Planta
Planta 134, 229- 240 (1977)
9 by Springer-Verlag 1977
The R61e of Membrane-bound Cytoplasmic Inclusions during Gametogenesis in Lilium longiflorum Thunb. H.G. Dickinson and L. Andrews* Departments of Botany and Agricultural Botany, Plant Science Laboratories, University of Reading, Whiteknights, Reading RG6 2AS, U.K.
Abstract. In the prophase of both mega- and micro-
sporogenesis, a sizeable proportion of the meiocyte cytoplasm becomes invested in double or multiple membrane-bround inclusions. This cytoplasm remains thus isolated from the rest of the cell until the completion of meiosis II in the female cells, or the 'young spore' stage in those of the male. Significantly this encapsulation proceeds immediately the elimination of the major part of the ribosome population from the cytoplasm and, further, the electron microscope reveals that those ribosomes contained in these membranous inclusions remain unaffected by the lytic enzymes active elsewhere in the cytoplasm at this time. This encapsulated cytoplasm is proposed to fulfill two r61es; one, that it carries reserves necessary for postmeiotic development through from the diplophase to the haplophase environment and, two, that it permits the continuity of protein synthesis throughout meiosis I and II, a period when the major part of the protein synthetic apparatus is absent. Key words: Alternation of generation - Gametogenesis - L i l i u m - Meiosis - Ribosomes.
Introduction
The suppositions from the early work of Guillermond (1920) and subsequently that of Py (1932) that events during meiosis in higher plants are in no way restricted to the nucleus have only comparatively recently been confirmed with the electron microscope. Apart from a cycle of de- and redifferentiation in plastid population (Dickinson and Heslop-Harrison, * Present address." Luddington E.H.S. (MAFF), Stratford-onAvon, Warwicks, U.K.
MI=membrane-bound inclusions; MMI=multimembraned inclusions Abbreviations."
1970) one of the most conspicuous of these cytoplasmic activities is the eradication of the ribosome population from the pollen mother cell, and its restoration during the tetrad stage (Heslop-Harrison et al., 1967; Dickinson and Heslop-Harrison, 1970; Williams et al., 1973). While this phenomenon was originally observed in L i l i u m , there is little doubt that similar events are characteristic of microsporogenesis in a wide range of plants (Gaudevan, 1948 ; Painter, 1943). The rationale behind this 'ribosome cycle' has yet to be understood, but it is nevertheless quite evident that a period of considerable duration exists, between the zygotene stage of prophase I until some point in meiosis II, where the cells are apparently considerably lacking in apparatus for cytoplasmic protein synthesis. This is especially difficult to understand since proteins not only must be necessary for the actual mechanics of meiosis and cytokinesis, but also large amounts are certainly required subsequently for the manufacture of the pollen grain wall. These latter compounds may, of course, be manufactured prior to elimination of the ribosomes, but it is still difficult to conceive how such active cells could survive without active protein synthesis within the cytoplasm. This apparent paradox is also particularly valid for the female tissue, in which a cycle of ribosome elimination and restoration also occurs (Dickinson and HeslopHarrison, 1976). Here large quantities of a paracrystalline protein arise in the cytoplasm during meiosis II, indicating that somewhere in the cell, if not in the ground cytoplasm itself, equipment for protein synthesis exists or at least reserves of protein are held, capable of being transferred to the cytoplasm. Since both the developing microspores and megaspores are invested in a callosic special wall during this period, a barrier that has been shown to be effectively impermeable to macromolecules (Heslop-Harrison and Mackenzie, 1967; Dickinson and Bell, 1976),
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H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
the likelihood that the materials employed in the early postmeiotic differentiation of these cells are derived from tapetally-supplied precursors is remote. A source of these materials utilised during, and immediately following meiosis may lie in a class of cytoplasmic inclusions peculiar to the sporogenous cells over this period. These are membrane bound inclusions which are formed during early prophase, remain largely unchanged during karyo-and cytokinesis, and disperse during the young spore stage in male cells (Dickinson, 1971) or late in the tetrad in female cells. Significantly, in their disintegration those in the young spore appear to become associated with the developing pollen grain wall (Dickinson and Heslop-Harrison, 1971). In female cells, these bodies may be bound by a large number of double unit membrane profiles, and it has been suggested that their content is employed in the subsequent development of the embryosac (Rodkiewcz, 1970). In the work reported here, the formation of these membrane-bound inclusions (MI) has been closely examined and their behaviour during ensuing stages of mega- and microsporogenesis investigated in detail. The results ensuing considerably reinforce the hypothesis that these inclusions contain an isolated proportion of cytoplasm capable of active protein synthesis, and, in the male cells, contain or provide compounds important in the formation of the inner layer of the pollen grain exine. Materials and Methods Electron Microscopy The techniques employed in the preparation of pollen of Lilium for electron microscopy are described in Dickinson (1970). Ovaries containing megaspore mother cells at suitable stages of development (calibrated by m e a n s of prior fixations followed by wax embedding) were excised from flowers of Lilium longiflorum and sliced transversely into sections approx. 1 m m thick, under the surface of the fixative (2.5% Glutaric dialdehyde in 0.03 M PO4 buffer, at p H 7). Following fixation for 5 h at r o o m temperature, the material was rinsed several times over 12 h in the buffer and then post-fixed in o s m i u m textroxide. Osmication and subsequent preparation followed the same course as that outlined for the male tissue (Dickinson, 1970).
Estimation of the Proportion of Cytoplasm Occupied by, and the Numbers and Sizes of the Membranous Inclusions throughout Microsporogenesis A perspex grid calibrated in 1 cm 2 squares was placed over appropriate electron micrographs, and the area occupied by MI estimated to the nearest a/4cm2. The n u m b e r of MI was also recorded. The total area o f cytoplasm was estimated in the same way. In order to calculate the volume occupied by the MI and relate this measure, and the n u m b e r of inclusions, to the volume of cytoplasm representative of each developmental stage, it was necessary to
follow the change in cell and nuclear size throughout microsporogenesis. This was effected with the aid of a camera lucida and phase contrast optics, by drawing twenty cells and their nuclei from sections of each stage. The paper 'cells' and 'nuclei' were cut out and weighed, and the weight used as a measure of area. In order to relate weight units to p m 2 for the calculation of the actual size of the cells and nuclei, an eyepiece graticule and micrometer slide were employed. Using methods outlined below, these measurements were then converted to volume. The cells and nuclei in the zygotene-early pachytene and late pachytene stages were regarded as spherical and a measure of their volume could be calculated using the formula ~nr3, and by subtraction a measure of the cytoplasmic volume could be derived. After diakinesis and the breakdown of the nuclear envelope the same method could not be used, and only a measure of overall cell size could be found. In addition estimations were not made during the second meiotic division stage, on account of asynchrony in division of the cells. In the early tetrad stage, when the nuclear m e m b r a n e s have been reformed, a simple formula was derived to allow for the volume occupied by the callose walls, and a similar method used to calculate a representative cytoplasmic volume as in prophase. It proved impractical to calculate the volume of the young microspores as sections show that these are not spherical, but it is known from electron microscope studies that the MI have disappeared by the vacuolate spore stage. The size of individual MI at each stage was calculated by dividing the total volume of cytoplasm occupied by M1 by the n u m b e r of MI present.
Estimation of the Proportion of Cytoplasm Occupied by the MI during Megasporogenesis The electron microscope reveals that a far greater proportion of the female cytoplasm is occupied by MI than is observed in male cells. However, the extreme vasiculation of the female cytoplasm during megasporogenesis makes precise measurement of cytoplasmic volume impossible. For this reason no quantitative studies have been carried out on the developing megaspore mothercell and its products.
