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The development of the pollen grain wall in Ipomoea purpurea (L.) Roth
Review of Palaeobotany and Palynology Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
THE DEVELOPMENT OF THE POLLEN GRAIN WALL IN IPOMOEA PURPUREA (L.) ROTH H. GODWIN, P. ECHLIN AND B. C H A P M A N
Department of Botany, University of Cambridge, Cambridge (Great Britain) (Received August 15, 1966)
SUMMARY
Light and electron microscope investigations of wall development in the polyporate pollen grains of Ipomoea purpurea indicate that distinction must be made between processes giving rise respectively to the outer primary exine and to the inner secondary exine. Inside the callose wall of the pollen mother cell meiosis occurs and each of the four microspores develops a callose wall. Each microspore develops outside the cell membrane a primexine, the precursor or template of the primary exine: the primexine has thin areas corresponding to the future pores, and gaps where bacula and spines will form. The latter seem to originate by injection of material (possibly sporopollenin) from vesicles passing the cell membrane whilst the callose wall persists. The callose disappears and the spines and bacula grow and increase in (electron) density, both acquiring large heads and sharing in the formation of a tectum enclosing an interbacular cavity. Below the primary exine, and parallel with it, there now arise outside the cell membrane numerous strands, most of which have a central "white line" of 50 A width running centrally along them. These strands progressively thicken with material (sporopollenin) of the same electron density as the bacula, they coalesce and ultimately form a dense almost homogeneous band under the primary exine and between the pores. As this arises later and by a process quite different from that of the primary exine, it can be regarded as secondary exine. There seems reason to regard this morphogenetic distinction between primary and secondary exine as characteristic of at least some other pollen types although the morphogenetic mechanisms and the nature of the "white lines" in the early stranded secondary exine still remain obscure.
MATERIALS AND METHODS
This paper describes an investigation of the microscopic anatomy and ontogeny of the pollen-grain exine in Ipomoeapurpurea (L.) ROTH. It is an elaborate, Rev. Palaeobotan. Palynol., 3 (1967) 181-195
181
large, spherical, polyporate, spinose pollen grain for which already the main features were known so far as they were accessible to careful light microscopy (BEER, 191 I ). Beer's results indeed, by drawing attention to '+kinoplasmic radiation'" from the young microspore nucleus, seemed to offer hope of an approach to the discovery of morphogenetic control of features in the pollen wall. Material was grown at the University Botanic Garden and at suitable stages anthers were excised and quickly fixed in 1.57/o gtutaratdehyde in 0.1 M phosphate buffer, pH 7.2, for 16 h at 4~C. After repeated washing in the same buffer the anthers were placed either in osmium tetroxide in veronal-acetate buffer, pH 6.1, or in 2 o; osmium tetroxide in 0.1 M phosphate buffer, pH 7.2 for 2h at 4°C.They were dehydrated through acetone, embedded in Araldite and microtomed. Suitable sections were stained for 60 minutes in l °/ooaqueous uranyl acetate, pH 5.0, washed well with distilled water, and floated on lead citrate stain for 30 min. Small variations in these techniques were employed from time to time. The sections were examined in an A.E.I. EM6 electron microscope: other thick sections were taken for mounting in balsam, for direct light microscope examination intended to provide general information on topography and development. At a later stage we took advantage of the courtesy of the Cambridge Instrument Company, to examine fresh pollen grains of Ipomoea in their recently developed "Stereoscan" scanning reflection electron microscope. At lower powers especially the highly stereoscopic views of the pollen-grain surface (Plate l A, B) most usefully complement the electron micrographs of thin sections. In the event the very heavy wall thickening made fixation of the pollen grain contents difficult, and it also proved extremely troublesome to identify and recover critical stages of pollen-grain development so that very large numbers of anthers had to be cut and examined and the developmental series was completed with some difficulty. The electron microscopic observations were supplemented by light-microscope examination of paraffin-embedded sections of fresh and ofacetolyzed material, stained to emphasize intine/exine differentiation in the pollen wall.
