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Sporogenesis in Conifers
Sporogenesis in Conifers
ROGER I. PENNELL
John Innes Institute and AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, UK
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11.
Sporogenesis in the Pollen-bearing Cone . . . . . . . . . A. The Archaesporium and Differentiation within the Sporangium B. Sporogenous Cells and Tapetum . . . . . . . . . . C. Meiosis . . . . . . . . . . . . . . . . . . D. ExinePatterningandtheFreeSporePeriod . . . . . .
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Megasporogenesis . . . . . . . . . . . . . . . . A. The Origin of the Reproductive Cell Lineage within the Ovule B. Mitochondria, Plastids and Planes of Division within the Megaspore Mother Cell . . . . . . . . . . . . . . . C. Megaspore Viability . . . . . . . . . . . . . .
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I.
Introduction
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I. INTRODUCTION Sexual reproduction is essential for the long-term vigour of all organisms. Although plants are capable of a variety of sexual methods of reproduction (e.g. apomixis, apogamy and vivipary), only sexual methods bring together genes from two parents in a blend which confers genetic uniqueness upon the progeny. The random reassortment of parental genes affords the chance that a proportion of the progeny will be pre-adapted to thrive in changing environments. Sexual reproduction therefore offers the means by which plants may spread globally and compete and succeed in conditions which might otherwise be exclusive. Copyright 01988 Academic Press Limited All rights of reproduction in any form reserved.
Advances in Botanical Research Vol. 15 ISBN 0-12-005915-0
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In land plants it is the spore that allows sexual reproduction, since in all instances, both male and female, the spore is the ultimate product of meiosis and hence contains a haploid complement of chromosomes. Seed plants bear spores which develop heterotrophically into unisexual gametophytes capable of bearing either sperms or eggs, as distinct from the hermaphroditic gametophytes of many pteridophytes. They are thus termed “heterosporous”, and the origin of heterospory itself today forms the basis of active research. In the majority of heterosporous plants (those that also form seeds), the spore that gives rise to the male gametophyte (the microspore; the term does not imply volume relationship to its female counterpart) is liberated from the parent sporophyte, while that giving rise to the female gametophyte (the megaspore) is retained within a specialized part of the parent plant which subsequently functions as a part of the seed. The “pollen grains” of the yews (Tuxus spp.) are more correctly microspores since the single nucleus contained within each is one of a tetrad of four produced during meiosis. The term “pollen grain” is accurate only when post-meiotic divisions of the haploid nucleus take place, giving rise to the bi- and tricellular grains characteristic of Pinus and many other conifers and of the flowering plants. Although these plants do not liberate microspores as such, their pollen grains are nevertheless the product of the same sequence of events, known as microsporogenesis. Together with comparable events which take place in the ovular tissues (megasporangium or carpel), the process of sporogenesis is a lengthy one and involves a series of now well-documented stages. The cell lineage which ultimately gives rise to the spore appears within the plant well before meiosis occurs. In Tuxus buccatu a period of approximately four months separates the two events (Pennell and Bell, 1985), although in flowering plants only a few days may pass between the appearance of the reproductive cell lineage and the reductive division of meiosis. The first cell which can with certainty be identified as the forerunner of the meiocyte is generally termed the archaesporial cell, and collectively a mass of these cells forms the archaesporium. In a microsporangium there are several hundred sporogenous cells which consequently yield very many pollen grains, while in a megasporangium the single tetrad is formed from only one. The term “sporogenous cell” may then be applied to any destined to give rise to a meiotic cell once the archaesporium has differentiated, the remainder forming the tapetum. The sporogenous cells in both microsporangia and megasporangia are, like those of the archaesporium, meristematic, and undergo a finite number of divisions. The final division gives rise to a spore mother cell or meiocyte within which meiosis takes place. This brings to a close the diploid phase of the life cycle, but the haploid spores subsequently acquire elaborate surface architecture. These subjects will be developed at more length in the following pages. It is noteworthy that the conifers are generally difficult subjects for study since
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the reproductive cells of interest are often inaccessible to experimental manipulation and difficult or impossible to isolate. For these reasons the most profitable approach has been an in situ one, using a variety of microscopical techniques. The Taxales have been regarded by many authors as a group closely allied to (or a part of) the Coniferales, and the behaviour of Taxus is here regarded as an example of this group as a whole.
11. SPOROGENESIS IN THE POLLEN-BEARING CONE A. THE ARCHAESPORIUM AND DIFFERENTIATION WITHIN THE SPORANGIUM
The origin of the cell which gives rise to the archaesporium is in all instances unclear. It is likely to be a hypodermal cell of the primordial microsporangium. Like meristematic cells generally, it gives rise to a number of others which divide rapidly, quickly filling the microsporangium with a mass of angular cells up to 10 p m in width (Moitra and Bhatnagar, 1982). The cytoplasms of these cells are interconnected by numerous plasmatic channels (a feature shared with the archaesporial cells of flowering plants) ,sometimes to such an extent that they become largely confluent. Nevertheless, the development of the archaesporial cells is characteristically asynchronous, and for this reason what is known of their metabolism comes from in situ studies following sectioning and staining. The archaesporial cells display many features characteristic of cells undergoing rapid metabolic reorganization, including fluxes in numbers of ribosomes (Pennell and Bell, 198S), extensive lytic activity (Pennell and Bell, 1985), and cyclical changes in the state of differentiation of the plastids (Moitra and Bhatnagar, 1982; Pennell and Bell, 1985). There are also changes in the affinity of the plastid envelopes for osmium (Dickinson and Bell, 1976; Pennell and Bell, 1985), which may represent increasing saturation of membrane lipids and accompanying enhanced permeability to metabolites (Bell, 1983), and changes in the structure and composition of the primary walls which precede the appearance within them of callose (Moitra and Bhatnagar, 1982). The transition from archaesporium to sporogenous tissue occurs in Pinus banksiana only a few days before meiosis takes place in the microspore mother cells (Dickinson and Bell, 1976), but in Taxus 4-5 weeks separate the two events (Pennell and Bell, 1985). The archaesporium differentiates into the sporogenous cells and tapetum in Taxus (Pennell and Bell, 1985) and Pinus banksiana (Dickinson and Bell, 1976), but the tapetum appears to arise from the sporangial wall cells in Pinus sylvestris (Walles and Rowley, 1982). The finding that in Taxus the cells destined to become tapetal contain in their inner radial walls thickenings that are unique to this site (Pennell and
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Bell, 1985) indicates that their metabolism is different from those elsewhere in the microsporangium at a very early stage, even though the fine structures of their cytoplasms are identical. The tapetum and sporogenous cells develop along independent lines soon after such thickenings appear within these walls, but the stimulus which initiates their developmental separation is not yet known. Radial gradients within the sporangium have been invoked as being responsible for the dichotomy (Dickinson and Bell, 1976), and the unique thickenings in the inner tangential walls may be active in preventing such gradients from traversing this barrier. The absence of symplastic connections between the peripheral layer of cells of the Tuxus archaesporium would therefore lead to the physiological isolation of these cells and might even promote their subsequent degeneration. B. SPOROGENOUS CELLS AND TAPETUM
In gymnosperms generally, the sporogenous cells develop in a loculus enclosed by one or two layers of tapetal cells. Tapeta such as this, which remain in a peripheral position throughout microsporogenesis, are termed “secretory”. Although invasive tapeta are common in the flowering plants (Maheshwari, 1950), the partially invasive tapetum of Aruucuriu (Hodcent, 1965) and of Lurix (Mikulska et ul., 1969) remain unusual features within the gymnosperms. The development of the tapetum takes place at a rate that coincides with the development of the sporogenous cells within, so that by the time meiosis is completed the tapetum itself is almost wholly disorganized. Structural changes in the tapetum at this time include an increase in numbers of dictyosomes and vesicles, and in endoplasmic reticulum, which gradually becomes organized into whorls containing up to 12 gyres of cisternae (Pennell and Bell, 1985). In Lilium these membranes are assembled at the expense of “storage” lipid (Reznickova and Dickinson, 1982), and this is probably also the case in Tuxus. Meanwhile, the sporogenous cells in Tuxus undergo changes in basophilia that are the result of fluxes in ribosomes. Lytic vesicles appear in all and are likely to be responsible for the diminution in ribosome numbers that takes place before the cells enter meiosis (Pennell and Bell, 1985). These changes are difficult to quantify since, like those which accompany the development of the archaesporium, they take place at different rates in different cells. However, the technique of microfluorometry has now been applied for the first time to the microsporangia of Tuxus (Pennell and Bell, 1985), and has given support to the notion that marked changes in cytoplasmic RNA do occur in the developing sporangium. This technique involves staining high molecular weight nucleic acids with a dye which fluoresces when excited by far-blue light. The fluorochrome Acridine Orange will, at pH 4.0, form a complex with RNA
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that fluoresces orange-red and with DNA that fluoresces green-blue. When the DNA has been removed by pretreatment of the tissue with DNase, the fluorescence of the RNA alone can be measured by a single photocell. Since the binding of the dye to the nucleic acid is stoichiometric (Bucknall and Sutcliffe, 1965; Darzynkiewicz et al., 1979), its intensity can be taken as indicative of the amount of RNA in the section of the cell and, by extrapolation, in the cell as a whole. In Tuxus the trend during the development of the sporogenous cells appears to be towards a general increase in RNA (Pennell and Bell, 1985), this being attributable to a proliferation of ribosomes. The significance of this is still unclear, but it is conceivable that it takes place in preparation for meiosis, when very special and specific proteins associated with the synaptonemal complex are assembled (Moens et ul., 1987). Other proteins are certain to include some that control the entry into prophase and the progression through meiosis, and, as during mitosis, those of the cytoskeleton which determine planes of division and cytokinesis (Wicket a l . , 1981). The rapid and timely assembly of these proteins may therefore necessitate the appearance within the sporogenous cells of additional ribosomes upon which their messenger RNAs may be translated. Once completed, the controlled elimination of ribosomes by the directed action of proteases and nucleases could then give rise to the fall of RNA levels observed immediately prior to meiotic prophase (Pennell and Bell, 1985). C. MEIOSIS
Meiosis within pollen mother cells has been best studied in flowering plants, and common features have appeared that may be significant controlling events in the life cycle (Dickinson and Heslop Harrison, 1977). One of the most important of these is the undoubted elimination of a major proportion of ribosomes during prophase (Mackenzie et al., 1967) and their subsequent restoration by the disintegration in the cytoplasm of “nucleoloids”, possibly assembled in the nucleus (Williams et al., 1973). This “ribosome cycle” is accompanied by the transient dedifferentiation of both the mitochondria and plastids (Dickinson and Heslop Harrison, 1977) and the formation of “nuclear vacuoles” within the prophase nuclei (Sheffield et a l . , 1979). These events have been interpreted as crucial in the reorganization of a diploid cell (a sporogenous cell) into one whose genome is haploid (the spore), and in which expression of that part of the genome which is concerned specifically with sporophytic growth must be replaced by that part concerned specifically with gametophytes (Dickinson and Heslop Harrison, 1977). The physiological isolation of the meiocytes by cell walls rich in callose [strictly those capable of forming a fluorescent complex with Aniline Blue (Mangin, l889), now proven to involve a variety of mixed P-linked glucans (Smith and McCully, 1978)], has been implicated as being significant in the change of phase (Heslop Harrison and Mackenzie, 1967).
