Planta
Planta 151, 180 188 (1981)
9 Springer-Verlag 1981
Structural Differentiation of Membranes Involved in the Secretion of Polysaccharide Slime by Root Cap Cells of Cress (Lepidium sativum L.) Dieter Volkmann Botanisches Institut der Universit/it Bonn, Venusbergweg 22, D-5300 Bonn, Federal Republic of Germany
Abstract. The peripheral secretion tissue of the root cap of Lepidiurn sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins- recognizable as IMPs - are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome Abbreviations: PF=protoplasmic fracture face; EF=exoplasmic
fracture face; IMP =intramembranous particle
0032-0935/81/0151/0180/$1.80
membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.
Key words: Exocytosis - Golgi membranes- L e p i d u m Membrane transformation - Plasma membrane - Recycling (membranes) - Secretion (slime).
Introduction The peripheral cells of the root cap produce polysaccharide slime, which is exported from the cell by exocytosis of Golgi vesicles (Mollenhauer et al. 1961; Northcote and Pickett-Heaps 1966; Morr6 etal. 1967). Whereas the physiology of the secretion process has been investigated in detail (Ltittge and Schnepf 1976), many problems regarding the cellular events, which occur during the exocytotic process, remain to be solved (Whaley and Dauwalder 1979): formation of vesicles at the cisternae of the dictyosomes; directed transport of vesicles to the plasma membrane; fusion of the vesicle membranes with the plasmalemma and incorporation into the same; extrusion of the vesicle content by fission of the fusion sites; and recycling of membrane constituents. On the basis of morphological, histochemical, and biochemical investigations, the Golgi apparatus is discussed as being an organelle of membrane differentiation and transformation (Mollenhauer and Morr6 1966; Grove et al. 1968; Schnepf 1969; Sievers 1973; Mollenhauer et al. 1976; Morr6 and Ovtracht 1977; Whaley and Dauwalder 1979). Starting from the forming face, the thickness of membranes and the uptake of staining material increase across the stack of dictyosome cisternae. The membrane of Golgi vesi-
D. Volkmann: Membrane Differentiation During Secretion
cles becomes increasingly similar in structure to the plasma membrane (Whaley 1966; Sievers 1967; Grove etal. 1968; Kiermayer 1970; Mollenhauer etal. 1976). By freeze-fracturing, Staehelin and Kiermayer (1970) showed that membrane differentiation occurs across the stacked membranes as well as within single cisternae. In connection with exocytotic processes in animals (for literature see Chailley 1979) and lower plants (Robinson and Preston 1971 ; Kiermayer and Staehelin 1972; Kiermayer and Dobberstein 1973; Peng and Jaffe 1976; Pinto da Silva and Nougeira 1977; Kiermayer and Sleytr 1979; Giddings et al. 1980), structural differentiation of the plasma membrane is a well recognized phenomenon, even if the existence of some structures observed is questioned (Chandler and Heuser 1979 and 1980). For higher plants, however, there are only a few data resulting from the early beginning of freeze-fracturing. Branton and Moor (1964) as well as Northcote and Lewis (1968) observed structural changes of the plasma membrane caused by exocytosis of peripheral vesicles in cells of the root tip. This communication deals with the differentiation of both the Golgi membranes and the plasmalemma in root cap cells during the granulocrine secretion of polysaccharide slime. Material and Methods Dry seeds of Lepidium sativum L. were soaked in tap water for 30 rain. The seeds were germinated on moist filter paper in saturated humidity at 24_+2 ~ C in the dark. Seedlings with radicles of the same length were fixed 28 h after the beginning of soaking.
TEM: Fixation was carried out in 3% glutaraldehyde buffered in 0.05 M phosphate buffer, pH 7.2, for 2 h at 20 ~ C. Postfixation was done in 2% osmium tetroxide in the same buffer solution for 2 h. Dehydration was done in an aceton series. Electronmicroscopy was carried out at 60 kV with a Zeiss EM 10. Freeze-fracturing: Prefixation was carried out in the same way as fixation for TEM, but only for 30 min. Cryoprotection was done in 10, 20, and 30% glycerin, each step for 20 min. Unfixed material was incubated for less than 3 s in 0.3 M sucrose or in 30% glycerin. Root tips with a maximum length of 1 m m were frozen in melting nitrogen. Freeze-fracturing was performed with Balzers BAF 300 according to Moor and Mfihlethaler (1963).
Cleaning of the Replicae." 24 h in chromic acid (20% for 2 h, 40% for 22 h); 48 h in sodium hypochlorite (5% for 2 h, 10% for 46 h). Electronmicroscopy was done as described above. The nomenclature of freeze-fractures follows Branton et al. (1975).
