Planta

Planta (1982) 155:140-145

9 Springer-Verlag 1982

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse N. Salhani 1, H. Schnabl 2, G. Kfippers 1, and U. Zimmermann 1 1 Arbeitsgruppe Membranforschung am Institut fiir Medizin, KernforschungsanlageJfilich GmbH, Postfach 1913, D-5170 Jfilich, and 2 Institut ffir Botanik und Mikrobiologie der Technischen Universit~it, Arcisstrasse 21, D-8000 Miinchen 2, Federal Republic of Germany

Abstract. The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Ar e n a sativa L. and of mesophyll and guard cell protoplasts of Vicia f a b a L. were found to be 1.9_+0.9.10 -6, 3 . 2 + 2 . 2 . 1 0 -6, and 0.8_+ 0.7.10-6 cm. b a r - t. s - 1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe. Key words: Ar en a - Electric field - Hydraulic conductivity - Membrane integrity - Protoplast fusion Vicia - Volume relaxation.

Introduction Fusion of plant protoplasts, animal cells, or lipid vesicles can be achieved by exposing them to an electric field (Zimmermann and Scheurich 1981a, 1981b; Zimmermann et al. 1981a; Scheurich and Zimmermann 198t; Scheurich etal. 1981; Vienken etal. 1981 ; Richter et al. 1982). The electric field - induced fusion process can be divided into two stages. In the first stage, close membrane contact between cells or lipid vesicles, is established by dielectrophoresis, i.e., the movement of particles in an alternating inhomogeAbbreviations: GCP=guard cell protoplast; Lp=hydraulic con-

ductivity; MCP = mesophyll cell protoplast

0032-0935/82/0155/0140/$01.20

neous field which results in the formation of "pearl chains" of varying lengths by mutual attraction between adjacent cells (Muth 1927; Pohl 1978; Pethig 1979; Saito et al. 1966). The fusion process is subsequently initiated by reversible electrical breakdown in the membrane contact zone between any two adjacent cells (Zimmermann et al. 1973, 1976, 1980b, 1981 a). Electrical breakdown is induced by application of very short pulses of high intensity. Not only is this procedure very gentle, but the fusion process is synchronized and gives very high yields of fused cells or lipid vesicles (up to 100%). The number of cells to be subjected to fusion can be preselected as desired (Zimmermann et al. 1981 a). In this communication we report on whether any change in membrane properties can be detected in protoplasts fused by using an electrical field pulse. To do this we measured the hydraulic conductivity of the membrane of fused protoplasts and compared the values obtained with those determined for individual unfused cells. The hydraulic conductivity was derived from the shrinking and swelling kinetics of protoplasts in response to external osmotic stress. Measurements of the volume kinetics were carried out on large, fused, mesophyll cell protoplasts of A r e n a sativa and Vicia f a b a and guard cell protoplasts of V. faba. Material and methods Plants of Vica faba L. were grown in peat in a greenhouse maintained at 23-24~ C. After about nine days the plants, which were about 5 7 cm in height, were transferred to a culture chamber at 20~ (day) and 17~ (night) with a photoperiod (8,000lx) of over 12 h. The humidity was 65% during the day and 80% at night. The plants were watered three times a week. Leaves from plants which were no more than three weeks old were used for the preparation of mesophyll cell protoplasts (MCP's) and guard cell protoplasts (GCP's). Arena sativa L. plants were kept in a light - dark rhythm of 14 to 10 h at 21~ C (day) and 18~ C (night) in a culture chamber. The humidity was 60% during the day and 85% at night. The plants used for the isolation of protoplasts were at least eleven days old. The preparations of MCP's and GCP's from leaves of V.faba are described in detail elsewhere (Schnabl et al. 1978). MCP's of A. sativa were obtained by the procedure of Hampp and Ziegler