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Meiosis in conifers has, in the absence of evidence to the contrary, been thought comparable. Indeed, the process in both angiosperms and gymnosperms shares some common features, including the partial dedifferentiation of the mitochondria and plastids and the appearance of vacuoles within the nuclei (Sheffield et al., 1979). Meiosis in many conifers differs significantly in the duration of prophase, however. In Lilium (which may be regarded as representative of the situation in floweringplants generally) meiosis lasts for just 2-3 days (Dickinson, personal communication), while in Juniperus (Singh et al., 1983) and Taxus (Pennell and Bell, 1987a) it lasts for approximately five weeks, and in Pseudotsuga, Tsuga, Thuja and Larix for almost two months (Owens and Molder, 1971). In all instances it is the prophase of meiosis which is extended, the remaining stages of meiosis I and I1 occurring within a few days. Not only does this set the conifers apart from the flowering plants, but it also makes them favourable subjects for the study of events regulating meiosis. Advantage has recently been taken of this, and a new technique applied to the pollen mother cells of Taxus has provided novel information for an entirely new concept of controlling mechanisms (Pennell and Bell, 1986a, 1987a). The technique is also based upon the preferential staining with Acridine Orange of RNA in de-embedded sections of microspore mother cells. However, to obtain greater resolution than afforded by microfluorometry per se, the photographed images of the cells have been enlarged sufficiently to allow investigation of cytoplasm and nucleus separately, and these enlargements have been used as the subject of study. The density of reduced halide on the film, like the intensity of the fluorescence itself, is a direct measure of RNA within the section. When each compartment of the image of the cell is scanned separately in a microdensitometer, the original intensity of fluorescence can be judged from the restriction of the scanning beam, and quantified. Once cells from each of the crucial stages of meiosis have been examined in this way, the data coming from cytoplasm, nucleus excluding nucleolus, and nucleolus alone, may be plotted separately. When applied to the pollen mother cells of Taxus, it is evident that there is only slight diminution in cytoplasmic RNA during prophase (Pennell and Bell, 1986a, 1987a) and this is matched by reciprocal changes within the nucleus (excluding the nucleolus). Throughout prophase, RNA levels in the nucleolus remain constant. The significance of these findings may be great. It is conceivable that in Taxus, and possibly in the conifers generally, it is the absence of widespread degeneration of cytoplasmic RNA that is responsible for the protracted nature of prophase. The controlled hydrolysis of RNA that occurs during prophase in flowering plants may liberate oligoribonucleotides into the cytoplasm which affect the rate of expression of nuclear genes and therefore the progression through meiosis. The absence of such putative signalling molecules from the Taxus meiocytes may thus explain the slow passage through prophase. Whatever the case, it now seems clear that the “nuclear vacuoles” that have in the past been
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invoked as the means by which information-carrying molecules move between nucleus and cytoplasm are induced only where the chromosomes exert traction on the nuclear envelope during contraction (Pennell and Bell, 1985, 1986b). Once prophase is completed the remainder of male meiosis proceeds in gymnosperms in much the same way as in angiosperms and heterosporous pteridophytes. Meiosis I gives rise to pairs of cells (diads), each of which quickly divides again into two spores. In Pinus these divisions take place without the formation of partitioning walls within the tetrad (Willemse, 1971; Moitra, 1980), but in Taxus a primary wall does develop during telophase I which separates the cells of the diad (Pennell and Bell, 1986b). It therefore seems likely that the sequence of partitioning walls within the pollen mother cell carries with it systematic significance, but it has no evident functional effect. What is perhaps more important functionally is the appearance of invaginations within the nuclei of post-meiotic cells of Pinus banksiana (Dickinson and Bell, 1970) and Podocarpus macrophylla (Aldrich and Vasil, 1970). These tube-like infoldings of the nuclear envelope have rightly been thought significant both because of the presence of highly ordered pore-like structures at their openings (Dickinson and Bell, 1972a), and by virtue of the accumulation within them of osmiophilic material (Dickinson and Bell, 1970), possibly RNA (Dickinson and Potter, 1975). When taken in the context of the change of phase which occurs during meiosis, these invaginations have been cited as specialized structures by which information-carrying molecules are provided en masse to the cytoplasm of the spore (Dickinson and Bell, 1972a). It has been thought conceivable, therefore, that these molecules are in some way specific to and crucial for the cytoplasm of the spore, and prime it for gametophytic growth (Dickinson and Bell, 1972a). Such a mechanism would find a parallel in the angiosperms, where “nucleoloids” become dispersed in the spore cytoplasm at the end of meiosis (Williams et al., 1973). In Taxus, however, there are neither nuclear invaginations nor nucleoloids, and there is no evidence of nucleocytoplasmic interaction of this or any other kind at the end of meiosis. This of course may be related to the limited flux of RNA between cytoplasm and nucleus during prophase described earlier.
