Planta (Berl.) 102, 302--323 (1972) 9 by Springer-Verlag 1972
Electrical Properties of Parenchymal Cell Membranes in the Oat Coleoptile MA~Y HELEN M. GOLDSMITH, HECTOI~ E. FEI~NXNDEZ, and TIMOTHY H. GOLDSMITH Department of Biology, Yale University, New Haven, Connecticut, and Marine Biological Laboratory, Woods Hole, Massachusetts Received July 28/September 29, 1971
Summary. Parenchymal cells of oat (Avena sativa) coleoptiles had an osmotic concentration of 410 mM (determined by plasmolysis); of this only 22 ram was K + and 1 mM Iqa+ (flame photometry). Cells were impaled with micropipette electrodes. Iontephoretic injection of the dye Niagara sky-blue from the micropipette showed that the tip of the electrode penetrated the vacuole. When sections of tissue were immersed in a solution of 22 mM KC1, 1 mM CaCl~, and 50 mM glucose, average membrane potential was found to be 38.5 mV inside negative, specific membrane resistance was ~-~5100 ~ cm2, and specific membrane capacitance, ~ 2 ~f cm-~. The cell membranes showed < 25 % rectification and no electrical excitability. Electrotonic coupling of adjacent cells could not be demonstrated. Introduction Plasmodesmata, intracellular bridges t h a t structurally couple the cytoplasm and cell membranes of adjacent cells, are ubiquitous in plant tissues (Voeller, 1964). For decades these connections, which are only 250-500/~ in diameter, have been assumed to function in transport and communication between cells t h a t would otherwise be isolated from each other b y non-living cell walls. Plasmodesmal channels in the cell wall are lined b y a membrane t h a t is continuous with the plasma membranes of the connected cells and contain a dense desmotubule similar to a cytoplasmic microtubulc (Robards, 1968). The suggestion t h a t communication and transport between nonspecialized parenchymal cells occurs via plasmodesmata has been based mainly on their existence, and only a small amount of direct physiological work bears on their function (Arisz, 1958; Voeller, 1964; Laties, 1969). The discovery b y electrophysiological methods of low-resistance connections between embryonic and certain other animal cells (Lowenstein und Kanno, 1964; Lowenstein, 1966; Pappas and Bennett, 1966; Potter et al., 1966; Furshpan and Potter, 1968), suggested t h a t similar techniques apphed to plant tissues might show whether plant cells are connected b y pathways through which small ions can readily move.
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Electrical coupling between plant cells is known to exist in those few cases where plant cells possess electrical excitability: between internodal cells of the giant green algae Nitella and Chara (Sibaoka, 1966; Spanswick and Costerton, 1967), and in the protoxylem as well as between phloem parenchymal cells of the Mimosa petiole (Sibaoka, 1962, 1966). I n the algae, action potentials appear to be transmitted electrotonically through plasmodesmata and intervening cells that are not excitable, and in some places the same occurs in Mimosa. Despite these striking examples in algae and sensitive plants, most plant cells are probably not electrically excitable; however they must still possess means of communication and integration with other cells. Therefore, it is of interest to explore the possibility of electrical coupling within different plant tissues. For the present work, we chose the oat eoleoptile because much is already known about its growth, photo- and geotropie responses, hormone transport, and the gross, variable, electrical potentials of its surface. Since the present study was undertaken, Spitzer (1970) has reported widespread electrical coupling in the cells of the developing anther of the lily flower. The measurements of membrane resistance and of electrical coupling were made on the parenehymal cells, which form the bulk of the eoleoptile. These cylindrical cells vary in length from one to several hundred micrometers depending on their age and location in the tissue, and in diameter from 15 to 60 ~m. They contain a large vacuole bounded b y the tonoplast and a thin peripheral layer of cytoplasm which is thickened at the base of the cell and around the nucleus but is only 0.5 to 2.0 ~m thick elsewhere. The cell walls are about 0.5 [~m thick and are penetrated by numerous plasmodesmata (O'Brien, personal communications; O'Brien and Thimann, 1965, 1967).
Materials and Methods
Preparation el Plants Hulled oat seeds (Avena sativa L., cv. Victory) were soaked 4-5 h and then placed on damp paper. After 48 h in the dark the seedlings were exposed to 12 h of red light (2 Sylvania :F20T12-R fluorescent tubes at about 75 cm distance). The developing roots were in contact with either distilled water or 10-4 M KC1. Coleoptiles were used at 80-104 h. The leaf was removed and the coleoptile decapitated several millimeters from the tip. In many experiments the sections were floated one or several hours in the solution in which they were mounted for recording before excising a shorter section to be studied. This time is not sufficient to produce large changes in the internal concentration of K + (Etherton, 1962; Higinbotham et al., 1967). For penetrating end walls, the decapitated section was mounted vertically on a glass peg. For penetrating side walls, intact coleoptiles were split lengthwise and held to the floor of the recording chamber by a small amount of silicone grease and
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two small, grooved lucite blocks placed over the ends. The mounted section was just covered with solution (see below). The point of recording was 0.5-1 cm from the original tip. Penetration of parenchymal cells was observed with a 100 X binocular dissecting microscope. Visibility was enhanced by use of substage illumination and a dark field condenser. The coleoptiles were in room light from the time they were harvested, and while in the recording chamber they were exposed to bright white light from two microscope lamps. Initially it seemed easier to penetrate end walls, and more measurements were obtained from this approach.
Osmotic Concentration The internal concentration of solute was determined by the plasmometric method, following Ray and Ruesink (1963). Coleoptiles were split lengthwise and soaked about 1 h in 0.6 M mannitol. The cells are quite impermeable for mannitol, and 0.6 M is hypertenic. Sections were mounted in the plasmolyzing solution and examined with phase-contrast optics at 500 • The cylindrical parenchymal cells tend to plasmolyze from the ends, and by measuring the lengths of the protoplasts, lv, and cells (i.e. walls), tc, with an ocular micrometer, osmotic concentration, ~, was calculated from the relation c =~ 0.6 lvflc.
Ionic Composition Coleoptiles were collected in a moist chamber, adhering drops of water blotted off, and the coleoptiles weighed, lyophilized, and reweighed. The dried tissues was digested in 2 mt of a 5:1 mixture of nitric and perchloric acids until dry. K + and Na + were determined with the flame photometry attachment of a Cary spectrophotometer, using an acetylene-oxygen flame and LiNO 3 as an internal standard to correct for self absorption. In preparing all samples for analysis of ions, blanks containing no tissue were prepared in parallel.