N. Salhani et al. : M e m b r a n e integrity of fused protoplasts (1980), except that the cellulysin concentration was reduced to 2%. The protoplasts were stored for no longer than 2 h in a 0.5 M mannitol solution containing 1 m M CaC12. For dielectrophoresis and electric field-induced fusion, the protoplasts were washed three times with a 0.5 M mannitol solution containing Ca 2 + ions only as trace contaminants. The conductivity of the solution should be less than 10-* ~ - ~ cm ~ in order to avoid excessively high electrical currents which would lead to heat development and, in turn, to turbulence in the solution (Zimmerm a n n et al. 1981 a). The setup used for dielectrophoresis and electrical breakdown is described elsewhere ( Z i m m e r m a n n and Scheurich 1981 a; Z i m m e r m a n n et al. 1981 a). To study the shrinking and swelling kinetics, the suspension of fused cells was rapidly mixed with a mannitol or sucrose solution of higher or lower osmolarity. The osmolarity was cryoscopically determined using a K n a u e r osmometer (Berlin, FRG). A droplet of the suspension was put onto a microscope slide and the kinetics of volume change followed using a Zeiss photomicroscope. On the average, the time interval between the first measurement and the addition of mannitol or sucrose to the protoplast suspension was about 30 s. The photographs of the ceils taken at different time intervals were magnified ten-fold, and the diameters of the spherical cells were measured.

Results and discussion

Figure 1 a shows pearl chain formation of MCP's of V.faba in an alternating inhomogeneous electric field between two cylindrical electrodes, 300 gm apart. The amplitude of the alternating voltage was 10 V (peak-to-peak value) and the frequency 500 kHz. Frequencies below 500 kHz, particularly in the range of between 20 and 40 kHz, were not used because of the rotation phenomenon observed in the cells

141

(Zimmermann et al. 1981b; Holzapfel et al. 1982), which would obviously disrupt close membrane contact between cells in a given pearl chain. In order to obtain large protoplasts, it is necessary to align several pearl chains parallel and close to each other. In some experiments, as in Fig. 1, pronase was added at a concentration of lmg/ml just prior to application of the field pulse to the suspension. Pronase stabilizes the protoplasts against higher electric field strengths and longer application times, as established in sea urchin eggs and cultured mammalian cells (Zimmermann et al. 1981; Pilwat et al. 1981; Zimmermann and Vienken 1982). The likely mechanism by which pronase stabilizes the cells in the presence of an electric field is described elsewhere (Zimmermann et al. 1982; Pilwat et al. 1981; Zimmermann and Vienken 1982). Pronase shifts the range of frequencies that induce protoplast rotation to higher values (500kHz1 MHz). It is therefore necessary to increase the frequency of the dieleetrophoretic voltage from 500 kHz to 1.5 MHz for a pronase incubation period of up to 30 min, in order to prevent rotation of the protoplasts. The field intensity of the alternating field is initially chosen to be 200 V c m - t , resulting in a small area of contact between the ceils within a chain. Since a larger membrane contact zone is required for fusion, the voltage between the electrodes is increased just

Fig. l a - e . Fusion of mesophyi1 cell protoplasts of Vicia faba by an eleclric field pulse, a Several cell chains formed in parallel on application of an alternating, non-uniform electric field (200 V cm 1, 1.5 MHz). Pronase (I mg/ml) was added to the isotonic mannitol solution just before field application. The gap between the two parallel cyclindrical electrodes was 300 l-tin, b-e Typical sequence of vertical and lateral fusion after application of the breakdown pulse (60 V, 40 las duration). Photographs were taken 1 rain (b), 10 min (c), 20 min (d) and 30 min (e) after field application. After 5 10 min isotonic mannitol solution containing 1 m M CaC12 was added. (Bar: 20 gm)

