ULTRASTRUCTURAL ASPECTS OF SPOROGENESIS IN THE APOGAMOUS FERN DRYOPTERIS BORRERI

Size: px

Start display at page:

Download "ULTRASTRUCTURAL ASPECTS OF SPOROGENESIS IN THE APOGAMOUS FERN DRYOPTERIS BORRERI"

Easter Paul
5 years ago
Views:

J. Cell Sci. 63, 125-134 (1983) 125 Printed in Great Britain The Company of Biologists Limited 1983 ULTRASTRUCTURAL ASPECTS OF SPOROGENESIS IN THE APOGAMOUS FERN DRYOPTERIS BORRERI E. SHEFFIELD*, S.

1 J. Cell Sci. 63, (1983) 125 Printed in Great Britain The Company of Biologists Limited 1983 ULTRASTRUCTURAL ASPECTS OF SPOROGENESIS IN THE APOGAMOUS FERN DRYOPTERIS BORRERI E. SHEFFIELD*, S. LAIRD AND P. R. BELL Department of Botany and Microbiology, University College London, Gotuer Street, London WC1E 6BT, U.K. SUMMARY The events that accompany sporogenesis in the apogamous fern Dryopteris borreri parallel those seen in sexually reproducing ferns. Organelles dedifferentiate and redifferentiate, and form a discrete band across the equator of dyads; nuclear vacuoles and lipid spherosomes appear during prophase, and the major part of the ribosome population is removed and subsequently replaced during meiosis. Similar events have been found to occur during sporogenesis in mosses, gymnosperms and angiosperms, and are therefore characteristic of the meiotic transition from sporophyte to gametophyte, even in the absence of a transition from diplophase to haplophase. The novel aspects of meiosis in D. borreri are largely those connected with the restitution event that precedes meiosis 1 and serves to maintain the sporophytic chromosome number throughout the life cycle of this fern. Pre-meiotic cells are regularly found to be cleaved by annular wall ingrowths, which traverse the cytoplasm but not the nuclei. The significance of these ingrowths in relation to theories concerning apogamy and plant cell division are discussed. INTRODUCTION The ultrastructural changes that occur during sporogenesis in sexually reproducing ferns have been described (e.g., see Sheffield & Bell, 1979; Marengo, 1979; Bell, 1981) and resemble those seen during sporogenesis in higher plants (e.g., see Dickinson & Heslop-Harrison, 1977). A regular sequence of nuclear events, organelle dedifferentiation and redifferentiation and ribosome elimination and replenishment characterize the process in all the ferns, gymnosperms and angiosperms investigated to date. These occurrences have been attributed to the change in phase from sporophytic to gametophytic that takes place during meiosis (Sheffield & Bell, 1979). Meiosis in these plants is characterized by a reduction from the diplophase to the haplophase chromosome number. No ultrastructural study has yet been made of meiosis in a life cycle in which the gametophytic chromosome number is unreduced. The sporophytic ploidy level has been shown to be maintained throughout the life cycle of apogamous ferns by means of a restitution event during sporogenesis and the subsequent generation of a sporophyte from the gametophyte without oogenesis (e.g., see Dopp, 1932; Manton, 1950). It is in such plants that one can expect to observe features associated with the phase change from sporophyte to gametophyte uncomplicated by the change from diplophase to haplophase. The aim of the present investigation was to examine meiosis in an apogamous species with the same chromosome Present address: Department of Botany, University of Manchester, Manchester M13 9PL, U.K.

