon addition of 50 µM Mg21 and steady current at different imposed voltages. Trace (c): inhibition of the current by EDTA. Only small current transitions such as those shown at a higher gain (enlarged section) were observed. (C) Current-voltage curve of the bilayer in the presence of B0. (a) Elementary currents (I) measured from records shown in (A). (b) Mean amplitude (I) of the steady-state macroscopic currents shown in (B). (D) Reversal potential in asymmetrical KCl solution. Current at different potential values, 10 min after the cis addition of B0 and 10 µM Mg21, in asymmetrical cis/trans KCl conditions (100 mM/500 mM KCl). For (A), (B) and (C), the data shown are representative of 10 experiments; for (D), the data shown are representative of four experiments; PCl/PK 5 0.30 6 0.06.
in the solution are roughly linear between –100 mV and 150 mV and crossed the zero-potential line at approximately 15 mV (Figure 1C(d)). The increase in membrane conductance of oocytes, induced by B0 in the presence of 1 mM Mg21, became significant when the B0 concentration was above 4 µM (data not shown, but see below the corresponding data with bilayer membranes, Figure 3).
Channel-like current transitions induced by beticolin 0 in protein-free bilayer membranes When no Mg21 was added to the solutions (estimated contaminant concentration 1–5 µM), discrete current transitions, similar to those occurring upon opening and closing of ion channels, could be observed 5–10 min after addition of 15 µM B0 to the cis solution Figure 2A). Such events were not observed in the absence of B0. The frequency of these events was initially low, but increased with time. In the steady state, several superimposed transitions could be observed, examples of which are illustrated in Figure 2(A), indicating that at least two channel-like structures were © Blackwell Science Ltd, The Plant Journal, (1998), 14, 359–364
organized in these conditions. The open time ranged from 1 to several tens of seconds. Several conductance levels were usually observed as in Figure 2(A). The maximal value of the current transitions recorded at each imposed potential, which corresponded also to the most frequent conductance level (Figure 2A), was plotted against voltage (Figure 2C(a)). The linear current–voltage relationship indicates that the current transitions corresponded to a main conductance of 12 pS.
Development of a macroscopic current through the bilayer upon addition of MgCl2 Addition of 50 µM Mg21 to the cis chamber solution (same bilayer as in Figure 2A) induced a large increase in the amplitude of the current within 2 min, which reached a steady level approximately 100-fold larger than the elementary transitions after about 6 min (Figure 2B(a)). When this effect started to develop, more and more jumps of current occurred simultaneously as shown in the enlarged section below the main trace (Figure 2B(a)). The steady
362 Cyril Goudet et al. not hyperbolic but rather sigmoidal, suggesting that the increase in membrane conductance involves a cooperative mechanism. Discussion
Beticolin 0 itself increases the membrane conductance B0 strongly increased the membrane conductance of Arabidopsis mesophyll protoplasts (Figure 1B) and Xenopus oocytes (Figure 1C). Although it cannot be ruled out that the endogenous conductances of these cells are responsible for the extra current recorded in the presence of B0, an increase in membrane conductance was also observed in artificial protein-free bilayers obtained from synthetic phospholipids (Figure 2). These observations strongly suggest that B0 alone can organize itself into ion-conducting structures within the animal and plant cell membranes (Figure 1). Figure 3. Increase in synthetic lipid membrane conductance induced by B0: dose–response curve in the presence of Mg21. Current through the bilayer was recorded at an imposed voltage of 1150 mV. Cis and trans media: 1.02 mM MgCl2, 1 mM EDTA (estimated free Mg21 concentration: 50 µM), 200 mM NaCl, 10 mM Hepes–NaOH. B0 concentration was varied from 1 to 12 µM. Bilayer instability and breakdown occurred when the B0 concentration was raised above 7 µM (four bilayers). The current values recorded through the same bilayer were normalized by the current value recorded in the presence of 7 µM B0 (100%). Vertical bars are standard deviations (the number of independent values is indicated by the figure in italics).
macroscopic currents recorded at different imposed voltages (Figure 2B(b)) varied linearly with voltage and reversed at 0 mV (Figure 2C(b)), as observed for the unitary current events shown in Figure 2(A) (Figure 2C(a)). Such a macroscopic current was not induced when substituting Ca21 for Mg21 (data not shown). The large and stable current induced by MgCl2 addition could be suppressed by adding 100 µM EDTA (estimated free Mg21 concentration: 3 µM). One min after the addition of 100 µM EDTA, the macroscopic current slowly decreased and disappeared totally within about 5 min (Figure 2B(c)). Only discrete and infrequent current transitions were then observed (Figure 2B(c), enlarged section below the main trace), which had the same amplitude as those shown in Figure 2(A). From other experiments in asymmetrical solutions of either KCl (Figure 2D), NaCl (0.1 M/0.5 M; not shown) or CaCl2 (0.2 M/1 M; not shown), the following permeability ratios were derived: PCl/PK 5 0.3 6 0.06 (n 5 4), PCl/PNa 5 0.5 6 0.09 (n 5 3), and PCl/PCa 5 0.8 6 0.1 (n 5 4), and therefore the PNa/PK and PCa/PK ratios were approximately 0.65 and 0.4, respectively.
