THE PRESENCE AND LOCATION OF SPOROPOLLENIN IN FRUITING BODIES OF THE CELLULAR SLIME MOULDS

Transcription

1 J. Cell Sci. 66, (1984) 297 I'riiited in Great Britain The Company of Biologists Limited 1984 THE PRESENCE AND LOCATION OF SPOROPOLLENIN IN FRUITING BODIES OF THE CELLULAR SLIME MOULDS YASUO MAEDA Biological Institute, /"'acuity of Science, Tohoku University, Aoba, Senclai 980, Japan SUMMARY The presence of an acetolysis-resistant polymer (sporopollenin) in the cellular slime moulds is demonstrated. This polymer is located on the stalk sheath of fruiting bodies as a bundle of fine fibrils (4 5 nm diameter). The location and structure of sporopollenin in spores are shown to vary considerably, depending upon the species. In Polysphondvltum violaceum spores, sporopollenin is composed of fine spicules (4-5 nm in diameter, nm long) that cover both the outermost layer of spore wall and the inner surface of the cell membrane. The sporopollenin of Dictyostelium discoicleum spores is located preferentially close to the inner surface of the cell membrane, forming a mass of electron-opaque fine granules (4-5 nm in diameter). D. mucoroides spores, however, appear not to possess a tight network of sporopollenin, since they were less resistant to acetolysis than those of the other species. The biological significance of the results is discussed with special reference to fruiting body formation. INTROD UCT1ON The presence and distribution of cell wall materials in plants are generally of special importance for the establishment and preservation of the shape of individual cells and cell masses. An acetolysis-resistant polymer (sporopollenin) is found widely in the pollen grains of angiosperms and gymnosperms and also in the spores of pteridophytes, fungi and algae (see Brooks et al. 1971). This polymer is undoubtedly responsible for giving physicochemical strength to the cell walls. Our primary interest in the present work was to discover if sporopollenin is also present in the cellular slime moulds. The cell aggregate (slug) of Dictyostelium discoideuvi migrates on the substratum and finally develops into a fruiting body, which consists of a mass of spores (sorus), a supporting stalk and a basal disc. During the culmination of the cell mass, a stalk sheath is continuously secreted at the tip of a cell mass, thus forming a cylinder-like structure. As the anterior pre-stalk cells migrate into the sheath cylinder, they become vacuolated and eventually differentiate into stalk cells, secreting their own cell wall. Similar processes are also observed at the tip of a cell mass during slug migration in D. mucoroides and PolysphondyHum violaceum. Differentiated spore cells germinate under proper conditions to release myxamoebae, which initiate their own life cycle. The fact that the stalk sheath as well as the stalk cell wall consists of cellulose has been suggested by several chemical and electron-microscopical studies (Raper & Fennell, 1952; Miihlethaler, 1956; Gezelius & RSnby, 1957). Differentiating spores