Results
The Formation of the Membrane Bound Inclusions
The electron microscopic image of the preprophase megaspore mother cell and pollen mother cell cytoplasm of Lilium is indicative of a period of intense protein synthesis. The ground matrix is electron opaque, and the protoplast is filled with banks of ribosome encrusted endoplasmic reticulum. Conspicuous features of this reticulum are the large numbers of structures, similar to nuclear pores, set into the membrane (Fig. 1). Numerous ribosomes, often in polyribosome conformation, also abound in the cytoplasm. At the onset of the leptotene stage and the beginning of the deposition of the callosic special wall, considerable reorganisation of this cytoplasm takes place. The endoplasmic reticulum, previously organised in banked arrays, becomes pleomorphic and corn-
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
231
Fig. 1. Pollen mother cell cytoplasm of Lilium in very early prophase. Endoplasmic reticulum (arrows) is freely distributed, and the inset reveals it to possess structures resembling nuclear pores, x 23,400; inset x 64,800 Fig. 2. The encapsulation of cytoplasm during prophase in pollen mother cells. Both double (D) and multiple (M) membraned inclusions are present, as are profiles suggesting that encapsulation is in progress (F). x 5598 Fig. 3. As Figure 2, but in the megaspore mother cell. Here the membranes often appear associated with a "membrane-centre" (C), and exclusively multiple membraned inclusions are formed. Note the mitochondrion (M) invested by the membrane, x 13,743
232
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
233
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis Ceil volume(~Jm 3) 9000-
Percentage of cytoplasm occupied by Mls 6-
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Nuclear volume jm 3)
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-500
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ProphaseI
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Meta-Ana- ~ MeiosisH ITetrad'Young' TetophaseI spore Developmenta[ stage
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Prophasel ' Meta-Ana- ' TetophaseI
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'Tetrad'Youn( spore Developmental stage
Fig. 6. The percentage of a "unit volume" of cytoplasm occupied by membrane bound inclusions (MI) [x and - standard error] throughout meiosis and early pollen development
Fig. 7. Changes in cell (o) and nuclear (e) volumes [+ and standard error] throughout meiosis and early pollen development
mences to form into spheres enclosing areas of cytoplasm. Since each element of the endoplasmic reticulum is formed of two appressed unit membrane profiles, the resultant structure is a double membrane bound inclusion, measuring between 1 and 3 gm in diameter, enclosing a portion of the premeiotic cytoplasm (Fig. 2). This encapsulation appears to occur at random, for not only are the cytoplasmic ribosomes invested, but often also small mitochondria and
indeed, other regions of included cytoplasm, thus giving rise to an assembly composed of up to 5 concentric layers of double unit membrane profiles (Fig. 3). Such multi-membraned inclusions (MMI are particularly characteristic of female tissue, whereas meiocytes contain mainly double membraned inclusions (DMI) (Fig. 4)). Structures with a diameter exceeding 1 gm appear not to be invested and it is thus rare to observe dedifferentiated plastids within the inclusions. During these early stages of formation the cytoplasm within and without the inclusions appears identical, and, similarly, ribosomes appear to remain attached to the membrane as they were when it constituted the banks of rough endoplasmic reticulum. The amount of cytoplasm encapsulated is considerable, as may be observed from the electron micrographs and, indeed from light micrographs of Azure B stained material (Fig. 5). By the end of the leptotene stage, just prior to the elimination of the ribosomes, it is estimated in the male cells that approaching some 4% of the protoplast is occupied by the inclusions (Fig. 6). These measurements also indicate that the actual size of the male cells and their nuclei begins to decrease during this period (Fig. 7). This shrinkage accelerates during meiosis I, and continues throughout meiosis II.
Fig. 4. Male cytoplasm at anaphase I showing the high concentration of ribosomes in the MI, (arrows) when compared with their frequency in the ground cytoplasm (C). A cytoplasmic 'nucleoloid' (N) is shown immediately prior to its disintegration to provide a fresh population of cytoplasmic responses, x 14,580 Fig. 5. Pollen mother cells of Lilium stained with Azure B. Note that inclusions, presumably the majority of them (deduced from electron micrographs) MI, occupy a considerable proportion of the prophase cytoplasm. Light micrograph x 780 Fig. 8. Inclusion invested by four layers of double membrane. Note the increase in concentration of ribosomes towards the centre of the inclusion, x 18,760 Fig. 9. Multimembrane bound inclusion in prophase II of megasporogenesis. The arrows indicate ribosomes in polysomal configurations, x 23,400
234
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
235
Number Mls 320-
Average vo[ume of Mls (pm3)
1
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[
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Fig. 11. Changes in volmne of membrane-bound inclusions (MI) [x and standard error] throughout meiosis and early pollen development
Fig. 12. The numbers of membrane-bound inclusions (MI) contained in a "unit volume" of cytoplasm [x and - standard error] throughout meiosis and early pollen development
The Behaviour of the Inclusions during Meiosis and Early Mega- and Microsporogenesis
membrane layer are probably eradicated, for the multimembraned inclusions assume a graded aspect, with a higher concentration of ribosomes within the centre compartment (Fig. 8). Early in meiosis the free ribosomes within the inclusions appear dispersed, but as reduction division proceeds an increasing proportion appear in polyribosomal configuration until, by the prophase of meiosis II, most of the ribosomes are so organised (Fig. 9). Eradication of the ribosomes appears to cease in pachytene stage and the inclusions, be they double or multimembrane bound, maintain their general aspect until their disintegration in the young spore stage, despite the repopulation of the cytoplasm with a new population of ribosomes following telophase I and telophase II. A characteristic of the female cytoplasm during these later stages is the appearance of paracrystalline arrays of protein. These gradually accumulate from the end of meiosis I, and, by the tetrad stage, may form masses some 1 2 ~tm in maximum dimension (Fig. 10). These accumulations are never observed either in, or in association with the membrane-bound inclusions. As may be seen from Figure 6, little further encap-
Early in the zygotene stage, coincident with removal of the free ribosomes from the cytoplasm, ribosomes are lost from the outer face of the inclusions. Ribosomes within the inclusions are not similarly affected, be they attached to the membrane or free. Some ribosomes within inclusions bounded by a single double
Fig. 10. Paracrystaltine arrays of proteins in the tetrad cytoplasm of a young megasporocyte of Lilium. x 27,250 Fig. 13. Young microspore cytoplasm showing the "splitting" of the double membranes (arrows) bonding the inclusions. Cytoplasmic ribosomes are still low in number Fig. 14. As Figure 13, but at a slightly later stage. The regions of membrane heavily encrusted with ribosomes (arrows) are well shown, and seen, in the main, to be orientated toward the cell surface (S). • 15,625 Fig. 15. Material at the same stage as that depicted in Figure 14, but revealing microtubules (arrows) running subjacent to the plasma membrane, x 51,380 Fig. 16. Material again as in Figure 14, but showing the apparent change from double to single (arrows) unit membrane profiles bounding the disintegrating inclusions, x 24,525
236
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
Fig. 17. Ribosome encrusted membranous plate (arrows) appressed to the lower face of the plasma membrane in developing pollen of Lilium. x 58,635 Fig. 18. Vacuolate young microspore showing the absence of any 'membrane-bound inclusions' in the cytoplasm layering the periphery of the cell. The exine (E) is almost fully formed, and the cytoplasm contains large numbers of amyloplasts (A). Vacuole: V. x 8,156 Fig. 19. Disintegration of multi membrane bound inclusions in the tetrad stage of megasporogenesis. The membranes bounding the inclusions (/) become irregular, and its content assumes a diffuse texture, x 16,200 Fig. 20. Final stages in the disintegration of multimembrane-bound inclusions in the tetrad stage of megasporogenesis. The bounding membranes (arrows) have now become discontinuous and the inclusion's content has mergedwith the cytoplasm Fig. 21. A general aspect of cytoplasm at the late tetrad stage of megasporogenesis in Lilium. Note the almost complete absence of multimembraned inclusions of the type characteristic of earlier stages, x 86,400
H.G. Dickinson and L. Andrews: CytoplasmicInclusions during Gametogenesis sulation takes place in the male cells following the zygotene stage when about 5.5% of the protoplast has been invested. The quantity of cytoplasm included does not change very significantly in subsequent stages, although very slight overall increase is indicated by statistical analysis of the results. The electron microscope indicates that these observations also apply to the female tissue. The measurements and, to a certain extent, the electron microscope suggest that a striking change takes place in the nature of the MI at the end of meiotic prophase in the male cells. At this point the MI increase in number (Fig. 12) and decrease considerably in size (Fig. 11), the volume of cytoplasm encapsulated remaining approximately the same (Fig. 6).