Dose–response curve The increase in conductance of the bilayer was observed when B0 concentration was raised above approximately 4 µM (Figure 3). The shape of the dose–response curve is
Mg21 ions are involved in the membrane conductance increase In Arabidopsis mesophyll protoplasts, the inhibitory effect of EDTA (Figure 1B(c)) suggested that either Mg21 or Ca21 ions were involved in the increase in conductance induced by B0 (Figure 1B(b)). The experiments performed with Xenopus oocytes showed that the presence of 1.8 mM Ca21 ions in the external medium did not induce macroscopic currents in the presence of B0 (Figure 1C(b)), whereas subsequent addition of Mg21 ions did (Figure 1C(c)). The data obtained using artificial bilayers (Figure 2) provided further evidence that Mg21 ions, and not Ca21 ions, potentiate the effect of B0 on the membrane conductance. Thus it can be concluded that Mg21 ions are specifically required for B0 to induce a significant increase in membrane conductance in each of the three types of membrane used.
The B0/Mg21-induced conductance shows poor selectivity The macroscopic current recorded through a bilayer facing symmetrical KCl but asymmetrical cis/trans Mg21 concentrations (Figure 2B(b)) reversed at zero potential (Figure 2C(b)). Establishing asymmetrical cis/trans KCl concentrations (Figure 2D), or NaCl or CaCl2 concentrations (not shown; see above PNa/PCl and PCa/PCl permeability ratio estimation) shifted the reversal potential from zero. We therefore assumed that K1, Cl–, Na1 and Ca21 ions (but not Mg21) were the main current carriers in these conditions.
The increase in membrane conductance results from incorporation of Beticolin/Mg21 complexes It has previously been reported that beticolins and Mg21 ions form a neutral dimeric complex which is the predomin© Blackwell Science Ltd, The Plant Journal, (1998), 14, 359–364
Beticolin channel-like structure 363 ant species in the pH 7 range (Gome`s et al., 1996; Mike`s et al., 1994). The partition coefficient of this complex into biological membranes is several 10-fold higher than that of monomeric beticolin (Mike`s et al., 1994). The data shown in Figure 2 support the hypothesis of a slow and reversible incorporation process of some B0–Mg21 complexes into the bilayer membrane. Indeed, in the experiment shown in Figure 2(B), although the cis solution was stirred to ensure rapid mixing after each addition, the increase in current was not observed immediately after the addition of Mg21: more than 5 min were required for the steady state to be reached (by the end of the clamp at 160 mV, Figure 2B(a)). Reciprocally, chelating Mg21 ions by adding EDTA to the medium did not immediately suppress the macroscopic current (Figure 2B(c)).
Beticolin/Mg21 complexes form channel-like structures In bilayer experiments, the discrete current transitions induced by B0 in the presence of contaminant Mg21 (Figure 2A) and the macroscopic current recorded after addition of 50 µM Mg21 (Figure 2B) displayed similar features. It can be assumed that the macroscopic current passed through the channel-like structures that mediated the discrete current transitions observed before Mg21 addition. This hypothesis is supported by the trace shown in the enlarged section in Figure 2(B)(a). The formation of ion-conducting pores is a striking feature for a non-peptide compound, but similar to previously reported observations for the carboxylic Ca21 ionophore A23187 (Balasubramanian et al., 1992) or for bis-macrocyclic bola-amphiphile (Fyles et al., 1996). From what is known about beticolin structure (the length of B0 is approximately 17 Å, Ducrot et al., 1996), a multimeric assembly would be required to form a pore across a bilayer. This hypothesis is further supported by the dose– response curve (Figure 3), the shape of which is indicative of a cooperative effect. We propose that Mg21 ions promote the assembly of such channel-like multimeric structures within the membrane. Since the chemical structure of B0 is relatively simple, the observed rapid oscillation of the current level (Figure 2) is unlikely to result from an internal on/off switch actually closing and opening the aqueous pore as described for proteinaceous ion channels (Jan and Jan, 1997). Alternative mechanisms may explain the discrete current jumps. Firstly, due to the amphiphilic behaviour of beticolins, the partitioning of B0 between aqueous solutions and the artificial membrane might fluctuate randomly during current recording: the current fluctuations would arise from B0 molecules or complexes jumping in and out of the membrane as previously described for gramicidin (Hladky and Haydon, 1984). Secondly, beticolin multimers may assemble and disassemble themselves rapidly within the © Blackwell Science Ltd, The Plant Journal, (1998), 14, 359–364