Electrical Recording The micropipettes were made and filled with 3 M KC1 by conventional techniques. Successful pipettes had resistances of 20-40 M ~ as measured in 22 mM KC1, and tip potentials of no more than a few millivolts. The electrodes were Ag:AgC1. For measurements of membrane potential they were matched to within 1-2 mV, imbedded in 2 per cent agar, and connected to the solutions through 3 M KCI-agar bridges. The recording chamber was a narrow (6.2 mm) Lucite trough 50 m m in length. The section was placed near the middle, and fresh solution was introduced at one end through a plastic manifold which allowed rapid alterations in the composition of the bath. The reference electrode (Ag:AgC1 connected via a 3 M KCl-agar bridge) was at the opposite end of the bath. During flow, solution was drawn from the surface of the bath by an aspirator placed on the downstream side of the coleoptile, midway between the tissue and the reference electrode. The recordings were done from one of several bathing solutions. The first contained 22 mM KC1, 1 mlVf CaCI~, and 50 mM glucose, with the K+ concentration chosen to match the internal concentration (see Results) so that changes in tip potential would be minimized as the electrode entered the cell. The solution used by Etherton (1962) and Higinbotham et at. (1967), which contained 10 mM KC1, 9.05 mM NaH~POa, 0.48 mM lga2HP04, 10 mM Ca(N03)~, 2.5 mM MgSOa, was used in some early experiments. I t seemed likely to us that the ionic concentration
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305
Fig. 1. Recording circuit employed in passing current through the micropipette electrode. With the electrode (re) in the bath, pulses of current divide over the two resistive paths. Points a and b are connected to the input terminals of a difference amplifier, and wiper b is adjusted until a and b remain at the same potential during passage of current. The subsequent addition of a membrane impedance, Zm, puts the bridge out of balance. Current was monitored by measuring the voltage at c. The resistances of the reference electrode and voltage calibrator were negligible. I current source; V voltage calibrator; A unity gain amplifier with capacitance neutralization
of both these solutions might be rather high compared to that which normally bathes parenchymal cell walls. Therefore in many later experiments solutions contained either 1 mM KC1 plus 1 mM CaC12 or 0.1 mM of both salts. The micropipette electrode was connected to a high-impedance amplifier (either an Electronics for Life Sciences, Roekville, Md. ELSA-1 or Bioelectric Instruments, Hastings-on-Hudson, N.Y. DS2C). The ELSA-1 unit employs a bridge at the input to permit passing currents through the micropipette without the i1~ drop in the mieropipette deflecting the record of voltage (X~ig.1). Use of this feature was very helpful in identifying impalements with single electrodes, and its reliability was checked by penetrating cells with pairs of electrodes (see Results). The records of voltage and applied current were displayed on an oscilloscope and photographed; in many ceils, membrane potential was also recorded continuously on an Esterline Angus strip chart. Electrode resistance was monitored regularly.
Dye Pipettes A number of cells were penetrated with micropipettes filled immediately prior to use with the anionic dye ~qiagara sky blue (3.5 % ). Each pipette was tested in the bath before inserting it into a cell. Satisfactory pipettes produced pulses of dye in response to brief (30-60 ms) current pulses. After penetrating the cell, the input resistance was measured by passing a 1 nA pulse of current through the bridge circuit. Then larger pulses of current were passed until the cell was visibly blue. The tissue containing the dyed cell was quickly cut out, mounted in a drop of solution, and observed with phase contrast at 200 or 500 x . The preparations were photographed with Plus-X film using a red filter to heighten the contrast between the stained cell and surrounding ones.
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Internal Concentration and Ionic Composition The osmotic c o n c e n t r a t i o n of p a r e n c h y m a l cells is given in T a b l e 1. I t was a b o u t 0.41 osmolar, a n d was n o t significantly a l t e r e d b y growing t h e coleoptiles in 10 -~ M KC1 i n s t e a d of distilled w a t e r . The results were also t h e s a m e w h e t h e r t h e sections were s i m p l y f l o a t e d on t h e solutions of m a n n i t o l or t h e e x t r a e e l l u l a r gas was e v a c u a t e d b y r e d u c e d pressure several t i m e s a t t h e s t a r t of t h e p e r i o d of equilibration.
Table 1. Osmotic concentration o/parenchymal cells Distilled-water grown, evacuate ~ (osmoles 1-1)
Distilled-water grown, floated (osmoles 1-1)
104 M KCl-grown, evacuated (osmoles 1-1)
0.41 4- 0.012 a 0.41 • 0.014 0.40 4- 0.023 0.44 4- 0.008 0.43 4- 0.015 0.38 4- 0.011
0.37 4- 0.013 0.444- 0.018
0.43 4- 0.014 0.424- 0.018 0.42 4- 0.014
Averages 0.41
0.405
0.42
a Each figure is an average of 10-12 cells from a single half coleoptile. 4- = standard error.
Table 2. Cationic composition o/parenchymal cells [K +] [Na +] (meq kg-lcell water) Distilled-water grown
21.4 23.7
0.45 0.98
10-4 M KCl-grown
16.9 24.0
1.18 0.99
Average
21.5
20 for a just detectable measurement of v2. With rt/ria the fraction of the input resistance that is accounted for by the tonoplast, then ra
which appears as the straight dashed line in Fig. 10. The significance of this graph is that ratios of r c t o r a could be detected only if they lie under (to the left of) the curve. Because re/r a increases as the coupling becomes weaker, detection of lower coupling requires that rt/rin be smaller. The graph can be made more readily interpretable by plotting rc/r m on the abscissa. This conversion can be made with reference to the equivalent circuit of Fig. 9 B. To do so, however, also requires an estimate of r s in terms of r m . It develops that as rc/r m varies over a range of 100, rs/r m changes slightly more than threefold, so a mean value of 0.1 r m was used. Therefore ra=
rm ~- (O'2 rc + rs) = 0"2 rcrm ~- O ' l rm 2 rm(O.2rc_~_rs ) 1.1rm @O.2r c
For various values of rc/ra, the equivalent values of rc/r m were calculated, and these are plotted as the solid curve in Fig. 10. :From this curve it can be seen, for example, that for detection of the moderate coupling that would exist if r c = 6 r m (roughly half of the injected current goes to the six nearest neighbors, half across rm), the tonoplast resistance would have to be less than 0.42 of the input resistance. Under these conditions r t > 0.38 r m. T h e r e l a t i v e r e s i s t a n c e s of t h e t o n o p l a s t (rt) a n d p l a s m a m e m b r a n e (rm) are n o t k n o w n for e o l e o p t i l e cells. I n N i t e l l a a n d C h a r a t h e r e s i s t a n c e of t h e p l a s m a m e m b r a n e is a b o u t 10 t i m e s h i g h e r t h a n t h e t o n o p l a s t , a n d m o r e o v e r m o s t of t h e p o t e n t i a l b e t w e e n t h e inside a n d o u t s i d e of t h e cell o c c u r s across t h e p l a s m a m e m b r a n e ( W a l k e r , 1960; F i n d l a y a n d H o p e , 1964). I n t h e r o o t hairs of A v e n a , t h e p l a s m a m e m b r a n e is also
Electrical Properties of Parenchymal Cell Membranes 1.o
I
I
I
I
I
I
I
I
3t9
I
O.8 ~ \\ \\ O.6 ~ X \ rt
rc
~\
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rin
0.4 rm
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0.2
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I 4
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Fig. 10. Theoretical curves showing the extent of coupling that could be detected under the conditions of these experiments, given different relative values of tonoplast resistance (rt), coupling resistance (rc), and membrane resistance (rm) as defined by the equivalent circuits of Fig. 9 and in the text. Ordinate gives the ratio of tonoplast to input resistance (rt/rin). The abscissa is either the ratio rc/ra (broken line; of. Fig. 9A) or rc/r m (solid curve; el. Fig. 9B). The degree of coupling that could be detected (region to the left of the curve) is less if the tonoplast accounts for a larger fraction of the input resistance. See the text for further discussion