MORPHOLOGYOF THE MATUREEXINE The light microscope shows that the wall of the Ipomoea pollen grain is strongly baculoid, the larger bacula ranging along the sides of the polygons into which the surface is divided, the centre of each polygon being occupied by a circular pore. At the corners of the polygons there are seen to be very large spines. Because of the thickness of the optical section it is hard to resolve the bacular pattern further, but this becomes entirely clear under electron-microscopic examination. The Stereoscan views (Plate 1A, B) make it apparent that a series of minor bacula lie inside the larger bacula of the muff and diminish in height 18 2
Rev. Palaeobotan. Palynol., 3 (I 967) 181-195
PLATE
I (pp.184-185)
A.
Surface "Stereoscan" view of fresh pollen grain of lpomoea showing polygons bounded by large bacula and spines (generally) at corners of polygons. Pores with small baculoid projections central in each polygon. B. Higher magnification than A, the occasional dark cracks in the surface are artefacts caused in preparation; no other breaks in the tectum are visible. C. Surface section cutting across seven polygons. The central one cuts the intine of the pore and round it the secondary exine: the bacula and spines (primary exine) of the surrounding polygons are cut transversely. D. Two young microspores each surrounded by callose wall and shewing the primexine interrupted by thin regions (pores) and gaps where later bacula and spines will develop. Legend: b = bacula; c = callose; ic = interbacular cavity; I = intine; M = microspore; Pb = probaculum; Pr = primexine; S = spine; S E = secondary exine; T = tapetum. The index line represents 1 # unless marked otherwise.
PLATE
II (pp.186-187)
A. B.
Young pollen grain inside the callose wall, and primexine with probacular gaps. Young pollen grain inside the callose wall with apparent injection of probacula with material from vesicles. C. Primary exine with bacula and spines well-developed: the callose wall has disappeared and the secondary exine is beginning to form below the primexine and between the pores. D. Primary exine with section shewing hollow base of a spine and the finely fibrillar material in the interbacular cavity. Strands of secondary exine are present most with a central "white line" of ca. 50/~ width. Legend: b = bacula; c = callose; ic = interbacular cavity; tc = tectum; P = pore; Pb = probaculum; PI = plasmalemma; Pr = primexine; S = spine; S E = secondary exine; T = tapetum. The index line represents 1/~ unless marked otherwise.
PLATE
III (pp.188-189)
A. B.
Larger magnification of stranded secondary exine with "white lines". Same stage as Plate II C and D, shewing the strands of secondary exine few and narrow round and over the pore. C. Stage of consolidation of secondary exine with threads of residual material indicating the strands: a spine and baculum in section in the primary exine. D. Pollen grain almost mature, shewing intine, fully consolidated and homogeneous secondary exine, section passing through heads of a spine and several bacula, and shewing fibrillar material still in the interbacular cavity. Legend: b = bacula; ic = interbacular cavity; tc = tectum; 1 = intine; P = pore; Pb = probaculum; S = spine; S E = secondary exine. The index line represents 1 tt unless marked otherwise.
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PLATE I
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PLATE II
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PLATE Ill
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towards the pore; minor quasi-baculoid projections occur on the outer surface of the pore itself. Electron micrographs of sections both tangential to the surface (Plate IC) and normal to it (Plate I1C, D) confirm this structure. The tectoid surface resembles a large tent, the largest tent poles being the spines with smaller poles supporting the tectum in its catenas between the spines and down to pore level. In the mature grain this tectum clearly overlies an extensive interbaculoid space that still contains an open fibrillar material. The spines themselves are baculoid in nature with strongly developed solid heads but hollow (forked) bases (Plate IID): the other bacula have capitate heads that do not become fully confluent with one another (Plate ilC). There is little if any tendency for the bacula to expand at their bases to form a foot layer of the kind apparent in many pollen types. The chances of orientation will naturally often produce oblique sections through the bacula (Plate IIIC)or shew their capitate heads without the supporting rods(Plate IIID). Radial sections of mature grains, even those seen under the light microscope, shew beneath the baculoid layer everywhere except below the pores, an extremely thick inner layer. This, that we propose to give reasons for calling "secondary exine" takes the same stains as the bacula and spines, has the same electron density and is equally resistant to acetolysis. There is no reason to doubt that it is also made of sporopollenin and is exinous in character (Plate IIID). The tangential surface section of a grain seen in Plate IC now becomes comprehensible: the pore in the centre of the picture is cut at a level where the obliquely cut intine is surrounded by the dense secondary exine, the polygons of adjacent pores are cut above pore level and transect both the spines and minor bacula, some of which form a close inner ring surrounding each pore. Within the secondary exine the mature grain has a substantial cellulosic intine which is susceptible to acetolysis and stains typically. It is thickened under the pores (Plate IIID), but we have not investigated its detailed structure.