2 126 E. Sheffield, S. Laird and P. R. Bell number as related sexual species in the expectation that differences would become apparent from the normal sequence that could be related to the elimination of the haplophase. MATERIALS AND METHODS Fertile fronds were taken from a cultivated plant of Dryopteris borreri, originally collected by Mrs P. J. Newbould from St Germains, Cornwall and determined by her to be of the 'diploid' (2n = 82) strain (Manton, 1950). Individual son were excised and fixed in 3 % glutaraldehyde in 0-05 M- phosphate buffer (ph 6-9) at room temperature for 3 h. The material was then washed twice and left overnight in ice-cold buffer. Post-fixation was in 2% aqueous osmium tetroxide for 2h at 0 C. Dehydration was in acetone and embedding in Durcupan or Epon. Material for electron microscopy was first sectioned at 4 fun and appropriate stages photographed with phase-contrast optics. These sections were remounted using the technique of Woodcock & Bell (1967), fine-sectioned and stained with uranyl acetate and lead citrate. They were examined in a Hitachi HS9, Siemens Elmiskop 1 or Siemens 101 electron microscope. Material for light microscopy was sectioned at 1-5 /Jm and stained either in 0-1 % Coomassie Brilliant Blue in 3: 1, ethanol/glacial acetic acid, or in a freshly made saturated solution of Sudan Black B in70%ethanol. RESULTS Pre-meiotic sporangia Successive divisions regularly gave rise to sporangia containing eight archesporial cells characterized by richly populated cytoplasm of the type shown in Fig. 1. There was an abundance of ribosomes in these cells, and profiles of endoplasmic reticulum and dictyosomes with attached vesicles were numerous. The plastids were large, often exceeding 7 /im in maximum length, and contained starch, several osmiophilic globuli and internal lamellae. Profiles of endoplasmic reticulum were frequently visible adjacent to the plastids. The mitochondria were smaller than the plastids, never exceeding 3 /zm in maximum diameter, although profiles indicating that both these classes of organelle were undergoing division were found at this stage. The mitochondria contained finger-like villi, and their stroma appeared less electron-opaque than the surrounding cytoplasm. The cell walls of this and of preceding stages were highly osmiophilic and generally formed a dark boundary around the cells, in both the light and electron microscope. Fig. 1. Cytoplasm of one of eight archesporial cells, which become spore mother cells. The organelles are well differentiated, ribosomes numerous, and the cell wall very osmiophilic. X In all micrographs :m, mitochondrion;/), plastid;«, nucleus ;«i, cell wall. Fig. 2. Portion of one of the spore mother cells from the inset, a slightly later stage of development than that of Fig. 1. The dark lines traversing the cells of the inset can be seen to consist of highly osmiophilic wall material. This material does not extend across the nucleus, although some discontinuities can be seen in the nuclear envelope. Fewer ribosomes and profiles of endoplasmic reticulum can be seen and the plastids have dedifferentiated. No envelope can be distinguished at the boundary of the plastids. X Inset: 4/Um section of the sporangium yielding Fig. 2. X220.

3 Sporogenesis in Dryopteris borreri 127 Figs 1-2

4 128 E. Sheffield, S. Laird and P. R. Bell Pre-leptotene /prophase The inset in Fig. 2 shows the phase-contrast appearance of the next stage of development of the eight-celled sporangia, during which lengths of wall material could be seen extending across the cells. Fig. 2 shows the electron microscopic view of this wall material, which traversed the cytoplasm but was never found to extend across the nuclei. The growth of this partitioning coincided with the return of the nuclei to an interphase condition after an apparently normal prophase and metaphase. Some discontinuity of the nuclear envelope could be detected at this time, but envelope dissolution and anaphase chromosome separation were never observed. The cytoplasm of these cells contained fewer ribosomes than the preceding stage, but the number of vacuolar and vesicular profiles had increased considerably. The plastids still contained starch and a few globuli, but no internal lamellae or plastid envelope could be resolved. The mitochondria remained unchanged, though appeared to have darker stroma in relation to the more diffuse cytoplasm of these cells. The bounding and partitioning walls were very highly osmiophilic and contained numerous multilayered regions. Late prophase Fig. 3 shows the stage of development that followed that of Fig. 2. The spore mother cells rounded off from one another, but remained connected by cytomictic channels measuring up to 20 nm in width. The cytoplasm contained large numbers of vacuoles, endoplasmic reticulum and membranous profiles, but very few ribosomes. The nuclei were characterized by invaginations of the inner membrane of the envelope (nuclear vacuoles), which frequently occupied almost half the volume of the nucleus. Fragments of electron-opaque material were visible in these vacuoles but their chemical nature was not determined. The plastids were of widely ranging shapes and sizes, contained no starch, and still showed no signs of a bounding envelope or internal lamellae. Electron-opaque inclusions, such as that illustrated by the bottom inset in Fig. 4, were found in the peripheral cytoplasm at this stage. These inclusions were similar in staining properties and appearance to aggregates of ribosomes and persisted throughout meiosis. A fine fibrillar layer was visible outside the plasmalemma (bottom inset, Fig. 4) that surrounded the spore mother cell. Another new class of inclusion appeared at this stage in the form of large, single Fig. 3. Spore mother cell, prophase, showing extensive development of nuclear vacuoles (nv). Plastids differ widely in shapes and sizes, stroma are very electron-opaque. Arrows indicate membrane-bound inclusions, cc, cytomictic channels; t, tapetum. X4000. Fig. 4. Portion of dyad cytoplasm showing band of organelles forming the equatorial plate. Envelope profiles distinguishable at some parts of plastid boundaries. Membranebound inclusion (arrow) resembles those of spore mother cells. Note scarcity of free ribosomes. X Top inset: 4ftfn section of spore mother cell in prophase showing phase-contrast appearance of membrane-bound inclusions (arrows). X780. Bottom inset: portion of spore mother cell peripheral cytoplasm showing aggregate of ribosome-like bodies. A fine fibrillar layer lies external to the plasmalemma. X

