9 Springer-Verlag 1981
Concanavalin A binds to the endoplasmic reticulum and the starch grain surface of root statocytes E. Marion Schneider and Andreas Sievers Botanisches Institut, Universit~.tBonn, Venusbergweg22, D-5300 Bonn, Federal Republic of Germany
Abstract. Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the anayloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of m e m b r a n e components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the m e m b r a n e is a glycoprotein tightly inserted in other components of the compartment. Key words: Concanavalin receptors - Endoplasmic reticulum - Lepidium - Statocytes.
affinity purification of molecules with alpha-D-glycosyl- or alpha-D-mannosyl-residues (Bittiger and Schnebli 1976). Histological studies mostly deal with receptors present at the surface of single cell suspensions (de Petris and R a f t 1974). A prerequisite for the localization of intracellular receptors by microscopical methods is a fixation procedure which immobilizes the molecules and makes them accessible to the staining reagents. Chemical fixatives, such as aldehydes, preserve cell structures, but further treatment is required to improve membrane permeability (Laurila et al. 1978). In order to obtain more information about the gravity perceiving cell structureg (Schneider et al. 1981) in root statocytes, we used aldehyde-fixed tissue samples for the intracellular identification of Concanavalin A receptor molecules in the sections and followed the results after treatment with solubilizing agents. As Lepidium statocytes have been extensively studied by electron microscopy (Volkmann and Sievers 1979), we could correlate light microscopical localizations with certain cell structures due to the polarity of the statocytes.
Introduction Materials and methods Lectins exhibiting various specificities for the sugar component of cellular protein and lipid substances have been characterized and purified (Lis and Sharon 1973). They are predominantly found in m a n y leguminous seeds but also in some animals such as Helix pomatia and Limuluspolyphenus. Because of their hemagglutinative properties lectins have obtained wide application in the functional evaluation of lymphocyte subpopulations after in vitro stimulation (Shou et al. 1976; Nowell 1960). Concanavalin A has become an important tool for the characterization and Abbreviations: Con A = Concanavalin A ; ER = endoplasmic reticulure; FITC=fluorescein isothiocyanate; NP 40=nonidet P40
Preparation of plant material. Roots of Lepidium sativum L. were normally grown for 24 h in the dark (control) or centrifuged for the last 20 rain at 1,000 g (Caspers and Sievers 1979) and then cut off of the seeds and immediately immersed in the fixatives (2% glutaraldehyde or 4% paraformaldehyde, in 0.1 M sodium phosphate buffer, pH 6.8). Fixation for 2 h, subsequent washing in phosphate buffer for 2 h, and dehydration in a series of alcohols (15, 30, 50, 70, 90, 100%, 15min each) were performed in an ice bath. The plant material was finally embedded in paraffin after not more than 24 h of infiltration. Longitudinal and transverse sections (2 gm thick) were stretched on poly-L-Iysine-coatedslides (0.05% poly-L-lysine, Sigma Biochem., FRG, MW 400,000). The paraffin was removed by 6-rain washing in xylene. Rehydration of the sections through a series of aIcohol was achieved within 5 rain.
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Fig. 1. Longitudinal section of the root cap after glutaraldehyde fixation and staining with FITC-Con A (2 gm thick, 0.01 mg ml- 1 FITC-Con A in sodium phosphate buffer, 0.05 M, pH 6.8). The nuclei (n, not sectioned in all the cells) located in the apical part of the statocytes are stained, as well as the distal complex of the endoplasmic reticulum (er). Amyloplasts are negative, lying as dark spots (a) within the fluoresceing matrix of the ER. x 373 Fig. 2. As Fig. 1 ; however, centrifuged at 1,000 g for 20 rain before fixation. The ER (er) is here situated in stacks above the amyloplasts (a) and again decorated with FITC-Con A. Fluorescence is very prominent where the cisternae are densely piled. Amyloplasts did not bind the marker molecule as in Fig. 1. x 373
Fluorescence labelingof Con A binding sites. FITC-conjugated Concanavalin A (FITC-Con A, Sigma Biochem., FRG) was used at a concentration of 0.01 mgm1-1 in 0.05 M sodium phosphate buffer, pH 6.8; 100 ~L1were used for one slide and incubation occurred for 30 to 40 min at room temperature. After 3 washes in buffer the sections were examined in a SM Lux Leitz fluorescence microscope equipped with an epi-illuminator.
