Planta 144, 209

Planta

215 (1979)

9 by Springer-Verlag 1979

Coneanavalin A Binding Sites on the Plasma Membrane of Leek Stem Protoplasts Francis A. Williamson* Laboratoire de Biologic V6g6tale, Cytologic Exp6rimentale, Ecole Normale Sup6rieure, 24 rue L h o m o n d , F-75231 Paris, France, and Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 314 1700, Tehran, Iran

A b s t r a c t . The binding of concanavalin A (con A) to leek ( A l t i u m p o r r u m L.) stem protoplasts has been investigated using sequential treatment with con A and haemocyanin and using con A covalently linked to ferritin. Prefixed protoplasts were evenly labelled. Unfixed protoplasts showed a clustered distribution of label. Low temperature and lanthanum reduced the clustering. Bound con A was lost from unfixed protoplasts incubated for 5 h after treatment, but con A/haemocyanin was not bound to nascent wall materials. Prefixed protoplasts treated with wall-removing enzymes before labelling showed only a small reduction of con A binding. These results indicate that con A is bound to plasma membrane components, but that binding is reduced by competition of nascent wall precursors.

Key words: A l l i u m - Concanavalin A - Fluidity - Lanthanum - Plasma Membrane - Protoplasts.

showed a very even distribution. These results were interpreted as indicative of cross-linking of binding sites in a fluid membrane by the multivalent lectin. Burgess and Linstead (1976) compared the binding characteristics of different species of protoplast with different propensities for cell wall regeneration and concluded that con A was more likely bound to nascent wall materials than to integral membrane glycoproteins. In a second publication, Burgess and Linstead (1977) concluded that clustering was dependent upon membrane fluidity, on the basis of differences of labelling patterns at different temperatures. The present paper describes experiments which attempt to distinguish between con A binding to the membrane per se and binding to wall components. Two main approaches are used: a reversible prevention of clustering by lanthanum (La) (Inbar et al., 1973) or by low temperature, and treatment with cell wallremoving enzymes after fixation, to remove any nascent wall materials.

Introduction M a t e r i a l s and M e t h o d s

The study of plant cell plasma membranes has been hampered by the presence of the cell wall which restricts the accessibility of large molecules such as lectins to the membrane. The availability of isolated protoplasts has recently allowed lectin labelling of plant plasma membranes (Williamson et al., 1975 and 1976a; Burgess and Linstead, 1976 and 1977). Williamson and coworkers found that soybean tissue culture protoplasts labelled with concanavalin A (con A) and haemocyanin before fixation showed a clustered distribution of binding sites and that protoplasts fixed with glutaraldehyde before labelling * Present address: 540 Reynolds St., S. Williamsport, PAl7701, USA Abbreviations: con A = c o n c a n a v a l i n A; con A - H = s e q u e n t i a l treatment with con A and haemocyanin; con A F = c o n A covalently linked to ferritin

a) Protoplast Isolation Mature plants of leek (Allium porrum, L.) weighing ca. 250 g were purchased in local markets. A transverse cut was made immediately above the roots which were discarded. A 2 cm length of the remaining base was excised and surface sterilised (Constabel, 1975). A cone of stem tissue was excised from the base as described by Morrg (1970) for green onions. A n y remaining leaf tissue was trimmed away and the truncated cone of stem was cut into 0.5 1.0 m m slices. The tissue was incubated for 16 h at 18-20 ~ in an enzyme solution obtained by mixing equal volumes of protoplast culture medium (Williamson et a l , 1976 a as modified by Prat a n d Williamson, 1976) and a solution of 1% Onozuka SS (Kinki Yakult, Nishinomiya), 1% Driselase (Kyowa H a k k a Kogyo, Tokyo) and 1% R h o z y m e HP-150 ( R o h m and Ha~s, Philadelphia) in 0.55 M sorbitol and 1 m M CaC12. This mixture was centrifuged at 10,000 g for 10 min and passed t h r o u g h a 0.45 BM Millipore filter before use. In some experiments, the tissue was incubated for 3~; h at 26 ~ in 1% Onozuka, 1% Driselase a n d