Results
At the investigated stage of development, the peripheral secretion tissue of Lepidium is formed by one
181
Fig. 1. Cross section through the root cap of cress (Lepidium satirum). Slime is visible at the periphery of the cap and in the distal region 4Mike region - of the anticlinal cell walls (arrow) of the peripheral cell layer. Amyloplasts (A) can be seen in central and peripheral cells. Section was stained with toluidin-blue. Bar 10 gm
cell layer (Fig. 1). Slime is located at the periphery of the root cap and in the distal region of the anticlinal cell walls (arrow) of the outer cell layer. With the extrusion of slime the anticlinals assume a qb-like shape. It was confirmed by lightmicroscopy of untreated material that this shape really exists and that it is not produced by swelling of the slime during the fixation process, when cells lose their turgor pressure. The dictyosomes of this developmental stage are extremely hypertrophied. Great parts of the cisternae are often dilated (Fig. 2). In this case the shape of the cisternae changes from flattened to spherical across the membrane stack. Besides the slime-transporting vesicles near the dictyosome cisternae at least two types of small vesicles are visible. The bigger one (large arrows), the diameter of which is approx. 90 nm, possesses an electronopaque centre. The diameter of the smaller one is approx. 40 nm (small arrows). The low diameter causes a dark and homogenous staining of the vesicles. Slime-transporting vesicles are visible particularly in the phi-like region and to a lesser degree in the region of the outer wall. The vesicle diameter is approx. 600 nm. The plasma membrane reveals an undulating course. It is vaulted
t82
D, Volkmann: Membrane Differentiation During Secretion
Fig. 2. qS-like region (compare Fig. 1) of a peripheral cell. Dictyosomes (D) are hypertrophied. Net-like slime (S) is located in the budding region (BR) of the dictyosomecisternae, in migrating vesicles (SV), and on both sides of the wall. Two types of small vesicles are visible near cisternae of dictyosomes. The smaller one (small arrows) is homogenously contrasted, the other one (large arrows) reveals an electronopaque centre. The plasmalemma (PL) shows an undulating course by slime (5) being extruded into the cell wall (CPO and by vesicles (SV) underlying the membrane. Cisternae of rough endoplasmic reticulum (ER) run parallel to the plasmalemma. Bar 1 gm
by vesicles adjacent to the plasmalemma in the direction of exocytosis. By extruded slime, however, it is vaulted against the direction of exocytosis. Often the original shape of the vesicle is nearly preserved. The characteristic net-like slime is visible in the region of dilated dictyosome cisternae, in migrating vesicles, and outside the cell on both sides of the wall. Often two or more cisternae of rough endoplasmic reticulum run parallel to the plasma membrane. When the plasma membrane is fractured in the phi-like region (Figs. 3a, b), the protoplasmic fracture face (PF) reveals differences in the distribution of intramembranous particles (IMPs), in comparison
with other cell types (data not shown). Large areas showing homogenously distributed IMPs are interrupted by small areas poor in particles. These areas are of different size; their diameter does not exceed 800 nm and their shape is mostly elipsoid or circular. Sometimes the areas are vaulted into the direction of exocytosis. In this case they are nearly free of IMPs. In order to exclude artificial structures which may result from prefixation or intensive cryoprotection (Chandler and Heuser 1979; Hasty and Hay 1978), freeze-fracturing was performed with unfixed material. Sucrose (0.3 M) or glycerin (30%) was used for less than three seconds. Figure 3 b shows the typi-
D. Volkmann: Membrane Differentiation During Secretion
183
Figs. 3a, b. Protoplasmic fracture face (PF) of the plasma membrane in the qS-like region. Large areas with homogenously distributed intramembranous particles (IMPs) are interrupted by areas poor in particles. The areas are of different size, their diameter does not exceed 800 nm. With prefixed material (a) the areas are often vaulted in the direction of exocytosis (arrow), cell wali (CW). With unfixed material (b) they are seldom vaulted. Bar 1 gm
cal appearance of the plasmalemma (PF) in the q)like region after short glycerination. It shows particlefree areas of the same number, size, and shape as described for prefixed material (see, however, Chandler and Heuser 1980). These areas, however, are seldom blebb-like vaulted. Similar results were found after freezing in sucrose. If parts of the plasma membrane are fractured away as shown in Fig. 4a, the membrane (EF) of underlying vesicles becomes visible in areas poor in particles. For the most part however, cross-fractured vesicles are observed. Figure 4b indicates that the particle-free area has exactly the same shape as the vesicle which underlies the plasma membrane. Although the distance between the vesicle and the plasma membrane is approx. 120 nm, the number of IMPs in the region nearest to the vesicle is lower than in other parts of the plasmalemma. If secretoric cells are cross-fractured (Fig. 5), a similar process of differentiation becomes visible in
the Golgi membranes and their derivatives. The number of IMPs is highest in membranes (PF) of the sacculus-like cisternae. In regions of budding cisternae the number of IMPs is lowered. Migrating vesicles, which underly ER cisternae or the plasmalemma, reveal only a few particles. The two types of small vesicles described above are observed in the surroundings of dictyosome cisternae. The membranes (PF) of the microvesicles (40 nm), in particular, possess a great density of particles (arrows). The exoplasmic fracture faces (EFs) of all membranes of the secretoric cells show much fewer IMPs than the PF, as is the case with most of the membranes after prefixation (Branton and Deamer 1972).