5 Sporogenesis in Dryopteris borreri 129 *< '. VM nv cc v la O Figs 3-4

6 E. Sheffield, S. Laird and P. R. Bell Figs 5-7

7 Sporogenesis in Dryopteris borreri 131 membrane-bound spherical bodies (arrows, Fig. 3; top inset, Fig. 4). The content of these inclusions was homogeneously electron-opaque and no sub-structure could be discerned. They showed an affinity for Sudan Black B, but no reaction to Coomassie Blue when stained and examined in the light microscope. Dyads The cytoplasm of spore mother cells altered very little during the remainder of meiosis I, and Fig. 4 shows the appearance of dyads. These cells were traversed by a band of organelles running between the telophase nuclei; Fig. 4 illustrates the similarity between the plastids and mitochondria at this time. Some of the plastids were found to contain starch and globuli, and most had regained an envelope profile and internal lamellae. Occasional profiles indicating organelle division were seen, and the single membrane-bound inclusions of early meiosis I were unchanged in number and appearance. Very few ribosomes could be detected, but some profiles of endoplasmic reticulum were found to bear ribosomes. Meiosis II Figs 5 and 6 illustrate the appearance of newly formed spores. Fig. 5 shows the thick layer of osmiophilic material that regularly lay outside the plasmalemma but internal to the fibrillar layer at this stage, some of which appeared to lie within the spore cytoplasm. Myelin figures could be discerned within this layer, but their fate could not be determined. The organelles had redifferentiated by this time, plastids showing starch, globuli and lamellae and mitochondria, large shelf-like cristae (Fig. 6). The membrane-bound inclusions remained unchanged in appearance and number. Endoplasmic reticulum profiles and ribosomes were slightly more frequent than in the preceding stage. Spores Fig. 7 shows a spore, surrounded by degenerating tapetum, inside which is the fine fibrillar layer seen in spore mother cells, which now lies around and between the spores. No sign of the myelin figures seen in the previous stage could be detected around the spores. The cytoplasm still contained the membrane-bound inclusions seen previously and the organellar components were slightly more differentiated but otherwise unchanged. Fig. S. Boundary between young spore and degenerating tapetum (t). A region occupied by highly osmiophilic myelin figures lies between the fibrillar layer and the spore plasmalemma, some profiles lie inside the cytoplasm. X Fig. 6. Cytoplasm showing redifferentiated organelles of young spore. Note distinct envelope of plastid, cristae within the mitochondrion and membrane-bound inclusion. Ribosomes are still scarce. X Fig. 7. One spore of tetrad showing no signs of osmiophilic material seen in Fig. 5. Organelles well differentiated, membrane-bound inclusions still present, also stacked endoplasmic reticulum on the far left and right of the spore. X7200.