D . EXINE PATTERNING AND THE FREE SPORE PERIOD
Once meiosis is completed a complex series of events occurs within the cytoplasm of the spores, and these are ultimately responsible for the patterning of the sporoderm. Exine formation in the pollen grain of Pinus banksiana has been studied in depth. The pollen grain bears two bladder-like
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sacci distally and a punctate sexine perforated by a single colpus. The reticulate pattern of the sexine is established while the spores remain enclosed in the callose wall, and pattern formation begins when a number of large vesicles make contact and fuse radially at the plasma membrane of the young spore (Dickinson, 1971). The vesicles arise from what appear to be enlarged dictyosomes, and fuse en masse with the plasma membrane (Dickinson, 1971). The outer radial faces of the vesicles then become diffuse and electron-opaque, although the inner faces, which represent the new plasma membrane of the cell, continue to stain normally. The outer surfaces of the vesicles act as pattern initials, and their subsequent thickening and elaboration with sporopollenin give rise to the surface topography of the grain. Similar events take place close to the plasma membranes of the young spores of Abies concolor (Kurmann, 1986), Tsuga canadensis (Kurmann, 1984a) and Taxodium distichum (Kurmann, 1985), and in each instance development within the tetrad proceeds until elements of both nexine and sexine are in place. Detailed information is also available for the flowering plants Cosmos and Lilium (Dickinson, 1976), and is again comparable although in the angiosperms generally the architecture of the sexine is considerably more complex than that of Pinus. The patterning of the Pinus pollen grain does not take place at that part of the plasma membrane which becomes “masked” by cisternae of endoplasmic reticulum, and this does not develop into the layered exine characteristic of other parts of the grain (Dickinson, 1976). In Taxodium, the granular layer of the sexine is absent from the distal surface at the region of the germinal papilla, and the number of layers in the nexine is reduced (Kurmann, 1985), but in Tsuga there is no evidence of a pre-formed germinal aperture in the sporoderm (Kurmann, 1984b). The situation in Tuxus is simpler. The contribution of the gametophyte (spore) to the pattern of the exine takes the form of a number of small osmiophilic globules (indistinguishable in the electron microscope from sporopollenin) that emerges from the protoplast and form a confluent layer (Pennell and Bell, 1986b), or layers (Rohr, 1977), at its surface. This thin smear of sporopollenin encapsulates the microspore during the early free spore period, and serves as a surface upon which tapetally synthesized sporopollenin is placed (Pennell and Bell, 1986b). The second phase of accretion then takes place as orbicules enter the loculus from the degenerating tapetal cells, and appears to involve no more than the transfer of sporopollenin from individual orbicules onto areas of exposed gametophytic sporopollenin. The sporopollenin of the orbicules appears to have greater affinity for that layered upon the spore surface by the gametophyte than for either the lipidic core of the orbicule or orbicular sporopollenin elsewhere. In consequence, orbicular sporopollenin is transferred en masse to the surface of the maturing microspore, first as a series of concentric layers and then as discrete globules which confer the punctate pattern upon the sporoderm. The number of globules attached to the microspore surface
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Fig. 1. Near-mature Taxus microspore, fixed inside a loculus of the microsporangium before dehiscence. The nexine (NX) consists of several distinct layers of sporopollenin and the sexine (SX) of a single layer of globules. The intine (I) is barely visible. N = nucleus, P = plastid. Scale bar = 1 pm.
increases until a complete layer is present, and no areas of layered sporopollenin remain available for transfer (Fig. 1).Similar events complete exine patterning in Tarodium (Kurmann, 1985). As in Tsuga, there is no evidence in Tuxus G f a germinal pore within the spore wall. Following the terminology of Erdtman (1966), the laminae of sporopollenin represents the nexine (of which several laminae can be resolved, presumably representing individual waves of synthesis from the tapetum), and the globules the sexine. It is notable that in Tuxus the entire sexine and much of the nexine are added to the microspores once the callose wall is dissipated, but in the members of the Pinaceae a large proportion of nexine and sometimes sexine is contributed by the spores themselves. Although the structural basis of patterning is now well-established for, amongst others, Abies, Pinus, Taxodium and Taxus, as well as for several angiosperms, there is still little information upon the means by which the subcellular events responsible for it are controlled. In Lilium it has now been demonstrated that neither the meiotic spindles nor the radial microtubules in the cytoplasm of the young spore control the positioning of pattern determinants in the plasma membrane of the young pollen grain (Sheldon
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Fig. 2. Junction between three tapetal cells of Taxus. The radial cell wall (CW) contains numerous lipidic globules (L). The plasma membranes (PM) of the cells are associated with osmiophilic globules of sporopollenin (S), and similar globules are also visible inside the protoplast upon small vesicles (V). Two such vesicles appear to have traversed the plasma membranes and are entering the cell wall (arrows). Scale bar = 1pm.
and Dickinson, 1986), as the microtubules of root (Lloyd and Wells, 1985) and seed hairs (Quader et al., 1986) appear to control the orientation of cellulose microfibrils in the primary cell wall. It seems more likely that patterning determinants are present ab initio within the plasma membrane of the young pollen mother cells (Sheldon and Dickinson, 1983), and these become reorganized into a pattern which predicts that which develops subsequently in the pollen grain wall (Sheldon and Dickinson, 1986). The control of positioning of the dictyosomes which add to the pattern initials in Pinus (Dickinson, 1971), Tsuga and Taxodium (Kurmann, 1984b), however, seems likely to be involved with the cytoskeleton of the spore. In contrast, the means by which the pattern becomes impregnated with sporopollenin from the tapetum appears clear. The vehicle responsible for the transport of sporopollenin from the tapetum into the loculus of the sporangium is the orbicule. The lipidic centre of each orbicule first appears as a spherical globule within a radial tapetal wall (Pennell and Bell, 1986b) (Fig. 2). The osmiophilic material which subsequently encloses each of these “pro-orbicular cores” appears concomitantly within the tapetal protoplasts close to the radial walls (Fig. 2), in association with small vesicles (Pennell and Bell, 1986b). This substance, judged from its affinity for osmium to be sporopollenin, also moves into the radial walls of the tapetum, apparently by forming a transient association with the plasma membranes at these sites.
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Fig. 3. Localization by lead capture of acid phosphatases in the tapetum of Taxus. Both the plasma membrane (PM) and the membrane of a small vesicle (V) appear to contain the enzyme. C = cytoplasm. Scale bar = 1 pm.
Several globules of sporopollenin then rapidly aggregate to form a sheet which enwraps a single pro-orbicular core (Pennell and Bell, 1986b). The composite body so formed is clearly identifiable as an orbicule. Many orbicules are released from the tapetal walls as the protoplasts degenerate and undergo hypertrophy. The formation of the orbicules appears therefore to owe much to the affinity of the sporopollenin sheets both for lipid and for sporopollenin elsewhere. Similarly, the transfer of sporopollenin from the orbicule to the developing exine seems to come about as a result of the differing affinity of orbicular sporopollenin for the lipidic centre of the orbicule and for the sporopollenin in place upon the surface of the microspore. Indeed, once the transfer has taken place, the pro-orbicular cores devoid of a sporopollenin encasement may be observed close by the microspores. The degeneration of the tapetum proceeds apace with the formation upon the microspores of the sporoderm. The complex membranous figures which all but fill the mature tapetal cells of Tuxus develop as the fine structure of the cells becomes disorganized, and may arise as a result of their increased physiological isolation by the peritapetal membrane. Such an investment has been observed in the sporangia of many conifers (Moitra and Bhatnagar,
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1982) and appears to be composed of saturated lipid (Dickinson and Bell, 1972b). Soon after the peritapetal membrane is formed in the microsporangia of Taxus acid phosphatases appear in the plasma membranes of the tapetal cells (Pennell and Bell, 1986b) (Fig. 3). These enzymes may lead to the generalized perturbation of the plasma membranes by dephosphorylating membrane phospholipids and phosphate-containing proteins. However, what is perhaps of more interest in the study of the development of the microspores is that these enzymes (and presumably other hydrolases which accompany them) are already present and active within the tapetal plasma membranes when the microspore mother cells enter meiosis, some eight weeks before the tissue disintegrates.
111. MEGASPOROGENESIS In comparison with the development of the microsporangium, the events which take place within the ovules of conifers are poorly explored by modern techniques. This is due both to the morphology of the female cone (or fertile axis in the Taxales) and, as in all seed plants, the presence of just a single meiocyte within the ovule. Indeed, so unyielding are the megaspores and their antecedents to experimental methods that all that is known about them has been reasoned from structural studies and comparison with megasporogenesis in heterosporous pteridophytes and flowering plants whose sporangia are more accessible. Nevertheless, it is clear that sporogenesis within the ovule differs from microsporogenesis in several important ways, most strikingly in the development of the tetrad and survival of the spores. A. THE ORIGIN OF THE REPRODUCTIVE CELL LINEAGE
WITHIN THE O W L E
Little is known about differentiation within the ovule in conifers. In Tuxus, whose ovules are borne singly on compressed fertile axes, the nucellus is initially homogeneous. The nucellus begins to differentiate about 12 weeks after the appearance upon the shoot apex of the primordium of the fertile axis, when a mass of basophilic cells appears in the middle of the nucellus. In the electron microscope they are seen to contain many ribosomes and conspicuous globules of lipid (Pennell and Bell, 1987b). Since it is within this mass of cells that the megaspore mother cell develops, they have been thought to participate in the nutrition of the meiocyte and the female gametophyte which succeeds it when meiosis is completed. There is, however, no experimental evidence in support of this view, and only structural comparison of the cells with those of the tapetum in the microsporangium suggests that both behave similarly. The antecedent of the megaspore mother cell has not been identified with
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certainty. That of Taxus may develop from a hypodermal cell inside the nucellus (Dupler, 1920). B . MITOCHONDRIA, PLASTIDS AND PLANES OF DIVISION WITHIN THE MEGASPORE MOTHER CELL
Meiosis in the megaspore mother cells of many conifers sees the unidirectional movement of the mitochondria and plastids into a single haploid cell. This was first observed at the beginning of the century as the appearance of a densely staining mass below the nuclei of the meiocytes of Larix, Taxus, Thuja, Taxodium (Coker, 1904) and Juniperus (Ottley, 1909). Since then the movement of amyloplasts within the megaspore mother cells of Pinus has been found to be similar (Willemse, 1968), and indeed in the zooidozamous Encephafartos (de Sloover, 1961) and Ginkgo (Stewart and Gifford, 1967) the distributions of both plastids and mitochondria become polarized in this way. Consequently, the partitioning of the megaspore mother cell during telophase I by a transverse wall gives rise to a pair of cells of which only one-that lying chalazally--contains either mitochondria or plastids, or both (Fig. 4). The organelles behave in a similar way during meiosis 11, so that in Taxus at least (the only genus to be examined thoroughly in this respect) the chalazal spore comes to contain the entire complement of organelles, the other three members of the tetrad remaining bereft of them (Pennell and Bell, 1987b). The mechanism responsible for the directed motion on the mitochondria and plastids is still unresolved. The belief that gravity is the motive force (Volkman and Sievers, 1975) seems untenable when chalazal accumulations are known to occur in the inclined or inverted ovules of both the cone-bearing conifers and taxads. More plausible is the possibility that the motion of the mitochondria and plastids is generated by their intimate association with the cytoskeleton. Comparable phenomena have been described in vivo (Inolie, 1981) and in vitro (Vale e t a f . , 1985b), and there now seems little doubt that microtubules are capable of directing the movement of membrane-bound organelles, possibly in association with the mechanochemical protein kinesin (Vale et a f . ,1985a). Although there is no direct evidence for such a mechanism in the megaspore mother cells of conifers (or zooidogamous gymnosperms), it is difficult to conceive of any hypothesis that is not in some way related to cytoplasmic structures akin to microtubules. The directed movement of the organelles may occur in all conifers, but the form of the tetrad of megaspores differs. In most, linear or T-shaped tetrads are formed (Maheshwari, 1950), but both isobilateral and tetrahedral tetrads occur in Sequoia (Looby and Doyle, 1942). In T-shaped tetrads the plane of division of the micropylar diad cell is perpendicular to its long axis and this is due to a similarly displaced spindle. The plane of division of plant
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Fig. 4. Diad produced by meiosis I within the ovule of T a u s . The chalaza1 diad cell (lower) contains numerous mitochondria and plastids, but the micropylar diad cell (upper), while rich in lipid (L), contains none of these organelles. The bounding wall of the diad is thickened adjacent to the plasma membrane in the same way as that of the prophase meiocyte (arrow, inset). Scale bar = 5 p m (0.5 p m inset).
