PLASMA MEMBRANE ULTRASTRUCTURE DURING PLANT PROTOPLAST PLASMOLYSIS, ISOLATION AND WALL REGENERATION: A FREEZE-FRACTURE STUDY

Transcription

1 J. Cell Sci. 42, (1980) Printed in Great Britain Company of Biologists Limited jg8o PLASMA MEMBRANE ULTRASTRUCTURE DURING PLANT PROTOPLAST PLASMOLYSIS, ISOLATION AND WALL REGENERATION: A FREEZE-FRACTURE STUDY M. J. WILKINSON AND D. H. NORTHCOTE Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CBz \QW, U.K. SUMMARY The freeze-fracture morphology of the plasma membrane of cells and isolated protoplasts of plant callus suspensions has been investigated. Plasmolysis of suspension cells leads to the formation of 2 types of hexagonal arrays of intramembrane particles situated on the inner fracture face (PF). These arrays are interpreted as proteins that have ' crystallized' in the plane of the membrane as the area of surrounding lipid bilayer is reduced during protoplast retraction from the cell wall. Time-course studies have revealed no positive relationship between the distribution of hexagonal arrays and the occurrence of microfibrils regenerated around isolated protoplasts during periods of culture. No evidence for the specialized transport functions attributed to hexagonal arrays of plant cells by previous workers has been found. INTRODUCTION Cellulose microfibrils of plant cell walls are probably synthesized at the plasma membrane. The molecular mechanism by which this membrane elaborates an intricate meshwork of microfibrils, forming a coherent wall with associated matrix polysaccharides and proteins, is poorly understood. Freeze-fracture replication is an excellent means of visualizing the macromolecular organization of biological membranes and the wall architecture of plant cells. Studies of the unicellular algae Oocystis opiculatum have demonstrated a relationship between the distribution of particles in the plasma membrane and wall microfibrils (Brown & Montezinos, 1976; Montezinos & Brown, 1976). Such clearly defined spatial associations between plasma membrane components and nascent microfibrils of higher plant cells have not yet been identified, though a number of studies on isolated plant protoplasts undergoing wall regeneration have been conducted (Willison & Cocking, 1972, 1975; Grout, 1975; Willison & Grout, 1978). The majority of these investigations employed isolated leaf mesophyll protoplasts of tomato and tobacco. In contrast, we have studied the plasma membrane ultrastructure of protoplasts isolated from fast-growing callus cell suspensions. This material was found to possess areas of regularly arranged intramembrane particles, a feature limited to the plasma membrane. While this investigation was in progress particle arrays were reported as characteristic of the plasma membrane of protoplasts isolated from Skimmia japonica callus