The Disintegration of the Membrane Bound Inclusions Here, considerable differences in detail were observed between male and female tissues. In the male, components of the MI appeared to be involved in the synthesis of wall components, while in the female the MI merely disintegrate within the tetrad cytoplasm. Detailed descriptions of the two processes follow below.
The fate of the inclusions in the male cytoplasm Once the young spores are released from the callosic special wall, the double membraned inclusions begin to disintegrate. This is not a simple process, and appears to commence with changes to the investing membrane. Over most of the periphery of the inclusion, the two elements of the membrane split, creating a considerable clear space (Fig. 13). This appears to take place at the expense of the content of the inclusion, rather than the cytoplasm. At the same time, certain regions of the remainder of the surface becomes highly electron opaque, and heavily encrusted with ribosomes on both its inner and outer face (Fig. 14 and 16). Areas of membrane not affected in either way become diffuse and subsequently may appear as single unit membrane profiles (Fig. 16). This disintegration is accompanied by a general increase in size of the inclusions, as evidenced by the measurements (Fig. 11). The precise fate of the inclusion content is not clear, it is not expelled directly into the cytoplasm, for no direct connection between the inclusion interior and the cytoplasm is ever seen. Instead, this content appears simply to become eradicated as the volume invested by the inner unit membrane profile becomes smaller. Some of the ribosomes (and, presumably other contents) may become associated with the
237
darkly staining ribosome-encrusted region of the membrane. A considerable proportion of the inclusions now begins to move towards the periphery of the cell, generally orientated in such a manner that the ribosome-encrusted areas are facing t h e plasma membrane (Fig. 14). We have not identified the agents responsible for the movement of the inclusions, but a considerable number of microtubules are present in the young spores at this stage (Fig. 15). In the course of this journey, the main body of the inclusions is generally lost, so that only the areas of ribosome coated membrane, which appear as plates, arrive subjacent to the plasma membrane (Fig. 17). How the remainder of the inclusion is removed is not evident, but the investing membrane, once the content is completely lost, appears to dissociate into vesicles. The membranous plates now sited under the plasma membrane are without doubt associated with the formation of an electron-lucent lamella, upon which acetolysis-resistant polymer of the nexine is deposited. This process is reported in detail elsewhere (Dickinson and Heslop-Harrison, 1971; Dickinson, 1975). This disintegration of the inclusions is rapid for, by the time the spore becomes vacuolate, which occurs only shortly after its release from the tetrad, no profiles of inclusions are visible within the protoplasts (Fig. 18).
The fate of the membrane bound inclusions in female tissue Here the demise of the membranes is far less spectacular than in the male cells. The contents of the MI first became diffuse, and the ribosomes contained therein difficult to detect. The membranes then become progressively more irregular (Fig. 19), in a manner reminiscent, but not so drastic, of those in the young spores. Large spaces are generally not formed between pairs of membranes, but elements do appear to fuse. The bounding membranes finally become discontinuous (Fig. 20), allowing mixture of the inclusion content with the haplophase cytoplasm, in which it is quickly dispersed. The membranes themselves are not eliminated, but remain free in the cytoplasm sometimes carrying polyribosomal clusters until, a t least, the end of the tetrad stage of megasporogenesis (Fig. 21). Discussion
The Encapsulation of the Cytoplasm and the Source of the Membrane Involved The apparent nuclear pores present in the rough endoplasmic reticulum of the proprophase mother cell
238
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
of both sexes indicate the nucleus to be the source of this membrane. Such a process has also been reported in the reproductive cells of Sciadopteris (Gianordoli, 1969), and appears also to take place throughout microsporogenesis in Lilium at the stages when there is a nuclear envelope present (Dickinson, 1968, 1971). No particular cytoplasmic event may conclusively be linked with the change in aspect of these membranes from the rough endoplasmic reticulure, often ' b a n k e d ' in the male cells, to the relatively ribosome-free dispersed sheets of membrane. Certainly this occurs at the beginning of a time of considerable cytoplasmic reorganisation; the plastids are commencing dedifferentiation (Dickinson and Heslop-Harrison, 1970, 1976), the cells are becoming isolated by the callosic special wall, and elimination of the ribosome population is about to occur. It is feasible that one of the first effects of the degredating agents is to disrupt the organisation of the ribosomecoated banks of membrane, but data to confirm such a supposition would be almost impossible to obtain. Equally, we are in ignorance of the factors controlling the actual process of encapsulation. F r o m the micrographs the sheets of membrane first form cuplike profiles and then close up, investing an area of cytoplasm. Clearly, while the forces involved there are undoubtedly biophysical, they must result from some chemical change in the cytoplasm. We have little indication as to the nature of this change, but simple observation of any plant cells held to be "badly-fixed" for the electron-microscope reveals a tendency for the formation of such structures. These 'artefacts' are presumably either the result of incorrect buffering of the specimen, or more probably from a period of activity of lytic enzymes permitted by slow or incomplete penetration of fixatives. Certainly considerable degradation of the cytoplasm is also occurring in these premeiotic cells, and it is perhaps not unreasonable to propose that such unfavourable conditions in the cytoplasm, be they naturally or artificially induced, result in the biophysical changes necessary to cause the transformation of free membrane to vesicles. It is important to point out that this process is no way completely analogous to 'bad fixation', for no single membraned vesicles are formed, but rather that some of the same factors may be involved here. While the encapsulation appears to take place at random, the nature of the inclusions formed is constant within cells of a particular sex. In the male cells, double-membraned inclusions are most frequently seen, with a diameter between 0.5 and 0.8 ~t, while, in the female, multi-membraned inclusions with a diameter of up to 3/am are most common. It is noteworthy that the central inclusion of the female
MMI is of the same order of dimension as the DMI in the male, and it may be that biophysical forces again dictate a minimal size for these inclusions. Presumably the final dimension of the inclusion depends upon the volume of cytoplasm and area of membrane available, and since there is more of both in the rapidly expanding megaspore mother cell, it is hardly surprising that M M I are more characteristic of this tissue than the male. While the most attractive explanation of the gradation of rfbosome numbers in the inclusions is that the membranes are conferring a partial immunity to their contents from the degradative agents, the fact that the MI do not seem to change in their gradation throughout development, and that they appear to be formed during the beginning of the eradication of ribosomes, suggests an alternative explanation. Since the ribosome-degrading agents are possibly active during the course of formation of a particular MMI, the last volume of cytoplasm encapsulated would contain correspondingly fewer ribosomes than those contained in the central inclusion. This would result in a gradation of ribosomes across the inclusion from the time of its formation. An implication of this proposal would be that the membranes confer total immunity to their content throughout meiosis I and II. The gradual decrease in volume of the protoplast throughout meiosis results from the deposition of the series of callose 'special walls' which clearly must displace t h e cytoplasm, particularly in the tetrad of young microspores.