Discussion From recent studies (Chandler and Heuser 1979 and 1980), is known that the arrangement of IMPs is
184
D. Volkmann: Membrane Differentiation During Secretion
Figs. 4a, b. PF of the plasma membrane and parts of a cross-fractured secretoric cell in the qMike region, a Vesicles (SV) underlying an area poor in IMPs. The EF of the vesicle membrane is nearly free of IMPs. Another vesicle is cross-fractured. Slime (53 is deposited between plasma membrane and cell wall (CW). b Shape and size of the underlying vesicle (SV) and the blebb-like area free of particles correspond very well. Although the distance between the vesicle and the membrane is approx. 120 nm, the number of IMPs is less dense in the region near the vesicle (arrow). Bar 1 gm
influenced in particular during fusion events by the use of fixatives prior to the process of freeze-fracturing. Control experiments prove the existence of particle-free areas in plasma membranes of secretoric cap cells: (i) the areas are observed with unfixed material treated with sucrose or glycerin for less than 3 s (Fig. 3 b), and (ii) these areas are found exclusively in cells showing secretoric activity. The blebb-like shape of particle-free areas, however, cannot be observed with unfixed material, so that this shape may depend on the pretreatment with fixatives, as was shown for the myocardial plasma membrane during development (Gros et al. 1980). Since it is difficult to obtain unbroken replicae and endo-
membranes well preserved from unfixed material, the routine investigations were performed with prefixed material (Figs. 3a, 4a, b, 5). The results presented above are compatible with the idea of the granulocrine secretion (Schnepf 1969) and the concept of membrane flow (Morr6 et al. 1971). Slime is transported outside the cell by formation of slime-transporting vesicles at the dictyosomes, by fusion of the vesicle membrane with the plasma membrane, by extrusion of the vesicle content into the cell wall, and by incorporation of the vesicle membrane into the accompanying plasma membrane. During these events a similar structural differentiation becomes visible in the membranes involved. Budding regions of the dictyosomes (Fig. 5) as well as parts of the plasmalemma are transformed by lateral displacement of IMPs into adjacent membrane regions. The result of this is migrating vesicles (Fig. 5) p o o r in IMPs,
D. Volkmann: Membrane Differentiation During Secretion
185
Fig. 5. Part of a cross-fractured secretoric cell showing cell wall (CW), plasmamembrane (PL), ER cisternae (ER), mitochondria (M), dictyosomes (D) and their derivatives. The PF of dictyosome cisternae is rich in IMPs whereas the PF of migrating vesicles (SV) underlying ER cisternae near the plasma membrane is poor in particles. The budding region (BR) of the dictyosome possesses fewer IMPs than the cisternae. Two types of small vesicles (small and large arrows) are visible near dictyosome cisternae. Especially the smaller one (small arrows) possesses a lot of IMPs in relation to the migrating vesicles. The EFs of the dictyosome cisternae and the budding region, of migrating vesicles, of ER cisternae and of the plasma membrane are poor in IMPs. Bar 1 gm
respectively, particle-free areas o f the plasma membrane (Figs. 3a, b; 4a, b). One i m p o r t a n t question arising f r o m the similar differentiation o f b o t h m e m b r a n e s concerns the particle-free areas o f the plasma membrane. Are they prospective sites o f m e m b r a n e fusion or do they represent the original vesicle m e m b r a n e after i n c o r p o r a t i o n into the p l a s m a l e m m a ? Three arguments support the idea that the areas are prospective sites o f fusion: (i) particle-free areas vaulted against the direction of exocytosis were never observed (Figs. 3a, b, see, however, Kiermayer and Staehelin 1972 with the alga Micrasterias), as one would expect f r o m i n c o r p o r a t e d vesicles (Fig. 2);
(ii) slime-transporting vesicles underlying the plasma m e m b r a n e (Figs. 4a, b) are always observed whenever parts o f the particle-free areas are fractured a w a y ; and (iii) the smaller the distance is between the vesicle and the plasmalemma, the larger is the diameter o f the particle-free areas (Figs. 4a, b). Since the EFs o f both m e m b r a n e s possess strikingly fewer IMPs, it is evident that areas p o o r in this special class o f proteins are involved in the fusion process. The ratio o f lipid/protein must therefore be comparatively high in sites o f m e m b r a n e fusion. Similar results are reported for secretoric processes
