J. Cell Sci. Si, 109-119 (IQ8I)Printed in Great Britain © Company of Biologists Limited 1081

MEGASPOROGENESIS IN A HETEROSPOROUS

FERN: FEATURES OF THE ORGANELLES

IN MEIOTIC CELLS AND YOUNG MEGASPORES

P. R. BELLDepartment of Botany and Microbiology, University College London,Gower Street, London WCiE 6BT

SUMMARY

Megasporogenesis in the heterosporous fern Marsilea (Hydropterideae) shows featuresintermediate between sporogenesis in homosporous fern3 and that in heterosporous seedplants. The plastids in the dyads and young spores were associated with tubules 30—35 nm indiameter, probably a specialized form of endoplasmic reticulum. No consistent differences insize or cytoplasmic components could be found between the megaspores of a tetrad that mightaccount for differential survival. The view that megaspore viability within the tetrad is gene-tically determined is thereby strengthened.

INTRODUCTION

The Hydropterideae are a small group of ferns distinguished by regular heterosporyaccompanied by strict segregation of sex. The formation of the female megaspores isnormally accompanied by resorption of 3 of the products of each meiosis. In thesefeatures they resemble seed plants, but differ from all living seed plants in producingmegaspores (as the microspores) in symmetrical tetrahedral tetrads, quite similar tothose characteristic of sporogenesis in ferns generally. Sporogenesis in homosporousferns has now been investigated at the ultrastructural level (Sheffield & Bell, 1979)and the process shows many parallels with microsporogenesis in flowering plants(e.g. see Dickinson & Heslop Harrison, 1977). Megasporogenesis in flowering plantsis less well known, but the indications are that it is similar in essentials (Dickinson &Potter, 1978). Although the homosporous ferns and flowering plants represent widelydifferent levels of evolutionary complexity, their methods of sporogenesis are nowknown to have many features in common. The principal differences lie in a regularloss of distinctness of the plastid envelopes during the first prophase of homosporoussporogenesis, and the absence of nucleoloids from the cytoplasm of the young spores.

In Marsilea vestita, the subject of the present investigation, 8 tetrads of megasporesare produced. Three megaspores in each tetrad regress rapidly as the spores separate.Of the 8 viable megaspores only one, which eventually fills the whole sporangium,comes to maturity. Previous reports of sporogenesis in Marsilea have all been of lightmicroscopic studies (Feller, 1953; Boterberg, 1956), and M. vestita itself has not beeninvestigated before in this respect. The present study was undertaken to discoverwhether, when viewed in greater detail, megasporogenesis in Marsilea showed

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features intermediate between sporogenesis in homosporous plants, and mega- andmicrosporogenesis in the seed plants. Further, the regularity of the megaspore tetradsin Marsilea provided an opportunity to assess whether the viability of only one of the4 megaspores in each instance could be attributed to a conspicuous inequality in thedistribution of cytoplasmic components. There is evidence in Gingko, an archegoniateseed plant with a single linear tetrad of megaspores, that the innermost viable sporereceives a greater volume of cytoplasm, and correspondingly more organelles, thanthose that degenerate (Stewart & Gifford, 1967). Nevertheless, only if such aninequality were found to be a general feature of megasporogenesis could it be confi-dently regarded as having causal significance.

The present account takes megasporogenesis in Marsilea as far as the formation ofthe megaspore tetrads. The events following the breaking open of the tetrads will beconsidered separately.

MATERIALS AND METHODS

Immature sporocarps of M. vestita from a plant of Californian origin, grown in a greenhouse,were dissected on an agar surface with a microscalpel. Short lengths of receptacle (< 1 mm)were removed and plunged immediately into 3 % glutaraldehyde (TAAB Laboratories,Reading, U.K.) in 005 M-phosphate buffer (pH 69) at 10 °C. Tapping was adequate toremove entrapped air. Fixation was continued for 4 h, and followed by overnight washing inice-cold buffer. Osmication (2 % aqueous) was for 2 h at o °C. Dehydration was in acetone andembedding in Durcupan AR (Fluka AG, Switzerland).