The Activities of the Inclusions during Meiosis I and H Apart from small changes which may be explained in terms of experimental error, no significant alteration in the amount of cytoplasm encased takes place during meiosis I and II. The reason for the fragmentation of the MI population in male cells to form a greater number of smaller inclusions is not easily explained. This event does, however, occur at the point at which the karyoplasm is mixed with the cytoplasm on the rupture of the nuclear envelope and there is evidence that pronounced biophysical changes do occur to the cytoplasmic inclusions at this point (He- . slop-Harrison and Dickinson, 1967). It is thus not unreasonable to propose that similar changes overcome the MI which result in the breakage and reformation of their bounding envelopes. Whatever the explanation of the graded aspect of the M M I those ribosomes on the surface of the outer membrane of the inclusions are certainly also affected and it is noteworthy that these organelles
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
are not completely degraded, but elements of their structure remain as a granular deposit, perhaps indicating that the active principle attacks only a particular component of the ribosome. Apart from these changes, the aspect of the cytoplasm contained in the inclusions hardly differs from that characteristic of the preprophase meiocyte throughout meiosis I and II. What changes there are appear to involve the organisation of the ribosomes, which become progressively associated into polysomal configurations. In female cells, where this association is most conspicuous, it is accompanied by an accumulation of paracrystalline proteins in the cytoplasm. Since the megaspore mother cell is invested in a callosic wall (Rodkiewcz, 1970), through which penetration of macromolecules is unlikely (HeslopHarrison and Mackenzie, 1967; Dickinson and Bell, 1976), and since the ground cytoplasm itself possess very few ribosomes, the evidence points to the MI as the source of this protein. Such a conclusion implies that the protein synthetic machinery situated within the inclusions could indeed be providing a supply of proteins for the cells throughout meiosis I and II, a time when its ground cytoplasm is free of any such apparatus.
The Fate of the Inclusions
While the fate of the inclusions does differ between the sexes, their demise appears to take place in approximately the same fashion, and at an identical point in the development of the cells. The main difference lies in the fate of their bounding membranes. In the first stage of degeneration, which occurs during the tetrad stage (Dickinson and Heslop-Harrison, 1970), elements of the investing membranes split, electron lucent spaces appearing between them. At this point development differs between the sexes, for in the female the membrane continues to degenerate, finally becoming discontinuous and permitting mixture of the inclusion content with the cytoplasm. The agents responsible for these events have not been identified, but it is interesting that, just prior to the mixture of the inclusion content with the cytoplasm, the former becomes diffuse, indicating that some elements of the diplophase cytoplasm may be eliminated before release into the 'haplophase' environment. More conclusive evidence is, however, available concerning the male cells. Here where it has been demonstrated that development of pollen wall in Lilium is maternally controlled (Heslop-Harrison, 1971), we find elements of the membranes investing the MI, clearly synthesised in the diplophase cytoplasm, contributing to the pollen wall. How this takes place
239
is not entirely clear at present, but the elements of membrane are first situated immediately subjacent to the plasma membrane by microtubules, where they control the deposition of acetolysis-resistant polymer upon an electron-lucent former. These layers of polymer are then packed into the wall to form the nexine layer (Dickinson and Heslop-Harrison, 1971; Dickinson, 1976).
The Rble of the Membranous Inclusions
While many aspects of the formation, behaviour and disintegration of these inclusions remain unclear, evidence is emerging that they (in female cells in any case) are capable of manufacturing protein for export to the main body of the cytoplasm and that they (in male cells) contribute elements to post-meiotic processes known to be under maternal control. Since the data from measurements indicate that encapsulation occurs over a very restricted period, just prior to the reorganisation of the sporophyte cytoplasm, it thus does appear likely that, at this point, a portion of'diplophase' cytoplasm is isolated and protected from the degradative agents that subsequently arise in the cytoplasm. Further, this cytoplasm seems not only richer in compounds required for the control of post-meiotic development, but also capable of synthesising protein for export to the dividing cells over the period when their main protein synthetic apparatus is in abeyance, during the fundamental reorganisation of the cytoplasm that is emerging as such a significant part of gametogenesis, and other changes of phase in the course of plant development (Dickinson and Heslop-Harrison, 1976). The statistical portion of this work was carried out by L.A. as part of the B.Sc. Course in the Department of Agricultural Botany. The authors thanks are due to the S.R.C. for financial support, and to Ursula Potter for valuable assistance with the illustrations.
References Dickinson, H.G.: Aspects of the physiology and ultrastructure of pollen development. Ph.D. thesis. Univ. Birmingham 1968 Dickinson, H.G.: Ultrastructural aspects of primexine formation in the microspore tetra of Lilium longiflorum. Cytobiologie 1(4), 437M49 (1970) Dickinson, H.G. : Nucleo cytoplasmic interaction following meiosis in the young microspores of Lilium longiJTorum. Events at the nuclear envelope. Grana 2, 107 127 (1971) Dickinson, H.G., Bell, P.R. : The changes in the tapetum of Pinus banksiana accompanying formation and maturation of the pollen. Ann. Bot. in press (1976) Dickinson, H.G. : Common Factors in exine deposition. In: Evolutionary significance of the exine K. Ferguson et al, (edit.). London: Academic Press 1976
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Dickinson, H.G., Heslop-Harrison, J. : The behaviour of plastids during meiosis in the microsporocytes of Lilium longiflorum. Thunb. Cytobios 6, 103-118 (1970) Dickinson, H.G., Heslop-Harrison, J.: The ribosome cycle, nucleoli and cytoplasmic nucleoloids in the meiocytes of Lilium. Protoplasma 69, 187-200 (1970) Dickinson, H.G., Heslop-Harrison, J.: The mode of growth of the inner layer of the pollen grain exine in Lilium. Cytobios. 4, 233-243 (1971) Dickinson, H.G., Heslop-Harrison, J.: Ribosomes in meiosis. Proc. roy. Soc. (B), in press (1976) Gaudevan, P.: l~change de materieux figures entre noyau et cytoplasm. Gatlica Biol. Acta 1, 205 226 (1948) Gianordoli, M.: Observations sur l'organisation du cytoplasm et sur la presence de lamelles annelees dans la cellule antiale du Sciadopteris verticillata. Rev. Cytol. Biol. v6g. 32, 183-202 (1969) Guillermond, A. : Sur l'evolution du condriome pendant la formation des grains de pollen de Lilium eandidum. C.R. Acad. Sci. Paris 170, 1003 1006 (1920) Heslop-Harrison, J.: Sporopollenin in the biological context. J. Brooks et al. (edit.). In: '~Sporopollenin ". London: Academic Press 197l
Heslop-Harrison, J., Dickinson, H.G.: A cycle of sphaerosome aggregation and disaggregation correlated with the meiotic divisions in Lilium. Phytomorphology 17, 195-199 (1967) Heslop-Harrison, J., Mackenzie, A.: Autoradiography of soluble (214C)-thymidine derivatives during meiosis and microsporogenesis in Lilium anthers. J. cell Sci. 2, 387-400 (1967) Mackenzie, A., Heslop-Harrison, J., Dickinson, H.G. : Elimination of ribosomes during meiotic prophase. Nature 215, 997-999 (1967) Painter, T.S. : Ceil growth and nucleic acid in the pollen of Rhoeo discolor. Bot. Gaz. 105, 58-68 (1943) Py, G. : Recherches cytologiques sur l'assise nourriciere des microspores et les microspores des plants vasculaires. Rev. G6n. Bot. 44, 316-368, 369-413, 450 462 (1932) Rodkiewcz, B.: Callose in cell walls during megasporogenesis in angiosperms. Planta 93, 39 47 (1970) Williams, E., Heslop-Harrison, J., Dickinson, H.G.: The activity of the nucleolus organising region and origin of the cytoplasmic nucleoloids in meiocytes of Lilium. Protoplasma 77, 79-93 (1973)
Received 25 September; accepted 12 November 1976
Planta 134, 229- 240 (1977)
9 by Springer-Verlag 1977
The R61e of Membrane-bound Cytoplasmic Inclusions during Gametogenesis in Lilium longiflorum Thunb. H.G. Dickinson and L. Andrews* Departments of Botany and Agricultural Botany, Plant Science Laboratories, University of Reading, Whiteknights, Reading RG6 2AS, U.K.