Treatment with non-ionic detergents. Either before or after the staining with FITC-Con A comparative sections were incubated in different dilutions of detergents. We used NP 40 (Sigma Biochem., FRG) and Triton X 100 (Serva, FRG) in concentrations of 0.05, 0.1, and 1% in sodium phosphate buffer, pH 6.8. The sections were incubated at room temperature for 30, 60, and 120 min or up to 6 h.
E.M. Schneider and A. Sievers: Con A binding sites in root statocytes
Fig. 3. As Fig. 1 ; however, fixed in formaldehyde. All the cellular components are negative in Con A-binding, except the surface of the starch grains (s) within the amyloplasts, x 373
Fig. 4. As Fig. 1; however, treated with 1% NP 40 in phosphate buffer, 0.1 M, pH 6.8, for 30 min before staining with FITC-Con A. Statocytes with fluoresceing endoplasmic reticulum (er) and positive starch grains (s) can be identified (n, nucleus), x 373 Bars: 20 ~m
Results
Con A receptor sites in glutaraldehyde-fixed statocytes. R o o t s t a t o c y t e s o f Lepidium sativum s h o w a h i g h l y r e g u l a r cell p o l a r i t y , as p r o v e n b y u l t r a s t r u c t u r a l inv e s t i g a t i o n s ( V o l k m a n n a n d S i e v e r s 1979). A s w e u s e d plant material grown under standardized conditions, l i g h t m i c r o s c o p i c a l f i n d i n g s w e r e c o r r e l a t e d w i t h ult r a s t r u c t u r a l l y - d e f i n e d cell c o m p a r t m e n t s . L o n g i t u d i nal sections of glutaraldehyde-fixed root caps were incubated in FITC-Con A and showed positive fluorescence of the nucleus and the distal region of the s t a t o c y t e s below t h e a m y l o p l a s t s (Fig. 1). A s w e u s e d
E.M. Schneiderand A. Sievers: Con A binding sites in root statocytes rather thin sections (2 lain) the nucleus is not sectioned in all the cells. Further identification of the Con Apositive structure below the amyloplasts was achieved by studying identically processed roots after shorttime centrifugation (1,000 g for 20 rain). Here, the statocytes show positive fluorescence linearly above the amyloplasts (Fig. 2). After centrifugation, the distal complex of the endoplasmic reticulum is displaced and found as a more or less round-shaped complex or in linear piles above the amyloplasts (Caspers and Sievers 1979; cf. Fig. 7: Volkmann and Sievers 1979). Intense fluorescence in the statocytes indicates regions of densely stacked ER cisternae in contrast to less fluorescence where the membranes are lessgstacked (Fig. 27. We proved this by serial sectioning.
Con A receptor sites in formaldehyde-fixed statocytes. Lepidium roots fixed in formaldehyde and further treated like the glutaraldehyde-fixed ones bind Con A at the surface of the starch grains within the amyloplasts only (Fig. 3). The nucleus as well as the endoplasmic reticulum do not bind Con A. Con A binding after treatment with non-ionic detergents. Changes of receptor activity in Lepidium roots were further studied after treatment with various concentrations of Triton X 100 and NP 40. The two detergents gave similar results in that concentrations up to 1% and incubation for less than 30 rain did not alter the binding specificities as described above. One % NP 40 applied to the sections for 30 rain or longer gave the most reproducible changes. The detergent incubation was made before staining with FITCCon A or after the test, but was then followed by three washes in buffer and another incubation in FITC-Con A. Glutaraldehyde-fixed specimens revealed the Con A receptor at the surface of the starch grains in addition to the positively stained nucleus and the distal complex of the endoplasmic reticulum (Fig. 4). The staining pattern in formaldehyde-fixed samples could not be altered by detergent treatment. Discussion
Phycical and chemical properties of Con A are generally well understood (Edelman et al. 1972). The lectin has been purified from Canavalia ensiformis seeds. Until now the physiological role of the lectin in the plant has remained unresolved (Lis and Sharon 1973). Lectins have been considered to act as attachment sites for glycoprotein enzymes, as mediators of sugar transport or - in view of their mitogenic properties