2 298 Y. Maeda secrete acid mucopolysaccharide, which ultimately constitutes the outermost layer of the spore cell wall; this is followed by the formation of cellulose between the outermost layer and the cell membrane (Hohl & Hamamoto, 1969; Maeda, 1971). During the morphogenetic events of culmination, the production of cell walls and sheath is essential for the final form and rigidity of the sorocarp. Therefore, knowledge of the composition of the walls and sheath, and the temporal and spatial patterns of their formation, is essential for an understanding of the mechanism of morphogenesis. In an attempt to elucidate the structural basis of cell shape and rigidity, we examined the sensitivity of cells or sheath materials to drastic treatments with alkali and acid. The present work describes and illustrates the presence and location of sporopollenin, particularly in the structures constituting fruiting bodies. The biological significance of sporopollenin formation is also discussed with special reference to the morphogenetic process at the tip of a culminating cell mass. MATERIALS AND METHODS Organisms and cultivation Dictyostelium discoideum NC-4, D. mucoroides no. 11, and Polysphondylium violaceum P6 were investigated. Spores of the above species were inoculated separately in association with Escherichia coli B/r on a standard nutrient agar medium (Bonner, 1947) and incubated for 6 days at C to obtain mature fruiting bodies. Treatments with alkali and acid Samples on the agar plates were suspended in distilled deionized water (DDW) and collected by centrifugation (3 min at 2500 rev./min). Treatments of the samples with alkali and acid were done using a slight modification of Erdtman's (1960) method, as follows. The collected samples were shaken in 10% (w/v) KOH in a boiling water-bath for 45 min, followed by two washings in DDW. The preparation thus obtained was designated as alkali-treated. Subsequently, the samples were dehydrated with acetic acid and then treated with a fresh mixture of nine parts anhydric acetic acid and one part concentrated H2SO4 by immersion for 10min in a boiling water-bath. After washing once in acetic acid, the samples were washed in DDW by centrifugation (5 min at rev./min). The resulting pellets were analysed as acetolysed samples. The gross morphology of samples at each step was monitored under a phase-contrast microscope. Electron microscopy For transmission electron microscopy (TEM), the specimens were fixed in 2% (v/v) glutaraldehyde in 10 mm-pipes buffer (ph 6-8) for 2 h at room temperature. After washing thoroughly in lomm-pipes buffer (ph68), they were post-fixed in 1% OsCU in salt solution (lomm-nacl, lomm-kcl, 25mM-CaCl2) for 20 min (Maeda & Takeuchi, 1969). Subsequently, the specimens were stained with saturated uranyl acetate in 50 % ethanol for 2 h, and were dehydrated in a graded series of ethanol solutions and then through acetone. They were finally embedded in an Epoxy resin. Ultrathin sections (60-80 nm thick) were stained with lead citrate according to Reynolds (1963) and were observed with a Hitachi HS-9 transmission electron microscope. For scanning electron microscopy (SEM), the specimens were fixed in 2% (v/v) glutaraldehyde, as described above. After removing the fixative, they were dehydrated with a graded series of ethanol solutions, transferred into isoamyl acetate, and then mounted on toluene-resistant plastic sheets. This was followed by critical-point drying. The specimens thus obtained were coated with gold by ion sputtering and were observed with a T-20 scanning electron microscope (Japan Electron Optics Lab., Tokyo).

3 Sporopollenin in the cellular slime moulds 299 RESULTS Wall constituents o/d. discoideum/rui/z'ng bodies Morphological changes during treatments of the three types of cells that comprise the fruiting body (spore, stalk and basal disc) with alkali and acid were firstly surveyed with a phase-contrast microscope. Following the treatment with alkali, the stalk and basal disc showed no appreciable changes, while a bright halo caused by the spore wall was slightly reduced as compared with non-treated spores. When the alkali-treated samples were acetolysed, remarkable changes were noticed as follows. During the first 2 3 min of acetolysis, stalk cells located within the stalk tube (sheath) began to dissolve, and were completely lost after 5 min of acetolysis. No further changes were observed in stalk components during a prolonged period (20 min) of acetolysis, the fragmentated stalk sheath being the only remaining structure. The basal disc also was completely lost after 5 min of acetolysis. The fragmentation of the stalk sheath seemed to be due to mechanical shearing force caused by pipetting during the preparation, because no fragmentation occurred when the stalk was acetolysed carefully without vigorous pipetting. In the spore, the bright halo associated with the wall began to decrease markedly during the first1 2 min of acetolysis. The major parts of spore cell wall and protoplasm seemed to be almost completely lost after 5 min of acetolysis, though a thin oval-shaped membrane that was located topologically close to the cell membrane of the original spore was retained. The morphological changes described above were then analysed electronmicroscopically. In a region of non-treated stalk located below the sorus three layers of extracellular structures were observed: (1) stalk cell wall; (2) stalk sheath; (3) electron-opaque membrane derived from the slime sheath surrounding a whole slug. From the longitudinal and cross-sections, it is evident that the stalk cell wall consists of fine fibrils (about 7 nm thick) oriented at random, while in the stalk sheath fine fibrils (4-5 nm thick) are longitudinally oriented in a parallel array (Fig. 1). When stalks were treated with KOH, the electron-opaque membrane (slime sheath), cell membrane and a major part of the protoplasm of the stalk cell were eliminated, only the stalk sheath and wall remaining. During subsequent acetolysis, the stalk cell wall was completely lost and only fine fibrils as seen in the stalk sheath remained (Fig. 2). As expected from previous electron-microscopic studies (see Hohl & Hamamoto, 1969), the spore cell wall was found to be composed of two distinct layers: (1) the outermost membrane derived originally from the lining membrane of pre-spore specific vacuoles (PSVs); (2) less electron-opaque wall materials, composed of cellulose (Fig. 3A). When the spores were treated with KOH, the outermost membrane was completely lost. In addition, the protoplasm of the spore became less dense, retaining fibrillar structures and oval-shaped crystalline bodies. Besides the above changes, fine electron-opaque granules (4 5 nm diameter) appeared, predominantly lining the inner surface of the original cell membrane (Fig. 3B). Similar granules were also distributed in the matrix of the cellulose wall and protoplasm. When the samples were acetolysed, structures other than the granules lining the cell membrane were completely eliminated (Fig. 3c).