reported to be the site of the potential (Etherton and Higinbotham, 1960). I t is therefore quite possible t h a t the resistance of the tonoplast is a small fraction of the m e m b r a n e resistance, and if so (c[. above and Figs. 9 and 10), experiments such as the one illustrated in Fig. 8 mean t h a t the cytoplasms of parenehymal cells are not strongly electrically coupled. Since electrical coupling has been detected between internodal cells of the giant green alga Nitella b u t n o t between oat p a r e n c h y m a l cells, it is worth while to compare the relative wall area occupied b y plasmodesmata in the two systems. I n Nitella the internodal cells are coupled t h r o u g h an intervening layer of nodal cells. According to Spanswiek and Costerton (1967), 3.6% of the wall area between i m m a t u r e N~tella cells and 0.59% of the wall area between m a t u r e cells is occupied b y plasmodesmata. I n oat p a r e n e h y m a there are a b o u t 100 p r i m a r y pit fields per cell (Wardrop, 1955) with about 10 plasmodesmata per pit field
320
M. It. M. Goldsmith, H. 1%.Fernandez, and T. H. Goldsmith:
(O'Brien and Thimann, 1967) for a total of 1000 per cell. Taking the diameter of an individual plasmodcsma as 500 •, the complement of 1000 would have an average cross-sectional area of about 2 ~m 2. Since the cell surface area of an average parenchymal cell (95 • 35 ~m) is about 1.2 • 104 ~m 2, only 0.016% of the wall between two adjacent cells is occupied by plasmodesmata. Thus contact between immature Nitella cells is 225 times greater and between mature Nitella cells 37 times greater than between adjacent oat parenchymal cells. Although electrical coupling exists between adjacent Nitella cells, Spanswiek and Costerton (1967) point out that the specific resistance of Nitella plasmodesmata is some 330 times the values predicted from a knowledge of plasmodesmal dimensions and cytoplasmic conductivity. Based on the considerations summarized in Fig. 10, it is possible to calculate lower limits of coupling resistance between oat coleoptile cells. From this, and the numbers and dimensions of plasmodesmata, one can also estimate lower limits for the resistivity of the plasmodesmal contents. These calculations, like the measurements on Nitella, suggest that diffusion of ions through plasmodesmata is restricted. Electron micrographs suggest a likely explanation for the high resistance of the plasmodcsmata. The internal desmotubule of the p]asmodesma, which bears some similarity to a cortical microtubule (Robards, 1968), virtually occludes the opening into the cell with a plug of electron dense material (O'Brien and Thimann, 1967; Robards, 1968). I t is a moot question whether the plasmodesmata are capable of specialized transport functions and/or whether restrictions on passage may be altered by physiological conditions. If diffusion of ions is limited in the plasmodesmata of coleoptiles, this raises the question whether these connections function as the pathways of transport for larger molecules. Of particular interest is the fact that oat eoleopti]es in common with many plant organs transport auxin via a polar, metabolically controlled system. The apparent lack of coupling between cells of the oat coleoptile suggests that in this organ, auxin may not travel from cell to cell via the symplast but may be exported across the cell membrane, diffuse through the intercellular space, and enter the adjacent cell through its plasmalemma. Spitzer (1970) has shown extensive coupling between cells in the lily anther. This is an interesting system because the cells lack large vacuoles, and the germinal cells in the early stages of meiosis that were examined in this study are interconnected by cytoplasmic bridges up to 1 ~ in diameter through which transfer of cytoplasmic organelles has been observed (tteslop-Harrison, 1966), whereas somatic cells have typical plasmodesmata. Coupling exists between all cells of the anther, even those separated by distances of 0.5 mm or more.
Electrical Properties of Parenchymal Cell Membranes
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Membrane Resistance The input resistance we obtained is a valid measure of membrane resistance only if coupling and leakage are negligible. We have shown that coupling cannot be detected under our conditions, but that some leakage is likely to occur when oat parenehymal cells are penetrated. How much leakage is not know,n, but it is well to keep in mind that if the membrane resistance is high, its measurement will be in error if only a small leakage occurs around the pipette. The specific membrane resistance of 5 kf2 cm 2 represents the tonoplast, plasma membrane, and cell wall in series. Our observations indicate that the wall contributes a minor portion of this value, and, as discussed above, where measured in other plants the resistance of the tonoplast is much lower than the plasma membrane. Consequently, in the absence of further information we suggest that the specific membrane resistance of 5 kf~ cm 2 is determined primarily by the plasma membrane. Most of the variation in input resistance from cell to cell can be accounted for by the distribution of surface areas; consequently there is little cause to hypothesize large variation in specific membrane resistance from cell to cell. Most of the information on specific membrane resistance of plant cells obtained by electrical methods has been from lower plants, but our value of 5 kf2 em 2 (22 mM KC1) is similar to other results. I n Neurospora resistances of 5 kf2 em 2 were found (tO mM KC1) (Slayman, 1965). In Nitella and Chara values range from 5 to 50 kf2 em 2 (Williams et al., 1964). The resistance increases and rectification becomes more obvious as the external concentration of potassium is lowered. Hope and Walker (1961) found that the average specific resistance of Chara cells rose from 5 to 14 kf2 em 2 as the external K + decreased from 1 to 0.1 mM. We are aware of only one other study of membrane resistance in higher plan~s. Higinbotham et al. (1964) used pairs of electrodes and reported a specific membrane resistance of cells of Avena eoleoptiles (in 1.0 mM KC1, 1.0 mM CaCI~) of 1.3 kf2 em "~. Their value is less than half what we measured with a similar two-electrode system at the same K+ and Ca++ concentrations and only about 25 % of what we consider our most reliable value. Although they used a different variety of oats, we are skeptical that this difference alone accounts for the discrepant results. Their penetrations may have been shunted by greater leakage. Recently Etherton (1970) has reported the existence of three distinct steps of change in potential as he penetrated oat parenchymal cells, which he presumes without additional evidence to correspond to a wall, cytoplasm, and vacuolar potential. Although such observations
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M . H . M . Goldsmith, H. R. Fernandez, and T. H. Goldsmith:
h a v e b e e n m a d e w i t h g i a n t algal cells ( N a g a i a n d K i s h i m o t o , 1964), t h e v a r i a t i o n s in tip p o t e n t i a l w h i c h we e n c o u n t e r e d in t h e walls of A v e n a cells a n d t h e low p r o b a b i l i t y of r e l i a b l y l o d g i n g a m i e r o p i p e t t e in a l a y e r of c y t o p l a s m o n l y s e v e r a l m i c r o n s t h i c k m ~ k e it i m p e r a t i v e to use a n i n d e p e n d e n t m e t h o d (such as d y e i n j e c t i o n ) for l o c a t i n g t h e site of r e c o r d i n g w i t h i n t h e cell. This work was supported by U.S. Public Health Service grants GM08886 and EY 00222. We thank Mr. J. Janosik for help with some of the experiments and Prof. M. V. L. Bennett for a critical reading of the manuscript.