179 - as trigger molecules for certain cell functions, i.e., cell division or differentiation (Sharon 1974). The lectin-binding molecules are polysaccharide chains, glycopeptides, glycoproteins, and glycolipids. With the advances of biochemistry it has become evident that the majority of cellular proteins in plants are glycoproteins (Sharon 1974). The preparation of our plant material includes several steps, where cellular components can be removed, i.e., dehydration in alcohol and xylene washing. We are aware of the fact that only structurally bound receptors are preserved in the cells. The use of rather thin sections and previous fixation of the specimen confirms accessibility and preservation of the receptors. Using glutaraldehyde-fixed roots, receptors for Con A were found at the nucleus and the distal complex of the endoplasmic reticulum in the statocytes (Fig. 1 and 2). In terms of the nucleus we obtained evidence from experiments not shown here that this receptor is also membrane-bound. The high binding capacity of Concanavalin A to the ER in root statocytes probably is a special property of the compartment in these cells and cannot be solely explained by the fact that the cisternae are stacked below the sedimented amyloplasts, as documented by electron microscopy (Sievers and Volkmann 1972). A comparison with similarly prepared human fibrocytes, in which the endoplasmic reticulum is highly elaborated, showed that the fluorescence of the ER after FITC-Con A labeling is much weaker than in Lepidium statocytes. Until now the function of the distal complex of rough endoplasmic reticulum in the central calyptra has remained a matter of discussion. It cannot be related to the secretory activity of the root cap. Morr6 et al. (1967) as well as Volkmann (1981) have shown that polysaccharide-secretion is limited to the very distal cell-layer of the root cap, and that it is ultrastructurally marked by hypertrophic dictyosome cisternae. The latter parameter, in particular, has not been verified for the statocytes of Lepidium (Volkmann and Sievers 1979). In the case of animal cells, different experiments strongly suggest that the rough endoplasmic reticulum is not solely active in protein synthesis but is also involved in the glycosylation of the polypeptide chains (Katz et al. 1977; Toneguzzo and Ghosh 1978). Katz even proposed that the protein is inserted through the membrane and that its carbohydrate chain is located within the cisternal space (Katz et al. 1977). Transfer of carbohydrate occurs via lipid-linked oligosaccharide (Struck and Lennarz 1980). In conclusion: If we accept that protein synthesis and mechanisms of glycosylation in the plant are analoguous to those described for animal cells, it seems
180
reasonable to find Con A receptor molecules in the endoplasmic reticulum. The extremely high amount of receptors, detected in statocytes, however, remains unresolved; it is probably due to the special function of this cell type. Different physiological studies of Lepidium root statocytes suggest that these cells are involved in graviperception (Volkmann and Sievers 1979). Changes of the g-vector result in the curvature of the root only if the statocytes are structurally organized, i.e., if sedimenting amyloplasts and underlying stacks of the rough endoplasmic reticulum are present (Sievers and Volkmann 1972). As we propose communication mechanisms between the ER and the amyloplast envelope, the characterization of membrane components is an important approach for the elucidation of graviperception. Glutaraldehyde fixation predominantly crosslinks proteins in menbranes, while the lipid components should be more or less extractable during alcohol and xylene treatment. The Con A binding activity, however, resisted these steps, as our results have proven. Non-ionic detergents, like NP 40 or Triton X 100, are rather well understood in terms of their solubilizing effect on glycoprotein and proteinlipid complexes (Helenius and Simons 1975; Pearlstein and Seaver 1976). Incubation in a rather high detergent concentration did not affect Con A binding at the endoplasmic reticulum (Fig. 4). The fact that membrane components were solubilized by this procedure is documented by the fact that the surface of the starch grains in the amyloplasts turned positive after FITCCon A treatment of the detergent step. The starch grains were also positive, if we used a formaldehydefixed specimen (Fig. 3), where membrane-associated Con A receptors could not be preserved. From these results we conclude that the receptor of the distal complex of the endoplasmic reticulum in statocytes of Lepidium roots is a large glycoprotein complex resistant to solubilizing organic solvents and non-ionic detergents after glutaraldehyde fixation.