The embedded material was sectioned at 4 /im with a glass knife and searched for megaspo-rangia. These were recognizable by their larger size and their location along the upper edge ofthe receptacle. The selected sections were photographed with phase-contrast optics andremounted by the technique of Woodcock & Bell (1967) for fine-sectioning. Staining was withsaturated aqueous uranyl acetate and lead citrate (Reynolds, 1963).

RESULTS

The developmental changes during sporogenesis are considered in relation to thecondition of the nucleus. This displayed the normal stages of meiosis, although thesynaptonemal complexes at zygotene/pachytene were very indistinct.

Fig. 1. Portion of a section of sporangium showing 2 megaspore mother cells andtapetal cell (t). a, possible autophagic region, x 25 000.

Insets: top left, thick resin section of sporangium yielding fine sections shown infigure. The spore mother cells lie grouped at the centre, x 315. Bottom right, commonwall between tapetum (below) and spore mother cell (above) at a slightly later stage.The position of the middle lamella (arrow) shows that the thickening is now princi-pally on the spore mother cell side. The plasmodesmata are mostly occluded, x 75 000.

In all micrographs: m, mitochondrion ;£, plastid; n, nucleus.Fig. 2. Portion of spore mother cell at slightly later state than Fig. 1. The boundaryof the plastid is now almost indistinguishable, that of the mitochondrion remainsdistinct, r, region rich in ribosomes surrounded by endoplasmic reticulum. x 45 000.

Inset: detail of boundary of plastid (ground cytoplasm, left) showing discontinuity,x 100000.

Fig. 3. Spore mother cell, early leptotene. Envelope of plastids again distinct. Arrowsindicate small imaginations of innermembraneof envelope into nucleoplasm. x 22 500.

Megasporogenesis in Marsilea

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i i2 P. R. Bell

Preleptotene

The megaspore mother cells, 8 in number, lay at the centre of the young sporan-gium. They were clearly set off as a group from the surrounding tapetum (Fig. i,upper inset), but each cell had i face in contact with it. It was evident from 4-/*msections that the organelles were largely adjacent to these faces, the regions towardsthe centre of the cluster being notably vesicular. This asymmetry was presumably aconsequence of the previous mitoses, the organelles remaining in the polar regions ofthe preceding spindles. Initially, the face in contact with the tapetum showed openplasmodesmata (Fig. i), but subsequently the common wall thickened, particularly onthe mother cell side (Fig. i, lower inset), and these plasmodesmata became occluded.Nodules of wall material, about 1-5 /im across and containing irregular profiles ofmembranes, were often seen at the boundaries of the mother cells (Fig. 1, right sporemother cell). Complex membranous profiles suggestive of autophagic regions werefrequent in the tapetal cells (Fig. 1).

As soon as the mother cells became recognizable a striking change was evident inthe plastid envelopes in the mother cells. Although these remained normal in thetapetal cells, in the mother cells the envelopes became progressively less distinct(Fig. 1). The loss of distinctness did not affect the whole envelope uniformly, butbegan locally, giving the envelope a discontinuous appearance (Fig. 2, inset). Ulti-mately the envelope could be distinguished only with difficulty (Fig. 2), and sometimesnot at all. The envelopes of the mitochondria by contrast remained unchanged inboth tapetum and mother cells (Figs. 1, 2).

Regions surrounded by paired membranes, containing high concentrations ofribosomes (Fig. 2), were occasionally seen at the stage at which the plastid envelopeswere barely detectable.

Prophase

The change in the response of the plastic envelope to fixation and osmication wasfound to be transient. By the beginning of leptotene the clarity of the envelopes wasre-established (Fig. 3). The asymmetry in the distribution of the organelles persisted.Some of the plasmodesmata in the common walls between the spore mother cells

Fig. 4. Portion of nucleus of a spore mother cell, late leptotene, showing extensivedevelopment of vesicular system from inner membrane of nuclear envelope. Arrowsindicate electron-opaque material (probably nucleolar). x 33 500.Figs. 5, 6. Comparison of representative areas of cytoplasm of spore mother cells atpre-leptotene (Fig. 5) and late leptotene (Fig. 6), showing fall in frequency of ribo-somes. Both, x 50000.Fig. 7. Telophase of first meiotic division. The mitochondria and plastids are congre-gated at the equatorial plate. The nuclear envelope of the daughter nuclei has notreformed, x 12500.Fig. 8. Plastids in a dyad associated with tubules in longitudinal section (upper arrow)and transverse (lower arrow) x 37 500.