MORPHOGENESIS:THE PRIMARY EXINE In his stimulating pioneer studies of the ontogeny of pollen in Silene pendula, Cannabis sativa and other species, HESLOP-HARRISON(1963) calls attention to the importance of the environment within which the pollen mothercell undergoes meiosis. He redirects attention to the development of what Beer called the "special mother-cell wall", a thick, hyaline, mucilaginous layer that surrounds the pollen mother cell, and subsequently found separately round each of the four newly formed microspore protoplasts. There appears to be good reason to think that this wall is of callose (HESLOP-HARRISON,1966) and it is plausibly conjectured that it serves to isolate the dividing germ cells from the influence of the surrounding tapetal tissues that are also engaged in considerable but quite different activity. The callose wall surrounding the young microspores is readily recognisable at 190
Rev. Palaeobotan. Palynol., 3 (1967) 181-195
the appropriate stage in Ipomoea (Plate ID, liB, C). It is whilst still isolated within this wall that the precursor structures of the pollen exine are laid down in Ipomoea as in Silene pendula; they can be seen in Plate ID and IIA, B. The primexine (to use Heslop-Harrison's term) consists of finely fibrillar material of moderate electron density, possibly cellulose. It clearly foreshadows the location of pores where it is thin, and interporal areas where it is thick and where there are gaps that in size and frequency correspond with what are to become bacula and spines. At first it seems that the gaps are empty or contain only the very loose textured material occupying the considerable space between the primexine and the well defined plasmalemma. Soon afterwards the gaps become filled by much more homogeneous material of increasing electron density (presumably sporopollenin) to form the bacula and spines. Occasionally, as in Plate IIB, one appears to have evidence that the sporopollenin is injected into the primexine gaps from vesicles that break through the plasmalemma. Until this stage the callose wall persists, but it now disappears and there follows the dramatic increase in height and thickness and density of the baculoid elements to which Heslop-Harrison has drawn attention (Plate IIC). Meanwhile the original primexine material becomes more open textured within the considerable interbacular voids, the bacula heads expand, and especially near the spines, tend to make a thick continuous tectum. Itis difficult to say how far a thicker or thinner tectum extends to the pores; the apparent continuity in the Stereoscan pictures is possibly due to a surface layer of typhine, since the preparatory metallic coating of the grains is itself electron transparent. One would hope to have found some organised pattern of organelles that might precede and determine the pore and bacula locations in the primexine such as the disposition of endoplasmic reticulum found by Heslop-Harrison in Silene. In Ipomoea however, despite fixation that reveals extreme organelle detail in the microspore and pollen mother cells (cf. ECHLIN, 1965), we have so far detected no trace of organelle precursors to the primexine. Since the primexine is however laid down within the compound callose wall, it seems likely that exine patterns are determined by the haploid microspore and not by the diploid parental material of the anther sac surrounding them. It must further be noted that whereas in Silene the first indication of primexine comes with the deposition of electron dense material as probacula, in Ipomoea the earliest stage has the character of a negative template into the gaps in which baculoid material is subsequently injected. Further investigations may well shew that the distinction between the two types of primexine is less clear than it now appears.