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cells generally is now known to be predicted by a cortical “pre-prophase band” (Wick and Duniec, 1983,1984), and hence it follows that it is the plane of assembly of this whorl of microtubules which is ultimately responsible for the opposed planes of division of the two diad cells during meiosis 11. Whatever the force responsible, the organization of the spores within the tetrad appears to be of little phylogenetic or developmental importance. C. MEGASPORE VIABILITY
Megasporogenesis in conifers is completed once the tetrad is fully formed and each spore within it is wholly enclosed by bounding and partitioning walls. However, in conifers only the chalazal megaspore develops further (eventually giving rise to the female gametophyte), the remaining spores ultimately degenerating. The promotion of the chalazal megaspore has been attributed to the presence within it of the entire complement of organelles of the mother cell from which it is derived, this conferring in some way “fitness” upon the cell. Although in the conifers the promotion of the chalazal megaspore is in all known instances prefaced by the directed movement into it of mitochondria and plastids, this is not the case in other plants. In Myosurus (Woodcock and Bell, 1968) and Epipactis (Bednara et al., 1981) the embryo sac develops from a single spore, but all the members of the tetrad receive more or less equal numbers of organelles during meiosis. Similarly, in the heterosporous fern Marsilea, megaspore viability cannot be related to any visible differences between the cytoplasms of the four spores of a tetrad (Bell, 1981). In Zeu the distribution of mitochondria and plastids between the four megaspores is only partially polarized, and again only one develops into an embryo sac (Russell, 1979). Clearly, the enriched endowment of a megaspore with mitochondria and plastids cannot be a general explanation of its preferred success, and must merely complement other factors. An alternative suggestion has been proposed by Lintilhac (1974a,b), who believes that the functional megaspore lies at a site within the nucellus where inwardly directed radial forces (established by repeated cell divisions) are opposed, giving rise to an area of “null pressure”. Since this is the only region within the ovule where there would be little or no resistance to expansion, the megaspores lying within would be able to enlarge whilst those elsewhere would not. This hypothesis is, however, difficult to reconcile with the situation in Taxus, where a pair of diametrically opposed vascular bundles in the chalazal region of the integument would be likely to displace the tectonic symmetry of the ovule as expounded by Lintilhac. Similarly, the presence of bisporic ovules in many conifers suggests that a single region of “null pressure” does not exist, or that two are present side by side. Indeed, the location of the functional megaspore within the tetrahedral tetrad of Mur-
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silea appears to bear no relation to its orientation within the radially symmetrical megasporangium (Bell, pers. comm.), and in this instance a tectonic explanation of megaspore viability is wholly unsatisfactory. The regular promotion of one member of a tetrad of four spores cannot, in fact, be readily explained by any contemporary model. This has led Bell (1981) to suggest that of the four genotypes present amongst the megaspores of Marsilea, only one is viable in the environment of the megasporangium, and this is invariably promoted at the expense of the remainder. The four genotypes would arise from recombination between two heterozygous loci which regularly experience crossing over. This hypothesis can only be applied at the present time to megaspore mother cells within which the formation of partitioning walls is delayed until both divisions of meiosis are completed. This is the case in Marsilea and the pteridophytes generally. In the linear and T-shaped tetrads of the conifers the genotype that becomes that of the functional megaspore is in all instances contained by telophase I in the chalaza1 diad cell, and is isolated from those that yield the pair of micropylar spores. For a Mendelian explanation to serve the spermatophytes it would therefore be necessary to invoke directed segregation of alleles during meiosis I, and this in itself is at variance with current thoughts upon genetic segregation preceding sexual reproduction.
ACKNOWLEDGEMENTS The author would like to express thanks to Professor P. R. Bell for helpful comments upon the manuscript.
REFERENCES Aldridge, H. C. and Vasil, I . K. (1970). J . Ultrastruct. Res. 32,307-315. Bednara, J., Kuras, M. and Rodkiewicz (1981). Acta SOC. Bot. Pol. 50, 127-130. Bell, P. R. (1981) J . Cell Sci. 51, 109-119. Bell, P. R. (1983). Eur. J . Cell Biol. 30,279-282. Bucknall, R. A . and Sutcliffe, J. F. (1965). J . Exp. Bot. 16,423-432. Coker, W. G. (1904). Bot. Gaz. 38,206-213. Darzynkiewicz, Z., Evenson, D . P . , Staiano-Coico, L., Sharpless, T. P. and Malamed, M. L. (1979). J. Cell Physiol. 100,425-438. de Sloover, J. L. (1961). La Cellule62,105-116. Dickinson, H. G. (1971). In “Sporopollenin” (J. Brooks et al., eds), pp. 31-68. Academic Press, London and New York. Dickinson, H. G. (1976). In “The Evolutionary Significance of the Exine” (K. Ferguson and J. Muller, eds.), pp. 67-90. Linnean Society Symposium, Series 1. Dickinson, H. G. and Bell, P. R. (1970). J . Ultrastruct. Res. 33,356-359. Dickinson, H. G. and Bell, P. R. (1972a). Dev. Biol. 27,425-429. Dickinson, H. G. and Bell, P. R. (1972b). Plants 107,205-215. Dickinson, H. G. and Bell, P. R. (1976). Ann. Bot. 40,103-113.