4 300 Y. Maeda Fig. 1. Fine structure of wall constituents in the stalk region of D. discoideum fruiting bodies. A. A longitudinal section; B, a cross-section. In A, the stalk sheath is mainly composed of fine fibrils (/) oriented in a parallel array, while the stalk cell wall (IU) consists of fibrous structures oriented at random. In B, the cross-sectioned fibrils (arrows) of the stalk sheath are indicated as granular structures. x

5 Sporopollenin in the cellular slime moulds 301.VV->4 V>, M *\ V 1.i* 2 A ' ' ^ '* Fig. 2. A. An electron micrograph of acetolysed D. discoideum stalks. Each sheet-like structure is composed of a bundle of fine fibrils. Note its structural resemblance to the stalk sheath of Fig. 1A. X B. An oblique section of the bundle in which fine fibrils are beautifully oriented in a parallel array. X According to SEM observations, two distinct structures were found to be retained after acetolysis of sorocarps, as expected from the above TEM observations. One was a folding sheet of stalk sheath, and the other was an oval-shaped structure, which might possibly be composed of the fine granules that line the spore cell membrane. Sporopollenin and cellulose in the culminating cell mass ofd. discoideum To ascertain the timing and topology of sporopollenin and cellulose formation, a cell mass at the late culmination stage was examined using the TEM. Within the uppermost region of the stalk sheath, at the point at which sheath materials were continuously being added, neither formation of the stalk cell wall nor conspicuous vacuolization of cells was noticed (Fig. 4). The stalk cell wall became detectable within a somewhat lower part of the stalk sheath; this was followed by a region showing considerable vacuolization of differentiating stalk cells. The thickness and morphology of the stalk sheath in this region was, for the most part, unchanged as the stalk penetrated down the central part of the rear cell mass; the stalk cell wall,

6 102 Y. Maeda -*., w Fig. 3

7 Sporopollenin in the cellular slime moulds 303 Fig. 4. A longitudinal section of the upper region of ad. discoideum culminating cell mass in which stalk sheath materials are continually formed. The stalk sheath is mainly composed of fine fibrils similar to those in the mature sheath shown in Fig. 1A. In the pre-stalk cells located within the stalk sheath (left of figure), the formation of their own cell wall cannot be seen. The prestalk cells (p) located outside the sheath are elongate. XSSOOO. however, increased further in thickness. The stalk cell wall facing the stalk sheath seemed to be always thicker than that between the cells. When the cell mass was treated with KOH and then acetolysed, the results obtained were essentially the same as those described in the previous section. Considering these Fig. 3. Electron micrographs showing structural changes in D. discoideum spores caused by treatment with alkali and acetolysis. A. A non-treated spore. The spore wall consists of the outermost electron-opaque membrane (ow) and a layer of less electron-opaque fibrils (w). B. An alkali-treated spore. The outermost membrane (ow, shown in A) and also the cell membrane are completely eliminated. The protoplasm contains a fibrillar matrix and oval-shaped crystalline bodies (cb) with a moderate electron density. Note highly electronopaque fine granules (arrows), which appear to be located predominantly on the inner surface of the original cell membrane. Similar granules also are observed in the matrix of the cellulose wall (w) and the protoplasm, c. An acetolysed spore. The cellulose wall and protoplasm are almost completely lost, only a layer of electron-opaque fine granules can be seen. X

