spectrum was regained by removing bound Ca with a Ca chelator. Polyeth- ylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in ...
Plant Physiol. (1980) 66, 835-837
0032-0889/80/66/0835/03/$00.50/0
Effects of Divalent Cations and Polyethylene Glycol on the Membrane Fluidity of Protoplast' Received for publication November 7, 1979 and in revised form May 27, 1980
WENDY F. BOSS AND RALPH L. MoTT Department of Botany, North Carolina State University, Raleigh, North Carolina 27650 were released enzymically from cells in the exponential phase of growth (3 to 4 days after transfer) (1). The protoplasts were filtered Calcium is often used to stabilize membranes and enhance membrane through glass wool and washed once by centrifugation (40g, 5 fusion. We have used the fatty acid spin label, 5-nitroxy steanc acid to min) through 1% (w/v) dextran in osmoticum A, which consisted measure fluidity changes in the plasma membrane of carrot suspension of 0.45 molal sorbitol and 0.5 millimolal Mes (pH 5.5). Debris culture celi protoplasts in response to divalent cations. Electron spin was further removed by three additional washes in osmoticum A resonance spectra from spin-labeled protoplasts showed no membrane without dextran. fluidity changes (as determined by the hyperfine splitting constant, 2Am.) Spin-Labeling of Protoplasts. A freshly prepared stock solution in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millinolar. of spin-label probe (SYVA Co. Palo Alto, CA) in methanol Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 (1.05-NS 1 ml) was kept in a darkened tube on ice during an mg/0. gauss in 2Am.a and evidence of exchange-broadening. The original (control) experiment. Aliquots (4 ,ul) were pipetted into 10-ml glass tubes; spectrum was regained by removing bound Ca with a Ca chelator. Polyeth- 0.1 ml settled volume of protoplasts (approximately 105 protoylene glycol, which enhances protoplast fusion, did not alter the membrane was added, and the tube was tapped gently to mix the fluidity in the region of the 5-nitroxy stearic acid probe if added simulta- plasts) and the spin-label. Labeled protoplasts were immediprotoplasts neously with or following 10 millimolar Ca. Pretreatment with polyethylene drawn up into a quartz capillary (1 mm i.d., Wilmad Glass ately glycol did, however, inhibit the Ca-induced phase separation. These data Co., Buena, N. J.). and the ESR spectrum was recorded. The on a lving system describe membrane structural changes under conditions protoplasts then were expelled from the capillary and washed once similar to those used for protoplast fusion. by centrifugation in an appropriate osmoticum with varying treatment (Table I) and rescanned. At least three replicate scans were made after each treatment. The sorbitol concentration of the treatment osmoticum was adjusted so that the osmolality remained the same in each treatment, except in the case of PEG. Protoplasts ESR2 has played an important role in the understanding of were gently manipulated at each stem to minimize breakage, and molecular motion of lipids in model and biological membrane the 40g centrifugation during the wash removed suspended debris. The spectra were recorded on a JEOL MES-3X spectrometer at systems (2, 4, 17). We chose the fatty acid spin label 5-NS to mw 2 power, 50- to 100-G scan width, 0.3- to 1.0-s response, 5- to susmembrane of carrot of the plasma measure fluidity changes pension culture cell protoplasts. The 5-NS probe has the nitroxide 10-min scan time, modulation frequency of 2 G, and a variable moiety on the fifth carbon from the carboxy terminus of stearic gain. The 2Amaz values were based on Tempo as a standard. acid and the probe intercalates into the membrane bilayer so that RESULTS the nitroxide is located in the region of the glycerol backbone of phospholipids (4, 15). The ESR signal from the probe is thus Effect of Ca and Mg on Membrane Fluidity. A typical firstsensitive to perturbations near the surface of the