Abstract. In the prophase of both mega- and micro-
sporogenesis, a sizeable proportion of the meiocyte cytoplasm becomes invested in double or multiple membrane-bround inclusions. This cytoplasm remains thus isolated from the rest of the cell until the completion of meiosis II in the female cells, or the 'young spore' stage in those of the male. Significantly this encapsulation proceeds immediately the elimination of the major part of the ribosome population from the cytoplasm and, further, the electron microscope reveals that those ribosomes contained in these membranous inclusions remain unaffected by the lytic enzymes active elsewhere in the cytoplasm at this time. This encapsulated cytoplasm is proposed to fulfill two r61es; one, that it carries reserves necessary for postmeiotic development through from the diplophase to the haplophase environment and, two, that it permits the continuity of protein synthesis throughout meiosis I and II, a period when the major part of the protein synthetic apparatus is absent. Key words: Alternation of generation - Gametogenesis - L i l i u m - Meiosis - Ribosomes.
Introduction
The suppositions from the early work of Guillermond (1920) and subsequently that of Py (1932) that events during meiosis in higher plants are in no way restricted to the nucleus have only comparatively recently been confirmed with the electron microscope. Apart from a cycle of de- and redifferentiation in plastid population (Dickinson and Heslop-Harrison, * Present address." Luddington E.H.S. (MAFF), Stratford-onAvon, Warwicks, U.K.
MI=membrane-bound inclusions; MMI=multimembraned inclusions Abbreviations."
1970) one of the most conspicuous of these cytoplasmic activities is the eradication of the ribosome population from the pollen mother cell, and its restoration during the tetrad stage (Heslop-Harrison et al., 1967; Dickinson and Heslop-Harrison, 1970; Williams et al., 1973). While this phenomenon was originally observed in L i l i u m , there is little doubt that similar events are characteristic of microsporogenesis in a wide range of plants (Gaudevan, 1948 ; Painter, 1943). The rationale behind this 'ribosome cycle' has yet to be understood, but it is nevertheless quite evident that a period of considerable duration exists, between the zygotene stage of prophase I until some point in meiosis II, where the cells are apparently considerably lacking in apparatus for cytoplasmic protein synthesis. This is especially difficult to understand since proteins not only must be necessary for the actual mechanics of meiosis and cytokinesis, but also large amounts are certainly required subsequently for the manufacture of the pollen grain wall. These latter compounds may, of course, be manufactured prior to elimination of the ribosomes, but it is still difficult to conceive how such active cells could survive without active protein synthesis within the cytoplasm. This apparent paradox is also particularly valid for the female tissue, in which a cycle of ribosome elimination and restoration also occurs (Dickinson and HeslopHarrison, 1976). Here large quantities of a paracrystalline protein arise in the cytoplasm during meiosis II, indicating that somewhere in the cell, if not in the ground cytoplasm itself, equipment for protein synthesis exists or at least reserves of protein are held, capable of being transferred to the cytoplasm. Since both the developing microspores and megaspores are invested in a callosic special wall during this period, a barrier that has been shown to be effectively impermeable to macromolecules (Heslop-Harrison and Mackenzie, 1967; Dickinson and Bell, 1976),
230
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
the likelihood that the materials employed in the early postmeiotic differentiation of these cells are derived from tapetally-supplied precursors is remote. A source of these materials utilised during, and immediately following meiosis may lie in a class of cytoplasmic inclusions peculiar to the sporogenous cells over this period. These are membrane bound inclusions which are formed during early prophase, remain largely unchanged during karyo-and cytokinesis, and disperse during the young spore stage in male cells (Dickinson, 1971) or late in the tetrad in female cells. Significantly, in their disintegration those in the young spore appear to become associated with the developing pollen grain wall (Dickinson and Heslop-Harrison, 1971). In female cells, these bodies may be bound by a large number of double unit membrane profiles, and it has been suggested that their content is employed in the subsequent development of the embryosac (Rodkiewcz, 1970). In the work reported here, the formation of these membrane-bound inclusions (MI) has been closely examined and their behaviour during ensuing stages of mega- and microsporogenesis investigated in detail. The results ensuing considerably reinforce the hypothesis that these inclusions contain an isolated proportion of cytoplasm capable of active protein synthesis, and, in the male cells, contain or provide compounds important in the formation of the inner layer of the pollen grain exine. Materials and Methods Electron Microscopy The techniques employed in the preparation of pollen of Lilium for electron microscopy are described in Dickinson (1970). Ovaries containing megaspore mother cells at suitable stages of development (calibrated by m e a n s of prior fixations followed by wax embedding) were excised from flowers of Lilium longiflorum and sliced transversely into sections approx. 1 m m thick, under the surface of the fixative (2.5% Glutaric dialdehyde in 0.03 M PO4 buffer, at p H 7). Following fixation for 5 h at r o o m temperature, the material was rinsed several times over 12 h in the buffer and then post-fixed in o s m i u m textroxide. Osmication and subsequent preparation followed the same course as that outlined for the male tissue (Dickinson, 1970).
Estimation of the Proportion of Cytoplasm Occupied by, and the Numbers and Sizes of the Membranous Inclusions throughout Microsporogenesis A perspex grid calibrated in 1 cm 2 squares was placed over appropriate electron micrographs, and the area occupied by MI estimated to the nearest a/4cm2. The n u m b e r of MI was also recorded. The total area o f cytoplasm was estimated in the same way. In order to calculate the volume occupied by the MI and relate this measure, and the n u m b e r of inclusions, to the volume of cytoplasm representative of each developmental stage, it was necessary to
follow the change in cell and nuclear size throughout microsporogenesis. This was effected with the aid of a camera lucida and phase contrast optics, by drawing twenty cells and their nuclei from sections of each stage. The paper 'cells' and 'nuclei' were cut out and weighed, and the weight used as a measure of area. In order to relate weight units to p m 2 for the calculation of the actual size of the cells and nuclei, an eyepiece graticule and micrometer slide were employed. Using methods outlined below, these measurements were then converted to volume. The cells and nuclei in the zygotene-early pachytene and late pachytene stages were regarded as spherical and a measure of their volume could be calculated using the formula ~nr3, and by subtraction a measure of the cytoplasmic volume could be derived. After diakinesis and the breakdown of the nuclear envelope the same method could not be used, and only a measure of overall cell size could be found. In addition estimations were not made during the second meiotic division stage, on account of asynchrony in division of the cells. In the early tetrad stage, when the nuclear m e m b r a n e s have been reformed, a simple formula was derived to allow for the volume occupied by the callose walls, and a similar method used to calculate a representative cytoplasmic volume as in prophase. It proved impractical to calculate the volume of the young microspores as sections show that these are not spherical, but it is known from electron microscope studies that the MI have disappeared by the vacuolate spore stage. The size of individual MI at each stage was calculated by dividing the total volume of cytoplasm occupied by M1 by the n u m b e r of MI present.