8 132 E. Sheffield, S. Laird and P. R. Bell The endoplasmic reticulum was found to be banked together in discrete regions, but the ground cytoplasm was otherwise rather empty, ribosomes still being infrequent. DISCUSSION There are many similarities between sporogenesis in D. borreri and the sexually reproducing fern Pteridium (Sheffield & Bell, 1979). These include the development of cytomictic channels, a fall and then a rise in ribosome number, the aggregation of organelles in a band between telophase nuclei at division I and a cycle of organelle dedifferentiation and redifferentiation; all of which have also been found to characterize sporogenesis in heterosporous ferns and higher plants (see Bell, 1981, for review). The formation of nuclear vacuoles during prophase also resembles that seen in a wide range of plants and animals (Sheffield, Cawood, Bell & Dickinson, 1979), although the functional significance of these inclusions, and their form in vivo is still unclear. The appearance of bodies resembling nucleoloids or aggregates of ribosomes in meiocytes of D. borreri also mirrors that seen in Pteridium at similar stages (Sheffield & Bell, 1979). The major difference between the cytoplasmic events during sporogenesis in D. borreri and that of non-apogamous plants lies in the restitution stage of meiosis. The incomplete wall formation that precedes meiosis in all the sporangia of the present study cannot be reconciled with previous studies of sporogenesis in apogamous ferns. In her "generalized description of the features of meiosis in apogamous ferns", Manton (1950) identified four distinct types of sporangial development occurring on the same plant. In the first, 16 spore mother cells result from four successive archesporial cleavages, and the 64 spores subsequently formed abort. In the second type, three archesporial cleavages are normal, but the last division is imperfect, anaphase separation does not occur, and interphase becomes re-established in the eight mother cells. The third type differs from the second in that "the cytoplasmic activities which normally result in cell cleavage may not be entirely suppressed, but may be present in unco-ordinated forms...". "The nuclei become irregularly lobed, cell walls partially crossing the cell may be laid down, and sometimes cleavage into two unequal portions containing different-sized pieces of the restitution nucleus may result". This type of sporangium is suggested to be an 'imperfect' version of the type two sporangia. A fourth type of sporangium contains only four giant mother cells as a consequence of two restitution events, and only 16 spores result. Exhaustive examination of the material used in the present study has so far failed to reveal sporangia of types one, two and four, yet there was no indication that sporangial development was in any way imperfect, or that abortive spores were formed as a result of the events observed. This may be because the 'diploid' material examined behaved in a more uniform manner than the largely 'triploid' material of Manton. Further apogamous species will clearly need to be investigated at the ultrastructural level before the present study can be incorporated into a new generalized scheme of apogamous sporangial development. The ingrowth of cell wall material observed during the restitution phase is of

9 Sporogenesis in Dryopteris borreri 133 considerable interest with regard to cell division in plants. Cytokinesis in somatic cells of D. borreri occurs as a result of phragmoplast, followed by cell plate formation, regarded as the usual mode of division in vascular plants (Pickett-Heaps, 1969). The annular furrowing seen during restitution in D. borreri mimics the mode of cell division of prokaryotic organisms, and early stages of division oispirogyra (Fowke & Pickett-Heaps, 1969). Peripheral extension of cell walls has been observed in wheat cells in which karyokinesis is inhibited by caffeine (Pickett-Heaps, 1969) and extensions have been observed during normal meiosis in archegoniates. A small wall extension can be seen in anaphase II meiocytes of Onoclea (Marengo, 1977, fig. 1) and substantial wall ingrowths characterize both divisions of moss meiosis (Brown & Lemmon, 1982a,b). It therefore seems that wall stubs are a normal feature of archegoniate meiotic divisions, but that the lack of a cell plate can result in their extension into deep ingrowths. After preliminary examination of D. borreri sporangia it was suggested that the membrane-bound cytoplasmic inclusions seen in spore mother cells were of causal significance to the apogamous life cycle (Bell, 1979). Such bodies are certainly absent from Pteridium meiocytes but have now been found in spore mother cells of the sexually reproducing ferns Dryopteris filix-mas (Sheffield & Bell, unpublished observations) and Onoclea (Marengo, 1979). Meiocytes and spores of mosses also contain similar bodies (Brown & Lemmon, 1982a), and they are now therefore thought to be unconnected with apogamy. Their appearance in D. borreri coincides with the disappearance of the highly osmiophilic wall ingrowths, and it is possible that the lipidic component of these extensions contributes to these inclusions. The chemical nature of these inclusions remains uncertain, but their affinity for osmium and Sudan Black B and lack of affinity for protein stains suggest that they are predominantly lipidic. Brown & Lemmon (1982a,b) and Marengo (1979) suggest that the inclusions of Rhynchostegium, Amblystegium and Onoclea are lipidic, and the D. borreri bodies are indistinguishable from these inclusions. One unique feature of sporogenesis inz). borreri remains, namely the highly osmiophilic cell walls. The exterior of the archesporial cells, meiocytes and young spores are strikingly more osmiophilic than those of Pteridium or Onoclea, and are thus more reminiscent of those of mosses (Brown & Lemmon, 1982a). The myelin figures exterior, and partially interior, to the young spores find no equivalent during the same stage in any other study of meiosis. Similar profiles characterize early prophase in spore mother cells of Pteridium (Sheffield & Bell, 1979) and Lycopodium (Pettit, 1978) but their significance and subsequent fate during later stages of spore development in D. borreri remains a matter for conjecture. In conclusion, the present study demonstrates that nuclear vacuole development, cycles of ribosome expunging and replenishment, and organellar dedifferentiation and redifferentiation do characterize the meiotic transition from sporophyte to gametophyte, even in the absence of a transition from diplophase to haplophase. Whether they inevitably accompany the change in phase from sporophyte to gametophyte in the absence of meiosis must await an ultrastructural study of apospory.