bilayer. Ca stabilizes membranes and enhances membrane fusion in both derivative spectrum of carrot suspension culture cell protoplasts (control). Protoplasts artificial and biological systems (3, 7, 14). Ca binds to the nega- labeled with 5-NS is shown in Figure I 0.5 washed in the presence of mm Ca(H2PO4)2 or tively charged phospholipids near the membrane surface resulting released and/or in osmoticum A in altered membrane fluidity and phase separation of the bilayer CaCl2 gave an ESR spectrum identical to thosewas no change in 5.0 there mm Even at Ca, Ca added. without on membrane effects of Ca fluidity are the here Reported (11-13). 5-NS 2Am.2 of the spin label as determined by membrane fluidity of living carrot protoplasts as determined through ESR using the (Table I). However, when spin-labeled protoplasts were washed 5-NS probe. in 10 mm Ca [either as CaCl2 or Ca(H2PO4)2J, there was a marked increase (5 G) in 2A,,1 indicative of decreased fluidity of the MATERIALS AND METHODS membrane lipid in the region of the fatty acid probe (Figure 1 Protoplast Isolation. A cell suspension culture of carrot, Daucus and Table I). carota L., was maintained as previously described (1). Protoplasts Along with the increase in 2Amax, the 10 mm Ca also caused a change in the spectrum indicated by the arrow (Fig. 1), This peak 1 This work was funded under Project No. 03585 of the North Carolina loss is indicative of exchange-broadening which is caused by Agricultural Research Service. This paper is No. 6187 in the Journal Series interaction between the nitroxide free radicals. Such exchangebroadening is commonly seen as the concentration of spin label in of the North Carolina Agricultural Research Service. 2Abbreviations: ESR, electron spin resonance; 5-NS, 5-nitroxy stearic protoplasts is increased in the absence of Ca (i.e. overlabeling). acid [2 -(3-carboxypropyl)-4, 4 dimethyl-2-tridecyl-3-oxazolidinyloxyl]; 16- The effect of Ca was not simply to increase the concentration of NS: 16-nitroxy stearic acid [2-(14-carboxy-tetradecyl-2 ethyl4, 4 dimethyl- spin label in the membrane since 2Am. did not increase when 3-oxazolidinyloxyl]; EGTA: ethylene glycol bis(JB-aminoethyl ether)N, N'- protoplasts were deliberately over labeled. These data suggest that Ca decreased membrane fluidity and, at the same time or as a tetraacetic acid; G, gauss; 2Am81, maximum hyperfme splitting constant. ABSTRACT
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Plant Physiol. Vol. 66, 1980
mM MgCl2 (Table I). To determine whether the Ca effect was localized at the surface of the bilayer, similar ESR studies using 16-NS-labeled protoplasts were conducted. The narrow three-line spectrum (32.5 G) was indicative of a fluid environment, suggesting that the nitroxide was intercalated deeply into the hydrophobic region of the bilayer. The 2Ama of the 16-NS-labeled protoplasts did not change in either 10 mM Ca or Mg. Effect of PEG on Membrane Fluidity. Fusion of plant proto.
COCI 2
2 A nox
2A
roX
FIG. 1. ESR spectrum of protoplasts from carrot suspension culture The arrow indicates peak loss due to exchange-broadening.
cells.
Table
I.
Maximum Hyperfine Splitting, 2A,. of Carrot Suspension Culture Cell Protoplasts
Treatment
2Amaxa
G 51.5 Control 51.5 0.5 mM CaCl2 51.5 0.5 mM Ca(H2PO4)2 51.5 5.0 mM CaCl2 56.5b 10 mM CaCl2 56.5b 10 mM Ca(H2PO4)2 51.5 10 mm CaCl2 followed by 1.6 mM EGTA 52 10 mM MgCl2 51.5 10 mM MgCl2 followed by 1.6 mM EGTA 56.5b 10 mM CaC12 followed by 15% PEG (0.9 M) 56.5b 10 mM CaCl2 followed by 32% PEG (2.1 M) 50.5 15% PEG 52 15% PEG followed by 10 mM CaC12 50.5 45% PEG (2.9 M) 51.5 32% PEG followed by 10 mm CaC12 56.5b 32% PEG in 10 mM CaCl2 a The values are based on Tempo as a standard and are a mean of a minimum of four different experiments. The SD for these data is I G or less. The large error in measuring 2A. is due partially to the width of the peaks in the spectrum and partially to the low signal to noise ratio. b Exchange-broadening is evident.