142

N. Salhani et al. : M e m b r a n e integrity of fused protoplasts

10-

uE O

k,

O

~8

O O O O

O O

O

O

O

O

O

6

~

8~

60

1~o"

t[sl Fig. 2. Plot of a typical volume relaxation of a fused protoplast of Arena sativa in response to an increase of the external osmolarity (6.25 bar). The fused cell consists of about 5 cells. Lp values were calculated from each point using Eq. (5a). The m e a n of these values is 1.5.10 6 cm s -1 bar -1

before the application of the breakdown pulse to a level not exceeding the breakdown voltage (Zimmermann et al. 1982). Under these conditions, the spherical protoplasts flatten out in the membrane contact zone, thus forming a larger area of contact. After this event, a pulse of 30 V amplitude and 20 ~ts duration is applied to the electrodes; in the presence of pronase, the voltage and duration were increased to 80 V and 60 gs, respectively. The field strengths are sufficiently high for breakdown to occur not only in the membrane contact zone but also in the membrane areas oriented at an angle up to 84 ~, with respect to the external field lines (Jeltsch and Zimmermann 1979), so that lateral fusion between cells of adjacent peral chains is possible (Scheurich and Zimmermann 1981; Zimmermann et al. 1981a). The result is the formation of large protoplasts. A typical sequence of events is shown in Fig. 1 vor V.faba protoplasts. On the average, 10 cells fuse together and the fusion process takes 3-30 min, depending on the species and number of cells which fuse together. The return to the spherical shape can be speeded up by transferring the fused cells to a 0.5 M mannitol solution containing 1 mM Ca 2 + ions, when they reach the stage illustrated in Fig. 1 c. Ca 2 + ions stabilize the cells mechanically. Prolonged absence of Ca 2 § ions results in very fragile fused aggregates. The same effect of Ca 2 § on the membranes is known to apply to immobilized protoplasts, dissolved out of a poly-

meric alginate matrix by the addition of citrate (Scheurich et al. 1980; Schnabl etal. 1980). Citrate chelates the Ca 2 + ions used for cross-linking the alginate and simultaneously removes Ca 2+ ions from the membrane, The addition of Ca 2 + ions will mechanically stabilize the cell membrane in this case as well. Figure 2 shows a typical volume relaxation of fused A. sativa protoplasts in response to an increase in the osmolarity of the mannitol solution (final osmolarity 0.75). The initial volume of the fused protoplast was about 1.1-10 .7 cm 3, which means that this fused cell consisted of about five individual protoplasts. The hydraulic conductivity, Lp, can be calculated from the volume relaxation curve as follows: According to the thermodynamics of irreversible process (Dainty 1963; Zimmermann and Steudle 1978), the following water transport equation holds: Ap ~dVt l- = L A ~ z = L p ( l r i ( t ) - ~ )

(1)

where A = mean membrane area (calculated from the arithmetic mean of the initial and final radius of the cell); Vt=the cell volume at a given time, t; A~= osmotic pressure difference between the cell and the external medium; rci(t)=the internal osmotic pressure and rc,=the experimentally established external osmotic pressure which is assumed to be constant during the relaxation process. If we assume that the protoplasts behave like ideal osmometers, then Eq. (2) holds:

Tel(l)= ZOoi"~

(2)

where TCoi=the initial intracellular osmotic pressure; Vo= initial volume. Substitution of Eq. (2) in Eq. (t) and rearrangement leads to : dVt Vt= LpA0coi Vo_ 7rr l/~)

(3)

dt

Integration of Eq. (3) with the boundary conditions Vo for t = 0 and V~for t yields:

Vt-Vo-V~176

n~176176

~

Lp~A

(4)

~oeVo-~V,

and, respectively, 1

Vo_ V~+nor Vo. in ~" 1 Lp =

7~r

hoe n"Vt 7~oe g 0

~e A t

(5)

N. Salhani et al. : M e m b r a n e integrity of fused protoplasts

143

where ~Zo~= ~Zo~= the initial extracellular osmotic pressure. Since V is not a simple function of time it seems to be worthwhile to calculate the Lp-value from the values of the volume, V, at a given time, t, and to average the Lp-values obtained in this way. The required value for V0 is calculated from Eq. (2) by insertion of the appropriate values for the initial and final steady state. Vo-