E.M. Schneider and A. Sievers: Con A binding sites in root statocytes
References
by centrifugation and subsequent regeneration of cell polarity : influence on graviresponse. Eur. J. Cell Biol. 20, 119 Edelman, G.M., Cunningham, B.A., Reeke, G.N. jr., Becker, J.W., Waxdal, M.J., Wang, J.L. (1972) The covalent and three dimensional structure of concanavalin A. Proc. Natl. Acad. Sci. USA 69, 2580-2584 Helenius, A., Simons, K. (1975) Solubilization of membranes by detergents. Biochim. Biophys. Acta 415, 29-79 Katz, F.N., Rothman, J.E., Lingappa, V.R., Blobel, G., Lodish, H.F. (1977) Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc. Natl. Acad. Sci. USA 74, 3278-3282 Laurila, P., Virtanen, I., Wartiovaara, J., Stenman, S. (1978) Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with nonionic detergent. J. Histochem. Cytochem. 26, 251-257 Lis, H., Sharon, N. (1973) The biochemistry of plant lectins (phytohemagglutinins). Annu. Rev. Biochem. 42, 541-574 Morr6, D.J., Jones, D.D., Mollenhauer, H.H. (1967) Golgi apparatus mediated polysaccharide secretion by outer root cap cells of Zea mays. I. Kinetics and secretory pathway. Planta 74, 286-301 Nowell, P. (1960) Phytohemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20, 462-466 Pearlstein, E., Seaver, J. (1976) Non@tic, non-ionic detergent extraction of plasma membrane constituents from normal and transformed fibroblasts. Biochim. Biophys. Acta 426, 589-597 Petris, de, S., Raft, M.C. (1974) Ultrastructural distribution and redistribution of alloantigens and concanavalin A receptors on the surface of mouse lymphocytes. Eur. J. Immunol. 4, 130-137 Schneider, E.M., Becker, J.U., Volkmann, D. (1981) Biochemical properties of potato phosphorylase change with its intracetlular localization as revealed by immunological methods. Planta 151, 124-134 Sharon, N. (1974) Glycoproteins of higher plants. In: Plant carbohydrate biochemistry, pp. 235-252, Pridham, J.B., ed., Academic Press, London New York Shou, L., Schwartz, S.A., Good, R.A. (1976) Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors. J. Exp. Med. 143, 1100-1110 Sievers, A., Volkmann, D. (1972) Verursacht differentieller Druck der Amyloplasten auf ein komplexes Endomembransystem die Geoperzeption in Wurzeln? Planta 102, 162-172 Struck, D.K., Lennarz, W.J. (1980) The function of saccharidelipids in synthesis of glycoproteins. In: The biochemistry of glycoproteins and proteoglycans, pp. 35 83, Lennarz, W.J., ed., Plenum Press, New York London Toneguzzo, F., Ghosh, H.P. (1978) In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes. Proc. Natl. Acad. Sci. USA 75, 715 719 Volkmann, D. (1981) Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.). Planta 151, 180-188 Volkmann, D., Sievers, A. (1979) Graviperception in multicellular organs. In: Encyclopedia of plant physiology, vol. 7, pp. 573600, Haupt, W., Feinleib, M.E., eds., Springer, Berlin Heidelberg New York
Bittiger, H., Schnebli, H.P. (1976) Concanavalin A as a tool. John Wiley and Sons, London New York Sydney Toronto Caspers, L., Sievers, A. (1979) Stratification of root statocytes
Received 26 August; accepted 28 November 1980
We are greatly indepted to Prof. Dr. I. Stroehmann from the Medical Department for helpful discussions. This study was supported by the Deutsche Forschungsgemeinschaft.