2 402 M. J. Wilkinson and D. H. Northcote tissues (Robenek & Peveling, 1977). It was suggested that particle arrays resulted from the incorporation of specific proteins from the endoplasmic reticulum into the plasma membrane during wall regeneration. Arrays were interpreted as forming specialized transport channels for the outward passage of wall precursors. Our investigations of the factors involved in the production of particle arrays and of their possible role in wall regeneration leads us to an alternative explanation of their presence in the plasma membrane of isolated protoplasts. MATERIALS AND METHODS Growth of cells Callus was initiated from tuber explants of potato (Solatium tiiberosum cv. Record) after the method of Anstis & Northcote (1973). Solid callus was subcultured every 4-6 weeks on PRL4 medium (Gamborg, 1966) containing 6 mg/1. 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures were established by dispersing 2 g of solid callus in 100 ml liquid medium (either PRL4 or the medium described by Upadhya, 1975) containing 3 mg/1. 2,4-D, 2 g/1. N.Z. Amine (KW-Revai Chemicals Ltd., London, U.K.) and incubating at 100 rev/min in an orbital shaker at 26 C. Suspension cultures grew slowly for several weeks but thereafter could be maintained by subculturing approximately half the cells into 100 ml of fresh growth medium, every 5-7 days. Cultures comprising large numbers of small, round cells growing in fine clumps were most suitable for protoplast isolation and could be obtained by subculturing at 3-day intervals for at least 2 weeks. These fast growing cultures were used throughout this investigation. They reduced the original ph of the growth medium ( ) to approximately ph 5-0 by the end of the 3-day growth period. Protoplast isolation Pectinase (Sigma, Poole, Dorset, U.K.) was purified before use (Hanke & Northcote, 1974). Driselase (a mixture of cellulase and pectinase obtained from Kyowa Hokko Kogyo Co., Tokyo, Japan) was used as supplied. Insoluble components were removed by centrifugation at g for 10 min prior to sterile filtration of enzyme solutions (Millipore filter, O'4S-/tm pore size). Cells were filtered onto 25-/tm steel mesh and washed once with fresh growth medium. They were mixed with enzyme solution (3 g fresh weight of cells to 10 ml solution) comprising 5 % (w/v) Driselase, 05 % (w/v) purified pectinase and 0-35 M sorbitol as the plasmolysing agent. This was prepared in growth medium (excluding sucrose, N.Z. Amine and 2,4-D) buffered with 20 mm 2 iv-morpholino ethanesulphonic acid (MES) at ph 5-7. The mixture was gently agitated on a reciprocal shaker for h at 25 C to effect plasmolysis, wall digestion and release of protoplasts. The crude protoplast preparation was then filtered through a 7-cm-wide, 25-/«n steel mesh. Isolated protoplasts, with diameters up to approximately 70 /(m passed through the mesh by constriction. They were centrifuged at 80 g for 3 min and then washed 3 times with osmoticum. Viability was estimated by staining with fluorescein diacetate (Larkin, 1976). Protoplast culture After washing, protoplasts were resuspended to a density of approximately 5 x io 5 /ml in fresh growth medium containing 0-35 M sorbitol and 1 g/1. N.Z. Amine. They were dispensed in o-5-ml aliquots in 50-ml plastic pots (Sterilin Ltd., Teddington, Middlesex, U.K.) in the dark at 25 C C. The presence of cellulose was detected by staining with 0-2 % (w/v) Calcofluor White (American Cyanamid Co., New Jersey, U.S.A.).

3 Plasma membrane of plant protoplasts 403 Digestion of the walls of prefixed cells Cells were first fixed in 1 % glutaraldehyde prepared in growth medium at ph 50 (i.e. the ph to which it had been conditioned by the rapidly growing cells) for 15 h at 25 C. More glutaraldehyde was then added to bring the concentration to 25 % and the ph readjusted to 5-0. Fixation was continued for a further 4 h at 25 C. Excess fixative was removed by rinsing the cells 3 times with 100-ml quantities of growth medium. The fixed cells were then treated in a similar manner to that used to isolate plasmolysed protoplasts, except that sorbitol was omitted from the enzyme solution and wall digestion was continued for 4 h. After cell wall removal fixed protoplasts retained their original shape and could not be purified by constriction through fine mesh. Wall fragments were removed by several washes in growth medium by centrifugation at 80 g for 3 min. Cell plasmolysis To test the effect of plasmolysis alone on the organization of the plasma membrane, cells (2 g fresh weight) were placed in 10 ml growth medium containing 0-35 M sorbitol and 10 mm MES at ph 50 or 5-7 for 25 h. The effect of using 035 M glucose or a salt mixture (i'7s % KC1, C75 % MgCl 2, 05 % KNO3) in place of sorbitol for plasmolysis was also examined. Freeze-fracture replication and electron microscopy Frceze-fracture replication was performed using a method developed specifically for this study (Wilkinson, 1978; Wilkinson & Northcote, 1980). Briefly, a small volume of cells or protoplasts, either alive or prefixed, was sandwiched between copper plates and frozen very rapidly in liquid propane. The samples were then loaded into a hinge device and fractured by separating the plates under high vacuum in a Balzers BA360 freeze-etching unit. Replication was by tungsten tantalum evaporation using an electron gun. Replicas mounted on gold grids were cleaned with 70 % (v/v) H 2 SO 4 for h at 35 C with the aid of a glass capillary apparatus. In experiments on wall regeneration etching was performed prior to replication on protoplast samples that had beenfixedin 2-5 % glutaraldehyde for 2-3 h at 25 C or overnight at 2 C C followed by washing with distilled water. Etching was carried out at a pressure of less than 133 /tpa (133 x io" 1 N m~ 2 ) and at 100 C for 3 min. Replicas were examined using an AEI 801 transmission electron microscope operating at 60 kv. Optical diffraction patterns were obtained using an optical diffractometer equipped with a laser source (Home & Markham, 1973). All reciprocal lattice points were used for image reconstruction. RESULTS The appearance of the plasma membrane of callus cells before and after plasmolysis Freeze-fracture replication of turgid suspension cells exposed only a small proportion of the total surface area of the plasma membrane. After enzymic digestion of the cell wall, however, the fracture characteristics of the remaining fixed protoplasts were similar to those of live plasmolysed protoplasts, with greater areas of plasma membrane exposed for replication and electron-microscope examination. The appearance of the plasma membrane of turgid suspension cells was typical of freezefractured biological membranes. Intramembrane particles, most prevalent on the inner fracture face (PF; Branton et al. 1975) occurred in an apparently random distribution (Fig. 1). The ultrastructure of the plasma membrane of cells sampled from growth medium at ph 5-0 was identical to that of cells that had been transferred to medium adjusted to ph 57 (i.e. the ph used for protoplast isolation) for 3 h.