membrane. The most frequent (and largest) conductance value of the channel-like structures formed by B0 (12 pS in symmetrical 100 mM KCl) is similar to those reported for proteinaceous ion channels with narrow pores within which ions are forced to position themselves in single file (Hille, 1992). Intermediate conductance levels were observed less frequently (see e.g. Figure 2A). This suggests that rearrangements within the multimeric structure, including variations in the number of monomers involved therein, may occur. The beticolins have previously been reported to inhibit the plasma membrane H1-ATPase activity (Simon-Plas et al., 1996). The large increase in membrane conductance induced by B0 in the presence of Mg21 ions could result in collapsing ionic and electrical gradients through biological membranes. This phenomenom could underlie the effects of beticolins on solute transport across the cell membrane (Gapillout et al., 1996; Macri and Vianello, 1979). Furthermore, because of its permeability to Ca21 ions, the toxinbuilt channel is likely to allow passive Ca21 influx into the cell. This may result in deleterious scrambling of cellular signalling. The ability to form channel-like structures is therefore likely to be involved in the biological activity of beticolins, as hypothesized for syringomycin, a necrosisinducing lipopeptide toxin secreted by the phytopathogenic bacteria Pseudomonas syringae (Hutchison et al., 1995). Other beticolins have been purified, their structure elucidated and their interaction with Mg21 ions studied (Ducrot et al., 1996; Gome`s et al., 1996). Comparison of their ionchannel forming abilities will enable conclusions about their structure–activity relationship to be reached. Experimental procedures
Beticolins Beticolins were extracted from the mycelium of the C. beticola strain CM and purified as previously described (Ducrot et al., 1996). One hundred micrograms of purified B0 were dissolved in an N,N-dimethylsulphoxide/water mixture (40/960 µl) and appropriate controls using N,N-dimethylsulphoxide/water without B0 were performed.
Protoplasts Arabidopsis thaliana (L. Heynh. cv. Landsberg) leaves were harvested from 3- to 4-week-old plants grown in potting compost in a growth chamber (day conditions 5 25°C and 52 W m–2 for 16 h; night conditions: 20°C for 8 h). Protoplasts were prepared as described by Spalding and Goldsmith (1993), except that the incubation time in the digesting solution was increased to 1.5 h. For patch–clamp recordings, protoplasts were perfused with a solution containing 50 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 380 mM mannitol and 5 mM Mes–Tris, pH 6. B0 and EDTA were added as indicated. Micropipettes were pulled from Kimax 51 glass capillaries (Kimble Products, Vineland, New Jersey), coated (Sylgard®, Dow Corning, Seneffe, Belgium) and fire-polished.
364 Cyril Goudet et al. Their resistance ranged around 20 MΩ when filled with the bath solution without MgCl2. Current recordings were performed with an Axopatch 200A electrometer (Axon Instruments Inc., Foster City, California) in the outside-out configuration (Hamill et al., 1981) at room temperature (20–22°C). Currents were filtered at 1 kHz (8-pole Bessel filter) and sampled at 2 kHz.
Xenopus oocytes Oocytes were obtained surgically from tricaine-anaesthetized Xenopus laevis, and voltage-clamped using the classical two-electrode technique, as previously described (Ve´ry et al., 1995). Currents were recorded in bath solutions containing 98 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 and 5 mM Hepes–NaOH, pH 7.4. B0 and MgCl2 were added as indicated.
Planar lipid bilayers Synthetic POPE (1-palmitoyl-2-oleoyl phosphatidylethanolamine) and POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) 1/1 (w/w) (Avanti Polar Lipids, Birmingham, Alabama) were dissolved in decane (30 mg ml–1) and painted across a 0.25 mm diameter hole. The capacity of the bilayer was 100–150 pF. Unless otherwise specified, the solution in both the cis and trans chambers (kept at 20–22°C) was 100 mM KCl and 10 mM Hepes–KOH (pH 7.4). B0, Mg21 and EDTA were introduced into the stirred cis solution. Currents were recorded with a RK-400 (Bio-Logic, Claix, France) or an Axopatch 200B (Axon instrument Inc., Foster City, California) patch–clamp amplifier. Trans and cis chamber solutions were connected (through 3 M KCl/AgCl/Ag half-cells) to headstage and signal-ground inputs of the amplifier, respectively. Potential values were thus defined as trans chamber (equivalent to intracellular compartment) minus cis chamber voltage. Recordings were sampled at 1 kHz and then filtered at 50 Hz.