Concanavalin A binds to the endoplasmic reticulum and the starch grain surface of root statocytes E. Marion Schneider and Andreas Sievers Botanisches Institut, Universit~.tBonn, Venusbergweg22, D-5300 Bonn, Federal Republic of Germany
Abstract. Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the anayloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of m e m b r a n e components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the m e m b r a n e is a glycoprotein tightly inserted in other components of the compartment. Key words: Concanavalin receptors - Endoplasmic reticulum - Lepidium - Statocytes.
affinity purification of molecules with alpha-D-glycosyl- or alpha-D-mannosyl-residues (Bittiger and Schnebli 1976). Histological studies mostly deal with receptors present at the surface of single cell suspensions (de Petris and R a f t 1974). A prerequisite for the localization of intracellular receptors by microscopical methods is a fixation procedure which immobilizes the molecules and makes them accessible to the staining reagents. Chemical fixatives, such as aldehydes, preserve cell structures, but further treatment is required to improve membrane permeability (Laurila et al. 1978). In order to obtain more information about the gravity perceiving cell structureg (Schneider et al. 1981) in root statocytes, we used aldehyde-fixed tissue samples for the intracellular identification of Concanavalin A receptor molecules in the sections and followed the results after treatment with solubilizing agents. As Lepidium statocytes have been extensively studied by electron microscopy (Volkmann and Sievers 1979), we could correlate light microscopical localizations with certain cell structures due to the polarity of the statocytes.
Introduction Materials and methods Lectins exhibiting various specificities for the sugar component of cellular protein and lipid substances have been characterized and purified (Lis and Sharon 1973). They are predominantly found in m a n y leguminous seeds but also in some animals such as Helix pomatia and Limuluspolyphenus. Because of their hemagglutinative properties lectins have obtained wide application in the functional evaluation of lymphocyte subpopulations after in vitro stimulation (Shou et al. 1976; Nowell 1960). Concanavalin A has become an important tool for the characterization and Abbreviations: Con A = Concanavalin A ; ER = endoplasmic reticulure; FITC=fluorescein isothiocyanate; NP 40=nonidet P40
Preparation of plant material. Roots of Lepidium sativum L. were normally grown for 24 h in the dark (control) or centrifuged for the last 20 rain at 1,000 g (Caspers and Sievers 1979) and then cut off of the seeds and immediately immersed in the fixatives (2% glutaraldehyde or 4% paraformaldehyde, in 0.1 M sodium phosphate buffer, pH 6.8). Fixation for 2 h, subsequent washing in phosphate buffer for 2 h, and dehydration in a series of alcohols (15, 30, 50, 70, 90, 100%, 15min each) were performed in an ice bath. The plant material was finally embedded in paraffin after not more than 24 h of infiltration. Longitudinal and transverse sections (2 gm thick) were stretched on poly-L-Iysine-coatedslides (0.05% poly-L-lysine, Sigma Biochem., FRG, MW 400,000). The paraffin was removed by 6-rain washing in xylene. Rehydration of the sections through a series of aIcohol was achieved within 5 rain.
0032-093 5/81/0152/0177/$01.00
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Fig. 1. Longitudinal section of the root cap after glutaraldehyde fixation and staining with FITC-Con A (2 gm thick, 0.01 mg ml- 1 FITC-Con A in sodium phosphate buffer, 0.05 M, pH 6.8). The nuclei (n, not sectioned in all the cells) located in the apical part of the statocytes are stained, as well as the distal complex of the endoplasmic reticulum (er). Amyloplasts are negative, lying as dark spots (a) within the fluoresceing matrix of the ER. x 373 Fig. 2. As Fig. 1 ; however, centrifuged at 1,000 g for 20 rain before fixation. The ER (er) is here situated in stacks above the amyloplasts (a) and again decorated with FITC-Con A. Fluorescence is very prominent where the cisternae are densely piled. Amyloplasts did not bind the marker molecule as in Fig. 1. x 373
Fluorescence labelingof Con A binding sites. FITC-conjugated Concanavalin A (FITC-Con A, Sigma Biochem., FRG) was used at a concentration of 0.01 mgm1-1 in 0.05 M sodium phosphate buffer, pH 6.8; 100 ~L1were used for one slide and incubation occurred for 30 to 40 min at room temperature. After 3 washes in buffer the sections were examined in a SM Lux Leitz fluorescence microscope equipped with an epi-illuminator.
