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Effect of ionophore A23187 on plasma membrane integrity in isolated protoplasts of Avena sativa
Plant Science, 69 (1990) 135-138 Elsevier Scientific Publishers Ireland Ltd.
135
EFFECT OF IONOPHORE A23187 ON PLASMA MEMBRANE INTEGRITY IN I S O L A T E D PROTOPLASTS OF A VENA SA TIVA
A. KELL* and E. DONATH Bereich Biophysil¢ Sektion Biologie der Humboldt-Universitdt zu Berli~ 1040Berli~ Invalidenstr. 4~ (Germany) (Received November 20th, 1989) (Revision received February 16th, 1990) (Accepted February 16th, 1990) It is argued that modulation of the cytoplasmic Ca2÷of plant cell protoplasts should be expected to decrease their ability to incorporate membrane material and thus to resist osmotic shock. Protoplasts from Avena sativa were incubated with ionophore A23187 under various conditions, and the extent of protoplast lysis was determined by the use of fluorescein diacetate. The amount of lysis was strongly dependent on both the extracellular Ca~*concentration and the extracellular pH. The influence of the ionophore concentration itself and the incubation time were also investigated. The results suggest that a certain level of intraceUular Ca2~is important for maintaining plasma membrane integrity, even under isotonic conditions. Key words:Arena sativa; plant protoplasts; ionophore A23187;calcium ions; membrane lysis
Introduction In the last decade, intracellular Ca 2+ has been shown to act as a second messenger in a variety of physiological responses in plants (for review see Refs. 1 -4). In investigating its role, the use of calcium-selective ionophores has proven an important tool since it allows one to change the intracellular concentration of Ca 2÷ and to disturb its distribution. Experiments using ionophore A23187 on pollen tubes showed that localized secretion was affected and tip growth was inhibited in the presence of the ionophore [5-7]. F r o m this, the authors concluded that cytoplasmic Ca 2÷ was involved in such directed exocytosis. Exocytosis is based on the fusion between cytoplasmic vesicles and the plasma membrane. A similar process is involved in the expansion of isolated protoplasts following hypotonic shock treatment. Here, a rapid incorporation of membrane material via membrane fusion is necessary to permit plasma membrane enlargement [8-10]. W e previously found that extracellular Ca 2÷ was necessary for the expan*To whom correspondenceshould be sentat:Department of BiologicalSciences,University College of Wales, Aberystwyth,Dyfed SY23 3DA, U.K.
sion of isolated p r o t o p l a s t s [11]. Moreover, t r e a t m e n t with several calmodulin a n t a g o n i s t s yielded an increase of lysis a f t e r hypotonic shock [12]. We t h e r e f o r e wished to d e t e r m i n e w h e t h e r t r e a t m e n t of p r o t o p l a s t s with ionophore A23187 influenced their ability to expand and hence to p e r f o r m rapid m e m b r a n e fusion. In comparison to plant tissues in which cells are s u r r o u n d e d by a Ca2÷-containing wall, isolated protoplasts provide the a d v a n t a g e t h a t the extracellular Ca 2÷ concentration is defined, and m a y be varied, and thus the intracellular Ca 2÷ concentration can be influenced in a controlled manner. Whilst doing the experiments, however, we u n e x p e c t e d l y found t h a t the incubation of protoplasts with ionophore A23187 itself caused m e m b r a n e lysis to occur, even u n d e r isotonic conditions. The results s u g g e s t t h a t a certain level of intracellular Ca 2÷ is n e c e s s a r y for maintaining plasma m e m b r a n e stability. Materials and M e t h o d s P l a n t material and pro toplast isolation Seeds from A. sativa were g e r m i n a t e d in the
0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
136 dark for 2 days and then the plants were grown under continuous light at a constant temperature of 295 K for another 6 days. Protoplasts were isolated as described previously [13]. The epidermis of the primary leaf of approx. 50 plants was removed. Cell wall digestion was performed for 3 h by incubation in 0.5 M sorbitol containing 0.5% (w/v) each of cellulase TC and pectinase 5S (both from Serva) and 0.5 mM CaCl~. The protoplasts were centrifuged at 60 × g for 5 rain and resuspended in 0.5 M sucrose containing 0.5 mM CaC12. 5 ml of this suspension were placed in a centrifuge tube and overlaid with 1 ml 0.5 M sorbitol containing 0.5 mM CaC12. The suspension was recentrifuged under the same conditions, and the protoplasts recovered at the interface between the two solutions.