4 M. jf. Wilkinson and D. H. Northcote

5 Plasma membrane of plant protoplasts 405 The plasma membrane of cells plasmolysed in growth medium containing 0-35 M sorbitol at ph 5-0 was similar to that of unplasmolysed control samples and exhibited randomly spaced particles. When plasmolysis was performed at ph 57, however, particles clustered to form discrete arrays (Fig. 2). These became apparent within 30 min of transferring cells to plasmolyticum, that is, by the time plasmolysis was complete. The arrays were irregular in outline and comprised rows of particles (approx. 10 nm diameter) located on the inner half (PF) of the plasma membrane. Particle arrays were formed regardless of which osmoticum was used to plasmolyse the protoplasts, i.e. sorbitol, glucose (Fig. 3) or salts. Arrays were evident in freeze-fixed specimens, where no pretreatment with fixatives and cryoprotectants was used, and in those fixed with glutaraldehyde and then cryoprotected with 20% glycerol before freeze-fracturing (Fig. 4). The appearance of the plasma membrane of isolated protoplasts Fast-growing potato callus cells treated simultaneously with a plasmolysing agent and a cellulase-pectinase mixture yielded large numbers of viable protoplasts in 2-3I1 (approx. io B /g fresh weight of cells). The viability of protoplast preparations was %. The appearance of the plasma membrane was unaltered by treating protoplasts with wall-degrading enzyme at ph 5-7. Particle arrays still characterized the inner fracture face (PF) of the plasma membrane of isolated protoplasts. Isolated protoplasts generally provided better quality plasma membrane fracture faces than plasmolysed cells. The more common array morphology, seen particularly clearly in isolated protoplasts, was that of a hexagonal lattice (Type I) (Fig. 5). A more closely packed arrangement of particles was occasionally evident (Type II, Fig. 6) and this was also based on a hexagonal pattern. The density of particles in this type of array was approximately Figs The circled arrow on each photograph incidates the direction of shadow. All specimens (except that shown in Fig. 4) were freeze-fixed, i.e. preserved in culture medium or water without the addition of glycerol. Figs. 1-7, 9-12 show the plasma membrane of potato callus cells (Figs. 1-4) and isolated protoplasts (Figs. 5-7, 9-12). Figs. 13 and 14 are of bean callus protoplasts. Fig. 1. The inner fracture face (PF) of a potato callus suspension cell sampled directly from the growth medium (ph S - o). The intramembrane particles exhibit no marked clustering, x Fig. 2. The plasma membrane PF of a suspension cell that was plasmolysed by the addition of sorbitol (0-35 M) to the growth medium at ph 5-7. A proportion of the intramembrane particles have clustered into discrete arrays. An area of the outer surface (ES) is visible at top right, x Fig. 3. An intramembrane particle array present on the plasma membrane, PF, after plasmolysis at ph 5-7 with 0-35 M glucose instead of sorbitol. x Fig. 4. The plasma membrane, PF, of a specimen that was cryoprotected with 20 % glycerol following plasmolysis (035 M sorbitol, ph 57). Randomly spaced particles in the area surrounding the array are clearly visible as this area has suffered no deformation (compare with Figs. 5, 6). x