186 particularly in cells of animals and lower plants (de Camilli et al. 1976; Chi etal. 1976; Lawson et al. 1977; Orci et al. 1977; Pinto da Silva and Nougeira 1977; Chailley 1979; see, however, Vian 1974 for higher plants). By binding studies with IgE and lectins as well as by freeze-fracturing, Lawson et al. (1977) gave convincing evidence that membrane proteins are displaced from the "interacting zones" when granule membranes fuse with the plasma membrane. Although the authors cannot exclude that some proteins may be left back in areas of fusion they are convinced that membrane fusion occurs between protein-depleted areas in mast cells. From investigations with artificial lipid vesicles it is known that calcium ions enhance the fusing process between negatively charged vesicles (Papahadjopoulos et al. 1974). In every cap cell some cisternae of the endoplasmic reticulum run parallel to the plasma membrane. They possibly function as a pool for calcium ions, as is suggested for the fusion process in encysting zoospores of Phytophthora (Hemmes and Pinto da Silva 1980). In secretory cells of the root cap, membrane differentiation was observed not only in the plasma membrane but also simultanously in membranes of the Golgi apparatus. At least the prominent classes of membrane proteins - recognizable as IMPs are not transported to the plasma membrane but left behind in the cisternae of the dictyosomes. This indicates: (i) that the enrichment of membrane proteins occurs in dictyosome cisternae rather than in the plasmalemma, and (ii) that lipids may be enriched in the plasma membrane. (i) is in good agreement with results of Staehelin and Kiermayer (1970), who convincingly showed the increase of particles across the stacked membranes of the dictyosomes from the forming to the maturing face. Especially in cisternae near the maturing face do the number of particles decrease from the centre to the periphery. Golgi vesicles are nearly free of IMPs (compare also Giddings et al. 1980). Already Staehelin and Kiermayer discussed the possibility that the increase of membrane staining and thickness in stacks might be caused by the increasing number of particles. (ii) is compatible with investigations of Keenan and Morr6 (1970) and Yunghans et al. (1970), who reported for rat liver cells that the lipid/protein ratio is higher in the plasma membrane than in membranes of the Golgi apparatus. The concept of membrane flow assumes recycling of membrane constituents from the plasma membrane
D. Volkmann: MembraneDifferentiationDuring Secretion and reutilization, in particular, of informational molecules (Morr~ and Ovtracht 1977). This is realized by endocytotic vesicles, especially in animal cells. The results presented above indicate that in cap cells the molecular composition of the plasma membrane is already regulated during the formation of slime-transporting vesicles in the Golgi apparatus, at least as far as the IMPs are concerned. This is supported by results from epithelia of urinary bladder (Severs and Hicks 1979) and from the alga Micrasterias (Giddings et al. 1980), which were investigated during the biogenesis of the plasma membrane. The authors showed Golgi vesicles characterized by a specific arrangement of IMPs. In the plasma membrane the same arrangement was observed by which the plasma membrane may be able to realize a specific function. One can conclude that in cap cells recycling of proteins from the plasma membrane does not occur to such an extent as has been assumed for secretoric cells up till now. Above all there is no indication of endocytotic vesicles in cap cells. Only little is known about the recycling of lipid components. Although secretoric cap cells do not grow anymore, their plasma membrane increases by extrusion of slime. Surely, lipids are necessary to some extent during these events, otherwise the plasma membrane would become thinner during secretion, which contradicts all morphological data. On the other hand, the amount of membranes incorporated into the plasma membrane during extrusion of slime is higher than the increase of the plasma membrane. At least in this region the plasma membrane may be enriched by lipids. With the membrane transformation in the Golgi apparatus by the selection of specific membrane components the problem of recycling is shifted from the plasma membrane to the dictyosome membranes. The fate of the particle-rich cisternae is unclear. It is interesting, however, that particle-rich microvesicles are visible in the surroundings of dictyosome cisternae. If both types are involved in recycling processes between the cisternae of the dictyosomes and the endoplasmic reticulum, or if one type represents transitoric vesicles which are responsible for the transfer of membrane material between the ER and the dictyosomes, is unclear up til now. The results confirm the Golgi apparatus as the important organelle of membrane transformation (Morr~ and Ovtracht 1977; Whaley and Dauwalder 1979). The organelle regulates the molecular composition of the plasma membrane by selection of specific membrane components. In this way it is possibly an important site for the distribution of information (Whaley and Dauwalder 1979). The mechanism, however, by which the selection of membrane components
D. Volkmann: Membrane Differentiation During Secretion t a k e s place, e.g., h o w the l a t e r a l d i s p l a c e m e n t o c c u r s a n d h o w it is r e g u l a t e d , is so far u n k n o w n . The results support the concept of membrane flow ( M o r r 6 et al. 1971) d u r i n g s e c r e t i o n e v e n t s in r o o t c a p cells, at least c o n c e r n i n g t h e step b e t w e e n t h e d i c t y o s o m e s a n d the p l a s m a m e m b r a n e . This research was supported by the Deutsche Forschungsgemeinschaft. 1 wish to thank Mrs. Petra Peters for skilfuI assistance. I am grateful to Professor A. Sievers for stimulating discussions.