Treatment with ionophore A23187 Ionophore A23187 (Calbiochem) was dissolved in ethanol/dimethyl formamide (2:1, v/v). After isolation, protoplasts were first washed three times in CaC12-free 0.5 M sorbitol and then washed once more in the same, containing the CaC12 concentration and pH (5 mM MES + 5 mM Tris, KOH/HC1) under investigation. Finally A23187 was added. The solvent concentration did not exceed 0.1%. The protoplast concentration was about 3"105 per ml. During incubation with A23187 the solutions were stirred gently every 10 min to avoid sedimentation. After 1 h, the samples were centrifuged (20 × g) and the sediment was used immediately for the fluorescein diacetate test [14]. About 500 protoplasts were evaluted in each sample. Each series of experiments was performed with at least three different protoplast preparations. The sensitivity of protoplasts to A23187 was somewhat different in different preparations. To allow a quantitative comparison between various conditions, curves shown in one diagram represent the behaviour of one and the same protoplast preparation. Results Following incubation with A23187, many
protoplasts which appeared quite intact when observed in the light microscope showed no fluorescence after addition of fluorescein diacetate. The effect of the ionophore on plasma membrane integrity was dependent both on the Ca 2÷ concentration and the pH of the external solution. Figure 1A shows the percentage of fluorescein-stained protoplasts, following a 1-h incubation with A23187 (10 ~M), as a function of the external Ca 2÷concentration. If the external pH was adjusted to 6.2 the ionophore increased the percentage of lysis in a manner that was strongly dependent on the external Ca ~÷concentration in the range between 5-10 -4 and 5.10 -3 M Ca 2÷. In comparison, on decreasing the pH to 5.2, A23187 caused significant destabilisation of membrane integrity at external Ca 2÷ concentrations up to 10 -2 M. In Fig. 1B the dependence of ionophore-induced proto~ plast lysis on the external pH is shown, in the presence of 10-4 M CaC12 and 5.10 -4 M EDTA, respectively. With 10-4 M Ca 2÷ outside, low pH induced lysis, whilst at pH values above 6 the protoplasts became more and more stable. In the absence of external Ca 2., only at very high external pH did A23187 not induce membrane destabilization. Furthermore, the effect of A23187 on plasma membrane integrity was found to be strongly dependent on ionophore concentration itself (up to 30 ~M) at any external Ca 2÷ concentration used (Fig. 1C). Finally, the time-dependence of the ionophore action was investigated (Fig. 1D). An incubation of about 30 min was sufficient to induce maximal membrane destabilization. Discussion Ionophore A23187 catalyzes the electroneutral exchange of divalent cations for protons across biological membranes [15--17]. Since one divalent cation is exchanged for two protons, the equilibrium is given, to a first approximation, by the following equation: [Me~*]~•[H*I~ = [MeZ~]~,"[H*]~
(1)
where Me 2. stands for divalent metal cation and
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Fig. 1. The percentage of protoplasts which were able to accumulate fluorescein after treatment with A23187. Protoplasts were prepared and incubated as described in the Materials and Methods. Except where stated, protoplasts were preineubated with A23187 for 1 h. (A) Dependence of the extent of protoplast lysis on the external CaC12concentration during incubation with 10 ~M A23187 at external pH values of 5.2 ( o - o ) and 6.2 ( m - . ) , respectively. Open symbols represent the control experiments with no added ionophore, but to which ethanol/dimethyl formamide (2:1, v/v) was added at a final concentration of 0.1%. (B) Dependence of the extent of protoplast lysis on the external pH. 0.1 mM CaC12 (m - n ) or 0.5 mM disodium EDTA ( o - • ),'respectively, were present during the incubation with 10 ~M A23187. The open square represents a control experiment in which A23187 was replaced by solvent as in Fig. 1A, and in which 0.1 mM CaC12 was also present. (C) Dependence of the extent of protoplast lysis on the ionophore concentration at pH 6.2, at external CaC12 concentrations of 0.5 mM (B -- m), 5 mM( • -- • ~ or 10 mM (A -- A), or in the presence of 0.5 mM disodium EDTA ( • -- • ). (D) Dependence of the extent of protoplast lysis on the time of incubation with 20/~M A23187 at pH 5.2, in the presence of 0.5 mM concentrations of CaCl 2 (B - , ) or disodium EDTA ( • -- • ), respectively•