8 304 Y. Maeda Fig. 5. A schematic demonstration of a D. discoideum culminating cell mass and structural changes in the spore and stalk caused by treatment with alkali and acetolysis. Both a bundle of fine fibrils derived from the stalk sheath and a mass of fine granules originally lining the spore cell membrane remain after acetolysis. 1, stalk cell wall; 2, stalk sheath; 3, slime sheath. facts, the presence and location of wall constituents in a culminating cell mass are schematically shown in Fig. 5. Sporopollenin in P. violaceum fruiting bodies When mature fruiting bodies of P. violaceum were acetolysed after treatment with alkali, tubular fragments derived from the stalk sheath and also spore capsules remained as two distinct structures (Fig. 6). The purple pigment in the fruiting bodies was retained well even after acetolysis, being a constituent of sporopollenin. The fibrillar structure of the stalk sheath was similar to that of D. discoideum, while the location and fine structure of sporopollenin in the spores were different in the two species. During treatment with alkali, fine spicules (4 5 nm in diameter, nm long) appeared predominantly on the outermost layer of the spore wall (Fig. 7). Similar spicules were observed close to the inner surface of the cell membrane and also in the protoplasmic matrix of the spore. Subsequent acetolysis of the spores showed that only the fine spicules are retained (Fig. 8).

9 Sporopollenin in the cellular slime moulds 305 Fig. 6. A phase-contrast micrograph showing two types of structures retained after acetolysis of P. violaceum fruiting bodies. In addition to a mass of oval-shaped spore residues (arrow), fragmented and folded sheets (/) derived from the stalk sheath can be observed. X1100. Sporopollenin in D. mucoroides fruiting bodies The stalk sheath of D. mucoroides was found to consist mainly of sporopollenin, as found for D. discoideum and P. violaceum. However, the spores were less resistant to acetolysis, since they were almost completely lysed by a prolonged period (20 min) of acetolysis. This suggests that D. mucoroides spores must lack a tight network of sporopollenin; sporopollenin forming a loose network would be lost during the preparation of samples. DISCUSSION This is the first report of the presence of sporopollenin in the fruiting bodies of the cellular slime moulds. The results further demonstrate that sporopollenin is a major component of the stalk sheath in the three species examined, while the distribution and fine structure of sporopollenin in the spores varies from species to species. The fact that the stalk sheath is composed of sporopollenin is of particular importance for understanding the mechanism of sorocarp construction, because it is regularly formed at the tip region of a culminating cell mass, possibly prior to the cellulose wall of the

10 Y. Maeda Fig. 7. An electron micrograph of a P. violaceum spore treated with alkali. A large number of fine spicules (arrows) are located on the outermost layer of spore wall, and some of them appear to line the inner surface of the original cell membrane. Similar spicules also are distributed in the protoplasmic matrix. X stalk cells. Using chemical tests and X-ray diffraction, several investigators have claimed that the stalk sheath as well as the stalk cell wall consists of cellulose (Raper &Fennell, 1952; Gezelius&Ranby, 1957; Freeze &Loomis, 1978). This discrepancy is probably due to the failure to separate the stalk sheath completely from the cellulose-containing stalk cell wall. The presence of sporopouenin has been widely reported in pollen exines of angiosperms and gymnosperms and also in spores of pteridophytes, fungi and algae. The sequence of sporopouenin deposition has now been studied in detail in several plants, including Lilium (Heslop-Harrison & Dickinson, 1968), Pinus (Dickinson & Bell, 1972) and algae (Atkinson, Gunning & John, 1972), and differences seem to exist between the deposition processes in the different plants. In D. discoideum spores, it is possible that sporopouenin is formed predominantly on the inner surface of the cell membrane. Alternatively, it might accumulate on the cell membrane through preferential deposition of sporopouenin synthesized in the cytoplasm, since