References Arisz, W. H. : Influence of inhibitors on the uptake and the transport of chloride ions in leaves of Vallisaeria spiralis. Acts bot. neerl. 7, 1-32 (1958). Bennett, M. V. L. : Physiology of eteetrotonic junctions. Ann. N.Y. Acad. Sci. 137, 509-539 (1966). Etherton, B. : The relationship of cell transmembrane electro-potential to potassium and sodium accumulation ratios in oat and pea seedlings. P h . D . Thesis, Washington State University (1962). - - Effect of indole-3-acetic acid on membrane potentials of oat coleoptile cells. Plant Physiol. 45, 527-528 {1970). - - Higinbotham, N. : Transmembrane potential measurements of cells of higher plants as related to salt uptake. Science 131, 409410 (1960). Findlay, O. P., Hope, A. B. : Ionic relations of cells of Chars australia. VII. The separate electrical characteristics of the plasmalemma and tonoplast. Aust. J. biol. Sci. 17, 62-77 (1964). Furshpan, E. J., Potter, D. D. : Low-resistance junctions between cells in embryos and tissue culture. Curr. Top. Develop. Biol. 3, 95-127 (1968). Heslop-Harrison, J.: Cytoplasmic connections between angiosperm meiocytes. Ann. Bot. (Load.) 30, 221-230 (1966). Higinbotham, N., Etherton, B., Foster, R. J. : Mineral ion contents and cell transmembrane electropotcntials of pea and oat seedling tissue. Plant Physiol. 42, 3 7 4 6 (1967). - - Hope, A. B., Findlay, G. P.: Electrical resistance of cell membranes of Arena coleoptiles. Science 143, 1448-1449 (1964). Hope, A. B., Walker, N. A. : Ionic relations of Chars australia ~. Br. IV. Membrane potential differences and resistance. Aust. J. biol. Sci. 14, 2 6 4 4 (1961). Laties, G. G.: Dual mechanisms of salt uptake in relation to eompartmentation and long distance transport. Ann. Rev. Plant Physiol. 20, 89-116 (1969). Loewenstein, W. R.: Permeability of membrane junctions. Ann. N.Y. Acad. Sci. 187, 441472 (1966). - - Kamm, Y.: Studies on an epithelial (gland) cell junction. I. Modifications of surface membrane permeability. J. Cell Biol. 25, 565-586 (1964). Nagai, R., Kishimoto, U.: Cell wall potential in Nitella. Plant Cell Physiol. 5, 21-31 (1964). O'Brien, T. P., Thimann, K. V.: Histological studies on the eoleoptile. I. Tissue and cell types in the coleoptile tip. Amer. J. Bot. 52, 910-918 (1965). - - - - Observations on the fine structure of the oat coleoptile. II. The parenchymal cells of the apex. Protoplasms (Wien) 68, 417442 (1967). Pappas, G.D., Bennett, M. V. L. : Specialized junctions involved in electrical transmission between neurons. Ann. N . Y . Acad. Sei. 187, 495-508 (1966).
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Potter, D. D., Furshpan, E. J., Lennox, E. S.: Connections between cells of the developing squid as revealed by eleetrophysiological methods. Proc. nat. Aead. Sci. (Wash.) 55, 328-336 (1966). Ray, P. M., Ruesink, A. W. : Osmotic behavior of oat coleoptile tissue in relation to growth. J. gen. Physiol. 47, 83-101 (1963). Robards, A. W.: A new interpretation of plasmodesmatal ultrastructure. Planta (]3erl.) 82, 200-210 (1968). Sibaoka, T.: Excitable cells in Mimosa. Science 137, 226 (1962). Action potentials in plant organs. Syrup. Soc. exp. Biol. 20, 49-73 (1966). Slayman, C. L.: Electrical properties of Neurospora crassa. Respiration and the intracellnlar potential. J. gen. Physiol. 49, 93-116 (1965). Spanswick, R.M., Costerton, J. W. F. : Plasmodesmata in Nitella translucens: structure and electrical resistance. J. Cell Sci. 2, 451464 (1967). Spitzer, N. : Low resistance connections between cells in the developing anther of the lily. J. Cell Biol. 4.~, 565-575 (1970). Voeller, ]3. R. : The plant cell: aspects of its form and function. In: The cell, p. 257267 (J. Brachet and A. E. Mirsky, eds.). New York: Academic Press, Inc. 1964. Walker, N. A. : Mieroelectrode experiments on Nitella. Aust. J. biol. Sci. 8, 476489 (1955). - - The electric resistance of the cell membranes in a Chara and a Nitella species. Aust. J. biol. Sci. 13, 468478 (1960). Wardrop, A. B.: The mechanism of surface growth in parenchyma of Avena coleoptiles. Aust. J. Bot. 3, 137-148 (1955). Williams, E. J., Johnston, R. J., Dainty, J. : The electrical resistance and capacitance of the membranes of Nitella translucens. J. exp. ]3ot. 15, 1-14 (1964). Mary Helen M. Goldsmith Timothy H. Goldsmith Department of Biology Kline Biology Tower Yale University New Haven, Connecticut 06520, U.S.A.
Hector R. FernAndez Department of Biological Sciences University of Southern California Los Angeles, California 90007, U.S.A.