Inset: an array of tubules in the vesicular area of the cytoplasm, a few above cuttransversely, x 60000.

Megasporogenesis in Marsilea

P. R. Bell

13

Megasporogenesis in Marsilea 115

enlarged to form cytomictic channels about 25 nm wide, the remainder disappeared.The channels were themselves not seen following leptotene.

Striking changes occurred at the nuclear envelope during prophase. At the beginningof leptotene the inner membrane of the envelope began to invaginate into the nucleo-plasm (Fig. 3, arrows) and these imaginations became more conspicuous as prophaseproceeded (Fig. 4). Membrane profiles also appeared within the imaginations (Fig. 4).indicating a complex folding of their surfaces as the invaginations extended throughoutthe nucleus. By diplotene/diakinesis, connections between the vesicular formations,now occupying much of the nucleus, and the inner membranes of the envelope wererarely seen, suggesting that structures beginning as invaginations were now free inthe nucleoplasm. No single nucleolus could be recognized at any time during meiosisibut aggregates of electron-opaque material, probably nucleolar, about o-i fim indiameter, were scattered amongst the invaginations (Fig. 4, arrows). The synapto-nemal complexes were similarly distributed.

By the end of prophase the cytoplasm was markedly less dense. Although therewere insufficient areas of ground cytoplasm clear of membrane to make meaningfulcounts, sections of pre-leptotene cells and those at the end of prophase showed a clearfall in the frequency of ribosomes (Figs. 5, 6). This was not accompanied by anysubstantial change in the volume of the cells.

Dyads

At telophase of the first division of meiosis the plastids and mitochondria, despitetheir asymmetrical distribution in prophase, were congregated in the region of theequatorial plate (Fig. 7). The cytoplasm in the polar regions was again largely vesicular.

A conspicuous new feature in the dyads was the association of the plastids, now oftencontaining prominent starch, with tubules 30-35 nm in diameter. These lay very closeto the outer membrane of the envelope and sometimes appeared continuous with it(Fig. 8, upper arrow). Arrays of these tubules were occasionally seen elsewhere in thecytoplasm (Fig. 8, inset). Sometimes single tubules could be seen continous withprofiles of endoplasmic reticulum (Fig. 9); but the width of the tubules (here about47 nm) was always less than the external dimension of the sheet (about 60 nm). Theboundary of the tubules frequently had a clear unit membrane profile (Fig. 8). Thiswas about 13 nm in width, slightly wider than the reticular membrane with which (inFig. 8) it was continuous.

Fig. 9. Profile of endoplasmic reticulum in dyad cytoplasm continuous with tubule.x 75 coo.Fig. 10. Section of tetrahedral tetrad passing through median plane of 3 spores. Thesurrounding tapetum is beginning to lose its cellular structure. The arrow indicatesthe strongly osmiophilic plasmalemma of the young spore, x 4625.Fig. 11. Plastid in young spore associated with tubules. Arrows indicate wheretubules are possibly fusing with the envelope, x 100000.Fig. 12. Possibly autophagic area in cytoplasm of young spore in tetrad, x 22 750.Fig. 13. Possible nucleoloids in cytoplasm of young spore, x 60000.

n6 P.R. Bell

Tetrads

The final division of meiosis, despite much searching, was never encountered. Itappeared to take place in the spore mother cells of one sporangium more or lesssimultaneously, since all of the tetrads encountered in sectioning were at closelysimilar stages of development. The tetrads were uniformly tetrahedral and there wasno evidence that the tetrads were oriented in any particular way in relation to thelongitudinal axis of the sporangium. The youngest tetrad found is shown in Fig. 10.The plasmalemma of the young spores was already notably osmiophilic, possiblyindicating the beginning of the secretion of exine.