138 I and II r e p r e s e n t t h e two p h a s e s s e p a r a t e d by the r e s p e c t i v e m e m b r a n e . The net direction of the cation e x c h a n g e is thus d e t e r m i n e d by both the cation g r a d i e n t itself and the p H difference [18,19]. In the p r e s e n t w o r k we h a v e s h o w n t h a t the incubation of isolated p r o t o p l a s t s with ionophore A23187 d i s t u r b e d p l a s m a m e m b r a n e i n t e g r i t y , and in a m a n n e r t h a t was s t r o n g l y d e p e n d e n t b o t h on the e x t e r n a l Ca 2" concentration and the e x t e r n a l pH. T h u s it s e e m e d t h a t the e x c h a n g e of Ca 2÷ and p r o t o n s b e t w e e n the c y t o p l a s m and the e x t e r n a l solution was responsible for producing lysis. The c y t o p l a s m i c a c t i v i t y of Ca 2÷in plant cells is v e r y low, and falls in the r a n g e b e t w e e n 10 -7 and 10 -5 M [1]. The c y t o p l a s m i c p H is in the r a n g e b e t w e e n 7.0 and 7.5. A s s u m i n g a c y t o p l a s m i c Ca 2÷ activity of 10 -6 M and a c y t o p l a s m i c p H of 7.2, and considering an e x t e r n a l Ca 2~ concentration of 10 -4 M, it follows f r o m Eqn.1 t h a t Ca 2÷ is e x c h a n g e d f r o m the c y t o p l a s m to the e x t e r n a l solution if the e x t e r n a l p H is l o w e r t h a n 6.2 (a c y t o p l a s m i c Ca z+ c o n c e n t r a t i o n of 10 -7 M yields a r e s p e c t i v e 'critical' pH of 5.7}. F i g u r e 1B indeed shows t h a t the p e r c e n t a g e of d i s t u r b e d p r o t o p l a s t s increased s t r o n g l y if the e x t e r n a l p H was d e c r e a s e d f r o m 6.5 to 5.6. T h u s we infer t h a t the d i s t u r b a n c e of p l a s m a m e m b r a n e i n t e g r i t y was caused b y the depletion of cytoplasmic Ca 2÷. H o w e v e r , w h e n discussing the effects of ionophore A23187, one should r e m e m b e r t h a t the ionophore is not specific for Ca 2÷ ions, b u t also f o r m s c o m p l e x e s with Mg 2÷ of a l m o s t the s a m e stability [17]. T h e c o n c e n t r a t i o n of Mg 2÷ in the c y t o p l a s m is higher t h a n t h a t of Ca 2+ by s e v e r a l o r d e r s of m a g n i t u d e [1] and hence the Mg 2÷ efflux carried b y A23187 should be much g r e a t e r t h a n t h a t of Ca 2÷. So the loss of Mg 2÷ m a y also c o n t r i b u t e to p r o t o p l a s t lysis. Furt h e r m o r e , the d e p e n d e n c e of lysis on the ionophore concentration, t h a t was o b s e r v e d in the r e l a t i v e l y high r a n g e up to 30 ~M, could be explained by t h e excess of intracellular Mg 2÷
and its c o m p e t i t i o n with Ca '~÷ for the A23187 binding sites. I t has a l r e a d y b e e n r e p o r t e d t h a t t r e a t m e n t with ionophore A23187 affected s e c r e t i o n and s t o p p e d tip g r o w t h in pollen t u b e s [5-- 7], which was a t t r i b u t e d to a specific effect of the ionophore on e x o c y t o t i c p r o c e s s e s . Our r e s u l t s now indicate t h a t incubation with A23187 causes m e m b r a n e lysis in isolated p r o t o p l a s t s , and t h a t this effect is r e l a t e d to a depletion of cytoplasmic Ca 2÷. I t is possible t h a t in isolated p r o t o p l a s t s m e m b r a n e fusion also t a k e s place u n d e r isotonic conditions, as a continuous m e m b r a n e flow, for which a c e r t a i n level of c y t o p l a s m i c Ca 2÷is required.