Ve~e

in which the non-osmotic volume, b, is also considered (Kauss 1977). The intercept yields the non-osmotic volume; the average values are 3.10 .9 cm 3, 2.10 .9 cm 3 and 5.10 - t ~ cm a for MCP's of A. saliva, MCP's of V. faba and for GCP's of V. faba, respectively. Taking the modified Eq. (2a) into account, the theory yields the following equation for the calculation of Lp, by analogy with Eq. (5) :

(6)

TCoe

1

Vo-Vt+~e (Vo-b ) 9In Derivation of Eqs. (4) and (5) assumes that the reflection coefficients for mannitol and sucrose are a = 1, that the non-osmotic volume within the protoplasts can be neglected, and that the membrane area is constant during the shrinking and swelling kinetics. The assumption of reflection coefficients of a = 1 seems to be justified because cell membranes are not permeable to mannitol or sucrose. The use of both osmotically active substances also leads to identical Lpvalues within the limits of error (see Table 1). The non-osmotic volume was determined by measuring the volume of the protoplasts as a function of the external osmotic pressure in the steady state. A plot of V versus 1/roe yields a straight line (Fig. 3), as expected from the modified Eq. (2) V0-b ~zi(t) = ~oe V~-b

(2a)

Lp=

1

~e 7~oe

~ ( v , - b) 7C~176

G At

(5 a)

The values listed in Table 1 are calculated on the basis of Eq. (5 a). The assumption of a constant membrane area seems to be justified when the experimental procedure is considered. The protoplasts were subjected to a hypertonic shock followed after equilibration by a hypotonic shock. Furthermore, calculation of the Lp-values from the volume relaxation curves, assuming that A = f i t ) , lead only to deviations of the Lp-values by about 10%. We therefore refrain from stating a theory for the calculation of Lp using this assumption, since the equations are very complicated.

Table 1. Water relation parameters of fused and unfused guard and mesophyll cell protoplasts of Vicia Jaba and mesophyll cell protoplasts of Arena sativa. (GCP = guard cell protoplast; M C P = mesophyi1 cell protoplast) Cell system

Number of cells

Osmolarity range (mol/l)

G C P V. faba unfused G C P V. faba fused

9 11

0.5 0.5

G C P V. faba unfused G C P V. faba fused

Initial volume Vo(10 -9 cm 3)

Range of time-constant, v(s)

Average hydraulic Remarks conductivity, Lp, (10 6 cm b a r - l . s 1)

0.8 0.8

2.7- 5.4 6.1 81.7

22-39 20-55

0.5-+0.2 0.5 +0.4

8 7

0.8 -0.4 0.8 -0.4

0.5 2.0 3.0- 10.2

25-39 22-48

0.4+0.3 1.2-+0.6

M C P V. faba unfused M C P V. faba fused

9 5

0.4 0.7 0.4 -0.7

13 49

46 94

26-42 27-71

1.8_+0.7 1.4_+0.3

M C P V. faba unfused M C P V. faba fused

28 9

0.7 0.7

9 - 49 51 141

20-58 20-86

2.2_+ 1.0 4.2 + 2.2

M C P A. saliva unfused M C P A. sativa fused MCP MCP MCP MCP

A. A. A. A.

saliva saliva saliva sativa

unfused unfused fused fused

0.4 0.4

5 9

0.5 -0.75 0.5 -0.75

43 - 61 78 -250

22-39 25-43

1.1 +0.3 1.1 +_0.3

20 7 13 6

0.75 0.5 0.65 0.5 0.75-0.5 0.65-0.5

11 30 24 - 34 31 -154 38 63

20-38 31-60 25-53 30-48

2.1 _+0.5 1.2_+0.4 2.4_+0.7 1.4_+0.4

Lp-values are given with standard deviation

shrinking shrinking, fusion in the presence of pronase swelling swelling, fusion in the presence of pronase shrinking shrinking, fusion in the presence of pronase swelling swelling shrinking shrinking, fusion in the presence of pronase swelling swelling sucrose swelling swelling sucrose