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1% Rhozyme in 0.55 M sorbitol plus 10ram CaC12. At the end of the incubation, the petri dishes were gently swirled to break up remaining tissue, and the suspension filtered through a 67 gM stainless steel sieve. Protoplasts were recovered grom the filtrate by centrifugation at 50-150 g for 3-4 rain and washed twice with 0.55 M sorbitol containing 1 mM or 10 mM CaCI2 (SCa-1 or SCa10). The enzyme and wash solutions had a pH of 6.0-6.2 (unadjusted). All subsequent manipulations were performed in SCa-1 or SCa-10 buffered at pH 7.2 with 10 mM HEPES-KOH or 10 mM Na maleate, except as otherwise noted.

b) Con A and Haemocyanin Binding Sequential treatment with con A and haemocyanin (con A H) was as previously described (Williamson et al., 1976 a and b). Haemocyanin is bound to the membrane specifically at sites occupied by the tetravalent con A.

c) Concanavalin A Ferritin Binding Con A covalently linked to ferritin (con A-F) was purchased from Miles Research Laboratories (Laboratoire Miles, Tour Maine Montparnasse, 75755-Paris) and used at a concentration of 200 500pg/ml after transferring to the appropriate buffer by washing in Amicon cones (Amicon Corp., Lexington, Mass., U.S.A.). Protoptasts were exposed to con A-F for 30-45 min and washed twice before preparation for electron microscopy.

d) Fixation For fixation before con A treatment ("prefixation"), protoplasts were incubated in 1% glutaraldehyde, washed and incubated in 100 mM glycine to block any remaining aldehyde groups (Williamson et al., 1976 a)

e) Electron Microscopy Preparation for thin sectioning was as previously described (Williamson et al., 1976 a) except that ruthenium red, which enhances the contrast of haemocyanin, was used only in the osmium postfixation. Specimens were embedded in an Epon-Araldite mixture. Except where noted in the figure legends, sections were stained with uranyl acetate and/or lead citrate (Reynolds, 1963). Sections were viewed and photographed with a Philips EM 300 operating at 80 kV, or with a Hitachi HU-12A operating at 100kV.

f) Light Microscopy Zeiss photomicrosope and Reichert Univar microscopes were used. Images similar to Nomarski optics were obtained by placing the condenser slightly off axis (R. Prat, personal communication).

Fig. 1. Light micrograph of leek stem protoplasts, n-nucleus s-strand of cytoplasm

a n d were in various stages of v a c u o l a t i o n . Nuclei (n) were readily visible, sometimes being placed centrally a n d c o n n e c t e d to the peripheral c y t o p l a s m by cytoplasmic strands (s) in which vigorous streaming was visible. I n other protoplasts, the n u c l e u s was in the t h i n layer of peripheral c y t o p l a s m which surr o u n d e d a single large vacuole. Even t h o u g h highly vacuolated, the larger protoplasts did n o t float o n sucrose (20%) or Ficoll (10% in 0.55 M sorbitol). H o w e v e r the p r o p o r t i o n of dead cells was usually very low, so that further p u r i f i c a t i o n was unnecessary. The use of R h o z y m e (hemicellulase) was essential for reproducible, rapid p r o t o p l a s t p r o d u c t i o n . T h e use of the two cellulases was n o t essential: protoplasts were p r o d u c e d in a m i x t u r e of R h o z y m e with O n o z u k a alone, b u t m a c e r a t i o n was m o r e r a p i d if Driselase was included. I n the presence of 1% of each enzyme at 26 ~ p r o t o p l a s t s could be o b t a i n e d in a b o u t 2 h, b u t longer i n c u b a t i o n was always used to ensure complete wall removal.