References 1 P.K. Hepler and R.0. Wayne, Annu. Rev. Plant Physiol., 36 (1985)397. 2 A.J. Trewavas, Molecular and Cellular Aspects of Calcium in Plant Development, Plenum Press, New York and London, 1986. 3 H. Kauss, Annu. Rev. Plant Physiol., 38 (1987) 47. 4 E. Allan and P.K. Hepler, Biochem. Plants, 15 (1989) 455. 5 W. Herth, Protoplasma, 96 (1978) 275. 6 H.-D.Reiss and W. Herth, Planta, 145 (1979) 225. 7 J.M. Picton and M.W, Steer, Protoplasma, 115 (1983) 11, 8 J. Wolfe and P.L. Steponkus, Biochim. Biophys. Acta, 643 (1981)663. 9 J. Wolfe, M.F. Dowgert and P.L. Steponkus, J. Membrane Biol., 86 (1985) 127. 10 10 A. Glaser and E. Donath, J. Exp. Bot., 40 (1989) 1231. 11 A. Glaser and E. Donath, Stud. Biophys., 131 (1989}35. 12 A. Glaser and E. Donath, Stud. Biophys., 127 {1988) 129. 13 A. Glaser and E. Donath, Stud. Biophys., 119 (1987)93. 14 P.F. Larkin, Planta, 128 (1976}213. 15 D.R. Pfeiffer and H.A. Lardy, Biochemistry, 15 (1976) 935. 16 P.W. Reed and H.A. Lardy, J. Biol. Chem., 247 (1972) 6970. 17 D.R. Pfeiffer, P.W. Reed and H.A. Lardy, Biochemistry, 13 (1974)4007. 18 J. Wulf and W.G. Pohl, Biochim. Biophys. Acta, 465 (1977) 471. 19 W.G.Pohl, R. Kreikenbohm and K. Seuwen, Z. Naturforsch., 35c (1980) 562.