MORPHOGENESIS; THE SECONDARY EXINE
When the callose wall round the individual microspores has disappeared the spines and bacula are well developed and electron dense, there arise beneath Rev. Palaeobotan. PalynoL, 3 (1967) 181-195
191
the baculoid layer the precursors of tile thick inner layer of" sporopollenin. In the loose-textured material between the plasmalennna and the baculoid layer a stranded layer develops, the elements of which are seen in section as separate strands more or less parallel with the grain surface and composed of homogeneous material similar in density lind texture to the bacular material. Most of the strands are characterised by a very sharply defined central white line of rigidly constant width of about 4 5 1 5 0 A. [Plate liD, IIIA). ~fhere is no suggestion of continuity with the baculoid layer but an occasional continuity into the base of the spines, the separateness of which however from the secondary exine below is emphasized by the dark line commonly seen between the two in section(Plate l l l C ) a n d by the way in which acetylosed grains during sectioning in paraffin wax commonly shew spinules detached at the base and displaced from the exine. Electron dense material appears to accumulate progressively upon the strands and as they widen, the matrix is left as granular threads between them (Plate IID, IIlC). Ultimately the widening strands coalesce (Plate lllCt and finally the granular matrix threads disappear leaving an apparently homogeneous or near homogeneous mass of sporopollenin (Plate IIID). By this stage little or no trace of the "white lines" remains. From the gradation in width of the strands it seems probable that those outside arise first and that others originateprogressively within them. The "white lines" can be readily seen converging on the flanks of the pores but they are associated with less and less strand thickening as they approach the pore margin and those few "white lines" that cover the pore itself seem to lead to very little exine formation. The isolated lumps of exinous material scattered on the surface of the pore seem to be homologous with the bacula. Despite the most careful search we have thus far been unable to trace any clear origin in the cell protoplast or plasmalemma of the white-cored strands, nor it must be confessed, can we define precisely the solid shape of the strand or the "white lines". The lines are visible over such considerable lengths of strand and in so many strands in each section that one is inclined to conjecture a lamellar or tape form for them. They seem to behave as centres of condensation of strand material and their uniformity and dimensions recall those of the central layer of a unit membrane. Whatever view we take of their precise nature it seems to us that they are part of a mechanism of exine formation later in time than that leading to formation of the baculoid layer and essentially different in character. It is for these reasons that it seems correct to consider the one as "primary exine" and the other as "secondary exine". These are precisely the criteria suggested recently by ERr)mMAN (1966) as a basis for morphogenetic (as distinct from topographic) classification of strata in the pollen grain wall. Indeed he arrives at a separation of the tectum plus bacula plus foot layer on the one hand from the subjacent endexine on the other on the basis that "(ils) aient des origines diff~rentes en apparaissant des moments diff~rents." It seems thus that his use of "ectine" and "endine" is intended exactly as we employ "primary exine" and "secondary exine". 192
Rev. Palaeobotan. Palynol., 3 (1967) 181-195
It remains to enquire how general is the phenomenon of a banded secondary exine and to what extent the "white lines" have been observed in relation to it. Both are clearly displayed in unpublished electron micrographs made in this department of pollen of Humulus lupulus L., Helleborous foetidus L. and Tilia platyphyllos ScoP., in each instance fixed in glutaraldehyde and osmium tetroxide and stained with potassium permanganate or lead citrate. A structure strongly suggestive of the same organisation was described by LARSON and LEWIS(1961) in the grains of Parkinsonia aculeata where the aperture membranes are said to have "laminae which have thicknesses between 300 and 700 A", "composed of paired layered fibrils with diameters of approximately 50A. The paired fibrils are rigidly oriented along the long axis of the lamina and may represent longitudinal sections through tubular structures or cross-sections throughevenly spaced parallel sheets." LARSON and SKVARLA (1961) illustrated a banded or lamellated endexine in Krameria lanceolata. Later LARSONet al. (1962)figured a lamellated and discontinuous exine below the foot layer in Ricinus communis L., and similarly placed "lamellated endexine" in Saintpaulia ionantha and Ambrosia artemisioides. Again in 1963, LARSONand LEWISfigured in Parkinsonia aculeata the same structures as "microfibrils of sporopollenin in lamellae of the endexinous aperture membrane" and they are clearly beneath a baculoid layer. A secondary exine of this kind may be widespread, but almost certainly is absent in some species, such as Scilla nutans (R. Angold, unpublished). In view of the fact that bacula commonly are of the dimensions of the strands in the secondary exine and that structures such as the spines in Ipomoea sometimes seem to shew a faintly subcellular texture like that of the mature secondary exine, it might be expected that "white lines" might also appear in the early stages of the primary exine. No sign of this has been seen or apparently reported, although ROWLEY (1962a,b) described a bundle or stranded state in tectum, bacula and foot layer of the primary wall of Poa annua microspores, and described within the bundles "windows of low density having an axial separation of 50--80 A and a diameter up to 50 A". The primary exine in Poa annua becomes progressively homogeneous in texture just as the secondary exine in Ipomoea is seen to do.