144

N. Salhani et al. : Membrane integrity of fused protoplasts

to drastic changes in external osmolarity, as was also observed in experiments on walled giant algal cells (Zimmermann and Steudle 1974). The average Lp-values of unfused and fused protoplasts are:

L-" E L.a

L,

MCP, A. sativa: 1.9_+0.7-10 -6 cm bar -1 S - 1 MCP, V.faba: 2.4_+ 1.5-10 -6 cm bar -1 s -1 GCP, V.faba: 0.7_+0.6.10 -6 cm bar -1 s -1

>3 / ''if" o j o j

/ /

/

o

i

t

0

1.10-3

2.10-3

3.10-3 IT, e

[mosmo[ -I" l]

Fig. 3. Plot of the volume function V=f(I/~) measured in the steady state for mesophyll cell protoplasts of A. saliva. From the intercept of the extrapolated straight line the non-osmotic volume, b, can be calculated

Table 1 lists data obtained from various fused protoplast cells of A. sativa and V. faba under conditions of shrinking and swelling. It also gives the corresponding values for protoplasts which were not subjected to fusion, both in the presence and absence of pronase. It is evident from Table 1 that the presence of pronase during fusion has no influence on the water permeability of the fused cells. There is also apparently no difference between the values obtained from fused and unfused MCP's and GCP's of the two cell species. No significant change in the Lp-values is observed when sucrose instead of mannitol was used as the osmotic agent. It has to be noted that the data for Lp derived from volume relaxations in response to osmotic stress induced by sucrose were obtained from protoplasts of plants cultivated November 1981, whereas the other measurements were performed on plants grown between June and October 1981. Physiological changes in the plant growth may explain the slight difference in the absolute values of the unfused and fused protoplasts. Furthermore, it was not possible to detect any dependence of the hydraulic conductivity on concentration. For experimental reasons the osmotic pressure could be varied by a maximum of 6.5 bar, particularly under swelling conditions: otherwise, bursting was observed. No trend in the Lp-values to increase with the volume of the fused protoplasts could be detected. Both results exclude the possibility that the Lp-values are influenced by small reversible leakages within the membrane in response to strong osmotic stress. The Lp-values of very large fused protoplast cells consisting of more than 10 protoplasts could not be accurately determined, since large cells are sensitive

The polarity of water flow observed in Characean algal cells (Kamija and Tazawa 1956; Kiyosawa and Tazawa 1973; Zimmermann and Steudle 1978) also seems to be present in the protoplasts studied here (see Table 1). Considering the procedure used for changing the osmolarity, we believe that the influence of sweepaway effects on the value of the hydraulic conductivity should be negligible (House 1974). During water flow the solute concentration, Cm, at the external surface of the membrane is given by (Dainty 1963): Cm=Cee- Jv'6 D

(7)

whereby Ce is the bulk concentration, Jv the water flow, D the diffusion coefficient, and 6 the thickness of the unstirred layer. If we consider the unfavourable case where the cell volume changes by a factor of 2 in response to external osmotic stress, the thickness of the unstirred layer corresponds, at a maximum, to the difference in the radii of the initial and final volume. With a typical value for the cell volume of 6.5.10 -s cm 3, we thus obtain 6 = 5 . 1 0 .4 cm. With J ~ = l . 1 0 - S c m s - t and D = l . 1 0 - S c m 2 s -1, equation (7) yields: Cm= 1,0005 Ce

This means that the concentration close to the membrane surface is nearly identical to that in the bulk solution. It should be noted that in analogous studies on plasmolysed walled cells (Stadelmann 1966; Url 1971), the error in the Lp-determination may be much larger due to the larger unstirred layers within the plasmolysed cells. The values for the hydraulic conductivity of the protoplasts reported here are in the same range as those measured for suspended walled cells of Chenopodium rubrum (Biichner et al. 1981) and for individual cells from leaves of higher plants with the aid of the modified pressure probe (Steudle et al. 1980; Zimmermann et al. 1980a; Tomos et al. 1982; Zimmermann and Steudle 1980). The agreement of the Lp-values of fused cells with