6 406 M. jf. Wilkinson and D. H. Northcote Fig. 5. The more common, open lattice structure based on a repeated hexagonal pattern (particle density approx. 5oo//im2) is shown particularly clearly in the array (Type I). Deformation of the membrane due to fracturing is limited to the area surrounding the array, x Fig. 6. A more tightly packed array (Type II, particle density approx. iooo//im2) also based on a hexagonal pattern. In this example the particles are triangular. Membrane deformation is again limited to the region outside the array, x

7 Plasma membrane of plant protoplasts Fig. 7. Optical diffraction patterns of intramembrane particle arrays. Types I and II (top left and right, x and , respectively) are shown, together with their corresponding filtered image (middle row) obtained using all the reciprocal lattice points in the diffraction patterns (bottom row). Averaged lattice constants are 267 nm (Type 1) and 18-1 nm (Type II).

8 408 M. J. Wilkinson and D. H. Northcote

9 Plasma membrane of plant protoplasts 409 twice that of the Type I arrangement. The optical diffraction patterns of these 2 types of array together with their reconstructed images are presented in Fig. 7. Though the tonoplast contained numerous intramembrane particles no arrays were ever observed in this membrane either before or after plasmolysis (Fig. 8). Plasma membrane ultrastructure during protoplast wall regeneration When isolated protoplasts were returned to growth medium containing 0-35 M sorbitol they rapidly began to regenerate a new wall. Cell shape resumed within 2 days and protoplasts then stained positively with Calcofluor White. Budding occurred during the first 2 weeks of culture and this was followed by the onset of cell division. After 3-4 weeks small aggregates of cells developed. Particle arrays in the plasma membrane were most common in freshly isolated protoplasts and in a number of preparations were detectable on almost every inner fracture face (PF) examined. They continued to be a regular feature of the plasma membrane for up to 48 h and accounted for up to 19% of the surface area of plasma membrane (PF) visible in replicas, accommodating up to approximately 45 % of all particles. As protoplast wall regeneration continued the fracture characteristics of the material remained constant. The ph of the protoplast culture medium did not vary significantly. After 3 to 4 days, however, particle arrays became far less common, with only about 1 in 25 fracture faces (PF) showing evidence of such particle arrangements. After 7 days of protoplast culture arrays were rarely seen and they did not reappear during further periods of wall regeneration and cell growth up to 3 weeks, when cell aggregates developed. The progressive development of a network of microfibrils, probably of cellulose, over the plasma membrane surface during wall regeneration was visualized by etching of glutaraldehyde-fixed samples that had been resuspended in water before freezefracturing. Freshly isolated protoplasts were naked and smooth (Fig. 9). Microfibrils began to appear within 30 min of transferring protoplasts to culture medium. The production of microfibrils on the plasma membrane surface (ES) did not appear to depend upon the presence of particle arrays, as judged by inspection of adjacent regions of membrane where the inner fracture face (PF) was revealed. Where particle arrays did occur in conjunction with newly formed microfibrils the orientation of Fig. 8. Randomly distributed intramembrane particles on the inner fracture face (PF) of the tonoplast of a potato protoplast; such particles were never observed to aggregate, even after plasmolysis. A region of the outer surface is visible (top left), x Fig. 9. The etched outer surface (ES) of a freshly isolated potato callus protoplast showing the complete absence of wall material. An area of PF is visible (top left), x Fig. 10. The etched surface of a potato callus protoplast cultured for 2-5 h. A fibril (single arrow) runs across the outer surface (ES). The orientation of particle rows in the array present on the adjacent fracture face (PF) does not coincide with the direction in which the fibril has been deposited. A second fibril (double arrows) runs in a similar direction to the first and has been pulled away from the surface and hence is thickened with carbon, x CEL42