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Dickinson, H. G. andHeslopHarrison, J. (1977). Philos. Trans. R. SOC.London. B 277,327-342. Dickinson, H. G. and Potter, U. (1975). Pluntu 122,99-104. Dupler, A. W. (1920). Bot. Guz. 69,492-521. Erdtman, G. (1966). Grunu Pulynol. 6,318-323. Heslop Harrison, J. and Mackenzie, A. (1967). J. Cell Sci. 2,387400. Hodcent, E. (1965). Bull. SOC. Bot. France 112,121-127. Inout, S . (1981). J. Cell Biol. 89,346-356. Kurmann, M. H. (1984a). “Abst. 6th Int. Palynol. Conf.”, Calgary, Canada. Kurmann, M. H. (1984b). “Abst. 2nd Int. Org. Palaeobot. Conf.”, Edmonton, Canada. Kurmann, M. H. (1985). Pulynology 9,246 (abstract). Kurmann, M. H. (1986). A m . J. Bot. 73,632 (abstract). Lintilhac, P. M. (1974a). A m . J. Bot. 61,135-140. Lintilhac, P. M. (1974b). A m . J. Bot. 61,230-237. Lloyd, C. W. and Wells, B. (1985). J. Cell Sci. 75,225-238. Looby, W. J. and Doyle, J. (1942). Proc. R. Dublin SOC. 23,35-54. Mackenzie, A., Heslop Harrison, J. and Dickinson H. G. (1967). Nature 215, 997-999. Maheshwari, P. (1950). “An Introduction to the Embryology of Angiosperm.” McGraw-Hill, New York. Mangin, L. (1889). Bull. SOC.Bot. France 33,512-517. Mikulska, E., Zolincrowicz, W. and Walek-Czernecka, A. (1969). Actu SOC. Bot. Pol. 38,291-301. Moens, P. B., Heyting, C., Dietrich, A. J. J., Raarnsdonk, W. van and Chen., Q. (1987). J. Cell. Biol. 105,93-103. Moitra, A. (1980). Ph.D. Dissertation, University of Delhi. Moitra, A. andBhatnagar, S. P. (1982). Gum. Res. 5,71-112. Ottley, A. M. (1909). Bot. Guz. 48,3146. Owens, J. N. and Molder, M. (1971). Can. J. Bot. 49,2061-2064. Pennell, R. I. andBell, P. R. (1985). Ann. Bot. 56,415-427. Pennell, R. I. andBell, P. R. (1986a). ActuSoc. Bot. Pol. 55,ll-16. Pennell, R. I. and Bell, P. R. (1986b). Ann. Bot. 57,545-555. Pennell, R. I. and Bell, P. R. (1987a). A m . J. Bot. 74,444-450. Pennell, R. I. and Bell, P. R. (1987b). Ann. Bot. 59,693-704. Quader, H., Deichgraber, G. and Schnepf, E. (1986). Pluntu 168,l-10. Reznickova, S . A. and Dickinson, H. G. (1982). Plantu 155,400408. Rohr, R. (1977). Cytologiu 42,157-167. Russell, S . D. (1979). Can. J. Bot. 57,1093-1110. Sheffield, E., Cawood, A. H., Bell, P. R. and Dickinson, H. G. (1979). Pluntu 146, 597-601. Sheldon, J. M. and Dickinson, H. G. (1983). J. Cell Sci. 63,191-208. Sheldon, J. M. and Dickinson, H. G. (1986). Pluntu 168, 11-23. Singh, H., Owens, J. N. and Dietrich, H. F. (1983). A m . J. Bot. 70,1272-1280. Smith, M. M. and McCully, M. E. (1978). Protoplusmu 95,229-254. Stewart, K. D. and Gifford, Jr., E. M. (1967). A m . J. Bot. 54,375-383. Vale, R. D., Reese, T. S. and Sheetz, M. P. (1985a). Cell 41,39-50. Vale, R. D., Schnapp, B. J., Reese, T. S. and Sheetz, M. P. (1985b). Cell 40, 449454. Volkman, D. and Severs, A. (1975). Pluntu 127,ll-19. Walles, B. and Rowley, J. R. (1982). Nord. J. Bot. 2,53-70. Wick, S . M. and Duniec, J. (1983). J. Cell Biol. 97,235-243. Wick, S . M. and Duniec, J. (1984). Protoplusmu 122,45-55.
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Wick, S. M., Seagull, R. W . , Osborn, M., Weber, K. and Gunning, B . E. S. (1981). J . Cell Biol. 89,685-690. Willemse, M. T. M. (1968). Actu Bot. Neerl. 17,330-331. Willemse, M. T. M . (1971). Actu Bot. Neerl. 20,411-427. Williams, E., Heslop Harrison, J. and Dickinson, H. G . (1973). Protoplusmu 77, 79-83. Woodcock, C. L. F. and Bell, P. R. (1968). J. Ultrastruct. Res. 22,546-563.
ROGER I. PENNELL
John Innes Institute and AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, UK
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Sporogenesis in the Pollen-bearing Cone . . . . . . . . . A. The Archaesporium and Differentiation within the Sporangium B. Sporogenous Cells and Tapetum . . . . . . . . . . C. Meiosis . . . . . . . . . . . . . . . . . . D. ExinePatterningandtheFreeSporePeriod . . . . . .
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Megasporogenesis . . . . . . . . . . . . . . . . A. The Origin of the Reproductive Cell Lineage within the Ovule B. Mitochondria, Plastids and Planes of Division within the Megaspore Mother Cell . . . . . . . . . . . . . . . C. Megaspore Viability . . . . . . . . . . . . . .
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Introduction
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I. INTRODUCTION Sexual reproduction is essential for the long-term vigour of all organisms. Although plants are capable of a variety of sexual methods of reproduction (e.g. apomixis, apogamy and vivipary), only sexual methods bring together genes from two parents in a blend which confers genetic uniqueness upon the progeny. The random reassortment of parental genes affords the chance that a proportion of the progeny will be pre-adapted to thrive in changing environments. Sexual reproduction therefore offers the means by which plants may spread globally and compete and succeed in conditions which might otherwise be exclusive. Copyright 01988 Academic Press Limited All rights of reproduction in any form reserved.
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In land plants it is the spore that allows sexual reproduction, since in all instances, both male and female, the spore is the ultimate product of meiosis and hence contains a haploid complement of chromosomes. Seed plants bear spores which develop heterotrophically into unisexual gametophytes capable of bearing either sperms or eggs, as distinct from the hermaphroditic gametophytes of many pteridophytes. They are thus termed “heterosporous”, and the origin of heterospory itself today forms the basis of active research. In the majority of heterosporous plants (those that also form seeds), the spore that gives rise to the male gametophyte (the microspore; the term does not imply volume relationship to its female counterpart) is liberated from the parent sporophyte, while that giving rise to the female gametophyte (the megaspore) is retained within a specialized part of the parent plant which subsequently functions as a part of the seed. The “pollen grains” of the yews (Tuxus spp.) are more correctly microspores since the single nucleus contained within each is one of a tetrad of four produced during meiosis. The term “pollen grain” is accurate only when post-meiotic divisions of the haploid nucleus take place, giving rise to the bi- and tricellular grains characteristic of Pinus and many other conifers and of the flowering plants. Although these plants do not liberate microspores as such, their pollen grains are nevertheless the product of the same sequence of events, known as microsporogenesis. Together with comparable events which take place in the ovular tissues (megasporangium or carpel), the process of sporogenesis is a lengthy one and involves a series of now well-documented stages. The cell lineage which ultimately gives rise to the spore appears within the plant well before meiosis occurs. In Tuxus buccatu a period of approximately four months separates the two events (Pennell and Bell, 1985), although in flowering plants only a few days may pass between the appearance of the reproductive cell lineage and the reductive division of meiosis. The first cell which can with certainty be identified as the forerunner of the meiocyte is generally termed the archaesporial cell, and collectively a mass of these cells forms the archaesporium. In a microsporangium there are several hundred sporogenous cells which consequently yield very many pollen grains, while in a megasporangium the single tetrad is formed from only one. The term “sporogenous cell” may then be applied to any destined to give rise to a meiotic cell once the archaesporium has differentiated, the remainder forming the tapetum. The sporogenous cells in both microsporangia and megasporangia are, like those of the archaesporium, meristematic, and undergo a finite number of divisions. The final division gives rise to a spore mother cell or meiocyte within which meiosis takes place. This brings to a close the diploid phase of the life cycle, but the haploid spores subsequently acquire elaborate surface architecture. These subjects will be developed at more length in the following pages. It is noteworthy that the conifers are generally difficult subjects for study since
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the reproductive cells of interest are often inaccessible to experimental manipulation and difficult or impossible to isolate. For these reasons the most profitable approach has been an in situ one, using a variety of microscopical techniques. The Taxales have been regarded by many authors as a group closely allied to (or a part of) the Coniferales, and the behaviour of Taxus is here regarded as an example of this group as a whole.