Estimation of the Proportion of Cytoplasm Occupied by the MI during Megasporogenesis The electron microscope reveals that a far greater proportion of the female cytoplasm is occupied by MI than is observed in male cells. However, the extreme vasiculation of the female cytoplasm during megasporogenesis makes precise measurement of cytoplasmic volume impossible. For this reason no quantitative studies have been carried out on the developing megaspore mothercell and its products.
Results
The Formation of the Membrane Bound Inclusions
The electron microscopic image of the preprophase megaspore mother cell and pollen mother cell cytoplasm of Lilium is indicative of a period of intense protein synthesis. The ground matrix is electron opaque, and the protoplast is filled with banks of ribosome encrusted endoplasmic reticulum. Conspicuous features of this reticulum are the large numbers of structures, similar to nuclear pores, set into the membrane (Fig. 1). Numerous ribosomes, often in polyribosome conformation, also abound in the cytoplasm. At the onset of the leptotene stage and the beginning of the deposition of the callosic special wall, considerable reorganisation of this cytoplasm takes place. The endoplasmic reticulum, previously organised in banked arrays, becomes pleomorphic and corn-
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
231
Fig. 1. Pollen mother cell cytoplasm of Lilium in very early prophase. Endoplasmic reticulum (arrows) is freely distributed, and the inset reveals it to possess structures resembling nuclear pores, x 23,400; inset x 64,800 Fig. 2. The encapsulation of cytoplasm during prophase in pollen mother cells. Both double (D) and multiple (M) membraned inclusions are present, as are profiles suggesting that encapsulation is in progress (F). x 5598 Fig. 3. As Figure 2, but in the megaspore mother cell. Here the membranes often appear associated with a "membrane-centre" (C), and exclusively multiple membraned inclusions are formed. Note the mitochondrion (M) invested by the membrane, x 13,743
232
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
233
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis Ceil volume(~Jm 3) 9000-
Percentage of cytoplasm occupied by Mls 6-
T
2000 O
,yl
!Jr
4-
O
8000-
o
5-
Nuclear volume jm 3)
-1500 7000-
1 "1000
3No measurements possible during nuclear division 2-
~
r ~ , 01
6000-
-500
'
ProphaseI
'
Meta-Ana- ~ MeiosisH ITetrad'Young' TetophaseI spore Developmenta[ stage
5000
Prophasel ' Meta-Ana- ' TetophaseI
Meosisll
'Tetrad'Youn( spore Developmental stage
Fig. 6. The percentage of a "unit volume" of cytoplasm occupied by membrane bound inclusions (MI) [x and - standard error] throughout meiosis and early pollen development
Fig. 7. Changes in cell (o) and nuclear (e) volumes [+ and standard error] throughout meiosis and early pollen development
mences to form into spheres enclosing areas of cytoplasm. Since each element of the endoplasmic reticulum is formed of two appressed unit membrane profiles, the resultant structure is a double membrane bound inclusion, measuring between 1 and 3 gm in diameter, enclosing a portion of the premeiotic cytoplasm (Fig. 2). This encapsulation appears to occur at random, for not only are the cytoplasmic ribosomes invested, but often also small mitochondria and
indeed, other regions of included cytoplasm, thus giving rise to an assembly composed of up to 5 concentric layers of double unit membrane profiles (Fig. 3). Such multi-membraned inclusions (MMI are particularly characteristic of female tissue, whereas meiocytes contain mainly double membraned inclusions (DMI) (Fig. 4)). Structures with a diameter exceeding 1 gm appear not to be invested and it is thus rare to observe dedifferentiated plastids within the inclusions. During these early stages of formation the cytoplasm within and without the inclusions appears identical, and, similarly, ribosomes appear to remain attached to the membrane as they were when it constituted the banks of rough endoplasmic reticulum. The amount of cytoplasm encapsulated is considerable, as may be observed from the electron micrographs and, indeed from light micrographs of Azure B stained material (Fig. 5). By the end of the leptotene stage, just prior to the elimination of the ribosomes, it is estimated in the male cells that approaching some 4% of the protoplast is occupied by the inclusions (Fig. 6). These measurements also indicate that the actual size of the male cells and their nuclei begins to decrease during this period (Fig. 7). This shrinkage accelerates during meiosis I, and continues throughout meiosis II.
Fig. 4. Male cytoplasm at anaphase I showing the high concentration of ribosomes in the MI, (arrows) when compared with their frequency in the ground cytoplasm (C). A cytoplasmic 'nucleoloid' (N) is shown immediately prior to its disintegration to provide a fresh population of cytoplasmic responses, x 14,580 Fig. 5. Pollen mother cells of Lilium stained with Azure B. Note that inclusions, presumably the majority of them (deduced from electron micrographs) MI, occupy a considerable proportion of the prophase cytoplasm. Light micrograph x 780 Fig. 8. Inclusion invested by four layers of double membrane. Note the increase in concentration of ribosomes towards the centre of the inclusion, x 18,760 Fig. 9. Multimembrane bound inclusion in prophase II of megasporogenesis. The arrows indicate ribosomes in polysomal configurations, x 23,400
234
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
235
Number Mls 320-
Average vo[ume of Mls (pm3)
1
3001.5]
l/~
280260-
1.4 O
240220-
1.3-
2001.2-
[
O Absent
H
1/1
180160I&0120-
1.0
O ~
_____------- O 1
JTetophaseI ~ Meta-Ana- h Meiosis]I ITetrad~Young~ Telophase] spore Developmenta[stage
I00
/iProphasel1 " Meta-Ana- t Te[ophnsel
Meiosisff Tetrad~Young spore Developmental stage '
Fig. 11. Changes in volmne of membrane-bound inclusions (MI) [x and standard error] throughout meiosis and early pollen development
Fig. 12. The numbers of membrane-bound inclusions (MI) contained in a "unit volume" of cytoplasm [x and - standard error] throughout meiosis and early pollen development
The Behaviour of the Inclusions during Meiosis and Early Mega- and Microsporogenesis
membrane layer are probably eradicated, for the multimembraned inclusions assume a graded aspect, with a higher concentration of ribosomes within the centre compartment (Fig. 8). Early in meiosis the free ribosomes within the inclusions appear dispersed, but as reduction division proceeds an increasing proportion appear in polyribosomal configuration until, by the prophase of meiosis II, most of the ribosomes are so organised (Fig. 9). Eradication of the ribosomes appears to cease in pachytene stage and the inclusions, be they double or multimembrane bound, maintain their general aspect until their disintegration in the young spore stage, despite the repopulation of the cytoplasm with a new population of ribosomes following telophase I and telophase II. A characteristic of the female cytoplasm during these later stages is the appearance of paracrystalline arrays of protein. These gradually accumulate from the end of meiosis I, and, by the tetrad stage, may form masses some 1 2 ~tm in maximum dimension (Fig. 10). These accumulations are never observed either in, or in association with the membrane-bound inclusions. As may be seen from Figure 6, little further encap-
Early in the zygotene stage, coincident with removal of the free ribosomes from the cytoplasm, ribosomes are lost from the outer face of the inclusions. Ribosomes within the inclusions are not similarly affected, be they attached to the membrane or free. Some ribosomes within inclusions bounded by a single double
Fig. 10. Paracrystaltine arrays of proteins in the tetrad cytoplasm of a young megasporocyte of Lilium. x 27,250 Fig. 13. Young microspore cytoplasm showing the "splitting" of the double membranes (arrows) bonding the inclusions. Cytoplasmic ribosomes are still low in number Fig. 14. As Figure 13, but at a slightly later stage. The regions of membrane heavily encrusted with ribosomes (arrows) are well shown, and seen, in the main, to be orientated toward the cell surface (S). • 15,625 Fig. 15. Material at the same stage as that depicted in Figure 14, but revealing microtubules (arrows) running subjacent to the plasma membrane, x 51,380 Fig. 16. Material again as in Figure 14, but showing the apparent change from double to single (arrows) unit membrane profiles bounding the disintegrating inclusions, x 24,525
236
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
Fig. 17. Ribosome encrusted membranous plate (arrows) appressed to the lower face of the plasma membrane in developing pollen of Lilium. x 58,635 Fig. 18. Vacuolate young microspore showing the absence of any 'membrane-bound inclusions' in the cytoplasm layering the periphery of the cell. The exine (E) is almost fully formed, and the cytoplasm contains large numbers of amyloplasts (A). Vacuole: V. x 8,156 Fig. 19. Disintegration of multi membrane bound inclusions in the tetrad stage of megasporogenesis. The membranes bounding the inclusions (/) become irregular, and its content assumes a diffuse texture, x 16,200 Fig. 20. Final stages in the disintegration of multimembrane-bound inclusions in the tetrad stage of megasporogenesis. The bounding membranes (arrows) have now become discontinuous and the inclusion's content has mergedwith the cytoplasm Fig. 21. A general aspect of cytoplasm at the late tetrad stage of megasporogenesis in Lilium. Note the almost complete absence of multimembraned inclusions of the type characteristic of earlier stages, x 86,400
H.G. Dickinson and L. Andrews: CytoplasmicInclusions during Gametogenesis sulation takes place in the male cells following the zygotene stage when about 5.5% of the protoplast has been invested. The quantity of cytoplasm included does not change very significantly in subsequent stages, although very slight overall increase is indicated by statistical analysis of the results. The electron microscope indicates that these observations also apply to the female tissue. The measurements and, to a certain extent, the electron microscope suggest that a striking change takes place in the nature of the MI at the end of meiotic prophase in the male cells. At this point the MI increase in number (Fig. 12) and decrease considerably in size (Fig. 11), the volume of cytoplasm encapsulated remaining approximately the same (Fig. 6).