Electrical Properties of Parenchymal Cell Membranes in the Oat Coleoptile MA~Y HELEN M. GOLDSMITH, HECTOI~ E. FEI~NXNDEZ, and TIMOTHY H. GOLDSMITH Department of Biology, Yale University, New Haven, Connecticut, and Marine Biological Laboratory, Woods Hole, Massachusetts Received July 28/September 29, 1971
Summary. Parenchymal cells of oat (Avena sativa) coleoptiles had an osmotic concentration of 410 mM (determined by plasmolysis); of this only 22 ram was K + and 1 mM Iqa+ (flame photometry). Cells were impaled with micropipette electrodes. Iontephoretic injection of the dye Niagara sky-blue from the micropipette showed that the tip of the electrode penetrated the vacuole. When sections of tissue were immersed in a solution of 22 mM KC1, 1 mM CaCl~, and 50 mM glucose, average membrane potential was found to be 38.5 mV inside negative, specific membrane resistance was ~-~5100 ~ cm2, and specific membrane capacitance, ~ 2 ~f cm-~. The cell membranes showed < 25 % rectification and no electrical excitability. Electrotonic coupling of adjacent cells could not be demonstrated. Introduction Plasmodesmata, intracellular bridges t h a t structurally couple the cytoplasm and cell membranes of adjacent cells, are ubiquitous in plant tissues (Voeller, 1964). For decades these connections, which are only 250-500/~ in diameter, have been assumed to function in transport and communication between cells t h a t would otherwise be isolated from each other b y non-living cell walls. Plasmodesmal channels in the cell wall are lined b y a membrane t h a t is continuous with the plasma membranes of the connected cells and contain a dense desmotubule similar to a cytoplasmic microtubulc (Robards, 1968). The suggestion t h a t communication and transport between nonspecialized parenchymal cells occurs via plasmodesmata has been based mainly on their existence, and only a small amount of direct physiological work bears on their function (Arisz, 1958; Voeller, 1964; Laties, 1969). The discovery b y electrophysiological methods of low-resistance connections between embryonic and certain other animal cells (Lowenstein und Kanno, 1964; Lowenstein, 1966; Pappas and Bennett, 1966; Potter et al., 1966; Furshpan and Potter, 1968), suggested t h a t similar techniques apphed to plant tissues might show whether plant cells are connected b y pathways through which small ions can readily move.
Electrical Properties of Parenchymal Cell Membranes
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Electrical coupling between plant cells is known to exist in those few cases where plant cells possess electrical excitability: between internodal cells of the giant green algae Nitella and Chara (Sibaoka, 1966; Spanswick and Costerton, 1967), and in the protoxylem as well as between phloem parenchymal cells of the Mimosa petiole (Sibaoka, 1962, 1966). I n the algae, action potentials appear to be transmitted electrotonically through plasmodesmata and intervening cells that are not excitable, and in some places the same occurs in Mimosa. Despite these striking examples in algae and sensitive plants, most plant cells are probably not electrically excitable; however they must still possess means of communication and integration with other cells. Therefore, it is of interest to explore the possibility of electrical coupling within different plant tissues. For the present work, we chose the oat eoleoptile because much is already known about its growth, photo- and geotropie responses, hormone transport, and the gross, variable, electrical potentials of its surface. Since the present study was undertaken, Spitzer (1970) has reported widespread electrical coupling in the cells of the developing anther of the lily flower. The measurements of membrane resistance and of electrical coupling were made on the parenehymal cells, which form the bulk of the eoleoptile. These cylindrical cells vary in length from one to several hundred micrometers depending on their age and location in the tissue, and in diameter from 15 to 60 ~m. They contain a large vacuole bounded b y the tonoplast and a thin peripheral layer of cytoplasm which is thickened at the base of the cell and around the nucleus but is only 0.5 to 2.0 ~m thick elsewhere. The cell walls are about 0.5 [~m thick and are penetrated by numerous plasmodesmata (O'Brien, personal communications; O'Brien and Thimann, 1965, 1967).
Materials and Methods
Preparation el Plants Hulled oat seeds (Avena sativa L., cv. Victory) were soaked 4-5 h and then placed on damp paper. After 48 h in the dark the seedlings were exposed to 12 h of red light (2 Sylvania :F20T12-R fluorescent tubes at about 75 cm distance). The developing roots were in contact with either distilled water or 10-4 M KC1. Coleoptiles were used at 80-104 h. The leaf was removed and the coleoptile decapitated several millimeters from the tip. In many experiments the sections were floated one or several hours in the solution in which they were mounted for recording before excising a shorter section to be studied. This time is not sufficient to produce large changes in the internal concentration of K + (Etherton, 1962; Higinbotham et al., 1967). For penetrating end walls, the decapitated section was mounted vertically on a glass peg. For penetrating side walls, intact coleoptiles were split lengthwise and held to the floor of the recording chamber by a small amount of silicone grease and
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M. It. M. Goldsmith, H. R. Fernandez, and T. H. Goldsmith:
two small, grooved lucite blocks placed over the ends. The mounted section was just covered with solution (see below). The point of recording was 0.5-1 cm from the original tip. Penetration of parenchymal cells was observed with a 100 X binocular dissecting microscope. Visibility was enhanced by use of substage illumination and a dark field condenser. The coleoptiles were in room light from the time they were harvested, and while in the recording chamber they were exposed to bright white light from two microscope lamps. Initially it seemed easier to penetrate end walls, and more measurements were obtained from this approach.
Osmotic Concentration The internal concentration of solute was determined by the plasmometric method, following Ray and Ruesink (1963). Coleoptiles were split lengthwise and soaked about 1 h in 0.6 M mannitol. The cells are quite impermeable for mannitol, and 0.6 M is hypertenic. Sections were mounted in the plasmolyzing solution and examined with phase-contrast optics at 500 • The cylindrical parenchymal cells tend to plasmolyze from the ends, and by measuring the lengths of the protoplasts, lv, and cells (i.e. walls), tc, with an ocular micrometer, osmotic concentration, ~, was calculated from the relation c =~ 0.6 lvflc.
Ionic Composition Coleoptiles were collected in a moist chamber, adhering drops of water blotted off, and the coleoptiles weighed, lyophilized, and reweighed. The dried tissues was digested in 2 mt of a 5:1 mixture of nitric and perchloric acids until dry. K + and Na + were determined with the flame photometry attachment of a Cary spectrophotometer, using an acetylene-oxygen flame and LiNO 3 as an internal standard to correct for self absorption. In preparing all samples for analysis of ions, blanks containing no tissue were prepared in parallel.