Acknowledgements This work was supported by a MESR Doctoral fellowship (A.A.V.) and an INRA/Conseil Re´gional de Bourgogne Doctoral fellowship (C.G.). We thank Dr Helen Logan for critical reading of the manuscript.
References Balasubramanian, S.V., Sikdar, S.K. and Easwaran, K.R.K. (1992) Bilayers containing calcium ionophore A23187 form channels. Biochem. Biophys. Res. Commun. 189, 1038–1042. Ding, G., Maume, G., Milat, M.-L., Humbert, C., Blein, J.-P. and Maume, B.F. (1996) Inhibition of cellular growth and steroid 11b-hydroxylation in ras-transformed adrenocortical cells by the fungal toxins beticolins. Cell. Biol. Int. 20, 523–530. Ducrot, P.H., Einhorn, J., Kerhoas, L., Lallemand, J.Y., Milat, M.-L.,
Blein, J.P., Neuman, A. and Prange, T. (1996) Cercospora beticola toxins. Part XI. Isolation and structure of beticolin 0. Tetrahedron Lett. 37, 3121–3124. Fyles, T.M., Loock, D., van Straaten-Nijenhuis, W.F. and Zhou, X. (1996) Pores formed by bis-macrocyclic bola-amphiphiles in vesicle and planar bilayer membranes. J. Org. Chem. 61, 8866–8874. Gapillout, I., Mike`s, V., Milat, M.-L., Simon-Plas, F., Pugin, A. and Blein, J.-P. (1996) Cercospora beticola toxins. Part XIV: use of fluorescent cyanine dye to study their effects on tobacco cell suspensions. Phytochemistry, 43, 387–392. Gome`s, E., Gordon-Weeks, R., Simon-Plas, F., Pugin, A., Milat, M.-L., Leigh, R. and Blein, J.-P. (1996) Cercospora beticola toxins. Part XVII: the role of the beticolin/Mg21 complexes in their biological activity. Study of plasma membrane H1-ATPase, vacuolar H1-PPase, alkaline and acid phosphatases. Biochim. Biophys. Acta, 1285, 38–46. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J. (1981) Improved patch–clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflu¨gers Arch. 391, 85–100. Hille, B. (1992) Ionic Channels in Excitable Membranes, 2nd edn. Sunderland, Massachusetts: Sinauer Associates. Hladky, S.B. and Haydon, D.A. (1984) Ion movements in gramicidin channels. Curr. Topics Membr. Transp. 21, 327–372. Hutchison, M.L., Tester, M.A. and Gross, D.C. (1995) Role of biosurfactant and ion channel-forming activities of syringomycin in transmembrane ion flux: a model for the mechanism of action in the plant–pathogen interaction. Mol. Plant–Microbe Interact. 8, 610–620. Jan, Y.J. and Jan, Y.N. (1997) Cloned potassium channels from eukaryotes and prokaryotes. Annu. Rev. Neurosci. 20, 91–123. Macri, F. and Vianello, A. (1979) Inhibition of K1 uptake, H1 extrusion and K1-activated ATPase, and depolarization of transmembrane potential in plant tissues treated with Cercospora beticola toxin. Physiol. Plant Pathol. 15, 161–170. Macri, F., Dell’Antone, P. and Vianello, A. (1983) ATP-dependent proton uptake inhibited by Cercospora beticola toxin in pea stem microsomal vesicles. Plant Cell Environ. 6, 555–558. Mike`s, V., Milat, M.-L., Pugin, A. and Blein, J.-P. (1994) Cercospora beticola toxins. Part VII: fluorometric study of their interactions with biological membranes. Biochim. Biophys. Acta, 1195, 124–130. Schlo¨sser, E. (1962) U¨ber eine biologisch aktive Substanz aus Cercospora beticola. Phytopathol. Z. 44, 295–312. Simon-Plas, F., Gome`s, E., Milat, M.-L., Pugin, A. and Blein, J.-P. (1996) Cercospora beticola toxin. Part X: inhibition of plasma membrane H1-ATPase by beticolin-1. Plant Physiol. 111, 773– 779. Spalding, E.P. and Goldsmith, M.H.M. (1993) Activation of K1 channels in the plasma membrane of Arabidopsis by ATP produced photosynthetically. Plant Cell, 5, 477–484. Ve´ry, A.-A., Gaymard, F., Bosseux, C., Sentenac, H. and Thibaud, J.-B. (1995) Expression of a cloned plant K1-channel in Xenopus oocytes: analysis of macroscopic currents. Plant J. 7, 321–332.
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