N. Salhani et al. : Membrane integrity of fused protoplasts t h o s e o f u n f u s e d cells w i t h i n t h e l i m i t s o f e r r o r indic a t e s t h a t at least t h e w a t e r p e r m e a b i l i t y u n d e r t u r g o r less c o n d i t i o n s h a s n o t b e e n a f f e c t e d by t h e field a p p l i cation. This result paves the way toward measurem e n t s o n i m m o b i l i z e d f u s e d cells in o r d e r t o d e t e r mine the electrical membrane parameters, pressured e p e n d e n t fluxes, a n d t h e i n f l u e n c e o f i n h i b i t o r s a n d e c o t o x i c o l o g i c a l s u b s t a n c e s o n t h e s e cell p a r a m e t e r s . The authors are very grateful to Chr. Matschke, H. Jfickel (KFA Jiilich) and Chr. Elbert (TU Miinchen) for expert technical assistance, and to Dipl.-Phys. S. Wendler and Dr. E. Steudle for very critical discussion of the manuscript. This work was supported by grants of the BMFT to U.Z. (037266) and to H. Sch. (037269).

References Bfichner, K.-H., Zimmermann, U., Bentrup, F.-W. (1981) Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum. Planta 151, 95 102 Dainty, J. (1963) Water relations of plant cells. Adv. Bot. Res. 1, 279-326 Hampp, R., Ziegler, H. (1980) On the use of Arena protoplasts to study chloroplast development. Planta 147, 485-494 Holzapfel, Ch., Vienken, J., Zimmermann, U. (1982) Rotation of cells in an alternating electric field: Theory and experimental proof. J. Membr. Biol. 67, 13-26 House, C.R. (1974) Water transport in cells and tissues. Edward Arnold, London Jeltsch, E., Zimmermann, U. (1979) Particles in a homogeneous electrical field: a model for the electrical breakdown of living cells in a Coulter Counter. Bioelectrochem. Bioenerg. 6, 349-384 Kamija, N., Tazawa, M. (1956) Studies on water permeability of a single plant cell by means of transcellular osmosis. Protoplasma 46, 394~22 Kauss, H. (1977) Biochemistry of osmotic regulation. Int. Rev. Biochem. Plant Biochem. II, ed. D.H. Northcote 13, 119-140 Kiyosawa, K., Tazawa, M. (1973) Rectification characteristics of Nitella membranes in respect to water permeability. Protoplasma 78, 203-214 Muth, E. (1927) Ober die Erscheinung der Perlschnurketten von Emulsionspartikelchen unter Einwirkung eines Wechselfeldes. Kolloid Z. 41, 97 102 Pethig, R. (1979) Dielectric and electronic properties of biological materials. Wiley, Chichester Pilwat, G., Richter, H.-P., Zimmermann, U. (1981) Giant culture cells by electrical field-induced fusion. FEBS Lett. 133, 169-174 Pohl, H.A. (I978) Dielectrophoresis. Cambridge University Press, Cambridge Richter, J.-P., Scheurich, P., Zimmermann, U. (1981) Electric field induced fusion of sea urchin eggs. Dev. Growth Differ 23, (in press) Saito, M., Schwan, H.P., Schwarz, G. (1966) Response of nonspherical biological particles to alternating electric fields. Biophys. J. 6, 313-327 Scbeurich, P., Schuabl, H., Zimmermann, U., Klein, J. (1980) Immobilisation and mechanical support of individual protoplasts. Biochim. Biophys. Acta 598, 645-651 Scheurich, P., Zimmermann, U. (1981) Giant human erythrocytes by electric-field-induced cell-to-cell fusion. Naturwissenschaften 68, 45-46 Scheurich, P., Zimmermann, U., Schnabl, H. (1981) Electrically stimulated fusion of different plant cell protoplasts. Plant Physiol. 67, 849 853