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Received 26 August, accepted 7 October 1980
Planta 151, 180 188 (1981)
9 Springer-Verlag 1981
Structural Differentiation of Membranes Involved in the Secretion of Polysaccharide Slime by Root Cap Cells of Cress (Lepidium sativum L.) Dieter Volkmann Botanisches Institut der Universit/it Bonn, Venusbergweg 22, D-5300 Bonn, Federal Republic of Germany
Abstract. The peripheral secretion tissue of the root cap of Lepidiurn sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins- recognizable as IMPs - are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome Abbreviations: PF=protoplasmic fracture face; EF=exoplasmic
fracture face; IMP =intramembranous particle
0032-0935/81/0151/0180/$1.80
membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.
Key words: Exocytosis - Golgi membranes- L e p i d u m Membrane transformation - Plasma membrane - Recycling (membranes) - Secretion (slime).
Introduction The peripheral cells of the root cap produce polysaccharide slime, which is exported from the cell by exocytosis of Golgi vesicles (Mollenhauer et al. 1961; Northcote and Pickett-Heaps 1966; Morr6 etal. 1967). Whereas the physiology of the secretion process has been investigated in detail (Ltittge and Schnepf 1976), many problems regarding the cellular events, which occur during the exocytotic process, remain to be solved (Whaley and Dauwalder 1979): formation of vesicles at the cisternae of the dictyosomes; directed transport of vesicles to the plasma membrane; fusion of the vesicle membranes with the plasmalemma and incorporation into the same; extrusion of the vesicle content by fission of the fusion sites; and recycling of membrane constituents. On the basis of morphological, histochemical, and biochemical investigations, the Golgi apparatus is discussed as being an organelle of membrane differentiation and transformation (Mollenhauer and Morr6 1966; Grove et al. 1968; Schnepf 1969; Sievers 1973; Mollenhauer et al. 1976; Morr6 and Ovtracht 1977; Whaley and Dauwalder 1979). Starting from the forming face, the thickness of membranes and the uptake of staining material increase across the stack of dictyosome cisternae. The membrane of Golgi vesi-
D. Volkmann: Membrane Differentiation During Secretion
cles becomes increasingly similar in structure to the plasma membrane (Whaley 1966; Sievers 1967; Grove etal. 1968; Kiermayer 1970; Mollenhauer etal. 1976). By freeze-fracturing, Staehelin and Kiermayer (1970) showed that membrane differentiation occurs across the stacked membranes as well as within single cisternae. In connection with exocytotic processes in animals (for literature see Chailley 1979) and lower plants (Robinson and Preston 1971 ; Kiermayer and Staehelin 1972; Kiermayer and Dobberstein 1973; Peng and Jaffe 1976; Pinto da Silva and Nougeira 1977; Kiermayer and Sleytr 1979; Giddings et al. 1980), structural differentiation of the plasma membrane is a well recognized phenomenon, even if the existence of some structures observed is questioned (Chandler and Heuser 1979 and 1980). For higher plants, however, there are only a few data resulting from the early beginning of freeze-fracturing. Branton and Moor (1964) as well as Northcote and Lewis (1968) observed structural changes of the plasma membrane caused by exocytosis of peripheral vesicles in cells of the root tip. This communication deals with the differentiation of both the Golgi membranes and the plasmalemma in root cap cells during the granulocrine secretion of polysaccharide slime. Material and Methods Dry seeds of Lepidium sativum L. were soaked in tap water for 30 rain. The seeds were germinated on moist filter paper in saturated humidity at 24_+2 ~ C in the dark. Seedlings with radicles of the same length were fixed 28 h after the beginning of soaking.
TEM: Fixation was carried out in 3% glutaraldehyde buffered in 0.05 M phosphate buffer, pH 7.2, for 2 h at 20 ~ C. Postfixation was done in 2% osmium tetroxide in the same buffer solution for 2 h. Dehydration was done in an aceton series. Electronmicroscopy was carried out at 60 kV with a Zeiss EM 10. Freeze-fracturing: Prefixation was carried out in the same way as fixation for TEM, but only for 30 min. Cryoprotection was done in 10, 20, and 30% glycerin, each step for 20 min. Unfixed material was incubated for less than 3 s in 0.3 M sucrose or in 30% glycerin. Root tips with a maximum length of 1 m m were frozen in melting nitrogen. Freeze-fracturing was performed with Balzers BAF 300 according to Moor and Mfihlethaler (1963).
Cleaning of the Replicae." 24 h in chromic acid (20% for 2 h, 40% for 22 h); 48 h in sodium hypochlorite (5% for 2 h, 10% for 46 h). Electronmicroscopy was done as described above. The nomenclature of freeze-fractures follows Branton et al. (1975).