The Disintegration of the Membrane Bound Inclusions Here, considerable differences in detail were observed between male and female tissues. In the male, components of the MI appeared to be involved in the synthesis of wall components, while in the female the MI merely disintegrate within the tetrad cytoplasm. Detailed descriptions of the two processes follow below.
The fate of the inclusions in the male cytoplasm Once the young spores are released from the callosic special wall, the double membraned inclusions begin to disintegrate. This is not a simple process, and appears to commence with changes to the investing membrane. Over most of the periphery of the inclusion, the two elements of the membrane split, creating a considerable clear space (Fig. 13). This appears to take place at the expense of the content of the inclusion, rather than the cytoplasm. At the same time, certain regions of the remainder of the surface becomes highly electron opaque, and heavily encrusted with ribosomes on both its inner and outer face (Fig. 14 and 16). Areas of membrane not affected in either way become diffuse and subsequently may appear as single unit membrane profiles (Fig. 16). This disintegration is accompanied by a general increase in size of the inclusions, as evidenced by the measurements (Fig. 11). The precise fate of the inclusion content is not clear, it is not expelled directly into the cytoplasm, for no direct connection between the inclusion interior and the cytoplasm is ever seen. Instead, this content appears simply to become eradicated as the volume invested by the inner unit membrane profile becomes smaller. Some of the ribosomes (and, presumably other contents) may become associated with the
237
darkly staining ribosome-encrusted region of the membrane. A considerable proportion of the inclusions now begins to move towards the periphery of the cell, generally orientated in such a manner that the ribosome-encrusted areas are facing t h e plasma membrane (Fig. 14). We have not identified the agents responsible for the movement of the inclusions, but a considerable number of microtubules are present in the young spores at this stage (Fig. 15). In the course of this journey, the main body of the inclusions is generally lost, so that only the areas of ribosome coated membrane, which appear as plates, arrive subjacent to the plasma membrane (Fig. 17). How the remainder of the inclusion is removed is not evident, but the investing membrane, once the content is completely lost, appears to dissociate into vesicles. The membranous plates now sited under the plasma membrane are without doubt associated with the formation of an electron-lucent lamella, upon which acetolysis-resistant polymer of the nexine is deposited. This process is reported in detail elsewhere (Dickinson and Heslop-Harrison, 1971; Dickinson, 1975). This disintegration of the inclusions is rapid for, by the time the spore becomes vacuolate, which occurs only shortly after its release from the tetrad, no profiles of inclusions are visible within the protoplasts (Fig. 18).
The fate of the membrane bound inclusions in female tissue Here the demise of the membranes is far less spectacular than in the male cells. The contents of the MI first became diffuse, and the ribosomes contained therein difficult to detect. The membranes then become progressively more irregular (Fig. 19), in a manner reminiscent, but not so drastic, of those in the young spores. Large spaces are generally not formed between pairs of membranes, but elements do appear to fuse. The bounding membranes finally become discontinuous (Fig. 20), allowing mixture of the inclusion content with the haplophase cytoplasm, in which it is quickly dispersed. The membranes themselves are not eliminated, but remain free in the cytoplasm sometimes carrying polyribosomal clusters until, a t least, the end of the tetrad stage of megasporogenesis (Fig. 21). Discussion
The Encapsulation of the Cytoplasm and the Source of the Membrane Involved The apparent nuclear pores present in the rough endoplasmic reticulum of the proprophase mother cell
238
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
of both sexes indicate the nucleus to be the source of this membrane. Such a process has also been reported in the reproductive cells of Sciadopteris (Gianordoli, 1969), and appears also to take place throughout microsporogenesis in Lilium at the stages when there is a nuclear envelope present (Dickinson, 1968, 1971). No particular cytoplasmic event may conclusively be linked with the change in aspect of these membranes from the rough endoplasmic reticulure, often ' b a n k e d ' in the male cells, to the relatively ribosome-free dispersed sheets of membrane. Certainly this occurs at the beginning of a time of considerable cytoplasmic reorganisation; the plastids are commencing dedifferentiation (Dickinson and Heslop-Harrison, 1970, 1976), the cells are becoming isolated by the callosic special wall, and elimination of the ribosome population is about to occur. It is feasible that one of the first effects of the degredating agents is to disrupt the organisation of the ribosomecoated banks of membrane, but data to confirm such a supposition would be almost impossible to obtain. Equally, we are in ignorance of the factors controlling the actual process of encapsulation. F r o m the micrographs the sheets of membrane first form cuplike profiles and then close up, investing an area of cytoplasm. Clearly, while the forces involved there are undoubtedly biophysical, they must result from some chemical change in the cytoplasm. We have little indication as to the nature of this change, but simple observation of any plant cells held to be "badly-fixed" for the electron-microscope reveals a tendency for the formation of such structures. These 'artefacts' are presumably either the result of incorrect buffering of the specimen, or more probably from a period of activity of lytic enzymes permitted by slow or incomplete penetration of fixatives. Certainly considerable degradation of the cytoplasm is also occurring in these premeiotic cells, and it is perhaps not unreasonable to propose that such unfavourable conditions in the cytoplasm, be they naturally or artificially induced, result in the biophysical changes necessary to cause the transformation of free membrane to vesicles. It is important to point out that this process is no way completely analogous to 'bad fixation', for no single membraned vesicles are formed, but rather that some of the same factors may be involved here. While the encapsulation appears to take place at random, the nature of the inclusions formed is constant within cells of a particular sex. In the male cells, double-membraned inclusions are most frequently seen, with a diameter between 0.5 and 0.8 ~t, while, in the female, multi-membraned inclusions with a diameter of up to 3/am are most common. It is noteworthy that the central inclusion of the female
MMI is of the same order of dimension as the DMI in the male, and it may be that biophysical forces again dictate a minimal size for these inclusions. Presumably the final dimension of the inclusion depends upon the volume of cytoplasm and area of membrane available, and since there is more of both in the rapidly expanding megaspore mother cell, it is hardly surprising that M M I are more characteristic of this tissue than the male. While the most attractive explanation of the gradation of rfbosome numbers in the inclusions is that the membranes are conferring a partial immunity to their contents from the degradative agents, the fact that the MI do not seem to change in their gradation throughout development, and that they appear to be formed during the beginning of the eradication of ribosomes, suggests an alternative explanation. Since the ribosome-degrading agents are possibly active during the course of formation of a particular MMI, the last volume of cytoplasm encapsulated would contain correspondingly fewer ribosomes than those contained in the central inclusion. This would result in a gradation of ribosomes across the inclusion from the time of its formation. An implication of this proposal would be that the membranes confer total immunity to their content throughout meiosis I and II. The gradual decrease in volume of the protoplast throughout meiosis results from the deposition of the series of callose 'special walls' which clearly must displace t h e cytoplasm, particularly in the tetrad of young microspores.