Electrical Recording The micropipettes were made and filled with 3 M KC1 by conventional techniques. Successful pipettes had resistances of 20-40 M ~ as measured in 22 mM KC1, and tip potentials of no more than a few millivolts. The electrodes were Ag:AgC1. For measurements of membrane potential they were matched to within 1-2 mV, imbedded in 2 per cent agar, and connected to the solutions through 3 M KCI-agar bridges. The recording chamber was a narrow (6.2 mm) Lucite trough 50 m m in length. The section was placed near the middle, and fresh solution was introduced at one end through a plastic manifold which allowed rapid alterations in the composition of the bath. The reference electrode (Ag:AgC1 connected via a 3 M KCl-agar bridge) was at the opposite end of the bath. During flow, solution was drawn from the surface of the bath by an aspirator placed on the downstream side of the coleoptile, midway between the tissue and the reference electrode. The recordings were done from one of several bathing solutions. The first contained 22 mM KC1, 1 mlVf CaCI~, and 50 mM glucose, with the K+ concentration chosen to match the internal concentration (see Results) so that changes in tip potential would be minimized as the electrode entered the cell. The solution used by Etherton (1962) and Higinbotham et at. (1967), which contained 10 mM KC1, 9.05 mM NaH~POa, 0.48 mM lga2HP04, 10 mM Ca(N03)~, 2.5 mM MgSOa, was used in some early experiments. I t seemed likely to us that the ionic concentration
Electrical Properties of Parcnchymal Cell Membranes
305
Fig. 1. Recording circuit employed in passing current through the micropipette electrode. With the electrode (re) in the bath, pulses of current divide over the two resistive paths. Points a and b are connected to the input terminals of a difference amplifier, and wiper b is adjusted until a and b remain at the same potential during passage of current. The subsequent addition of a membrane impedance, Zm, puts the bridge out of balance. Current was monitored by measuring the voltage at c. The resistances of the reference electrode and voltage calibrator were negligible. I current source; V voltage calibrator; A unity gain amplifier with capacitance neutralization
of both these solutions might be rather high compared to that which normally bathes parenchymal cell walls. Therefore in many later experiments solutions contained either 1 mM KC1 plus 1 mM CaC12 or 0.1 mM of both salts. The micropipette electrode was connected to a high-impedance amplifier (either an Electronics for Life Sciences, Roekville, Md. ELSA-1 or Bioelectric Instruments, Hastings-on-Hudson, N.Y. DS2C). The ELSA-1 unit employs a bridge at the input to permit passing currents through the micropipette without the i1~ drop in the mieropipette deflecting the record of voltage (X~ig.1). Use of this feature was very helpful in identifying impalements with single electrodes, and its reliability was checked by penetrating cells with pairs of electrodes (see Results). The records of voltage and applied current were displayed on an oscilloscope and photographed; in many ceils, membrane potential was also recorded continuously on an Esterline Angus strip chart. Electrode resistance was monitored regularly.
Dye Pipettes A number of cells were penetrated with micropipettes filled immediately prior to use with the anionic dye ~qiagara sky blue (3.5 % ). Each pipette was tested in the bath before inserting it into a cell. Satisfactory pipettes produced pulses of dye in response to brief (30-60 ms) current pulses. After penetrating the cell, the input resistance was measured by passing a 1 nA pulse of current through the bridge circuit. Then larger pulses of current were passed until the cell was visibly blue. The tissue containing the dyed cell was quickly cut out, mounted in a drop of solution, and observed with phase contrast at 200 or 500 x . The preparations were photographed with Plus-X film using a red filter to heighten the contrast between the stained cell and surrounding ones.
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Internal Concentration and Ionic Composition The osmotic c o n c e n t r a t i o n of p a r e n c h y m a l cells is given in T a b l e 1. I t was a b o u t 0.41 osmolar, a n d was n o t significantly a l t e r e d b y growing t h e coleoptiles in 10 -~ M KC1 i n s t e a d of distilled w a t e r . The results were also t h e s a m e w h e t h e r t h e sections were s i m p l y f l o a t e d on t h e solutions of m a n n i t o l or t h e e x t r a e e l l u l a r gas was e v a c u a t e d b y r e d u c e d pressure several t i m e s a t t h e s t a r t of t h e p e r i o d of equilibration.
Table 1. Osmotic concentration o/parenchymal cells Distilled-water grown, evacuate ~ (osmoles 1-1)
Distilled-water grown, floated (osmoles 1-1)
104 M KCl-grown, evacuated (osmoles 1-1)
0.41 4- 0.012 a 0.41 • 0.014 0.40 4- 0.023 0.44 4- 0.008 0.43 4- 0.015 0.38 4- 0.011
0.37 4- 0.013 0.444- 0.018
0.43 4- 0.014 0.424- 0.018 0.42 4- 0.014
Averages 0.41
0.405
0.42
a Each figure is an average of 10-12 cells from a single half coleoptile. 4- = standard error.
Table 2. Cationic composition o/parenchymal cells [K +] [Na +] (meq kg-lcell water) Distilled-water grown
21.4 23.7
0.45 0.98
10-4 M KCl-grown
16.9 24.0
1.18 0.99
Average
21.5
20 for a just detectable measurement of v2. With rt/ria the fraction of the input resistance that is accounted for by the tonoplast, then ra
which appears as the straight dashed line in Fig. 10. The significance of this graph is that ratios of r c t o r a could be detected only if they lie under (to the left of) the curve. Because re/r a increases as the coupling becomes weaker, detection of lower coupling requires that rt/rin be smaller. The graph can be made more readily interpretable by plotting rc/r m on the abscissa. This conversion can be made with reference to the equivalent circuit of Fig. 9 B. To do so, however, also requires an estimate of r s in terms of r m . It develops that as rc/r m varies over a range of 100, rs/r m changes slightly more than threefold, so a mean value of 0.1 r m was used. Therefore ra=
rm ~- (O'2 rc + rs) = 0"2 rcrm ~- O ' l rm 2 rm(O.2rc_~_rs ) 1.1rm @O.2r c
For various values of rc/ra, the equivalent values of rc/r m were calculated, and these are plotted as the solid curve in Fig. 10. :From this curve it can be seen, for example, that for detection of the moderate coupling that would exist if r c = 6 r m (roughly half of the injected current goes to the six nearest neighbors, half across rm), the tonoplast resistance would have to be less than 0.42 of the input resistance. Under these conditions r t > 0.38 r m. T h e r e l a t i v e r e s i s t a n c e s of t h e t o n o p l a s t (rt) a n d p l a s m a m e m b r a n e (rm) are n o t k n o w n for e o l e o p t i l e cells. I n N i t e l l a a n d C h a r a t h e r e s i s t a n c e of t h e p l a s m a m e m b r a n e is a b o u t 10 t i m e s h i g h e r t h a n t h e t o n o p l a s t , a n d m o r e o v e r m o s t of t h e p o t e n t i a l b e t w e e n t h e inside a n d o u t s i d e of t h e cell o c c u r s across t h e p l a s m a m e m b r a n e ( W a l k e r , 1960; F i n d l a y a n d H o p e , 1964). I n t h e r o o t hairs of A v e n a , t h e p l a s m a m e m b r a n e is also
Electrical Properties of Parenchymal Cell Membranes 1.o
I
I
I
I
I
I
I
I
3t9
I
O.8 ~ \\ \\ O.6 ~ X \ rt
rc
~\
"~x~ia
rin
0.4 rm
\x
0.2
\\x\ \%% \\
uplinq
0
I
I 4
I
J 8
I rc or rc rm ra
I 12
"1
I ;6
I ~, 20
Fig. 10. Theoretical curves showing the extent of coupling that could be detected under the conditions of these experiments, given different relative values of tonoplast resistance (rt), coupling resistance (rc), and membrane resistance (rm) as defined by the equivalent circuits of Fig. 9 and in the text. Ordinate gives the ratio of tonoplast to input resistance (rt/rin). The abscissa is either the ratio rc/ra (broken line; of. Fig. 9A) or rc/r m (solid curve; el. Fig. 9B). The degree of coupling that could be detected (region to the left of the curve) is less if the tonoplast accounts for a larger fraction of the input resistance. See the text for further discussion