10 410 M. J. Wilkinson and D. H. Northcote

11 Plasma membrane of plant protoplasts 411 particle rows did not necessarily coincide with the direction in which microfibrils were deposited (Figs. 10, 11). The surface of the plasma membrane lying beneath the network of microfibrils was only rarely displayed. It appeared relatively smooth except for depressions left by the original microfibrils which had fractured away (Fig. 12). We have also observed hexagonal particle arrays on the PF of the plasma membrane of protoplasts isolated from bean (JPhaseolus vulgaris L.) callus suspensions. These suspensions were originally obtained from stem callus and protoplasts were isolated by the method used for potato callus suspensions. Two types of array, strikingly similar to Types I and II present in potato protoplasts, were evident (Figs. 13, 14). DISCUSSION Using freeze-fracture replication we have demonstrated the presence of hexagonally arranged intramembrane particles on the inner half (PF) of the plasma membrane of plasmolysed potato callus cells. These arrays occurred in glutaraldehyde-fixed specimens. It is therefore unlikely that they arose by lateral displacement of macromolecules due to lipid solidification during freezing (Bullivant, 1977). The presence of arrays in samples frozen live without pretreatment showed that chemical fixation and glycerination were not the cause of particle clustering. Particle arrays may therefore be taken as indicative of the distribution of macromolecules, probably integral proteins, within the plasma membrane in vivo. The lack of marked particle-pit complementarity between matching halves of fractured plasma membrane (Wilkinson & Northcote, 1980) suggested that the particles were composed of proteins rather than lipids (Verkleij & Ververgaert, 1978). Array formation depended on plasmolysis rather than the plasmolysing agent (sorbitol, glucose or salts). Plasmolysis has 2 major consequences for the plant cell plasma membrane. It breaks any links between the outer surface of the membrane and the wall and simultaneously reduces the surface area of lipid bilayer available to Fig. 11. A partially regenerated wall formed after 3 days of protoplast culture. Bundles of microfibrils, closely applied to the etched surface (ES) of the plasma membrane, are being produced at a significant rate at this time, yet the frequency of particle arrays is decreasing, so that areas of PF below the microfibrils (centre) often lack evidence of particle arrays, x Fig. 12. A rare view of the plasma membrane ES with all microfibrils fractured away. The membrane surface appears smooth except for depressions left by the original structures. In some cases these depressions can be traced back to the remaining microfibrils (arrow). The PF is exposed on the right of the picture, x Fig. 13. An intramembrane particle array present on the inner fracture face (PF) of a freshly isolated bean protoplast obtained from a callus suspension. The pattern is very similar to the Type I arrays seen in potato callus protoplasts (compare with F'g- 5)- x Fig. 14. A second type of intramembrane particle array evident on the PF of the plasma membrane of a freshly isolated bean callus protoplast. This more closely resembles the Type II arrays of potato callus protoplasts (compare with Fig. 6). x

12 412 M. J. Wilkinson and D. H. Northcote accommodate proteins. Breaking membrane-wall links may tend to set membrane proteins adrift in the lipid bilayer and so facilitate mutual interaction by increasing their lateral mobility. A concomitant reduction in membrane surface area would perhaps reinforce this effect by increasing the concentration of proteins per unit area of bilayer. We envisage the net result of these 2 processes to be the partial' crystallization' of proteins into the 2-dimensional arrays observed in plasmolysed material. Why only a proportion, up to approximately 45%, of intramembrane particles form arrays is unclear. There is at present no detailed information on the chemical identity of the intramembrane particles which might provide clues as to their capacity for aggregation. Plasma membrane-wall connexions would be broken by mechanical stress during plasmolysis but the fate of any transmembrane connexions on the inner face of the membrane, perhaps involving microtubules, is more difficult to determine. Microtubules are certainly present in close proximity to the inner plasma membrane face, probably appressed to it, in both turgid cells and plasmolysed protoplasts. Linear structures lying parallel to each other and interpreted as microtubule impressions were commonly visible on the plasma membrane fracture faces which exhibited arrays (Wilkinson & Northcote, 1980). These impressions did not, however, relate in any obvious way to the ordering of particles within arrays or to the grouping of arrays. Particle arrays were common in protoplasts plasmolysed at ph 5-7, the ph used for optimum yield and viability during protoplast isolation, but could not be detected in identical cell batches plasmolysed at ph 5-0. It was not possible to obtain good quality protoplasts at ph 5-0 as bursting occurred upon their release into the enzyme solution, possibly due to alterations in the permeability properties of the plasma membrane or to the toxicity of wall-degrading enzymes at this lower ph. Time-course experiments demonstrated the progressive loss of arrays during protoplast culture. After 7 days arrays were only rarely detected. During this period the effect of protoplast growth and expansion which occur despite the presence of osmoticum, would be to increase the surface area of the plasma membrane. The result of this increase may be to reduce the number of proteins per unit area of lipid bilayer. This may tend to reverse the original effect of plasmolysis and encourage the dissolution of 'crystalline' proteins back into their lipid 'solvent' by diffusion. All freeze-fractured biological membranes exhibit randomly spaced intramembrane particles. The occurrence of such particles in geometric arrays is often taken to indicate their role in some specialized transport function, such as in the case of arrays present in the junction between axons of crayfish ganglia (Peracchia, 1974). Hexagonal arrays very similar to the Type I arrays described here were found in the plasma membrane (PF) of cryoprotected transfer cells of clover root nodules (Briarty, 1973). It is particularly relevant that glutaraldehyde-fixed transfer cells that were subsequently glycerated did not exhibit particle arrays. Only those cells placed in 20 to 30% glycerol without prior fixation, and thereby suffering plasmolysis, possessed evidence of arrays. Briarty argued that aldehyde fixation might disrupt any naturally occurring arrays which were possibly involved in the specialized transport functions of transfer cells. We have found that glutaraldehyde fixation has no detrimental effect on the morphology of particle arrays present in potato callus. It seems more probable,