Results
At the investigated stage of development, the peripheral secretion tissue of Lepidium is formed by one
181
Fig. 1. Cross section through the root cap of cress (Lepidium satirum). Slime is visible at the periphery of the cap and in the distal region 4Mike region - of the anticlinal cell walls (arrow) of the peripheral cell layer. Amyloplasts (A) can be seen in central and peripheral cells. Section was stained with toluidin-blue. Bar 10 gm
cell layer (Fig. 1). Slime is located at the periphery of the root cap and in the distal region of the anticlinal cell walls (arrow) of the outer cell layer. With the extrusion of slime the anticlinals assume a qb-like shape. It was confirmed by lightmicroscopy of untreated material that this shape really exists and that it is not produced by swelling of the slime during the fixation process, when cells lose their turgor pressure. The dictyosomes of this developmental stage are extremely hypertrophied. Great parts of the cisternae are often dilated (Fig. 2). In this case the shape of the cisternae changes from flattened to spherical across the membrane stack. Besides the slime-transporting vesicles near the dictyosome cisternae at least two types of small vesicles are visible. The bigger one (large arrows), the diameter of which is approx. 90 nm, possesses an electronopaque centre. The diameter of the smaller one is approx. 40 nm (small arrows). The low diameter causes a dark and homogenous staining of the vesicles. Slime-transporting vesicles are visible particularly in the phi-like region and to a lesser degree in the region of the outer wall. The vesicle diameter is approx. 600 nm. The plasma membrane reveals an undulating course. It is vaulted
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D, Volkmann: Membrane Differentiation During Secretion
Fig. 2. qS-like region (compare Fig. 1) of a peripheral cell. Dictyosomes (D) are hypertrophied. Net-like slime (S) is located in the budding region (BR) of the dictyosomecisternae, in migrating vesicles (SV), and on both sides of the wall. Two types of small vesicles are visible near cisternae of dictyosomes. The smaller one (small arrows) is homogenously contrasted, the other one (large arrows) reveals an electronopaque centre. The plasmalemma (PL) shows an undulating course by slime (5) being extruded into the cell wall (CPO and by vesicles (SV) underlying the membrane. Cisternae of rough endoplasmic reticulum (ER) run parallel to the plasmalemma. Bar 1 gm
by vesicles adjacent to the plasmalemma in the direction of exocytosis. By extruded slime, however, it is vaulted against the direction of exocytosis. Often the original shape of the vesicle is nearly preserved. The characteristic net-like slime is visible in the region of dilated dictyosome cisternae, in migrating vesicles, and outside the cell on both sides of the wall. Often two or more cisternae of rough endoplasmic reticulum run parallel to the plasma membrane. When the plasma membrane is fractured in the phi-like region (Figs. 3a, b), the protoplasmic fracture face (PF) reveals differences in the distribution of intramembranous particles (IMPs), in comparison
with other cell types (data not shown). Large areas showing homogenously distributed IMPs are interrupted by small areas poor in particles. These areas are of different size; their diameter does not exceed 800 nm and their shape is mostly elipsoid or circular. Sometimes the areas are vaulted into the direction of exocytosis. In this case they are nearly free of IMPs. In order to exclude artificial structures which may result from prefixation or intensive cryoprotection (Chandler and Heuser 1979; Hasty and Hay 1978), freeze-fracturing was performed with unfixed material. Sucrose (0.3 M) or glycerin (30%) was used for less than three seconds. Figure 3 b shows the typi-
D. Volkmann: Membrane Differentiation During Secretion
183
Figs. 3a, b. Protoplasmic fracture face (PF) of the plasma membrane in the qS-like region. Large areas with homogenously distributed intramembranous particles (IMPs) are interrupted by areas poor in particles. The areas are of different size, their diameter does not exceed 800 nm. With prefixed material (a) the areas are often vaulted in the direction of exocytosis (arrow), cell wali (CW). With unfixed material (b) they are seldom vaulted. Bar 1 gm
cal appearance of the plasmalemma (PF) in the q)like region after short glycerination. It shows particlefree areas of the same number, size, and shape as described for prefixed material (see, however, Chandler and Heuser 1980). These areas, however, are seldom blebb-like vaulted. Similar results were found after freezing in sucrose. If parts of the plasma membrane are fractured away as shown in Fig. 4a, the membrane (EF) of underlying vesicles becomes visible in areas poor in particles. For the most part however, cross-fractured vesicles are observed. Figure 4b indicates that the particle-free area has exactly the same shape as the vesicle which underlies the plasma membrane. Although the distance between the vesicle and the plasma membrane is approx. 120 nm, the number of IMPs in the region nearest to the vesicle is lower than in other parts of the plasmalemma. If secretoric cells are cross-fractured (Fig. 5), a similar process of differentiation becomes visible in
the Golgi membranes and their derivatives. The number of IMPs is highest in membranes (PF) of the sacculus-like cisternae. In regions of budding cisternae the number of IMPs is lowered. Migrating vesicles, which underly ER cisternae or the plasmalemma, reveal only a few particles. The two types of small vesicles described above are observed in the surroundings of dictyosome cisternae. The membranes (PF) of the microvesicles (40 nm), in particular, possess a great density of particles (arrows). The exoplasmic fracture faces (EFs) of all membranes of the secretoric cells show much fewer IMPs than the PF, as is the case with most of the membranes after prefixation (Branton and Deamer 1972).