reported to be the site of the potential (Etherton and Higinbotham, 1960). I t is therefore quite possible t h a t the resistance of the tonoplast is a small fraction of the m e m b r a n e resistance, and if so (c[. above and Figs. 9 and 10), experiments such as the one illustrated in Fig. 8 mean t h a t the cytoplasms of parenehymal cells are not strongly electrically coupled. Since electrical coupling has been detected between internodal cells of the giant green alga Nitella b u t n o t between oat p a r e n c h y m a l cells, it is worth while to compare the relative wall area occupied b y plasmodesmata in the two systems. I n Nitella the internodal cells are coupled t h r o u g h an intervening layer of nodal cells. According to Spanswiek and Costerton (1967), 3.6% of the wall area between i m m a t u r e N~tella cells and 0.59% of the wall area between m a t u r e cells is occupied b y plasmodesmata. I n oat p a r e n e h y m a there are a b o u t 100 p r i m a r y pit fields per cell (Wardrop, 1955) with about 10 plasmodesmata per pit field
320
M. It. M. Goldsmith, H. 1%.Fernandez, and T. H. Goldsmith:
(O'Brien and Thimann, 1967) for a total of 1000 per cell. Taking the diameter of an individual plasmodcsma as 500 •, the complement of 1000 would have an average cross-sectional area of about 2 ~m 2. Since the cell surface area of an average parenchymal cell (95 • 35 ~m) is about 1.2 • 104 ~m 2, only 0.016% of the wall between two adjacent cells is occupied by plasmodesmata. Thus contact between immature Nitella cells is 225 times greater and between mature Nitella cells 37 times greater than between adjacent oat parenchymal cells. Although electrical coupling exists between adjacent Nitella cells, Spanswiek and Costerton (1967) point out that the specific resistance of Nitella plasmodesmata is some 330 times the values predicted from a knowledge of plasmodesmal dimensions and cytoplasmic conductivity. Based on the considerations summarized in Fig. 10, it is possible to calculate lower limits of coupling resistance between oat coleoptile cells. From this, and the numbers and dimensions of plasmodesmata, one can also estimate lower limits for the resistivity of the plasmodesmal contents. These calculations, like the measurements on Nitella, suggest that diffusion of ions through plasmodesmata is restricted. Electron micrographs suggest a likely explanation for the high resistance of the plasmodcsmata. The internal desmotubule of the p]asmodesma, which bears some similarity to a cortical microtubule (Robards, 1968), virtually occludes the opening into the cell with a plug of electron dense material (O'Brien and Thimann, 1967; Robards, 1968). I t is a moot question whether the plasmodesmata are capable of specialized transport functions and/or whether restrictions on passage may be altered by physiological conditions. If diffusion of ions is limited in the plasmodesmata of coleoptiles, this raises the question whether these connections function as the pathways of transport for larger molecules. Of particular interest is the fact that oat eoleopti]es in common with many plant organs transport auxin via a polar, metabolically controlled system. The apparent lack of coupling between cells of the oat coleoptile suggests that in this organ, auxin may not travel from cell to cell via the symplast but may be exported across the cell membrane, diffuse through the intercellular space, and enter the adjacent cell through its plasmalemma. Spitzer (1970) has shown extensive coupling between cells in the lily anther. This is an interesting system because the cells lack large vacuoles, and the germinal cells in the early stages of meiosis that were examined in this study are interconnected by cytoplasmic bridges up to 1 ~ in diameter through which transfer of cytoplasmic organelles has been observed (tteslop-Harrison, 1966), whereas somatic cells have typical plasmodesmata. Coupling exists between all cells of the anther, even those separated by distances of 0.5 mm or more.
Electrical Properties of Parenchymal Cell Membranes
321
Membrane Resistance The input resistance we obtained is a valid measure of membrane resistance only if coupling and leakage are negligible. We have shown that coupling cannot be detected under our conditions, but that some leakage is likely to occur when oat parenehymal cells are penetrated. How much leakage is not know,n, but it is well to keep in mind that if the membrane resistance is high, its measurement will be in error if only a small leakage occurs around the pipette. The specific membrane resistance of 5 kf2 cm 2 represents the tonoplast, plasma membrane, and cell wall in series. Our observations indicate that the wall contributes a minor portion of this value, and, as discussed above, where measured in other plants the resistance of the tonoplast is much lower than the plasma membrane. Consequently, in the absence of further information we suggest that the specific membrane resistance of 5 kf~ cm 2 is determined primarily by the plasma membrane. Most of the variation in input resistance from cell to cell can be accounted for by the distribution of surface areas; consequently there is little cause to hypothesize large variation in specific membrane resistance from cell to cell. Most of the information on specific membrane resistance of plant cells obtained by electrical methods has been from lower plants, but our value of 5 kf2 em 2 (22 mM KC1) is similar to other results. I n Neurospora resistances of 5 kf2 em 2 were found (tO mM KC1) (Slayman, 1965). In Nitella and Chara values range from 5 to 50 kf2 em 2 (Williams et al., 1964). The resistance increases and rectification becomes more obvious as the external concentration of potassium is lowered. Hope and Walker (1961) found that the average specific resistance of Chara cells rose from 5 to 14 kf2 em 2 as the external K + decreased from 1 to 0.1 mM. We are aware of only one other study of membrane resistance in higher plan~s. Higinbotham et al. (1964) used pairs of electrodes and reported a specific membrane resistance of cells of Avena eoleoptiles (in 1.0 mM KC1, 1.0 mM CaCI~) of 1.3 kf2 em "~. Their value is less than half what we measured with a similar two-electrode system at the same K+ and Ca++ concentrations and only about 25 % of what we consider our most reliable value. Although they used a different variety of oats, we are skeptical that this difference alone accounts for the discrepant results. Their penetrations may have been shunted by greater leakage. Recently Etherton (1970) has reported the existence of three distinct steps of change in potential as he penetrated oat parenchymal cells, which he presumes without additional evidence to correspond to a wall, cytoplasm, and vacuolar potential. Although such observations
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M . H . M . Goldsmith, H. R. Fernandez, and T. H. Goldsmith:
h a v e b e e n m a d e w i t h g i a n t algal cells ( N a g a i a n d K i s h i m o t o , 1964), t h e v a r i a t i o n s in tip p o t e n t i a l w h i c h we e n c o u n t e r e d in t h e walls of A v e n a cells a n d t h e low p r o b a b i l i t y of r e l i a b l y l o d g i n g a m i e r o p i p e t t e in a l a y e r of c y t o p l a s m o n l y s e v e r a l m i c r o n s t h i c k m ~ k e it i m p e r a t i v e to use a n i n d e p e n d e n t m e t h o d (such as d y e i n j e c t i o n ) for l o c a t i n g t h e site of r e c o r d i n g w i t h i n t h e cell. This work was supported by U.S. Public Health Service grants GM08886 and EY 00222. We thank Mr. J. Janosik for help with some of the experiments and Prof. M. V. L. Bennett for a critical reading of the manuscript.