GENERALCONSIDERATIONS Our presentation of the evidence of the manner of exine formation in lpornoea has been deliberately restricted in scope and we have purposely omitted large areas of observation and interpretation where the data cannot yet be fitted to a precise developmental sequence. We thus omit reference to the abundant organelles (some of an unusual kind) and complex cellular organisation in the pollen mother cells and microspores (see ECnLIN, 1965), and we omit consideration of the intine and its relationship to the pores. Finally we have not reported upon Rev. Palaeobotan. Palynol., 3 (1967) 181-195
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tile tapetum, although our preparattons have been such as generally to include sections of it. l'his layer, so strongly cv>m..plementary to the devcloping sporogenom; material pursues a developmental course totally out of phase with that of the pollen forming cells. It divides mitotically at an early stage and enters a condition o1 apparent autolysis during the meiotic tetrad t\~rmation of the microspores: the cellulose walls thin and disappear and the protoplasts retract by a process which appears to involve the dischalge of large vesicles at the f~ee cell su~ face. In/pomoea there appears to be no Ubisch body or plaque formation, nor have we seen any evidence for a particulate transpoit of tapetal matelial to the exines of the young microspores, either in the form of the fine fiblillar th:eads, described by ROWLEY (1962a) in Poa anmea, or in larger solid bodies. The whole subject of tapetum-microspore relationships is clearly one of great complexity and importance, to the study of which HESLOP-HARRISON(1963) has given an impressive primary consideration. We would only add the suggestion that features of exine formation may well relate to the juxtaposition of the tapetal contents in a late phase of senescent autolysis and breakdown, with the microspore surface in an early phase of ontogenetic metabolic drift with the strongest anabolic tendencies. If the earliest "'template" of the primexine is indeed cellulose, it is laid down when presumably isolated from the disorganising tapetal material by the callose wall. So far as the tectum continues to interpose such a barrier the cellulose will remain, but in those pollen grains where the tectum is perforate it is reasonable to expect the cellulose to hydrolyse under the influence of the tapetal cellulases, so producing the interbacular voids which either remain empty or are injected by the tapetal material generally if mysteriously referred to as "tryphine". The sporopollenin by contrast appears not to be susceptible to hydrolysis in the tapetal autolysis (and is indeed synthesised in plaques and l~lbish bodies at this time), so that when the callose wall disappears from the young microspores the sporopollenin already present in the exine as bacula, tectum, etc., is not attacked and indeed continues active growth, possibly by condensation from soluble precursors derived from the disorganising tapetum. Such a situation calls strongly for elucidation of the biochemistry of the sporopollenin and the role of its possible organisers including the "white lines" to which we have drawn attention.
ACK NOWLEDGEMENTS
We wish to thank the Department of Scientific and Industrial Research (now the Science Research Council) for providing the A.E.I. electron microscope and ancillary equipment used in the research, and the Cambridge Instrument Company for their kindness in allowing us to use their newly developed"Stereoscan" electron microscope for pictures of the surface of the mature grain. We are indebted to Mr. R. D. H. C. Whybrow and Miss R. Andrew for help with paraffin-wax 194
Rev. Palaeobotan. Palynol., 3 (1967) 181-195
sectioning and preparation, but most particularly to Mr. Paul Curtis who has been responsible for the photographic processing throughout.