135
EFFECT OF IONOPHORE A23187 ON PLASMA MEMBRANE INTEGRITY IN I S O L A T E D PROTOPLASTS OF A VENA SA TIVA
A. KELL* and E. DONATH Bereich Biophysil¢ Sektion Biologie der Humboldt-Universitdt zu Berli~ 1040Berli~ Invalidenstr. 4~ (Germany) (Received November 20th, 1989) (Revision received February 16th, 1990) (Accepted February 16th, 1990) It is argued that modulation of the cytoplasmic Ca2÷of plant cell protoplasts should be expected to decrease their ability to incorporate membrane material and thus to resist osmotic shock. Protoplasts from Avena sativa were incubated with ionophore A23187 under various conditions, and the extent of protoplast lysis was determined by the use of fluorescein diacetate. The amount of lysis was strongly dependent on both the extracellular Ca~*concentration and the extracellular pH. The influence of the ionophore concentration itself and the incubation time were also investigated. The results suggest that a certain level of intraceUular Ca2~is important for maintaining plasma membrane integrity, even under isotonic conditions. Key words:Arena sativa; plant protoplasts; ionophore A23187;calcium ions; membrane lysis
Introduction In the last decade, intracellular Ca 2+ has been shown to act as a second messenger in a variety of physiological responses in plants (for review see Refs. 1 -4). In investigating its role, the use of calcium-selective ionophores has proven an important tool since it allows one to change the intracellular concentration of Ca 2÷ and to disturb its distribution. Experiments using ionophore A23187 on pollen tubes showed that localized secretion was affected and tip growth was inhibited in the presence of the ionophore [5-7]. F r o m this, the authors concluded that cytoplasmic Ca 2÷ was involved in such directed exocytosis. Exocytosis is based on the fusion between cytoplasmic vesicles and the plasma membrane. A similar process is involved in the expansion of isolated protoplasts following hypotonic shock treatment. Here, a rapid incorporation of membrane material via membrane fusion is necessary to permit plasma membrane enlargement [8-10]. W e previously found that extracellular Ca 2÷ was necessary for the expan*To whom correspondenceshould be sentat:Department of BiologicalSciences,University College of Wales, Aberystwyth,Dyfed SY23 3DA, U.K.
sion of isolated p r o t o p l a s t s [11]. Moreover, t r e a t m e n t with several calmodulin a n t a g o n i s t s yielded an increase of lysis a f t e r hypotonic shock [12]. We t h e r e f o r e wished to d e t e r m i n e w h e t h e r t r e a t m e n t of p r o t o p l a s t s with ionophore A23187 influenced their ability to expand and hence to p e r f o r m rapid m e m b r a n e fusion. In comparison to plant tissues in which cells are s u r r o u n d e d by a Ca2÷-containing wall, isolated protoplasts provide the a d v a n t a g e t h a t the extracellular Ca 2÷ concentration is defined, and m a y be varied, and thus the intracellular Ca 2÷ concentration can be influenced in a controlled manner. Whilst doing the experiments, however, we u n e x p e c t e d l y found t h a t the incubation of protoplasts with ionophore A23187 itself caused m e m b r a n e lysis to occur, even u n d e r isotonic conditions. The results s u g g e s t t h a t a certain level of intracellular Ca 2÷ is n e c e s s a r y for maintaining plasma m e m b r a n e stability. Materials and M e t h o d s P l a n t material and pro toplast isolation Seeds from A. sativa were g e r m i n a t e d in the
0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
136 dark for 2 days and then the plants were grown under continuous light at a constant temperature of 295 K for another 6 days. Protoplasts were isolated as described previously [13]. The epidermis of the primary leaf of approx. 50 plants was removed. Cell wall digestion was performed for 3 h by incubation in 0.5 M sorbitol containing 0.5% (w/v) each of cellulase TC and pectinase 5S (both from Serva) and 0.5 mM CaCl~. The protoplasts were centrifuged at 60 × g for 5 rain and resuspended in 0.5 M sucrose containing 0.5 mM CaC12. 5 ml of this suspension were placed in a centrifuge tube and overlaid with 1 ml 0.5 M sorbitol containing 0.5 mM CaC12. The suspension was recentrifuged under the same conditions, and the protoplasts recovered at the interface between the two solutions.