13 Plasma membrane of plant protoplasts 413 therefore, that glycerol-mediated plasmolysis of unfixed cells created the arrays and that they do not occur under normal conditions in the plasma membrane of clover transfer cells. The presence of particle arrays in the plasma membrane of protoplasts isolated from Skimmia japonica callus has been reported (Robonek & Peveling, 1977). It was suggested that the arrays represent specific proteins incorporated into the plasma membrane from the highly active endoplasmic reticulum (which characterized protoplasts in culture), so forming specialized channels for the passage of cellulose precursors during wall regeneration. In the case of Skimmia protoplasts, intramembrane particles began to form arrays after 24 h of protoplast isolation and particles in hexagonal arrangements became more clearly defined after some h of culture. This contrasts with our observations that hexagonal particle arrays are apparent as soon as protoplasts are plasmolysed, well before there has been any opportunity for the activation of endoplasmic reticulum. Furthermore, observations of thin sections (unpublished results) show that the endoplasmic reticulum also appears to proliferate in isolated potato callus protoplasts during periods of culture when arrays are becoming less rather than more frequent. From the observations on potato and bean protoplasts we suggest that there is no need to implicate the endoplasmic reticulum and the incorporation of specific proteins into the plasma membrane to explain the formation of particle arrays. In the absence of positive evidence of protein incorporation the simplest explanation is that plasmolysis is the essential trigger that brings about the clustering of intramembrane particles. Though particle arrays have been viewed here as a product of plasmolysis rather than as specialized regions of plasma membrane involved in wall precursor transport, we nevertheless considered the possibility that the individual macromolecules comprising the arrays function in wall production and that their geometrical arrangement would facilitate the investigation of such a role. Freeze-etching of protoplasts at various stages in the process of wall regeneration was performed to uncover any similarities in orientation between nascent microfibrils and particles aligned within arrays. A number of time courses were performed. The synthesis and orientation of microfibrils always seemed independent of particle arrays. Even when occurring in close proximity to each other, the arrangement of particles within arrays was not necessarily reflected in the orientation of newly formed microfibrils. At present, therefore, there seems to be no definite indication of a role for hexagonal particle arrays in any aspect of wall regeneration in higher plant cells. What is clear, however, is that plasmolysis may markedly influence the ultrastructural appearance of the plasma membrane of plant cells. Whether plasmolysis has been intentional (e.g. when conferring osmotic stability on isolated protoplasts) or whether it has been due to the use of a cryoprotectant such as glycerol without prior chemical fixation, its potential for causing alterations in plasma membrane organization should encourage caution when interpreting the results of ultrastructural studies on plasmolysed cells and isolated protoplasts. During the completion of this manuscript, Davy & Mathias (1979) reported that hexagonal particle arrays in the plasma membrane of isolated higher plant protoplasts