REFERENCES
BEER, R., 1911. Studies in spore development. Ann. Botany (London), 25: 199-214. ECHLIS, P., 1965. An apparent helical arrangement of ribosomes in developing pollen mother cells of lpomoeapurpurea (L). ROTH. J. Cell Biol., 24: 150-153. ERDTMAN, G., 1966. A p r o p o s de la stratification de l'exine. Pollen Spores, 8: 5-7. HESLOP-HARRISON,J., 1962. Origin of exine. Nature, 195: 1069-1071. HESLOP-HARRISON, J., 1963. Ultrastructural aspects of differentiation in sporogenous tissue. Cell Differentiation--Symp. Soc. Exptl. Biol., 17:315-340. HESLOP-HARRISON, J., 1966. Cytoplasmic connections between angiosperm meiocytes. Ann. Botany (London), 30: 221-230. LARSON,D. A. and LEWIS, C. W., 1961. Fine structure ofParkinsonia aculeata, I. The pollen wall. Ann. J. Botany, 48: 934-943. LARSOr~,D. A. and LEWIS, C. W., 1963. Pollen wall development in Parkinsonia aculeata. Grana Palynologica, 3: 21-27. LARSON,D. A. and SKVARLA,J. J., 1961. The morphology and fine structure of pollen of Polygala alba NtrrT. and P. incarnata L. Pollen Spores, 3: 21-32. LARSON, D. A., SKVARLA,J. J. and LEwis JR., C. W., 1962. An electron microscope study of exine stratification and fine structure. Pollen Spores, 4: 233-246. ROWLEY, J. R., 1962a. Stranded arrangement of sporopollenin in the exine of microspores of Poa annua. Science, 137: 526-528. ROWLEY, J. R., 1962b. Non-homogeneous sporopollenin in microspores of Poa annua L. Grana Palynologica, 3: 3-19.
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THE DEVELOPMENT OF THE POLLEN GRAIN WALL IN IPOMOEA PURPUREA (L.) ROTH H. GODWIN, P. ECHLIN AND B. C H A P M A N
Department of Botany, University of Cambridge, Cambridge (Great Britain) (Received August 15, 1966)
SUMMARY
Light and electron microscope investigations of wall development in the polyporate pollen grains of Ipomoea purpurea indicate that distinction must be made between processes giving rise respectively to the outer primary exine and to the inner secondary exine. Inside the callose wall of the pollen mother cell meiosis occurs and each of the four microspores develops a callose wall. Each microspore develops outside the cell membrane a primexine, the precursor or template of the primary exine: the primexine has thin areas corresponding to the future pores, and gaps where bacula and spines will form. The latter seem to originate by injection of material (possibly sporopollenin) from vesicles passing the cell membrane whilst the callose wall persists. The callose disappears and the spines and bacula grow and increase in (electron) density, both acquiring large heads and sharing in the formation of a tectum enclosing an interbacular cavity. Below the primary exine, and parallel with it, there now arise outside the cell membrane numerous strands, most of which have a central "white line" of 50 A width running centrally along them. These strands progressively thicken with material (sporopollenin) of the same electron density as the bacula, they coalesce and ultimately form a dense almost homogeneous band under the primary exine and between the pores. As this arises later and by a process quite different from that of the primary exine, it can be regarded as secondary exine. There seems reason to regard this morphogenetic distinction between primary and secondary exine as characteristic of at least some other pollen types although the morphogenetic mechanisms and the nature of the "white lines" in the early stranded secondary exine still remain obscure.
MATERIALS AND METHODS
This paper describes an investigation of the microscopic anatomy and ontogeny of the pollen-grain exine in Ipomoeapurpurea (L.) ROTH. It is an elaborate, Rev. Palaeobotan. Palynol., 3 (1967) 181-195
181
large, spherical, polyporate, spinose pollen grain for which already the main features were known so far as they were accessible to careful light microscopy (BEER, 191 I ). Beer's results indeed, by drawing attention to '+kinoplasmic radiation'" from the young microspore nucleus, seemed to offer hope of an approach to the discovery of morphogenetic control of features in the pollen wall. Material was grown at the University Botanic Garden and at suitable stages anthers were excised and quickly fixed in 1.57/o gtutaratdehyde in 0.1 M phosphate buffer, pH 7.2, for 16 h at 4~C. After repeated washing in the same buffer the anthers were placed either in osmium tetroxide in veronal-acetate buffer, pH 6.1, or in 2 o; osmium tetroxide in 0.1 M phosphate buffer, pH 7.2 for 2h at 4°C.They were dehydrated through acetone, embedded in Araldite and microtomed. Suitable sections were stained for 60 minutes in l °/ooaqueous uranyl acetate, pH 5.0, washed well with distilled water, and floated on lead citrate stain for 30 min. Small variations in these techniques were employed from time to time. The sections were examined in an A.E.I. EM6 electron microscope: other thick sections were taken for mounting in balsam, for direct light microscope examination intended to provide general information on topography and development. At a later stage we took advantage of the courtesy of the Cambridge Instrument Company, to examine fresh pollen grains of Ipomoea in their recently developed "Stereoscan" scanning reflection electron microscope. At lower powers especially the highly stereoscopic views of the pollen-grain surface (Plate l A, B) most usefully complement the electron micrographs of thin sections. In the event the very heavy wall thickening made fixation of the pollen grain contents difficult, and it also proved extremely troublesome to identify and recover critical stages of pollen-grain development so that very large numbers of anthers had to be cut and examined and the developmental series was completed with some difficulty. The electron microscopic observations were supplemented by light-microscope examination of paraffin-embedded sections of fresh and ofacetolyzed material, stained to emphasize intine/exine differentiation in the pollen wall.