Treatment with ionophore A23187 Ionophore A23187 (Calbiochem) was dissolved in ethanol/dimethyl formamide (2:1, v/v). After isolation, protoplasts were first washed three times in CaC12-free 0.5 M sorbitol and then washed once more in the same, containing the CaC12 concentration and pH (5 mM MES + 5 mM Tris, KOH/HC1) under investigation. Finally A23187 was added. The solvent concentration did not exceed 0.1%. The protoplast concentration was about 3"105 per ml. During incubation with A23187 the solutions were stirred gently every 10 min to avoid sedimentation. After 1 h, the samples were centrifuged (20 × g) and the sediment was used immediately for the fluorescein diacetate test [14]. About 500 protoplasts were evaluted in each sample. Each series of experiments was performed with at least three different protoplast preparations. The sensitivity of protoplasts to A23187 was somewhat different in different preparations. To allow a quantitative comparison between various conditions, curves shown in one diagram represent the behaviour of one and the same protoplast preparation. Results Following incubation with A23187, many
protoplasts which appeared quite intact when observed in the light microscope showed no fluorescence after addition of fluorescein diacetate. The effect of the ionophore on plasma membrane integrity was dependent both on the Ca 2÷ concentration and the pH of the external solution. Figure 1A shows the percentage of fluorescein-stained protoplasts, following a 1-h incubation with A23187 (10 ~M), as a function of the external Ca 2÷concentration. If the external pH was adjusted to 6.2 the ionophore increased the percentage of lysis in a manner that was strongly dependent on the external Ca ~÷concentration in the range between 5-10 -4 and 5.10 -3 M Ca 2÷. In comparison, on decreasing the pH to 5.2, A23187 caused significant destabilisation of membrane integrity at external Ca 2÷ concentrations up to 10 -2 M. In Fig. 1B the dependence of ionophore-induced proto~ plast lysis on the external pH is shown, in the presence of 10-4 M CaC12 and 5.10 -4 M EDTA, respectively. With 10-4 M Ca 2÷ outside, low pH induced lysis, whilst at pH values above 6 the protoplasts became more and more stable. In the absence of external Ca 2., only at very high external pH did A23187 not induce membrane destabilization. Furthermore, the effect of A23187 on plasma membrane integrity was found to be strongly dependent on ionophore concentration itself (up to 30 ~M) at any external Ca 2÷ concentration used (Fig. 1C). Finally, the time-dependence of the ionophore action was investigated (Fig. 1D). An incubation of about 30 min was sufficient to induce maximal membrane destabilization. Discussion Ionophore A23187 catalyzes the electroneutral exchange of divalent cations for protons across biological membranes [15--17]. Since one divalent cation is exchanged for two protons, the equilibrium is given, to a first approximation, by the following equation: [Me~*]~•[H*I~ = [MeZ~]~,"[H*]~
(1)
where Me 2. stands for divalent metal cation and
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Fig. 1. The percentage of protoplasts which were able to accumulate fluorescein after treatment with A23187. Protoplasts were prepared and incubated as described in the Materials and Methods. Except where stated, protoplasts were preineubated with A23187 for 1 h. (A) Dependence of the extent of protoplast lysis on the external CaC12concentration during incubation with 10 ~M A23187 at external pH values of 5.2 ( o - o ) and 6.2 ( m - . ) , respectively. Open symbols represent the control experiments with no added ionophore, but to which ethanol/dimethyl formamide (2:1, v/v) was added at a final concentration of 0.1%. (B) Dependence of the extent of protoplast lysis on the external pH. 0.1 mM CaC12 (m - n ) or 0.5 mM disodium EDTA ( o - • ),'respectively, were present during the incubation with 10 ~M A23187. The open square represents a control experiment in which A23187 was replaced by solvent as in Fig. 1A, and in which 0.1 mM CaC12 was also present. (C) Dependence of the extent of protoplast lysis on the ionophore concentration at pH 6.2, at external CaC12 concentrations of 0.5 mM (B -- m), 5 mM( • -- • ~ or 10 mM (A -- A), or in the presence of 0.5 mM disodium EDTA ( • -- • ). (D) Dependence of the extent of protoplast lysis on the time of incubation with 20/~M A23187 at pH 5.2, in the presence of 0.5 mM concentrations of CaCl 2 (B - , ) or disodium EDTA ( • -- • ), respectively•