Discussion From recent studies (Chandler and Heuser 1979 and 1980), is known that the arrangement of IMPs is
184
D. Volkmann: Membrane Differentiation During Secretion
Figs. 4a, b. PF of the plasma membrane and parts of a cross-fractured secretoric cell in the qMike region, a Vesicles (SV) underlying an area poor in IMPs. The EF of the vesicle membrane is nearly free of IMPs. Another vesicle is cross-fractured. Slime (53 is deposited between plasma membrane and cell wall (CW). b Shape and size of the underlying vesicle (SV) and the blebb-like area free of particles correspond very well. Although the distance between the vesicle and the membrane is approx. 120 nm, the number of IMPs is less dense in the region near the vesicle (arrow). Bar 1 gm
influenced in particular during fusion events by the use of fixatives prior to the process of freeze-fracturing. Control experiments prove the existence of particle-free areas in plasma membranes of secretoric cap cells: (i) the areas are observed with unfixed material treated with sucrose or glycerin for less than 3 s (Fig. 3 b), and (ii) these areas are found exclusively in cells showing secretoric activity. The blebb-like shape of particle-free areas, however, cannot be observed with unfixed material, so that this shape may depend on the pretreatment with fixatives, as was shown for the myocardial plasma membrane during development (Gros et al. 1980). Since it is difficult to obtain unbroken replicae and endo-
membranes well preserved from unfixed material, the routine investigations were performed with prefixed material (Figs. 3a, 4a, b, 5). The results presented above are compatible with the idea of the granulocrine secretion (Schnepf 1969) and the concept of membrane flow (Morr6 et al. 1971). Slime is transported outside the cell by formation of slime-transporting vesicles at the dictyosomes, by fusion of the vesicle membrane with the plasma membrane, by extrusion of the vesicle content into the cell wall, and by incorporation of the vesicle membrane into the accompanying plasma membrane. During these events a similar structural differentiation becomes visible in the membranes involved. Budding regions of the dictyosomes (Fig. 5) as well as parts of the plasmalemma are transformed by lateral displacement of IMPs into adjacent membrane regions. The result of this is migrating vesicles (Fig. 5) p o o r in IMPs,
D. Volkmann: Membrane Differentiation During Secretion
185
Fig. 5. Part of a cross-fractured secretoric cell showing cell wall (CW), plasmamembrane (PL), ER cisternae (ER), mitochondria (M), dictyosomes (D) and their derivatives. The PF of dictyosome cisternae is rich in IMPs whereas the PF of migrating vesicles (SV) underlying ER cisternae near the plasma membrane is poor in particles. The budding region (BR) of the dictyosome possesses fewer IMPs than the cisternae. Two types of small vesicles (small and large arrows) are visible near dictyosome cisternae. Especially the smaller one (small arrows) possesses a lot of IMPs in relation to the migrating vesicles. The EFs of the dictyosome cisternae and the budding region, of migrating vesicles, of ER cisternae and of the plasma membrane are poor in IMPs. Bar 1 gm
respectively, particle-free areas o f the plasma membrane (Figs. 3a, b; 4a, b). One i m p o r t a n t question arising f r o m the similar differentiation o f b o t h m e m b r a n e s concerns the particle-free areas o f the plasma membrane. Are they prospective sites o f m e m b r a n e fusion or do they represent the original vesicle m e m b r a n e after i n c o r p o r a t i o n into the p l a s m a l e m m a ? Three arguments support the idea that the areas are prospective sites o f fusion: (i) particle-free areas vaulted against the direction of exocytosis were never observed (Figs. 3a, b, see, however, Kiermayer and Staehelin 1972 with the alga Micrasterias), as one would expect f r o m i n c o r p o r a t e d vesicles (Fig. 2);
(ii) slime-transporting vesicles underlying the plasma m e m b r a n e (Figs. 4a, b) are always observed whenever parts o f the particle-free areas are fractured a w a y ; and (iii) the smaller the distance is between the vesicle and the plasmalemma, the larger is the diameter o f the particle-free areas (Figs. 4a, b). Since the EFs o f both m e m b r a n e s possess strikingly fewer IMPs, it is evident that areas p o o r in this special class o f proteins are involved in the fusion process. The ratio o f lipid/protein must therefore be comparatively high in sites o f m e m b r a n e fusion. Similar results are reported for secretoric processes