MORPHOLOGYOF THE MATUREEXINE The light microscope shows that the wall of the Ipomoea pollen grain is strongly baculoid, the larger bacula ranging along the sides of the polygons into which the surface is divided, the centre of each polygon being occupied by a circular pore. At the corners of the polygons there are seen to be very large spines. Because of the thickness of the optical section it is hard to resolve the bacular pattern further, but this becomes entirely clear under electron-microscopic examination. The Stereoscan views (Plate 1A, B) make it apparent that a series of minor bacula lie inside the larger bacula of the muff and diminish in height 18 2
Rev. Palaeobotan. Palynol., 3 (I 967) 181-195
PLATE
I (pp.184-185)
A.
Surface "Stereoscan" view of fresh pollen grain of lpomoea showing polygons bounded by large bacula and spines (generally) at corners of polygons. Pores with small baculoid projections central in each polygon. B. Higher magnification than A, the occasional dark cracks in the surface are artefacts caused in preparation; no other breaks in the tectum are visible. C. Surface section cutting across seven polygons. The central one cuts the intine of the pore and round it the secondary exine: the bacula and spines (primary exine) of the surrounding polygons are cut transversely. D. Two young microspores each surrounded by callose wall and shewing the primexine interrupted by thin regions (pores) and gaps where later bacula and spines will develop. Legend: b = bacula; c = callose; ic = interbacular cavity; I = intine; M = microspore; Pb = probaculum; Pr = primexine; S = spine; S E = secondary exine; T = tapetum. The index line represents 1 # unless marked otherwise.
PLATE
II (pp.186-187)
A. B.
Young pollen grain inside the callose wall, and primexine with probacular gaps. Young pollen grain inside the callose wall with apparent injection of probacula with material from vesicles. C. Primary exine with bacula and spines well-developed: the callose wall has disappeared and the secondary exine is beginning to form below the primexine and between the pores. D. Primary exine with section shewing hollow base of a spine and the finely fibrillar material in the interbacular cavity. Strands of secondary exine are present most with a central "white line" of ca. 50/~ width. Legend: b = bacula; c = callose; ic = interbacular cavity; tc = tectum; P = pore; Pb = probaculum; PI = plasmalemma; Pr = primexine; S = spine; S E = secondary exine; T = tapetum. The index line represents 1/~ unless marked otherwise.
PLATE
III (pp.188-189)
A. B.
Larger magnification of stranded secondary exine with "white lines". Same stage as Plate II C and D, shewing the strands of secondary exine few and narrow round and over the pore. C. Stage of consolidation of secondary exine with threads of residual material indicating the strands: a spine and baculum in section in the primary exine. D. Pollen grain almost mature, shewing intine, fully consolidated and homogeneous secondary exine, section passing through heads of a spine and several bacula, and shewing fibrillar material still in the interbacular cavity. Legend: b = bacula; ic = interbacular cavity; tc = tectum; 1 = intine; P = pore; Pb = probaculum; S = spine; S E = secondary exine. The index line represents 1 tt unless marked otherwise.