The Activities of the Inclusions during Meiosis I and H Apart from small changes which may be explained in terms of experimental error, no significant alteration in the amount of cytoplasm encased takes place during meiosis I and II. The reason for the fragmentation of the MI population in male cells to form a greater number of smaller inclusions is not easily explained. This event does, however, occur at the point at which the karyoplasm is mixed with the cytoplasm on the rupture of the nuclear envelope and there is evidence that pronounced biophysical changes do occur to the cytoplasmic inclusions at this point (He- . slop-Harrison and Dickinson, 1967). It is thus not unreasonable to propose that similar changes overcome the MI which result in the breakage and reformation of their bounding envelopes. Whatever the explanation of the graded aspect of the M M I those ribosomes on the surface of the outer membrane of the inclusions are certainly also affected and it is noteworthy that these organelles
H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
are not completely degraded, but elements of their structure remain as a granular deposit, perhaps indicating that the active principle attacks only a particular component of the ribosome. Apart from these changes, the aspect of the cytoplasm contained in the inclusions hardly differs from that characteristic of the preprophase meiocyte throughout meiosis I and II. What changes there are appear to involve the organisation of the ribosomes, which become progressively associated into polysomal configurations. In female cells, where this association is most conspicuous, it is accompanied by an accumulation of paracrystalline proteins in the cytoplasm. Since the megaspore mother cell is invested in a callosic wall (Rodkiewcz, 1970), through which penetration of macromolecules is unlikely (HeslopHarrison and Mackenzie, 1967; Dickinson and Bell, 1976), and since the ground cytoplasm itself possess very few ribosomes, the evidence points to the MI as the source of this protein. Such a conclusion implies that the protein synthetic machinery situated within the inclusions could indeed be providing a supply of proteins for the cells throughout meiosis I and II, a time when its ground cytoplasm is free of any such apparatus.
The Fate of the Inclusions
While the fate of the inclusions does differ between the sexes, their demise appears to take place in approximately the same fashion, and at an identical point in the development of the cells. The main difference lies in the fate of their bounding membranes. In the first stage of degeneration, which occurs during the tetrad stage (Dickinson and Heslop-Harrison, 1970), elements of the investing membranes split, electron lucent spaces appearing between them. At this point development differs between the sexes, for in the female the membrane continues to degenerate, finally becoming discontinuous and permitting mixture of the inclusion content with the cytoplasm. The agents responsible for these events have not been identified, but it is interesting that, just prior to the mixture of the inclusion content with the cytoplasm, the former becomes diffuse, indicating that some elements of the diplophase cytoplasm may be eliminated before release into the 'haplophase' environment. More conclusive evidence is, however, available concerning the male cells. Here where it has been demonstrated that development of pollen wall in Lilium is maternally controlled (Heslop-Harrison, 1971), we find elements of the membranes investing the MI, clearly synthesised in the diplophase cytoplasm, contributing to the pollen wall. How this takes place
239
is not entirely clear at present, but the elements of membrane are first situated immediately subjacent to the plasma membrane by microtubules, where they control the deposition of acetolysis-resistant polymer upon an electron-lucent former. These layers of polymer are then packed into the wall to form the nexine layer (Dickinson and Heslop-Harrison, 1971; Dickinson, 1976).
The Rble of the Membranous Inclusions
While many aspects of the formation, behaviour and disintegration of these inclusions remain unclear, evidence is emerging that they (in female cells in any case) are capable of manufacturing protein for export to the main body of the cytoplasm and that they (in male cells) contribute elements to post-meiotic processes known to be under maternal control. Since the data from measurements indicate that encapsulation occurs over a very restricted period, just prior to the reorganisation of the sporophyte cytoplasm, it thus does appear likely that, at this point, a portion of'diplophase' cytoplasm is isolated and protected from the degradative agents that subsequently arise in the cytoplasm. Further, this cytoplasm seems not only richer in compounds required for the control of post-meiotic development, but also capable of synthesising protein for export to the dividing cells over the period when their main protein synthetic apparatus is in abeyance, during the fundamental reorganisation of the cytoplasm that is emerging as such a significant part of gametogenesis, and other changes of phase in the course of plant development (Dickinson and Heslop-Harrison, 1976). The statistical portion of this work was carried out by L.A. as part of the B.Sc. Course in the Department of Agricultural Botany. The authors thanks are due to the S.R.C. for financial support, and to Ursula Potter for valuable assistance with the illustrations.
References Dickinson, H.G.: Aspects of the physiology and ultrastructure of pollen development. Ph.D. thesis. Univ. Birmingham 1968 Dickinson, H.G.: Ultrastructural aspects of primexine formation in the microspore tetra of Lilium longiflorum. Cytobiologie 1(4), 437M49 (1970) Dickinson, H.G. : Nucleo cytoplasmic interaction following meiosis in the young microspores of Lilium longiJTorum. Events at the nuclear envelope. Grana 2, 107 127 (1971) Dickinson, H.G., Bell, P.R. : The changes in the tapetum of Pinus banksiana accompanying formation and maturation of the pollen. Ann. Bot. in press (1976) Dickinson, H.G. : Common Factors in exine deposition. In: Evolutionary significance of the exine K. Ferguson et al, (edit.). London: Academic Press 1976
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H.G. Dickinson and L. Andrews: Cytoplasmic Inclusions during Gametogenesis
Dickinson, H.G., Heslop-Harrison, J. : The behaviour of plastids during meiosis in the microsporocytes of Lilium longiflorum. Thunb. Cytobios 6, 103-118 (1970) Dickinson, H.G., Heslop-Harrison, J.: The ribosome cycle, nucleoli and cytoplasmic nucleoloids in the meiocytes of Lilium. Protoplasma 69, 187-200 (1970) Dickinson, H.G., Heslop-Harrison, J.: The mode of growth of the inner layer of the pollen grain exine in Lilium. Cytobios. 4, 233-243 (1971) Dickinson, H.G., Heslop-Harrison, J.: Ribosomes in meiosis. Proc. roy. Soc. (B), in press (1976) Gaudevan, P.: l~change de materieux figures entre noyau et cytoplasm. Gatlica Biol. Acta 1, 205 226 (1948) Gianordoli, M.: Observations sur l'organisation du cytoplasm et sur la presence de lamelles annelees dans la cellule antiale du Sciadopteris verticillata. Rev. Cytol. Biol. v6g. 32, 183-202 (1969) Guillermond, A. : Sur l'evolution du condriome pendant la formation des grains de pollen de Lilium eandidum. C.R. Acad. Sci. Paris 170, 1003 1006 (1920) Heslop-Harrison, J.: Sporopollenin in the biological context. J. Brooks et al. (edit.). In: '~Sporopollenin ". London: Academic Press 197l
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Received 25 September; accepted 12 November 1976