11. SPOROGENESIS IN THE POLLEN-BEARING CONE A. THE ARCHAESPORIUM AND DIFFERENTIATION WITHIN THE SPORANGIUM
The origin of the cell which gives rise to the archaesporium is in all instances unclear. It is likely to be a hypodermal cell of the primordial microsporangium. Like meristematic cells generally, it gives rise to a number of others which divide rapidly, quickly filling the microsporangium with a mass of angular cells up to 10 p m in width (Moitra and Bhatnagar, 1982). The cytoplasms of these cells are interconnected by numerous plasmatic channels (a feature shared with the archaesporial cells of flowering plants) ,sometimes to such an extent that they become largely confluent. Nevertheless, the development of the archaesporial cells is characteristically asynchronous, and for this reason what is known of their metabolism comes from in situ studies following sectioning and staining. The archaesporial cells display many features characteristic of cells undergoing rapid metabolic reorganization, including fluxes in numbers of ribosomes (Pennell and Bell, 198S), extensive lytic activity (Pennell and Bell, 1985), and cyclical changes in the state of differentiation of the plastids (Moitra and Bhatnagar, 1982; Pennell and Bell, 1985). There are also changes in the affinity of the plastid envelopes for osmium (Dickinson and Bell, 1976; Pennell and Bell, 1985), which may represent increasing saturation of membrane lipids and accompanying enhanced permeability to metabolites (Bell, 1983), and changes in the structure and composition of the primary walls which precede the appearance within them of callose (Moitra and Bhatnagar, 1982). The transition from archaesporium to sporogenous tissue occurs in Pinus banksiana only a few days before meiosis takes place in the microspore mother cells (Dickinson and Bell, 1976), but in Taxus 4-5 weeks separate the two events (Pennell and Bell, 1985). The archaesporium differentiates into the sporogenous cells and tapetum in Taxus (Pennell and Bell, 1985) and Pinus banksiana (Dickinson and Bell, 1976), but the tapetum appears to arise from the sporangial wall cells in Pinus sylvestris (Walles and Rowley, 1982). The finding that in Taxus the cells destined to become tapetal contain in their inner radial walls thickenings that are unique to this site (Pennell and
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Bell, 1985) indicates that their metabolism is different from those elsewhere in the microsporangium at a very early stage, even though the fine structures of their cytoplasms are identical. The tapetum and sporogenous cells develop along independent lines soon after such thickenings appear within these walls, but the stimulus which initiates their developmental separation is not yet known. Radial gradients within the sporangium have been invoked as being responsible for the dichotomy (Dickinson and Bell, 1976), and the unique thickenings in the inner tangential walls may be active in preventing such gradients from traversing this barrier. The absence of symplastic connections between the peripheral layer of cells of the Tuxus archaesporium would therefore lead to the physiological isolation of these cells and might even promote their subsequent degeneration. B. SPOROGENOUS CELLS AND TAPETUM
In gymnosperms generally, the sporogenous cells develop in a loculus enclosed by one or two layers of tapetal cells. Tapeta such as this, which remain in a peripheral position throughout microsporogenesis, are termed “secretory”. Although invasive tapeta are common in the flowering plants (Maheshwari, 1950), the partially invasive tapetum of Aruucuriu (Hodcent, 1965) and of Lurix (Mikulska et ul., 1969) remain unusual features within the gymnosperms. The development of the tapetum takes place at a rate that coincides with the development of the sporogenous cells within, so that by the time meiosis is completed the tapetum itself is almost wholly disorganized. Structural changes in the tapetum at this time include an increase in numbers of dictyosomes and vesicles, and in endoplasmic reticulum, which gradually becomes organized into whorls containing up to 12 gyres of cisternae (Pennell and Bell, 1985). In Lilium these membranes are assembled at the expense of “storage” lipid (Reznickova and Dickinson, 1982), and this is probably also the case in Tuxus. Meanwhile, the sporogenous cells in Tuxus undergo changes in basophilia that are the result of fluxes in ribosomes. Lytic vesicles appear in all and are likely to be responsible for the diminution in ribosome numbers that takes place before the cells enter meiosis (Pennell and Bell, 1985). These changes are difficult to quantify since, like those which accompany the development of the archaesporium, they take place at different rates in different cells. However, the technique of microfluorometry has now been applied for the first time to the microsporangia of Tuxus (Pennell and Bell, 1985), and has given support to the notion that marked changes in cytoplasmic RNA do occur in the developing sporangium. This technique involves staining high molecular weight nucleic acids with a dye which fluoresces when excited by far-blue light. The fluorochrome Acridine Orange will, at pH 4.0, form a complex with RNA
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that fluoresces orange-red and with DNA that fluoresces green-blue. When the DNA has been removed by pretreatment of the tissue with DNase, the fluorescence of the RNA alone can be measured by a single photocell. Since the binding of the dye to the nucleic acid is stoichiometric (Bucknall and Sutcliffe, 1965; Darzynkiewicz et al., 1979), its intensity can be taken as indicative of the amount of RNA in the section of the cell and, by extrapolation, in the cell as a whole. In Tuxus the trend during the development of the sporogenous cells appears to be towards a general increase in RNA (Pennell and Bell, 1985), this being attributable to a proliferation of ribosomes. The significance of this is still unclear, but it is conceivable that it takes place in preparation for meiosis, when very special and specific proteins associated with the synaptonemal complex are assembled (Moens et ul., 1987). Other proteins are certain to include some that control the entry into prophase and the progression through meiosis, and, as during mitosis, those of the cytoskeleton which determine planes of division and cytokinesis (Wicket a l . , 1981). The rapid and timely assembly of these proteins may therefore necessitate the appearance within the sporogenous cells of additional ribosomes upon which their messenger RNAs may be translated. Once completed, the controlled elimination of ribosomes by the directed action of proteases and nucleases could then give rise to the fall of RNA levels observed immediately prior to meiotic prophase (Pennell and Bell, 1985). C. MEIOSIS
Meiosis within pollen mother cells has been best studied in flowering plants, and common features have appeared that may be significant controlling events in the life cycle (Dickinson and Heslop Harrison, 1977). One of the most important of these is the undoubted elimination of a major proportion of ribosomes during prophase (Mackenzie et al., 1967) and their subsequent restoration by the disintegration in the cytoplasm of “nucleoloids”, possibly assembled in the nucleus (Williams et al., 1973). This “ribosome cycle” is accompanied by the transient dedifferentiation of both the mitochondria and plastids (Dickinson and Heslop Harrison, 1977) and the formation of “nuclear vacuoles” within the prophase nuclei (Sheffield et a l . , 1979). These events have been interpreted as crucial in the reorganization of a diploid cell (a sporogenous cell) into one whose genome is haploid (the spore), and in which expression of that part of the genome which is concerned specifically with sporophytic growth must be replaced by that part concerned specifically with gametophytes (Dickinson and Heslop Harrison, 1977). The physiological isolation of the meiocytes by cell walls rich in callose [strictly those capable of forming a fluorescent complex with Aniline Blue (Mangin, l889), now proven to involve a variety of mixed P-linked glucans (Smith and McCully, 1978)], has been implicated as being significant in the change of phase (Heslop Harrison and Mackenzie, 1967).
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Meiosis in conifers has, in the absence of evidence to the contrary, been thought comparable. Indeed, the process in both angiosperms and gymnosperms shares some common features, including the partial dedifferentiation of the mitochondria and plastids and the appearance of vacuoles within the nuclei (Sheffield et al., 1979). Meiosis in many conifers differs significantly in the duration of prophase, however. In Lilium (which may be regarded as representative of the situation in floweringplants generally) meiosis lasts for just 2-3 days (Dickinson, personal communication), while in Juniperus (Singh et al., 1983) and Taxus (Pennell and Bell, 1987a) it lasts for approximately five weeks, and in Pseudotsuga, Tsuga, Thuja and Larix for almost two months (Owens and Molder, 1971). In all instances it is the prophase of meiosis which is extended, the remaining stages of meiosis I and I1 occurring within a few days. Not only does this set the conifers apart from the flowering plants, but it also makes them favourable subjects for the study of events regulating meiosis. Advantage has recently been taken of this, and a new technique applied to the pollen mother cells of Taxus has provided novel information for an entirely new concept of controlling mechanisms (Pennell and Bell, 1986a, 1987a). The technique is also based upon the preferential staining with Acridine Orange of RNA in de-embedded sections of microspore mother cells. However, to obtain greater resolution than afforded by microfluorometry per se, the photographed images of the cells have been enlarged sufficiently to allow investigation of cytoplasm and nucleus separately, and these enlargements have been used as the subject of study. The density of reduced halide on the film, like the intensity of the fluorescence itself, is a direct measure of RNA within the section. When each compartment of the image of the cell is scanned separately in a microdensitometer, the original intensity of fluorescence can be judged from the restriction of the scanning beam, and quantified. Once cells from each of the crucial stages of meiosis have been examined in this way, the data coming from cytoplasm, nucleus excluding nucleolus, and nucleolus alone, may be plotted separately. When applied to the pollen mother cells of Taxus, it is evident that there is only slight diminution in cytoplasmic RNA during prophase (Pennell and Bell, 1986a, 1987a) and this is matched by reciprocal changes within the nucleus (excluding the nucleolus). Throughout prophase, RNA levels in the nucleolus remain constant. The significance of these findings may be great. It is conceivable that in Taxus, and possibly in the conifers generally, it is the absence of widespread degeneration of cytoplasmic RNA that is responsible for the protracted nature of prophase. The controlled hydrolysis of RNA that occurs during prophase in flowering plants may liberate oligoribonucleotides into the cytoplasm which affect the rate of expression of nuclear genes and therefore the progression through meiosis. The absence of such putative signalling molecules from the Taxus meiocytes may thus explain the slow passage through prophase. Whatever the case, it now seems clear that the “nuclear vacuoles” that have in the past been
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invoked as the means by which information-carrying molecules move between nucleus and cytoplasm are induced only where the chromosomes exert traction on the nuclear envelope during contraction (Pennell and Bell, 1985, 1986b). Once prophase is completed the remainder of male meiosis proceeds in gymnosperms in much the same way as in angiosperms and heterosporous pteridophytes. Meiosis I gives rise to pairs of cells (diads), each of which quickly divides again into two spores. In Pinus these divisions take place without the formation of partitioning walls within the tetrad (Willemse, 1971; Moitra, 1980), but in Taxus a primary wall does develop during telophase I which separates the cells of the diad (Pennell and Bell, 1986b). It therefore seems likely that the sequence of partitioning walls within the pollen mother cell carries with it systematic significance, but it has no evident functional effect. What is perhaps more important functionally is the appearance of invaginations within the nuclei of post-meiotic cells of Pinus banksiana (Dickinson and Bell, 1970) and Podocarpus macrophylla (Aldrich and Vasil, 1970). These tube-like infoldings of the nuclear envelope have rightly been thought significant both because of the presence of highly ordered pore-like structures at their openings (Dickinson and Bell, 1972a), and by virtue of the accumulation within them of osmiophilic material (Dickinson and Bell, 1970), possibly RNA (Dickinson and Potter, 1975). When taken in the context of the change of phase which occurs during meiosis, these invaginations have been cited as specialized structures by which information-carrying molecules are provided en masse to the cytoplasm of the spore (Dickinson and Bell, 1972a). It has been thought conceivable, therefore, that these molecules are in some way specific to and crucial for the cytoplasm of the spore, and prime it for gametophytic growth (Dickinson and Bell, 1972a). Such a mechanism would find a parallel in the angiosperms, where “nucleoloids” become dispersed in the spore cytoplasm at the end of meiosis (Williams et al., 1973). In Taxus, however, there are neither nuclear invaginations nor nucleoloids, and there is no evidence of nucleocytoplasmic interaction of this or any other kind at the end of meiosis. This of course may be related to the limited flux of RNA between cytoplasm and nucleus during prophase described earlier.