186 particularly in cells of animals and lower plants (de Camilli et al. 1976; Chi etal. 1976; Lawson et al. 1977; Orci et al. 1977; Pinto da Silva and Nougeira 1977; Chailley 1979; see, however, Vian 1974 for higher plants). By binding studies with IgE and lectins as well as by freeze-fracturing, Lawson et al. (1977) gave convincing evidence that membrane proteins are displaced from the "interacting zones" when granule membranes fuse with the plasma membrane. Although the authors cannot exclude that some proteins may be left back in areas of fusion they are convinced that membrane fusion occurs between protein-depleted areas in mast cells. From investigations with artificial lipid vesicles it is known that calcium ions enhance the fusing process between negatively charged vesicles (Papahadjopoulos et al. 1974). In every cap cell some cisternae of the endoplasmic reticulum run parallel to the plasma membrane. They possibly function as a pool for calcium ions, as is suggested for the fusion process in encysting zoospores of Phytophthora (Hemmes and Pinto da Silva 1980). In secretory cells of the root cap, membrane differentiation was observed not only in the plasma membrane but also simultanously in membranes of the Golgi apparatus. At least the prominent classes of membrane proteins - recognizable as IMPs are not transported to the plasma membrane but left behind in the cisternae of the dictyosomes. This indicates: (i) that the enrichment of membrane proteins occurs in dictyosome cisternae rather than in the plasmalemma, and (ii) that lipids may be enriched in the plasma membrane. (i) is in good agreement with results of Staehelin and Kiermayer (1970), who convincingly showed the increase of particles across the stacked membranes of the dictyosomes from the forming to the maturing face. Especially in cisternae near the maturing face do the number of particles decrease from the centre to the periphery. Golgi vesicles are nearly free of IMPs (compare also Giddings et al. 1980). Already Staehelin and Kiermayer discussed the possibility that the increase of membrane staining and thickness in stacks might be caused by the increasing number of particles. (ii) is compatible with investigations of Keenan and Morr6 (1970) and Yunghans et al. (1970), who reported for rat liver cells that the lipid/protein ratio is higher in the plasma membrane than in membranes of the Golgi apparatus. The concept of membrane flow assumes recycling of membrane constituents from the plasma membrane
D. Volkmann: MembraneDifferentiationDuring Secretion and reutilization, in particular, of informational molecules (Morr~ and Ovtracht 1977). This is realized by endocytotic vesicles, especially in animal cells. The results presented above indicate that in cap cells the molecular composition of the plasma membrane is already regulated during the formation of slime-transporting vesicles in the Golgi apparatus, at least as far as the IMPs are concerned. This is supported by results from epithelia of urinary bladder (Severs and Hicks 1979) and from the alga Micrasterias (Giddings et al. 1980), which were investigated during the biogenesis of the plasma membrane. The authors showed Golgi vesicles characterized by a specific arrangement of IMPs. In the plasma membrane the same arrangement was observed by which the plasma membrane may be able to realize a specific function. One can conclude that in cap cells recycling of proteins from the plasma membrane does not occur to such an extent as has been assumed for secretoric cells up till now. Above all there is no indication of endocytotic vesicles in cap cells. Only little is known about the recycling of lipid components. Although secretoric cap cells do not grow anymore, their plasma membrane increases by extrusion of slime. Surely, lipids are necessary to some extent during these events, otherwise the plasma membrane would become thinner during secretion, which contradicts all morphological data. On the other hand, the amount of membranes incorporated into the plasma membrane during extrusion of slime is higher than the increase of the plasma membrane. At least in this region the plasma membrane may be enriched by lipids. With the membrane transformation in the Golgi apparatus by the selection of specific membrane components the problem of recycling is shifted from the plasma membrane to the dictyosome membranes. The fate of the particle-rich cisternae is unclear. It is interesting, however, that particle-rich microvesicles are visible in the surroundings of dictyosome cisternae. If both types are involved in recycling processes between the cisternae of the dictyosomes and the endoplasmic reticulum, or if one type represents transitoric vesicles which are responsible for the transfer of membrane material between the ER and the dictyosomes, is unclear up til now. The results confirm the Golgi apparatus as the important organelle of membrane transformation (Morr~ and Ovtracht 1977; Whaley and Dauwalder 1979). The organelle regulates the molecular composition of the plasma membrane by selection of specific membrane components. In this way it is possibly an important site for the distribution of information (Whaley and Dauwalder 1979). The mechanism, however, by which the selection of membrane components
D. Volkmann: Membrane Differentiation During Secretion t a k e s place, e.g., h o w the l a t e r a l d i s p l a c e m e n t o c c u r s a n d h o w it is r e g u l a t e d , is so far u n k n o w n . The results support the concept of membrane flow ( M o r r 6 et al. 1971) d u r i n g s e c r e t i o n e v e n t s in r o o t c a p cells, at least c o n c e r n i n g t h e step b e t w e e n t h e d i c t y o s o m e s a n d the p l a s m a m e m b r a n e . This research was supported by the Deutsche Forschungsgemeinschaft. 1 wish to thank Mrs. Petra Peters for skilfuI assistance. I am grateful to Professor A. Sievers for stimulating discussions.
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Received 26 August, accepted 7 October 1980