b) Con A-Haemocyanin Labelling at 18-20 ~ Results a) Protoplasts F i g u r e 1 shows a light m i c r o g r a p h of the p r o t o p l a s t s o b t a i n e d . They r a n g e d f r o m 25 to 70 laM in diameter,

T h e p a t t e r n of labelling was similar to that previously f o u n d in s o y b e a n tissue culture protoplasts (Williamson et al., 1976): Cells fixed before c o n A t r e a t m e n t were evenly covered by h a e m o c y a n i n (Fig. 2). Cells treated with con A a n d h a e m o c y a n i n before fixation

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Fig. 2. Protoplast fixed with glutaraldehyde and blocked with glycine before treatment with con A~H at 20 ~ Haemocyanin is evenly bound to the plasma membrane Fig. 3. Protoplast treated with con A-H at 20 ~ then fixed immediately (45 min after first exposure to con A). Haemocyanin is unevenly distributed Fig. 4. Protoplast treated with con A-H, then incubated at 20 ~ for 1 h before fixation (1 h 45 min exposure to con A). Haemocyanin is tightly clustered (arrows)

and then fixed immediately were unevenly labelled (Fig. 3). If left a further 1 or 2 h before fixation (Fig. 4), the haemocyanin was in tight clusters (arrows) with large areas of bare membrane. After 5 h incubation between labelling and fixation, the amount of haemocyanin (molecules per linear micron per section) on the plasma membrane was greatly reduced. When protoplasts were incubated for 6 h before con A treatment haemocyanin was not bound either to the plasma membrane or to the nascent wall.

c) Con A-Haemocyanin Labelling al 0-4 ~ Cells labelled at low temperature (ice-bath) with con A (1 h) and haemocyanin (1 h) before fixation showed a pattern intermediate between those obtained at 18 20 ~ before and after fixation, i.e. some tight clusters of haemocyanin but no large areas free of label. Prefixed cells labelled at low temperature showed an even distribution, but at lower density than at room temperature.

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Fig. 5. Protoplast treated with con A-F at 20~ before fixation. Ferritin shows a clustered distribution (arrows). (Section not stained; no ruthenium red in osmium) Fig. 6. Protoplast treated with con A-F at 20~ after fixation. Ferritin is evenly distributed. (Section not stained; no ruthenium red in osmium) Fig. 7. Protoplast fixed, treated with cellulases and hemicellulase before treatment with con A-F. Ferritin is evenly bound at slightly lower density than without enzyme treatment (fig. 6). (Section not stained; no ruthenium red in osmium)

d) Con A-Ferritin Labelling at 18-20 ~ T h e p a t t e r n o f l a b e l l i n g was i d e n t i c a l to t h a t o b t a i n e d w i t h c o n A - H : u n f i x e d cells h a d discrete p a t c h e s o f f e r r i t i n ( a r r o w s , Fig. 5); prefixed cells were e v e n l y l a b e l l e d (Fig. 6).

e) Con A Labelling of Prefixed Cells Treated with Wall-Removing Enzymes W h e n p r e f i x e d cells were t r e a t e d for 1 h w i t h 1% O n o z u k a , 1% D r i s e l a s e p l u s 1% R h o z y m e b e f o r e labelling, c o n A - F (Fig. 7) a n d c o n A - H ( n o t s h o w n ) b i n d i n g was

F.A. Williamson: Concanavalin Binding Sites on the Plasma Membrane

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Fig. 8. Protoplast treated with con A-H at 20 ~ in presence of 10 mM La. Haemocyanin appears evenly bound, but is partially obscured by dark deposits of La on the plasma membrane Fig. 9. Protoplast treated with haemocyanin (without con A) in presence of 10 mM La. Haemocyanin is not bound Fig. 10. Protoplast treated with 10 mM La (no haemocyanin or con A). La is deposited on the plasma membrane. (Section not stained) Fig. 11. Protoplast treated with con A-H at 20 ~ in presence of 10 m M La, then washed (1 h) with 10 mM Ca before fixation. La is not deposited; haemocyanin is clustered (arrows)