D . EXINE PATTERNING AND THE FREE SPORE PERIOD
Once meiosis is completed a complex series of events occurs within the cytoplasm of the spores, and these are ultimately responsible for the patterning of the sporoderm. Exine formation in the pollen grain of Pinus banksiana has been studied in depth. The pollen grain bears two bladder-like
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sacci distally and a punctate sexine perforated by a single colpus. The reticulate pattern of the sexine is established while the spores remain enclosed in the callose wall, and pattern formation begins when a number of large vesicles make contact and fuse radially at the plasma membrane of the young spore (Dickinson, 1971). The vesicles arise from what appear to be enlarged dictyosomes, and fuse en masse with the plasma membrane (Dickinson, 1971). The outer radial faces of the vesicles then become diffuse and electron-opaque, although the inner faces, which represent the new plasma membrane of the cell, continue to stain normally. The outer surfaces of the vesicles act as pattern initials, and their subsequent thickening and elaboration with sporopollenin give rise to the surface topography of the grain. Similar events take place close to the plasma membranes of the young spores of Abies concolor (Kurmann, 1986), Tsuga canadensis (Kurmann, 1984a) and Taxodium distichum (Kurmann, 1985), and in each instance development within the tetrad proceeds until elements of both nexine and sexine are in place. Detailed information is also available for the flowering plants Cosmos and Lilium (Dickinson, 1976), and is again comparable although in the angiosperms generally the architecture of the sexine is considerably more complex than that of Pinus. The patterning of the Pinus pollen grain does not take place at that part of the plasma membrane which becomes “masked” by cisternae of endoplasmic reticulum, and this does not develop into the layered exine characteristic of other parts of the grain (Dickinson, 1976). In Taxodium, the granular layer of the sexine is absent from the distal surface at the region of the germinal papilla, and the number of layers in the nexine is reduced (Kurmann, 1985), but in Tsuga there is no evidence of a pre-formed germinal aperture in the sporoderm (Kurmann, 1984b). The situation in Tuxus is simpler. The contribution of the gametophyte (spore) to the pattern of the exine takes the form of a number of small osmiophilic globules (indistinguishable in the electron microscope from sporopollenin) that emerges from the protoplast and form a confluent layer (Pennell and Bell, 1986b), or layers (Rohr, 1977), at its surface. This thin smear of sporopollenin encapsulates the microspore during the early free spore period, and serves as a surface upon which tapetally synthesized sporopollenin is placed (Pennell and Bell, 1986b). The second phase of accretion then takes place as orbicules enter the loculus from the degenerating tapetal cells, and appears to involve no more than the transfer of sporopollenin from individual orbicules onto areas of exposed gametophytic sporopollenin. The sporopollenin of the orbicules appears to have greater affinity for that layered upon the spore surface by the gametophyte than for either the lipidic core of the orbicule or orbicular sporopollenin elsewhere. In consequence, orbicular sporopollenin is transferred en masse to the surface of the maturing microspore, first as a series of concentric layers and then as discrete globules which confer the punctate pattern upon the sporoderm. The number of globules attached to the microspore surface
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Fig. 1. Near-mature Taxus microspore, fixed inside a loculus of the microsporangium before dehiscence. The nexine (NX) consists of several distinct layers of sporopollenin and the sexine (SX) of a single layer of globules. The intine (I) is barely visible. N = nucleus, P = plastid. Scale bar = 1 pm.
increases until a complete layer is present, and no areas of layered sporopollenin remain available for transfer (Fig. 1).Similar events complete exine patterning in Tarodium (Kurmann, 1985). As in Tsuga, there is no evidence in Tuxus G f a germinal pore within the spore wall. Following the terminology of Erdtman (1966), the laminae of sporopollenin represents the nexine (of which several laminae can be resolved, presumably representing individual waves of synthesis from the tapetum), and the globules the sexine. It is notable that in Tuxus the entire sexine and much of the nexine are added to the microspores once the callose wall is dissipated, but in the members of the Pinaceae a large proportion of nexine and sometimes sexine is contributed by the spores themselves. Although the structural basis of patterning is now well-established for, amongst others, Abies, Pinus, Taxodium and Taxus, as well as for several angiosperms, there is still little information upon the means by which the subcellular events responsible for it are controlled. In Lilium it has now been demonstrated that neither the meiotic spindles nor the radial microtubules in the cytoplasm of the young spore control the positioning of pattern determinants in the plasma membrane of the young pollen grain (Sheldon
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Fig. 2. Junction between three tapetal cells of Taxus. The radial cell wall (CW) contains numerous lipidic globules (L). The plasma membranes (PM) of the cells are associated with osmiophilic globules of sporopollenin (S), and similar globules are also visible inside the protoplast upon small vesicles (V). Two such vesicles appear to have traversed the plasma membranes and are entering the cell wall (arrows). Scale bar = 1pm.
and Dickinson, 1986), as the microtubules of root (Lloyd and Wells, 1985) and seed hairs (Quader et al., 1986) appear to control the orientation of cellulose microfibrils in the primary cell wall. It seems more likely that patterning determinants are present ab initio within the plasma membrane of the young pollen mother cells (Sheldon and Dickinson, 1983), and these become reorganized into a pattern which predicts that which develops subsequently in the pollen grain wall (Sheldon and Dickinson, 1986). The control of positioning of the dictyosomes which add to the pattern initials in Pinus (Dickinson, 1971), Tsuga and Taxodium (Kurmann, 1984b), however, seems likely to be involved with the cytoskeleton of the spore. In contrast, the means by which the pattern becomes impregnated with sporopollenin from the tapetum appears clear. The vehicle responsible for the transport of sporopollenin from the tapetum into the loculus of the sporangium is the orbicule. The lipidic centre of each orbicule first appears as a spherical globule within a radial tapetal wall (Pennell and Bell, 1986b) (Fig. 2). The osmiophilic material which subsequently encloses each of these “pro-orbicular cores” appears concomitantly within the tapetal protoplasts close to the radial walls (Fig. 2), in association with small vesicles (Pennell and Bell, 1986b). This substance, judged from its affinity for osmium to be sporopollenin, also moves into the radial walls of the tapetum, apparently by forming a transient association with the plasma membranes at these sites.
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Fig. 3. Localization by lead capture of acid phosphatases in the tapetum of Taxus. Both the plasma membrane (PM) and the membrane of a small vesicle (V) appear to contain the enzyme. C = cytoplasm. Scale bar = 1 pm.
Several globules of sporopollenin then rapidly aggregate to form a sheet which enwraps a single pro-orbicular core (Pennell and Bell, 1986b). The composite body so formed is clearly identifiable as an orbicule. Many orbicules are released from the tapetal walls as the protoplasts degenerate and undergo hypertrophy. The formation of the orbicules appears therefore to owe much to the affinity of the sporopollenin sheets both for lipid and for sporopollenin elsewhere. Similarly, the transfer of sporopollenin from the orbicule to the developing exine seems to come about as a result of the differing affinity of orbicular sporopollenin for the lipidic centre of the orbicule and for the sporopollenin in place upon the surface of the microspore. Indeed, once the transfer has taken place, the pro-orbicular cores devoid of a sporopollenin encasement may be observed close by the microspores. The degeneration of the tapetum proceeds apace with the formation upon the microspores of the sporoderm. The complex membranous figures which all but fill the mature tapetal cells of Tuxus develop as the fine structure of the cells becomes disorganized, and may arise as a result of their increased physiological isolation by the peritapetal membrane. Such an investment has been observed in the sporangia of many conifers (Moitra and Bhatnagar,
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1982) and appears to be composed of saturated lipid (Dickinson and Bell, 1972b). Soon after the peritapetal membrane is formed in the microsporangia of Taxus acid phosphatases appear in the plasma membranes of the tapetal cells (Pennell and Bell, 1986b) (Fig. 3). These enzymes may lead to the generalized perturbation of the plasma membranes by dephosphorylating membrane phospholipids and phosphate-containing proteins. However, what is perhaps of more interest in the study of the development of the microspores is that these enzymes (and presumably other hydrolases which accompany them) are already present and active within the tapetal plasma membranes when the microspore mother cells enter meiosis, some eight weeks before the tissue disintegrates.