Treatment with non-ionic detergents. Either before or after the staining with FITC-Con A comparative sections were incubated in different dilutions of detergents. We used NP 40 (Sigma Biochem., FRG) and Triton X 100 (Serva, FRG) in concentrations of 0.05, 0.1, and 1% in sodium phosphate buffer, pH 6.8. The sections were incubated at room temperature for 30, 60, and 120 min or up to 6 h.
E.M. Schneider and A. Sievers: Con A binding sites in root statocytes
Fig. 3. As Fig. 1 ; however, fixed in formaldehyde. All the cellular components are negative in Con A-binding, except the surface of the starch grains (s) within the amyloplasts, x 373
Fig. 4. As Fig. 1; however, treated with 1% NP 40 in phosphate buffer, 0.1 M, pH 6.8, for 30 min before staining with FITC-Con A. Statocytes with fluoresceing endoplasmic reticulum (er) and positive starch grains (s) can be identified (n, nucleus), x 373 Bars: 20 ~m
Results
Con A receptor sites in glutaraldehyde-fixed statocytes. R o o t s t a t o c y t e s o f Lepidium sativum s h o w a h i g h l y r e g u l a r cell p o l a r i t y , as p r o v e n b y u l t r a s t r u c t u r a l inv e s t i g a t i o n s ( V o l k m a n n a n d S i e v e r s 1979). A s w e u s e d plant material grown under standardized conditions, l i g h t m i c r o s c o p i c a l f i n d i n g s w e r e c o r r e l a t e d w i t h ult r a s t r u c t u r a l l y - d e f i n e d cell c o m p a r t m e n t s . L o n g i t u d i nal sections of glutaraldehyde-fixed root caps were incubated in FITC-Con A and showed positive fluorescence of the nucleus and the distal region of the s t a t o c y t e s below t h e a m y l o p l a s t s (Fig. 1). A s w e u s e d
E.M. Schneiderand A. Sievers: Con A binding sites in root statocytes rather thin sections (2 lain) the nucleus is not sectioned in all the cells. Further identification of the Con Apositive structure below the amyloplasts was achieved by studying identically processed roots after shorttime centrifugation (1,000 g for 20 rain). Here, the statocytes show positive fluorescence linearly above the amyloplasts (Fig. 2). After centrifugation, the distal complex of the endoplasmic reticulum is displaced and found as a more or less round-shaped complex or in linear piles above the amyloplasts (Caspers and Sievers 1979; cf. Fig. 7: Volkmann and Sievers 1979). Intense fluorescence in the statocytes indicates regions of densely stacked ER cisternae in contrast to less fluorescence where the membranes are lessgstacked (Fig. 27. We proved this by serial sectioning.
Con A receptor sites in formaldehyde-fixed statocytes. Lepidium roots fixed in formaldehyde and further treated like the glutaraldehyde-fixed ones bind Con A at the surface of the starch grains within the amyloplasts only (Fig. 3). The nucleus as well as the endoplasmic reticulum do not bind Con A. Con A binding after treatment with non-ionic detergents. Changes of receptor activity in Lepidium roots were further studied after treatment with various concentrations of Triton X 100 and NP 40. The two detergents gave similar results in that concentrations up to 1% and incubation for less than 30 rain did not alter the binding specificities as described above. One % NP 40 applied to the sections for 30 rain or longer gave the most reproducible changes. The detergent incubation was made before staining with FITCCon A or after the test, but was then followed by three washes in buffer and another incubation in FITC-Con A. Glutaraldehyde-fixed specimens revealed the Con A receptor at the surface of the starch grains in addition to the positively stained nucleus and the distal complex of the endoplasmic reticulum (Fig. 4). The staining pattern in formaldehyde-fixed samples could not be altered by detergent treatment. Discussion
Phycical and chemical properties of Con A are generally well understood (Edelman et al. 1972). The lectin has been purified from Canavalia ensiformis seeds. Until now the physiological role of the lectin in the plant has remained unresolved (Lis and Sharon 1973). Lectins have been considered to act as attachment sites for glycoprotein enzymes, as mediators of sugar transport or - in view of their mitogenic properties