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diminished by 20-30% (comparing the number of Iigand molecules per linear micron per section).

f) Effect of Lanthanum on Con A Binding Protoplasts labelled with con A-H in the presence of 10 mM La(NO3)3 (in 10 mM HEPES-KOH, pH 6.8, 0.55 M sorbitol) showed an even distribution of haemocyanin similar to that of prefixed cells (Fig. 8). Although some precipitation of haemocyanin occurred in the presence of La, haemocyanin alone (no con A) was not bound to the plasma membrane (Fig. 9). Electron-dense deposits which can obscure or be confused with haemocyanin were found on, the plasma membrane of cells treated with La in the absence of haemocyanin (Fig. 10). Cells labelled with con A-H in the presence of La, but washed (1 h) with buffer containing 10 mM Ca before fixation showed the tight clusters of haemoeyanin, normally seen on unfixed cells (arrows, Fig. 1i). Treatment with con A-ferritin in the presence of La gave ambiguous results because of precipitation of con A-F by La and the obscuring of ferritin by La deposits on the plasma membrane.

Discussion

The stem of leek was found to be a very convenient tissue for the isolation of protoplasts of meristematic cells. In spite of the fact that leeks were purchased from a variety of sources, results obtained with the stem protoplasts were always consistent and reproducible, except during the bolting period (May to June in Paris), when stems were totally unsusceptible to protoplast isolation. A single large leek contains up to 1.5 g stem tissue - ample to enable various treatments to be compared using protoplasts obtained from a single plant. The clustering of labelled con A on animal membranes has been attributed to the cross-linking by the tetravalent con A molecule of binding sites which are mobile in a fluid membrane (Singer and Nicolson, 1972; Nicolson, 1974). The results presented here are consistent with this hypothesis. Fixation before labelling prevents clustering. Labelling at low temperature, while not preventing clustering, appears to slow it down, which may be explained by a reduction of the fluidity of the lipid phase of the membrane. However all the differences between fixed and unfixed cells cannot be explained by this simple clustering hypothesis. In addition to being clustered, the apparent number of sites is also much lower in cells labelled before fixation than in prefixed cells. Haemocyanin

is not bound to prefixed cells in the absence of con A, indicating that any free aldehyde groups left by the glutaraldehyde were effectively blocked by the subsequent glycine treatment. Zones of membrane between agglutinated protoplasts were always more highly labelled than exposed areas of the same cells. These results indicate that labelled con A may be sloughed from the membrane of unfixed cells, or simply not fixed to the plasma membrane during fixation, as proposed by Rowlatt et al. (1973) to explain similar observations on various animal cells. The study of lectin binding to plant protoplasts is complicated by their ability to re-synthesise cell wall materials, a process which may be very rapid (Williamson et al., 1977; Prat and Williamson, 1976). This presents two distinct complications: 1) Rapid secretion of (carbohydrate) wall materials might push con A molecules away from the membrane either mechanically, or by competing for binding sites on the con A. This does not appear to occur in soybean tissue culture cells where haemocyanin remains tightly attached to the plasma membrane for at least 16 h in the presence of very active wall synthesis (Williamson et al., 1976 a). However, on the leek stem protoplasts studied here, there was a considerable loss of label after a 5 h incubation. This is probably due to differences in the type of wall materials secreted. The leek protoplasts appear to synthesize a large amount of amorphous material which completely masks any fibrils seen in metal-shadowed replicas (Prat and Williamson, 1976). 2) The labelling observed may not be due to binding to intrinsic membrane components, but rather to nascent wall materials as advocated by Burgess and Linstead (1976). When leek stem protoplasts were treated with con A after wall resynthesis, no haemocyanin was bound to the wall. However, this may be because of the absence of any groups in the wall which could react with glutaraldehyde to cross-link to the con A/haemocyanin. In this case, the con A would be inactivated by the glutaraldehyde and the con A/haemocyanin lost during subsequent washing. However, the reversible fixation of binding sites by La and by labelling at low temperature described here are more easily explained in terms of membrane fluidity than by a labelling of nascent wall materials. In addition, binding to nascent wall materials presupposes a propensity for rapid wall synthesis. We have removed wall materials from protoplasts under conditions where no re-synthesis is possible, i.e. from fixed (dead) cells, and show only a small reduction of labelling by con A-H or con A-F. Similar conditions pertain in isolated plasma membranes which have also been found to bind con A-H and con A-F (Williamson, Lamant and Roland, in preparation).