145 Schnabl, H., Bornman, Ch., Ziegler, H. (1978) Studies on isolated starch-containing (Viciafaba) and starch-deficient (Allium cepa) guard cell protoplasts. Planta 143, 33-39 Schnabl, H., Scheurich, P., Zimmermann, U. (1980) Mechanical stabilization of guard cell protoplasts of Vicia faba. Planta 149, 280 282 Stadelmann, E.J. (1966) Evaluation of turgidity, plasmolysis and deplasmolysis of plant cells. In: Methods in cell physiology II, pp. 143~i6, Prescott, D.M., ed. Academic Press, New York Steudle, E., Smith, J.A.C., Lfittge, U. (1980) Water-relation parameters of individual mesophyll cells of the crassulacean acid metabolism plant Kalanchoe daigremontiana. Plant Physiol. 66, 1155 1163 Tomos, A.D., Steudle, E., Zimmermann, U., Schulze, E.-D. (1981) Water relations of leaf epidermal cells of Tradescantia virginiana. Plant Physiol., 68, 1135 1143 Url, W.G. (1971) The site of penetration resistance to water in plant protoplast. Protoplasma 72, 427-447 Vienken, J., Ganser, R., Hampp, R., Zimmermann, U. (1981) Electric-field induced fusion of isolated vacuoles and protoplasts of different developmental and metabolic provenance. Physiol. Plant. 53, 64-70 Zimmermann, U., Hfisken, D., Schulze, E. (1980a) Direct turgor pressure measurements in individual leaf cells of Tradescantia virginiana. Planta 149, 445-453 Zimmermann, U., Pilwat, G., Beckers, F., Riemann, E. (1976) Effects of external electrical fields on cell membranes. Bioelectrochem. Bioenerg. 3, 58-83 Zimmermann, U., Richter, H.-P., Pilwat, G. (1981) Electric fieldstimulated fusion: increased field stability of cells induced by pronase. Naturwissenschaften 68, 577 Zimmermann, U., Scheurich, P. (1981a) High frequency fusion of plant protoplasts by electric fields. Planta 151, 26 32 Zimmermann, U., Scheurich, P. (1981b) Fusion of Arena sativa mesophyll protoplasts by electrical breakdown. Biochim. Biophys. Acta 641, 160-165 Zimmermann, U., Scheurich, P., Pilwat, G., Benz, R. (1981 a) Zellen mit manipulierten Funktionen: Neue Perspektiven fiir Zellbiologie, Medizin und Technik. Angew. Chem. 93, 332 351 Cells with manipulated functions: New perspectives for cell biology, medicine and technology. Angew. Chem. Int. Ed. Engl. 20, 325 344 Zimmermann, U., Schulz, J., Pilwat, G. (1973) Transcellular ion flow in E. coli B. and electrical sizing of bacteria. Biophys. J. 13, 1005-1013 Zimmermann, U., Steudle, E. (1974) The pressure-dependence of the hydraulic conductivity, the membrane resistance and membrane potential during turgor pressure regulation. J. Membr. Biol. 16, 331-352 Zimmermann, U., Steudle, E. (1978) Physical aspects of water relations of plant cells. Adv. Bot. Res. 6, 45-117 Zimmermann, U., Steudle, E. (1980) Fundamental water relation parameters. In: Plant membrane transport: current conceptual issues, pp. 113-128, Spanswick, R.E., Lucas, W.J., Dainty, J., eds. Elsevier/North-Holland Biomedical Press, Amsterdam Zimmermann, U., Vienken, J. (1982) Electric field induced cell-tocell fusion. Topical Review. J. Membr. Biol. 67, 158-175 Zimmermann, U., Vienken, J., Pilwat, G. (1980b) Development of drug carrier systems: Electrical field induced effects in cell membranes. Bioelectrochem. Bioenerg. 7, 553-574 Zimmermann, U., Vienken, J., Pilwat, G. (198ib) Rotation of cells in an alternating electric field: the occurrence of a resonance frequency. Z. Naturforsch. 36e, 173-177

Received 21 December 1981; accepted 17 March 1982

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse.

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify p...
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