14 414 M.J. Wilkinson and D. H. Northcote were essentially an artefact of 20% glycerol treatment rather than the direct result of plasmolysis. In contrast to our results, they did not detect any arrays in protoplasts which had been only plasmolysed by the osmoticum during isolation. In the absence of evidence from control experiments where the extensive plasmolysis of the glycerol treatment (i.e. additional to that of the osmoticum) is paralleled by, for example, exposing protoplasts to an increased concentration of osmoticum, we view any possible effects of glycerol on array formation as being secondary to the macromolecular rearrangements related directly to the retraction of the plasma membrane and its significant reduction in surface area during plasmolysis. Dr K. Roberts of the John Innes Institute kindly performed the optical-diffraction analysis shown in Fig. 7. We thank Mr D. Knights for assistance in operating the freeze-fracture unit and Mr L. Jewitt for help with photography. M. J.W. is grateful to the Potato Marketing Board for a studentship during the tenure of which this work was performed. REFERENCES ANSTIS, P. J. P. & NORTHCOTE, D. H. (1973). The initiation, growth and characteristics of a tissue culture from potato tubers. J. exp. Bot. 24, BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOORE, H., MOHLETHALER, K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHLIN, L. A., STEERE, R. L. & WEINSTEIN, R. S. (1975). Freeze-etching nomenclature. Science, N.Y. 190, BRIARTY, L. G. (1973). Repeating particles associated with membranes of transfer cells. Planta 113, BROWN, R. M. JR. & MONTEZINOS, D. (1976). Cellulose microflbrils: Visualisation of biosynthetic and orienting complexes in association with the plasma membrane. Proc. natn. Acad. Sci. U.S.A. 73, BULLIVANT, S. (1977). Evaluation of membrane structure facts and artefacts produced during freeze-fracturing. J. Microscopy 111, DAVY, M. R. & MATHIAS, R. J. (1979). Close-packing of plasma membrane particles during wall regeneration by isolated higher plant protoplasts - fact or artefact? Protoplasma 100, GAMBORG, O. (1966). Aromatic metabolism in plants. II. Enzymes of the shikimate pathway in suspension cultures of plant cells. Can. J. Biochem. 44, GROUT, B. W. W. (1975). Cellulose microfibril deposition at the plasmalemma surface of regenerating tobacco mesophyll protoplasts: a deep etch study. Planta 123, HANKE, D. E. & NORTHCOTE, D. N. (1974). Cell wall formation by soyabean callus protoplasts. J. Cell Sci. 14, HORNE, R. W. & MARKHAM, R. (1973). Applications of optical diffraction and image reconstruction techniques to electron micrographs. In Practical Methods in Electron Microscopy, vol. 11 (ed. A. M. Glauert), pp Amsterdam: North-Holland. LARKIN, P. J. (1976). Purification and viability determinations of plant protoplasts. Planta 128, MONTEZINOS, D. & BROWN, R. M. JR (1976). Surface architecture of the plant cell: biogenesis of the cell wall with special emphasis on the role of the plasma membrane in cellulose biosynthesis. J. supramolec. Struct. 5, PERACCHIA, C. (1974). Excitable membrane ultrastructure. I. Freeze-fracture of crayfish axons. J. Cell Biol. 61, ROBENEK, H. & PEVELING, E. (1977). Ultrastructure of the cell wall of isolated protoplasts of Skimmia japonica Thunb. Planta 136, UPADHYA, M. D. (1975). Isolation and culture of mesophyll protoplasts of potato (Solonum tuberosum L.). Potato Res. 18,

15 Plasma membrane of plant protoplasts 415 VEKKLEIJ, A. J. & VERVERGAERT, P. H. J. TH. (1978). Freeze-fracture morphology of biological membranes. Biochim. biophys. Ada 515, WILKINSON, M. J. (1978). Studies on Tissue Cultures and Isolated Protoplasts of Potato. Ph.D. thesis, University of Cambridge, U.K. WILKINSON, M. J. & NORTHCOTE, D. H. (1980). A reliable method for obtaining matched replicas of freeze-fractured cell suspensions. J. Cell Sci. 42, WILLISON, J. H. M. & COCKING, E. C. (1972). The production of microfibrils at the surface of isolated tomato-fruit protoplasts. Protoplasma 75, WILLISON, J. H. M. & COCKING, E. C. (1975). Microfibrils synthesis at the surface of tobacco mesophyll protoplasts, a freeze-etch study. Protoplasma 84, WILLISON, J. H. M. & GROUT, B. W. W. (1978). Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato. Planta 140, {Received 14 August 1979)

16