F.A. Williamson: Concanavalin Binding Sites on the Plasma Membrane I am grateful to the D.G.R.S.T. for financial support under the programme of "Echanges avec l'6tranger", and by research contract 76 7 1640 from the D.G.R.S.T. to Prof. J.-C. Roland. I wish to thank Prof. Roland and his group, particularly Dr. R, Prat, for their collaboration. I am also very grateful for a grant from the Minister of Science and Higher Education of Iran.

References Burgess, J., Linstead, P.J.: Ultrastructural studies of the binding of Concanavalin A to the plasmalemma of higher plant protoplasts. Planta 130, 73-79 (1976) Burgess, J., Linstead, P.J. : Membrane mobility and the Concanavalin A binding system of higher plant protoplasts. Planta 136, 253-259 (1977) Constabel, F.C.: Isolation and culture of plant protoplasts. In: Plant tissue culture methods. Gamborg O.L. Wetter L.R., eds. Ottawa: National Research Council of Canada (1975) Inbar, M., Ben-Bassat, H., Huet, C., Oseroff, A.R., Sachs, L.: Inhibition of lectin agglutinability by fixation of the cell surface membrane. Biochim. Biophys. Acta 311, 594-599 (1973) Morr6, D.J. : In vivo incorporation of radioactive metabolites by Golgi apparatus and other cell fractions of onion stem. Plant Physiol. 45, 791-799 (I970) Nicolson, G.L.: The interactions of lectins with animal cell surfaces. Int. Rev. Cytol. 39, 89-190 (1974)

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Prat, R., Williamson, F.A.: Chronologie de la s6cr6tion de parois par les protoplastes v6g6taux. Soc. bot. Fr., Coll. S)cr~t. V~g6t., 123, 33-45 (1976) Reynolds, E.S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Rowlatt, Ch., Wicker, R., Bernhard, W. : Ultrastructural distribution of concanavalin A receptors on hamster embryo and adenovirustumour cell culture. Int. J. Cancer 11,314 326 (1973) Singer, S.J., Nicolson, G.L. : The fluid mosaic model of the structure of cell membranes. Science 175, 720-731 (1972) Williamson, F.A., Fowke, L.C., Constabel, F.C., Gamborg, O.L.: Localization of carbohydrates on the plasma membrane of plant protoplasts with concanavalin A and haemocyanin. Plant Physiol. 56, 88 (suppl.) (1975) Williamson, F.A. Fowke, L.C., Constabel, F.C., Gamborg, O.L.: Labelling of Concanavalin A sites on the plasma membrane of soybean protoplasts. Protoplasma 89, 305-316 (1976 a) WilIiamson, F.A., Fowke, L.C., Weber, G., Constabel, F., Gainborg, O. : Microfibril deposition on cultured protoplasts of Vicia hajastana. Protoplasma 91, 213~19 (1977) Williamson, F.A., Morr6, D.J., Shen-Miller, J.: Inhibition of 5'nucleotidase by concanavalin A: evidence for localization on the outer surface of the plasma membrane. Cell Tissue Res. 70, 477484 (I976b)

Received 21 March; accepted 26 June 197.8

Concanavalin A binding sites on the plasma membrane of leek stem protoplasts.

The binding of concanavalin A (con A) to leek (Allium porrum L.) stem protoplasts has been investigated using sequential treatment with con A and haem...
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