2A.,
consequence of the decreased fluidity, caused the probe to become concentrated into localized, discrete regions of the membrane, thus fostering interaction between the nitroxide free radicals. There was no visible change in the Ca-treated protoplasts as determined by light microscopy. The ESR spectrum changes due to Ca remained even after the protoplasts were extensively washed free of unbound Ca with osmoticum A. Removal of bound Ca by washing the Ca-treated protoplasts in osmoticum A containing the Ca chelator EGTA (1.6 mM) restored the original spectrum with no exchange-broadening (Table I). Since concentrations of Ca (0.5 to 5 mm) which should have decreased the surface charge (3, 10) did not alter the ESR spectra of 5-NS-labeled protoplasts, it was felt that the effects of 10 mm Ca were not simply due to charge neutralization. To further test this hypothesis, 10 mm MgCl2 was used. There was no change in 2Amax or exchange-broadening of 5-NS-labeled protoplasts in 10
plasts is enhanced by a transient treatment with 15 to 45% PEG (15,400 mol wt) following or coincidental with exposure to Ca (5, 6). When 5-NS-labeled protoplasts were treated with PEG in osmoticum A, the 2A.., did not change (Table I); however, the intensity of the signal was increased to as much as twice that of the control, followed by rapid signal loss with time. The increased intensity was probably due to closer packing of protoplasts in the ESR tube brought about by PEG-induced plasmolysis. The plasmolyzed protoplasts swelled to regain a spherical shape within 10 min, but the loss of signal continued. It is not clear whether the continued, gradual signal loss was due to electrochemical reduction of the 5-NS within the bilayer or to mobilization of the probe either into or out of the cell by the amphiphilic compound PEG. When 5-NS-labeled protoplasts were exposed first to 10 mM CaCl2, a subsequent wash in 15 or 32% (w/v) PEG did not cause further change in 2Ama (Table I), nor were exchange-broadening or rate of signal loss altered. That is, the restricted membrane fluidity in the region of the 5-NS probe established by Ca was not altered. Both the decreased membrane fluidity and exchangebroadening were established by Ca even when PEG and 10 mM Ca were added simultaneously. However, the fluidity changes and exchange-broadening of 10 mm Ca could not be obtained if the protoplasts were first pretreated with PEG (Table I). Protoplasts Breakage. One must always be cautious of breakage when working with protoplasts. Possible interference by debris from burst protoplasts in the sample was tested using samples of debris obtained by mechanical or osmotic (abrupt dilution of osmoticum A) disruption. Burst protoplasts were centrifuged at 40g and the pellet and supernatant were then labeled with 5-NS in the usual manner. The resulting spectra (2Ama. = 54 G for the pellet and 53 G for the supernatant) were different from the 51.5 G of intact protoplasts. After treating the 40g pellet with 10 mM Ca as one would treat intact protoplasts, the ESR spectra showed exchange-broadening and the 2Am.1 increased to 59 G. This was again different from intact protoplasts. If protoplasts were first labeled and then disrupted as above, the ESR signal was completely lost within 10 min. The 2A.. of the signal in the 40g supernatant was approximately 53 G. Precise measurement was difficult due to rapid signal loss. The 40g pellet gave a signal in which only the center peak was detectable above the background noise, and this was also rapidly lost. Signal loss was too rapid to permit washing the pellet free of supernatant prior to scanning and some contribution to the pellet signal could be expected from the residual supematant. The fact that intact protoplast preparations gave a relatively stable signal (t1/2 = 20 min) with 2Amax less than 53 G suggests that the 5-NS probe is in intact protoplasts.
DISCUSSION The spin-labeled fatty acid 5-NS was used to measure structural changes in the membranes of carrot suspension culture cell protoplasts. Mg concentrations of 10 mm and Ca concentrations up to 5 mM did not alter membrane fluidity in the region of the 5-NS probe. However, at Ca concentrations of 10 mm and greater, which are commonly used for protoplast fusion (5, 7), there was marked decrease in fluidity. The nitroxide moiety of the 16-NS probe intercalates more deeply into the bilayer than does the 5-NS probe (2, 4). The lack of response of the 16-NS probe to Ca suggests that, in the system used here, the response is limited to the surface
Plant Physiol. Vol. 66, 1980
PROTOPLAST MEMBRANE FLUIDITY
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With the 5-NS probe we measure an average of protoplast of the bilayer. Nagata and Melchers (10) have a shown a reduction in protoplast surface charge in the presence of 10 mm Ca; however, membrane fluidity in vivo. The studies presented here comparing inasmuch as 10 mm Mg did not cause fluidity changes in the intact and burst protoplasts show that the structural changes system used here, the effects of Ca on membrane fluidity seem to reported can be attributed to intact protoplast. The dramatic decrease in membrane fluidity in the presence of 10 mM Ca may extend beyond simple charge neutralization. Along with the measured decreased in fluidity, there was evi- be due to Ca binding to phospholipids (10) or to glycolipids (8) on dence of Ca-induced exchange-broadening. Exchange-broadening the membrane surface. Further studies using isolated membrane results from free radical interaction at high local concentrations of vesicles are in progress. spin label. Repeated washes in osmoticum without Ca (osmoticum Acknowledgments-The authors would like to thank Dr. Keith DeArmond and A) did not alter the Ca induced changes in membrane structure, Dr. Mary Ortner for the use of their electron spin resonance spectrometers and Dr. Frank was not irreversible Landsberger and Dr. Lawrence Altstiel for their helpful discussions and for but this an phenomenon because treating the protoplasts with EGTA restored the original signal with 2A. of the samples of Tempo. 