Examination of 6 tetrads, including that shown in Fig. 10 in which the section layclose to the median plane of 3 of the spores, gave no indication of convincing inequalityof volume or of frequency of plastids and mitochondria in the visible spores. All thespores appeared equally well formed. It seems unlikely, in view of the irregulararrangement of the tetrads, that the fourth spore out of the plane of the section wouldin each of these 6 instances have been strikingly different in size and contents. Therewas no evidence of elimination or degeneration of organelles so long as the sporesremained adhering to each other within the boundary of the original spore mother cell.

The plastids in the young spore continued to be closely associated with tubules(Fig. 11), and some profiles again indicated actual fusion with the envelope (e.g. Fig.11, above). Regions of convoluted membrane enclosing cytoplasm (Fig. 12) wereoccasionally seen towards the distal part of the young spore. A feature of particularinterest was the presence of electron-opaque bodies 0-3-0-6 ftm in diameter, resemblingnucleolar material (Fig. 13), in both nucleus and cytoplasm. They resembled, butwere larger than, the bodies seen only in the nuclei during prophase. The cytoplasmof the young spores (Fig. 10) began to increase in density and resemble that of thepre-leptotene spore mother cells.

DISCUSSION

There are close similarities between megasporogenesis in Marsilea and sporogenesisin the homosporous fern Pteridium (Sheffield & Bell, 1979). These includetheocclusionof the plasmodesmata between the spore mother cells and their replacement bycytomictic channels, also subsequently eliminated, and the presence in the mothercells of regions of cytoplasm surrounded by membranes and rich in ribosomes. Theoccurrence of nodules of wall material with membranous inclusions, the temporaryabsence from the plastids of well defined envelopes, and the progressive fall inribosome frequency during prophase are also all features of sporogenesis in Pteridium.

There are however undoubted differences in the extent of these features and theirtiming. In Pteridium the cytomictic channels persist into late prophase, in Marsileathey were not seen following leptotene. The aggregates of ribosomes found in thepre-leptotene spore mother cells, although resembling the pseudo-nucleoloids ofSheffield & Bell (1979), were not so conspicuous, and there was no evidence that theywere involved in replenishing the ribosome population at a later stage of sporogenesis.

Megasporogenesis in Marsilea 117

The alteration in the nature of the plastid envelope was also more transient; inPtertdium it persisted into prophase but in Marsilea the envelopes were fully restoredin early leptotene, similar to the situation during microsporogenesis in Pinus (Dickinson& Bell, 1976). The possible nature of this striking change in the envelope has beendiscussed (Sheffield & Bell, 1979). The several instances of discontinuous envelopesseen in the present work show that the entire envelope was not affected simultaneously.Although ultimately the whole was uniformly indistinct, Marsilea may indicate thebeginning of the weakening of this effect. In megasporogenesis in Lilium, for example,the plastid envelopes diminish in distinctness at diplotene, but nevertheless remainreadily detectable (Dickinson & Potter, 1978).

The formation of the vesicular nuclear inclusions during prophase was closelysimilar in Marsilea and Pteridium. This phenomenon is now known to accompanymeiosis in several plants and animals (Sheffield, Cawood, Bell & Dickinson, 1979;Fouquet & Dang, 1980), but its significance is not yet known.

The tubules adjacent to the plastid envelopes in the dyads, and subsequently inyoung spores, have not been observed before in sporogenesis. They were consistentlylarger than microtubules, and there was no evidence that their walls had a similarsubunit structure. Further, although microtubules may have lateral connections withmembranes (Cronshaw, 1967; Bell, 1978), there are no reports of microtubules havingdirect continuity with membrane profiles. Despite their arising from endoplasmicreticulum, the Marsilea tubules are clearly different from the tubules described byJensen (1968) in cotton, by Quan, Chi & Caplin (1974) in cultured broccoli and byHoefert (1975) in Thlaspi, since in all these instances the tubules were never observedoutside endoplasmic reticulum cisternae. The Marsilea tubules much more closelyresemble tubules arising from the endoplasmic reticulum in the leaf gland of Phaseolus(Steer & Newcomb, 1969). Nevertheless they are not identical. The diameter of theMarsilea tubules falls between that of the 'small' (29 nm) and 'large' (56-66 nm)tubules in Phaseolus. The boundary of the Phaseolus tubules also shows no clear unit-membrane profile, and the tendency of the small tubules to disaggregate, and give riseto the large, finds no parallel in Marsilea. Although the Phaseolus tubules arise fromthe endoplasmic reticulum, the absence of any clear unit-membrane profile in thewall, and the ability of the wall to disaggregate, suggests that any lipid present is indiscrete micelles, and not as a bi-molecular leaflet, now generally accepted as thenormal conformation in cell membranes. The wall of the Marsilea tubule, by contrast,retains a clear membrane profile. The function of the tubules can at present only bespeculative, but it is conceivable that they are a specialized form of endoplasmicreticulum, facilitating a particularly active metabolic interchange between plastids,interlamellar space and cisternae of the normal reticulum at this stage of spore develop-ment. It is striking that the tubules show no similar association with the mitochondria.