11 Sporopollenin in the cellular slime moulds 307 Fig. 8. An electron micrograph showing the location of sporopollenin in acetolysed P. violaceum spores. A mass of fine spicules consisting of sporopollenin are predominantly localized on the outermost layer of the spore wall and possibly on the inner surface of the original cell membrane. X sporopollenin-like structures are present in the cytoplasm of the alkali-treated spores. The reason why the sporopollenin of P. violaceum spores is localized extensively on the outermost layer of the wall is not evident. In morphological terms, sporopollenin seems typically to be deposited on special lipid surfaces, but in some pollen grains sporopollenin is known to be deposited around fibrillar masses consisting of mucopolysaccharide (Dickinson, 1976). In this connection, it is interesting to note the presence of acid mucopolysaccharide in the outermost layer of the slime mould spore wall (Maeda, 1971). There are other questions related to sporopollenin that arise. It is possible that a tight network of sporopollenin might function as a barrier to spore germination. If so, it would be of interest to know if there are specific enzymes that can degrade sporopollenin, particularly during spore germination. The presence of sporopollenin in the macrocysts and microcysts - other cellulose-containing fruiting structures - should also be investigated. While the chemical nature of sporopollenin is still not fully characterized, the work of Shaw and his collaborators (see Shaw, 1971) indicates that it is composed of polymers of carotenoid and/or carotenoid esters. In the cellular slime moulds,

12 308 Y. Maeda sporopollenin seems to assume multiple-forms: (1) fine granules in D. discoideum spores; (2) fine spicules in P. violaceum spores; and (3) fine fibrils in the stalk sheath of the three species examined. Such structural differences might be attributable merely to the degree of polymerization of the molecule, or there might be differences in the type of monomer synthesized by different cells. The separation and identification of each type of sporopollenin structure will be necessary in order to distinguish between the above possibilities and also to investigate the biosynthesis of sporopollenin. I thank Dr M. Filosa of University of Toronto, Dr M. Furuya of Tokyo University, and Dr K. Soma of Tohoku University for reading and criticizing the manuscript. This research was in part supported by grants-in-aid (nos , ) from the Ministry of Education of Japan. REFERENCES ATKINSON, A. W. JR, GUNNING, B. E. S. & JOHN, P. C. L. (1972). Sporopollenin in the cell wall olchlorella and other algae: Ultrastructure, chemistry, and incorporation of 14 C-acetate, studied in synchronous cultures. Planla 107, BONNER, J. T. (1947). Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Diclyostelium discoideum. J. exp. Zool. 106, BROOKS, J., GRANT, P. R., MUIR, M. D., VAN GIJZEL, P. & SHAW, G. (1971). Sporopollenin. London, New York: Academic Press. DICKINSON, H. G. (1976). The deposition of acetolysis-resistant polymers during the formation of pollen. Pollen & Spores 18, DICKINSON, H. G. & BELL, P. R. (1972). The role of tapetum in the formation of sporopollenincontaining structures during microsporogenesis in Pinus banksiana. Planta 107, ERDTMAN, G. (1960). The acetolysis method. A revised description. Svensk bol. Tidskr. 54, FREEZE, H. & LOOMIS, W. F. (1978). Chemical analysis of stalk components of Diclyostelium discoideum. Biochim. biophys. Ada 539, GEZELIUS, K. & RA'NBY, B. G. (1957). Morphology and fine structure of the slime mold Dictvostelium discoideum. Expl Cell Res. 12, HESLOP-HARRISON, J. & DICKINSON, H. G. (1968). A common mode of deposition of the sporopollenin of sexine and nexine. Nature, Land. 213, HOHL, H. R. &HAMAMOTO, S. T. (1969). Ultrastructure of spore differentiation in Dictyostelium: the prespore vacuole. J. Ultrastruct. Res. 26, MAEDA, Y. (1971). Studies on a specific structure in differentiating slime mold cells. Mem. Fac. Set., Kyoto Univ. 4, MAEDA, Y. & TAKEUCHI, I. (1969). Cell differentiation and fine structures in the development of the cellular slime molds. Deo. Growth & Differ. 11, MCHLETHALER, K. (1956). Electron microscopic study of the slime mold Dictyostelium discoideum. Am. J. Bol. 43, RAPER, K. B. & FENNELL, D. I. (1952). Stalk formation in Dictvostelium. Bull. Torrev hot. Club 79, REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SHAW, G. (1971). The chemistry of sporopollenin. Sporopollenin (ed. J. Brooks et al.), pp London, New York: Academic Press. (Received 6 July 1983-Accepted 12 September 1983)