51.5 G with no exchange-broadening. Similar Ca-induced exLITERATURE CITED change-broadening has been observed by Onishi and lto (11, 12) using artificial membrane vesicles. They proposed that, as a 1. Boss WF, AW RUESINK 1979 Isolation and characterization of concanavalin Alabeled plasma membranes of carrot protoplasts. Plant Physiol 65: 1005-1011 consequence of Ca binding to negatively charged phospholipids, WL, HM MCCONNEL 1969 Orientation and motion of amphiphilic spin there was a clustering of lipids into discrete regions on the bilayer. 2. HUBBELL labels in membranes. Proc Natl Acad Sci USA 64: 20-27 The formation of these lipid regions could result in locally in- 3. JACOBSON K, D PAPAHADJOPOULOS 1975 Phase transitions and phase separation creased concentrations of spin label, or Ca binding could enhance in phospholipid membranes induced by changes in temperature, pH, and concentration of bivalent cations. Biochemistry 14: 152-161 free radical interaction by simply restricting mobility of the fatty P, LJ LIBERTINI, VC HERBERT, OH GRIFFITH 1971 Lipid spin labels in acid in the bilayer. Either would result in increased exchange- 4. JOST lecithin multilayers. A study of motion along fatty acid chains. J Mol Biol 59: broadening. Our ESR data with protoplasts and studies by others 77-98 using artificial membranes (3, 12-14) and isolated membrane 5. KAo KN, F CONSTABEL, MR MICHAYLAK, OL GAMBORG 1974 Plant protoplast fusion and growth of intergeneric hybrid cells. Planta 120: 215-227 vesicles (9) demonstrate that high Ca concentrations can dramat- 6. KAo KN, MR MICHAYLAK 1974 A method for high-frequency intergeneric fusion ically alter membrane structure. Ca-induced phase separation has of plant protoplasts. Planta 115: 355-367 been proposed to be necessary for membrane fusion (13, 14) of 7. KELLER WA, G MELCHERS 1974 The effect of high pH and calcium on tobacco leaf protoplast fusion. Z Naturforsch 28c: 737-741 phospholipid vesicles. KS, A HAUG 1979 Regulation of zoospore development: electron spin The concentration of Ca needed to bring about decreased 8. LEONARDS resonance studies of the isolated lipid components from Blastocladiella emerfluidity will depend on the composition of the membrane (1 1, 12, sonil zoospores. Plant Physiol 63: S- 145 14) and, therefore, may be different for protoplasts of differing 9. MoRRE DJ, CE BRACKER 1974 Influence of calcium ions on the plant cell surface: membrane fusion and conformation changes. Proc Electron Microscopy Soc cell types. Changes in the ESR signal from the 5-NS probe gave Am 32: 154-155 evidence of changes in the membrane structure which occurred 10. NAGATA T, G MELCHERS 1978 Surface charge of protoplasts and their significance under conditions (10 mm Ca) conducive to protoplast fusion. Plant in cell-cell interaction. Planta 142: 235-238 protoplast fusion is generally enhanced by Ca treatment and 11. OHNISHI S-I, T ITO 1973 Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine. Biochem transient exposure to PEG (5, 6). Although the role of PEG in Biophys Res Commun 51: 132-138 is evidence that PEG alone protoplasts fusion unclear, suggests 12. OHNISHI S-I, T ITO 1974 Calcium-induced phase separations in phosphatidylserine-phosphatidylcholine membranes. Biochemistry 13: 881-887 will not enhance fusion but rather promotes phagocytosis (16). D, 1976 Effects of bivalent cations and proteins on thermoThe ESR studies presented here give evidence which also fits this 13. PAPAHADJOPOULOS tropic properties of phospholipid membranes. Implications for the molecular pattern. PEG alone did not alter membrane structure, as determechanism of fusion and endocytosis. J Coil Interface Sci 58: 459-470 mined by the 5-NS probe, and PEG pretreatment precluded an 14. PAPAHADJOPOULOS D, WJ VAIL, C NEWTON, S NIR, K JACOBSON, G PosTE, R LAZO 1977 Studies on membrane fusion. III. The role of calcium-induced immediate response to Ca. However, the Ca-induced fluidity phase changes. Biochim Biophys Acta 465: 579-598 change occurred and was persistent when protoplasts were given 15. SANSON A, M PTAK, JL RIGAUD, CM GARY-BOBO 1976 An ESR study of the simultaneous treatment of PEG and Ca or post-treatment with hydration steps of lecithin multilayers. Chem Phys Lipids 17: 445-455 PEG. The specific effect of PEG on Ca binding to membranes 16. UEDA K, K TAN, F SATO, Y YAMADA 1978 Phagocytosis in plant protoplasts. Cell Struct Funct 3: 25-30 has not been determined; however, preliminary experiments in- 17. VIERSTRA R, A HAUG 1978 The effect of A'3 on the physical properties of dicate that PEG does not reduce the uptake of 4 Ca by protoplasts membrane lipids in Thermoplasma acidophilum. Biochem Biophys Res Commun 84: 138-143 but, rather, at least doubles it.