The electron-opaque bodies in the young megaspores of M. vestita are probablyidentical with those staining densely with haematoxylin in the nucleus and cytoplasmat a similar stage of megasporogenesis in M. diffusa (Boterberg, 1956). They closelyresemble the nucleoloids described in micro- and megasporogenesis in Lillium(Dickinson & Heslop Harrison, 1977; Dickinson & Potter, 1978) and are believed to

n 8 P. R. Bell

be the source of many of the ribosomes appearing in the young spores. These nucleo-loids are regarded as being produced in the nucleus and left in the cytoplasm attelophase. This may be true of the similar bodies in the young megaspore of Marsilea,but electron-opaque aggregates visually identical to nucleoli can also appear in thecytoplasm without rupture of the nuclear envelope (e.g. in egg cells of the fernLygodium; Hutchinson & Bell, unpublished). Since there is no evidence that the fewpseudo-nucleoloids seen in prophase in Marsilea persist, a nucleoloid mechanism forreplenishing the ribosomes of the young spores is very probable. The membranousregions in young spores (Fig. 9) do not resemble the dispersing pseudo-nucleoloids ofPteridium (Sheffield & Bell, 1979; Plate 4B) and may be sites of local autophagy.The evolution of regular, sexually differentiated heterospory may therefore have beenaccompanied by direct passage of nucleolar material into the cytoplasm of the formingspores, with the concurrent loss of the pseudo-nucleoloids characteristic of homospory.

The early stages of megasporogenesis have given no explanation as to why only onemegaspore in each tetrad survives. Dyads always appeared equally matched and noconsistent differences between the 4 spores of the tetrad could be established. Therewas thus no evidence that the polarized distribution of the organelles in the megasporemother cells (also detected by Pettitt (1970) in an un-named species of Marsilea)persisted, and to regard it as effective in determining megaspore viability is unwarran-ted. The same doubt can be cast on Pettitt's (1977) interpretation of the cytoplasmicgradient in the megaspore mother cells of SelagineUa sulcata. Here, however, megas-porogenesis does not lead to the regular 1 :3 ratio of M. vestita, and the situation inthe lycopods generally may not be directly comparable to that in Marsilea and theseed plants. Gradients have also been held responsible for the failure of all but one ofthe microspores in each tetrad to develop in certain species of the Epacridaceae(Ford, 1972 a, b). In this instance the explanation is more satisfactory since there is noclear formation of dyads, and the gradient in organelle frequency in the pollen mothercell persists during meiosis. Three of the haploid nuclei move into the less-densecytoplasm and are resorbed. The formation of monad pollen in the Cyperaceae has beenascribed to spatial and physiological constraints arising from the radial packing of thewedge-shaped microspore mother cells (Strandhede, 1973). These considerations alsoseem inappropriate in Marsilea since the 8 tetrads of megaspores, separated by theintrusive tapetum, are arranged in no recognizable order, and the position of the viablespore in each is unrelated to the axis of the sporangium. As far as megaspore survivalin the tetrad is concerned, the present observations are in line with the argumentdeveloped elsewhere (Bell, 1979) that differential viability expressed asa 1:3 ratiohas a Mendelian basis.

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(Received 27 January 1981)