References Arisz, W. H. : Influence of inhibitors on the uptake and the transport of chloride ions in leaves of Vallisaeria spiralis. Acts bot. neerl. 7, 1-32 (1958). Bennett, M. V. L. : Physiology of eteetrotonic junctions. Ann. N.Y. Acad. Sci. 137, 509-539 (1966). Etherton, B. : The relationship of cell transmembrane electro-potential to potassium and sodium accumulation ratios in oat and pea seedlings. P h . D . Thesis, Washington State University (1962). - - Effect of indole-3-acetic acid on membrane potentials of oat coleoptile cells. Plant Physiol. 45, 527-528 {1970). - - Higinbotham, N. : Transmembrane potential measurements of cells of higher plants as related to salt uptake. Science 131, 409410 (1960). Findlay, O. P., Hope, A. B. : Ionic relations of cells of Chars australia. VII. The separate electrical characteristics of the plasmalemma and tonoplast. Aust. J. biol. Sci. 17, 62-77 (1964). Furshpan, E. J., Potter, D. D. : Low-resistance junctions between cells in embryos and tissue culture. Curr. Top. Develop. Biol. 3, 95-127 (1968). Heslop-Harrison, J.: Cytoplasmic connections between angiosperm meiocytes. Ann. Bot. (Load.) 30, 221-230 (1966). Higinbotham, N., Etherton, B., Foster, R. J. : Mineral ion contents and cell transmembrane electropotcntials of pea and oat seedling tissue. Plant Physiol. 42, 3 7 4 6 (1967). - - Hope, A. B., Findlay, G. P.: Electrical resistance of cell membranes of Arena coleoptiles. Science 143, 1448-1449 (1964). Hope, A. B., Walker, N. A. : Ionic relations of Chars australia ~. Br. IV. Membrane potential differences and resistance. Aust. J. biol. Sci. 14, 2 6 4 4 (1961). Laties, G. G.: Dual mechanisms of salt uptake in relation to eompartmentation and long distance transport. Ann. Rev. Plant Physiol. 20, 89-116 (1969). Loewenstein, W. R.: Permeability of membrane junctions. Ann. N.Y. Acad. Sci. 187, 441472 (1966). - - Kamm, Y.: Studies on an epithelial (gland) cell junction. I. Modifications of surface membrane permeability. J. Cell Biol. 25, 565-586 (1964). Nagai, R., Kishimoto, U.: Cell wall potential in Nitella. Plant Cell Physiol. 5, 21-31 (1964). O'Brien, T. P., Thimann, K. V.: Histological studies on the eoleoptile. I. Tissue and cell types in the coleoptile tip. Amer. J. Bot. 52, 910-918 (1965). - - - - Observations on the fine structure of the oat coleoptile. II. The parenchymal cells of the apex. Protoplasms (Wien) 68, 417442 (1967). Pappas, G.D., Bennett, M. V. L. : Specialized junctions involved in electrical transmission between neurons. Ann. N . Y . Acad. Sei. 187, 495-508 (1966).
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Potter, D. D., Furshpan, E. J., Lennox, E. S.: Connections between cells of the developing squid as revealed by eleetrophysiological methods. Proc. nat. Aead. Sci. (Wash.) 55, 328-336 (1966). Ray, P. M., Ruesink, A. W. : Osmotic behavior of oat coleoptile tissue in relation to growth. J. gen. Physiol. 47, 83-101 (1963). Robards, A. W.: A new interpretation of plasmodesmatal ultrastructure. Planta (]3erl.) 82, 200-210 (1968). Sibaoka, T.: Excitable cells in Mimosa. Science 137, 226 (1962). Action potentials in plant organs. Syrup. Soc. exp. Biol. 20, 49-73 (1966). Slayman, C. L.: Electrical properties of Neurospora crassa. Respiration and the intracellnlar potential. J. gen. Physiol. 49, 93-116 (1965). Spanswick, R.M., Costerton, J. W. F. : Plasmodesmata in Nitella translucens: structure and electrical resistance. J. Cell Sci. 2, 451464 (1967). Spitzer, N. : Low resistance connections between cells in the developing anther of the lily. J. Cell Biol. 4.~, 565-575 (1970). Voeller, ]3. R. : The plant cell: aspects of its form and function. In: The cell, p. 257267 (J. Brachet and A. E. Mirsky, eds.). New York: Academic Press, Inc. 1964. Walker, N. A. : Mieroelectrode experiments on Nitella. Aust. J. biol. Sci. 8, 476489 (1955). - - The electric resistance of the cell membranes in a Chara and a Nitella species. Aust. J. biol. Sci. 13, 468478 (1960). Wardrop, A. B.: The mechanism of surface growth in parenchyma of Avena coleoptiles. Aust. J. Bot. 3, 137-148 (1955). Williams, E. J., Johnston, R. J., Dainty, J. : The electrical resistance and capacitance of the membranes of Nitella translucens. J. exp. ]3ot. 15, 1-14 (1964). Mary Helen M. Goldsmith Timothy H. Goldsmith Department of Biology Kline Biology Tower Yale University New Haven, Connecticut 06520, U.S.A.
Hector R. FernAndez Department of Biological Sciences University of Southern California Los Angeles, California 90007, U.S.A.