134 E. Sheffield, S. Laird and P. R. Bell REFERENCES BELL, P. R. (1979). The contribution of the ferns to an understanding of the life cycles of vascular plants.

10 134 E. Sheffield, S. Laird and P. R. Bell REFERENCES BELL, P. R. (1979). The contribution of the ferns to an understanding of the life cycles of vascular plants. In Tlie Experimental Biology of Ferns (ed. A. F. Dyer), Experimental Botany, vol. 14, pp London: Academic Press. BELL, P. R. (1981). Megasporogenesis in a heterosporous fern: features of the organelles in meiotic cells and young megaspores. J. Cell Sci. 51, BROWN, R. C. & LEMMON, B. E. (1982a). Ultrastructure of sporogenesis in the moss, Amblystegium riparum. 1. Meiosis and cytokinesis. Am.J. Bot. 69, BROWN, R. C. & LEMMON, B. E. (19826). Ultrastructural aspects of moss meiosis: cytokinesis and organelle apportionment in Rkynchostegitan serrulatum.j. Hattori Bot. Lab. 53, DICKINSON, H. G. & HESLOP-HARRISON, J. (1977). Ribosomes, membranes and organelles during meiosis in angiosperms. Phil. Trans. R. Soc. B. 277, DOPP, W. (1932). Die apogamie bei Aspidium remotum Al. Br. Planta 17, FOWKE, L. C. & PICKETT-HEAPS, J. D. (1969). Cell division in Spirogyra. II. Cytokinesis. J. Phycol. 5, MANTON, I. (1950). Problems ofcytology and Evolution in the Pteridophyta. Cambridge University Press. MARENGO, N. (1977). Ultrastructural features of the dividing meiocyte of Onoclea sensibihs. Am. J. Bot. 64, MARENGO, N. (1979). The fine structure of the pre-meiotic stages of sporogenesis in Onoclea sensibilis. Am. FernJ. 69, PETTTT, J. M. (1978). Regression and elimination of cytoplasmic organelles during meiosis in Lycopodium. Grana 17, PICKETT-HEAPS, J. D. (1969). The evolution of the mitotic apparatus. An attempt at comparative ultrastructural cytology in dividing plant cells. Cytobios 3, SHEFFIELD, E. & BELL, P. R. (1979). Ultraatructural aspects of sporogenesis in a fern, Pteridium aquilinum (L.) Kuhn. Ann. Bot. 44, SHEFFIELD, E., CAWOOD, A. H., BELL, P. R. & DICKINSON, H. G. (1979). The development of nuclear vacuoles during meiosis in plants. Planta 146, WOODCOCK, C. L. F. &BELL, P. R. (1967). A method for mounting 4 fi resin sections routinely for ultrathin sectioning. Jl R. microsc. Soc. 87, (Received 9 February 1983-Accepted 28 February 1983)

THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L.

New Phytol (1974) 73, 139-142. THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L. BY JEAN M. WHATLEY Botany School, University of Oxford (Received 2 July 1973) SUMMARY Cells in