179 - as trigger molecules for certain cell functions, i.e., cell division or differentiation (Sharon 1974). The lectin-binding molecules are polysaccharide chains, glycopeptides, glycoproteins, and glycolipids. With the advances of biochemistry it has become evident that the majority of cellular proteins in plants are glycoproteins (Sharon 1974). The preparation of our plant material includes several steps, where cellular components can be removed, i.e., dehydration in alcohol and xylene washing. We are aware of the fact that only structurally bound receptors are preserved in the cells. The use of rather thin sections and previous fixation of the specimen confirms accessibility and preservation of the receptors. Using glutaraldehyde-fixed roots, receptors for Con A were found at the nucleus and the distal complex of the endoplasmic reticulum in the statocytes (Fig. 1 and 2). In terms of the nucleus we obtained evidence from experiments not shown here that this receptor is also membrane-bound. The high binding capacity of Concanavalin A to the ER in root statocytes probably is a special property of the compartment in these cells and cannot be solely explained by the fact that the cisternae are stacked below the sedimented amyloplasts, as documented by electron microscopy (Sievers and Volkmann 1972). A comparison with similarly prepared human fibrocytes, in which the endoplasmic reticulum is highly elaborated, showed that the fluorescence of the ER after FITC-Con A labeling is much weaker than in Lepidium statocytes. Until now the function of the distal complex of rough endoplasmic reticulum in the central calyptra has remained a matter of discussion. It cannot be related to the secretory activity of the root cap. Morr6 et al. (1967) as well as Volkmann (1981) have shown that polysaccharide-secretion is limited to the very distal cell-layer of the root cap, and that it is ultrastructurally marked by hypertrophic dictyosome cisternae. The latter parameter, in particular, has not been verified for the statocytes of Lepidium (Volkmann and Sievers 1979). In the case of animal cells, different experiments strongly suggest that the rough endoplasmic reticulum is not solely active in protein synthesis but is also involved in the glycosylation of the polypeptide chains (Katz et al. 1977; Toneguzzo and Ghosh 1978). Katz even proposed that the protein is inserted through the membrane and that its carbohydrate chain is located within the cisternal space (Katz et al. 1977). Transfer of carbohydrate occurs via lipid-linked oligosaccharide (Struck and Lennarz 1980). In conclusion: If we accept that protein synthesis and mechanisms of glycosylation in the plant are analoguous to those described for animal cells, it seems
180
reasonable to find Con A receptor molecules in the endoplasmic reticulum. The extremely high amount of receptors, detected in statocytes, however, remains unresolved; it is probably due to the special function of this cell type. Different physiological studies of Lepidium root statocytes suggest that these cells are involved in graviperception (Volkmann and Sievers 1979). Changes of the g-vector result in the curvature of the root only if the statocytes are structurally organized, i.e., if sedimenting amyloplasts and underlying stacks of the rough endoplasmic reticulum are present (Sievers and Volkmann 1972). As we propose communication mechanisms between the ER and the amyloplast envelope, the characterization of membrane components is an important approach for the elucidation of graviperception. Glutaraldehyde fixation predominantly crosslinks proteins in menbranes, while the lipid components should be more or less extractable during alcohol and xylene treatment. The Con A binding activity, however, resisted these steps, as our results have proven. Non-ionic detergents, like NP 40 or Triton X 100, are rather well understood in terms of their solubilizing effect on glycoprotein and proteinlipid complexes (Helenius and Simons 1975; Pearlstein and Seaver 1976). Incubation in a rather high detergent concentration did not affect Con A binding at the endoplasmic reticulum (Fig. 4). The fact that membrane components were solubilized by this procedure is documented by the fact that the surface of the starch grains in the amyloplasts turned positive after FITCCon A treatment of the detergent step. The starch grains were also positive, if we used a formaldehydefixed specimen (Fig. 3), where membrane-associated Con A receptors could not be preserved. From these results we conclude that the receptor of the distal complex of the endoplasmic reticulum in statocytes of Lepidium roots is a large glycoprotein complex resistant to solubilizing organic solvents and non-ionic detergents after glutaraldehyde fixation.
E.M. Schneider and A. Sievers: Con A binding sites in root statocytes
References
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Received 26 August; accepted 28 November 1980
We are greatly indepted to Prof. Dr. I. Stroehmann from the Medical Department for helpful discussions. This study was supported by the Deutsche Forschungsgemeinschaft.