111. MEGASPOROGENESIS In comparison with the development of the microsporangium, the events which take place within the ovules of conifers are poorly explored by modern techniques. This is due both to the morphology of the female cone (or fertile axis in the Taxales) and, as in all seed plants, the presence of just a single meiocyte within the ovule. Indeed, so unyielding are the megaspores and their antecedents to experimental methods that all that is known about them has been reasoned from structural studies and comparison with megasporogenesis in heterosporous pteridophytes and flowering plants whose sporangia are more accessible. Nevertheless, it is clear that sporogenesis within the ovule differs from microsporogenesis in several important ways, most strikingly in the development of the tetrad and survival of the spores. A. THE ORIGIN OF THE REPRODUCTIVE CELL LINEAGE
WITHIN THE O W L E
Little is known about differentiation within the ovule in conifers. In Tuxus, whose ovules are borne singly on compressed fertile axes, the nucellus is initially homogeneous. The nucellus begins to differentiate about 12 weeks after the appearance upon the shoot apex of the primordium of the fertile axis, when a mass of basophilic cells appears in the middle of the nucellus. In the electron microscope they are seen to contain many ribosomes and conspicuous globules of lipid (Pennell and Bell, 1987b). Since it is within this mass of cells that the megaspore mother cell develops, they have been thought to participate in the nutrition of the meiocyte and the female gametophyte which succeeds it when meiosis is completed. There is, however, no experimental evidence in support of this view, and only structural comparison of the cells with those of the tapetum in the microsporangium suggests that both behave similarly. The antecedent of the megaspore mother cell has not been identified with
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certainty. That of Taxus may develop from a hypodermal cell inside the nucellus (Dupler, 1920). B . MITOCHONDRIA, PLASTIDS AND PLANES OF DIVISION WITHIN THE MEGASPORE MOTHER CELL
Meiosis in the megaspore mother cells of many conifers sees the unidirectional movement of the mitochondria and plastids into a single haploid cell. This was first observed at the beginning of the century as the appearance of a densely staining mass below the nuclei of the meiocytes of Larix, Taxus, Thuja, Taxodium (Coker, 1904) and Juniperus (Ottley, 1909). Since then the movement of amyloplasts within the megaspore mother cells of Pinus has been found to be similar (Willemse, 1968), and indeed in the zooidozamous Encephafartos (de Sloover, 1961) and Ginkgo (Stewart and Gifford, 1967) the distributions of both plastids and mitochondria become polarized in this way. Consequently, the partitioning of the megaspore mother cell during telophase I by a transverse wall gives rise to a pair of cells of which only one-that lying chalazally--contains either mitochondria or plastids, or both (Fig. 4). The organelles behave in a similar way during meiosis 11, so that in Taxus at least (the only genus to be examined thoroughly in this respect) the chalazal spore comes to contain the entire complement of organelles, the other three members of the tetrad remaining bereft of them (Pennell and Bell, 1987b). The mechanism responsible for the directed motion on the mitochondria and plastids is still unresolved. The belief that gravity is the motive force (Volkman and Sievers, 1975) seems untenable when chalazal accumulations are known to occur in the inclined or inverted ovules of both the cone-bearing conifers and taxads. More plausible is the possibility that the motion of the mitochondria and plastids is generated by their intimate association with the cytoskeleton. Comparable phenomena have been described in vivo (Inolie, 1981) and in vitro (Vale e t a f . , 1985b), and there now seems little doubt that microtubules are capable of directing the movement of membrane-bound organelles, possibly in association with the mechanochemical protein kinesin (Vale et a f . ,1985a). Although there is no direct evidence for such a mechanism in the megaspore mother cells of conifers (or zooidogamous gymnosperms), it is difficult to conceive of any hypothesis that is not in some way related to cytoplasmic structures akin to microtubules. The directed movement of the organelles may occur in all conifers, but the form of the tetrad of megaspores differs. In most, linear or T-shaped tetrads are formed (Maheshwari, 1950), but both isobilateral and tetrahedral tetrads occur in Sequoia (Looby and Doyle, 1942). In T-shaped tetrads the plane of division of the micropylar diad cell is perpendicular to its long axis and this is due to a similarly displaced spindle. The plane of division of plant
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Fig. 4. Diad produced by meiosis I within the ovule of T a u s . The chalaza1 diad cell (lower) contains numerous mitochondria and plastids, but the micropylar diad cell (upper), while rich in lipid (L), contains none of these organelles. The bounding wall of the diad is thickened adjacent to the plasma membrane in the same way as that of the prophase meiocyte (arrow, inset). Scale bar = 5 p m (0.5 p m inset).
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cells generally is now known to be predicted by a cortical “pre-prophase band” (Wick and Duniec, 1983,1984), and hence it follows that it is the plane of assembly of this whorl of microtubules which is ultimately responsible for the opposed planes of division of the two diad cells during meiosis 11. Whatever the force responsible, the organization of the spores within the tetrad appears to be of little phylogenetic or developmental importance. C. MEGASPORE VIABILITY
Megasporogenesis in conifers is completed once the tetrad is fully formed and each spore within it is wholly enclosed by bounding and partitioning walls. However, in conifers only the chalazal megaspore develops further (eventually giving rise to the female gametophyte), the remaining spores ultimately degenerating. The promotion of the chalazal megaspore has been attributed to the presence within it of the entire complement of organelles of the mother cell from which it is derived, this conferring in some way “fitness” upon the cell. Although in the conifers the promotion of the chalazal megaspore is in all known instances prefaced by the directed movement into it of mitochondria and plastids, this is not the case in other plants. In Myosurus (Woodcock and Bell, 1968) and Epipactis (Bednara et al., 1981) the embryo sac develops from a single spore, but all the members of the tetrad receive more or less equal numbers of organelles during meiosis. Similarly, in the heterosporous fern Marsilea, megaspore viability cannot be related to any visible differences between the cytoplasms of the four spores of a tetrad (Bell, 1981). In Zeu the distribution of mitochondria and plastids between the four megaspores is only partially polarized, and again only one develops into an embryo sac (Russell, 1979). Clearly, the enriched endowment of a megaspore with mitochondria and plastids cannot be a general explanation of its preferred success, and must merely complement other factors. An alternative suggestion has been proposed by Lintilhac (1974a,b), who believes that the functional megaspore lies at a site within the nucellus where inwardly directed radial forces (established by repeated cell divisions) are opposed, giving rise to an area of “null pressure”. Since this is the only region within the ovule where there would be little or no resistance to expansion, the megaspores lying within would be able to enlarge whilst those elsewhere would not. This hypothesis is, however, difficult to reconcile with the situation in Taxus, where a pair of diametrically opposed vascular bundles in the chalazal region of the integument would be likely to displace the tectonic symmetry of the ovule as expounded by Lintilhac. Similarly, the presence of bisporic ovules in many conifers suggests that a single region of “null pressure” does not exist, or that two are present side by side. Indeed, the location of the functional megaspore within the tetrahedral tetrad of Mur-
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silea appears to bear no relation to its orientation within the radially symmetrical megasporangium (Bell, pers. comm.), and in this instance a tectonic explanation of megaspore viability is wholly unsatisfactory. The regular promotion of one member of a tetrad of four spores cannot, in fact, be readily explained by any contemporary model. This has led Bell (1981) to suggest that of the four genotypes present amongst the megaspores of Marsilea, only one is viable in the environment of the megasporangium, and this is invariably promoted at the expense of the remainder. The four genotypes would arise from recombination between two heterozygous loci which regularly experience crossing over. This hypothesis can only be applied at the present time to megaspore mother cells within which the formation of partitioning walls is delayed until both divisions of meiosis are completed. This is the case in Marsilea and the pteridophytes generally. In the linear and T-shaped tetrads of the conifers the genotype that becomes that of the functional megaspore is in all instances contained by telophase I in the chalaza1 diad cell, and is isolated from those that yield the pair of micropylar spores. For a Mendelian explanation to serve the spermatophytes it would therefore be necessary to invoke directed segregation of alleles during meiosis I, and this in itself is at variance with current thoughts upon genetic segregation preceding sexual reproduction.
ACKNOWLEDGEMENTS The author would like to express thanks to Professor P. R. Bell for helpful comments upon the manuscript.
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