138 I and II r e p r e s e n t t h e two p h a s e s s e p a r a t e d by the r e s p e c t i v e m e m b r a n e . The net direction of the cation e x c h a n g e is thus d e t e r m i n e d by both the cation g r a d i e n t itself and the p H difference [18,19]. In the p r e s e n t w o r k we h a v e s h o w n t h a t the incubation of isolated p r o t o p l a s t s with ionophore A23187 d i s t u r b e d p l a s m a m e m b r a n e i n t e g r i t y , and in a m a n n e r t h a t was s t r o n g l y d e p e n d e n t b o t h on the e x t e r n a l Ca 2" concentration and the e x t e r n a l pH. T h u s it s e e m e d t h a t the e x c h a n g e of Ca 2÷ and p r o t o n s b e t w e e n the c y t o p l a s m and the e x t e r n a l solution was responsible for producing lysis. The c y t o p l a s m i c a c t i v i t y of Ca 2÷in plant cells is v e r y low, and falls in the r a n g e b e t w e e n 10 -7 and 10 -5 M [1]. The c y t o p l a s m i c p H is in the r a n g e b e t w e e n 7.0 and 7.5. A s s u m i n g a c y t o p l a s m i c Ca 2÷ activity of 10 -6 M and a c y t o p l a s m i c p H of 7.2, and considering an e x t e r n a l Ca 2~ concentration of 10 -4 M, it follows f r o m Eqn.1 t h a t Ca 2÷ is e x c h a n g e d f r o m the c y t o p l a s m to the e x t e r n a l solution if the e x t e r n a l p H is l o w e r t h a n 6.2 (a c y t o p l a s m i c Ca z+ c o n c e n t r a t i o n of 10 -7 M yields a r e s p e c t i v e 'critical' pH of 5.7}. F i g u r e 1B indeed shows t h a t the p e r c e n t a g e of d i s t u r b e d p r o t o p l a s t s increased s t r o n g l y if the e x t e r n a l p H was d e c r e a s e d f r o m 6.5 to 5.6. T h u s we infer t h a t the d i s t u r b a n c e of p l a s m a m e m b r a n e i n t e g r i t y was caused b y the depletion of cytoplasmic Ca 2÷. H o w e v e r , w h e n discussing the effects of ionophore A23187, one should r e m e m b e r t h a t the ionophore is not specific for Ca 2÷ ions, b u t also f o r m s c o m p l e x e s with Mg 2÷ of a l m o s t the s a m e stability [17]. T h e c o n c e n t r a t i o n of Mg 2÷ in the c y t o p l a s m is higher t h a n t h a t of Ca 2+ by s e v e r a l o r d e r s of m a g n i t u d e [1] and hence the Mg 2÷ efflux carried b y A23187 should be much g r e a t e r t h a n t h a t of Ca 2÷. So the loss of Mg 2÷ m a y also c o n t r i b u t e to p r o t o p l a s t lysis. Furt h e r m o r e , the d e p e n d e n c e of lysis on the ionophore concentration, t h a t was o b s e r v e d in the r e l a t i v e l y high r a n g e up to 30 ~M, could be explained by t h e excess of intracellular Mg 2÷
and its c o m p e t i t i o n with Ca '~÷ for the A23187 binding sites. I t has a l r e a d y b e e n r e p o r t e d t h a t t r e a t m e n t with ionophore A23187 affected s e c r e t i o n and s t o p p e d tip g r o w t h in pollen t u b e s [5-- 7], which was a t t r i b u t e d to a specific effect of the ionophore on e x o c y t o t i c p r o c e s s e s . Our r e s u l t s now indicate t h a t incubation with A23187 causes m e m b r a n e lysis in isolated p r o t o p l a s t s , and t h a t this effect is r e l a t e d to a depletion of cytoplasmic Ca 2÷. I t is possible t h a t in isolated p r o t o p l a s t s m e m b r a n e fusion also t a k e s place u n d e r isotonic conditions, as a continuous m e m b r a n e flow, for which a c e r t a i n level of c y t o p l a s m i c Ca 2÷is required.
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