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PLATE I
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PLATE II
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PLATE Ill
O0
towards the pore; minor quasi-baculoid projections occur on the outer surface of the pore itself. Electron micrographs of sections both tangential to the surface (Plate IC) and normal to it (Plate I1C, D) confirm this structure. The tectoid surface resembles a large tent, the largest tent poles being the spines with smaller poles supporting the tectum in its catenas between the spines and down to pore level. In the mature grain this tectum clearly overlies an extensive interbaculoid space that still contains an open fibrillar material. The spines themselves are baculoid in nature with strongly developed solid heads but hollow (forked) bases (Plate IID): the other bacula have capitate heads that do not become fully confluent with one another (Plate ilC). There is little if any tendency for the bacula to expand at their bases to form a foot layer of the kind apparent in many pollen types. The chances of orientation will naturally often produce oblique sections through the bacula (Plate IIIC)or shew their capitate heads without the supporting rods(Plate IIID). Radial sections of mature grains, even those seen under the light microscope, shew beneath the baculoid layer everywhere except below the pores, an extremely thick inner layer. This, that we propose to give reasons for calling "secondary exine" takes the same stains as the bacula and spines, has the same electron density and is equally resistant to acetolysis. There is no reason to doubt that it is also made of sporopollenin and is exinous in character (Plate IIID). The tangential surface section of a grain seen in Plate IC now becomes comprehensible: the pore in the centre of the picture is cut at a level where the obliquely cut intine is surrounded by the dense secondary exine, the polygons of adjacent pores are cut above pore level and transect both the spines and minor bacula, some of which form a close inner ring surrounding each pore. Within the secondary exine the mature grain has a substantial cellulosic intine which is susceptible to acetolysis and stains typically. It is thickened under the pores (Plate IIID), but we have not investigated its detailed structure.
MORPHOGENESIS:THE PRIMARY EXINE In his stimulating pioneer studies of the ontogeny of pollen in Silene pendula, Cannabis sativa and other species, HESLOP-HARRISON(1963) calls attention to the importance of the environment within which the pollen mothercell undergoes meiosis. He redirects attention to the development of what Beer called the "special mother-cell wall", a thick, hyaline, mucilaginous layer that surrounds the pollen mother cell, and subsequently found separately round each of the four newly formed microspore protoplasts. There appears to be good reason to think that this wall is of callose (HESLOP-HARRISON,1966) and it is plausibly conjectured that it serves to isolate the dividing germ cells from the influence of the surrounding tapetal tissues that are also engaged in considerable but quite different activity. The callose wall surrounding the young microspores is readily recognisable at 190
Rev. Palaeobotan. Palynol., 3 (1967) 181-195
the appropriate stage in Ipomoea (Plate ID, liB, C). It is whilst still isolated within this wall that the precursor structures of the pollen exine are laid down in Ipomoea as in Silene pendula; they can be seen in Plate ID and IIA, B. The primexine (to use Heslop-Harrison's term) consists of finely fibrillar material of moderate electron density, possibly cellulose. It clearly foreshadows the location of pores where it is thin, and interporal areas where it is thick and where there are gaps that in size and frequency correspond with what are to become bacula and spines. At first it seems that the gaps are empty or contain only the very loose textured material occupying the considerable space between the primexine and the well defined plasmalemma. Soon afterwards the gaps become filled by much more homogeneous material of increasing electron density (presumably sporopollenin) to form the bacula and spines. Occasionally, as in Plate IIB, one appears to have evidence that the sporopollenin is injected into the primexine gaps from vesicles that break through the plasmalemma. Until this stage the callose wall persists, but it now disappears and there follows the dramatic increase in height and thickness and density of the baculoid elements to which Heslop-Harrison has drawn attention (Plate IIC). Meanwhile the original primexine material becomes more open textured within the considerable interbacular voids, the bacula heads expand, and especially near the spines, tend to make a thick continuous tectum. Itis difficult to say how far a thicker or thinner tectum extends to the pores; the apparent continuity in the Stereoscan pictures is possibly due to a surface layer of typhine, since the preparatory metallic coating of the grains is itself electron transparent. One would hope to have found some organised pattern of organelles that might precede and determine the pore and bacula locations in the primexine such as the disposition of endoplasmic reticulum found by Heslop-Harrison in Silene. In Ipomoea however, despite fixation that reveals extreme organelle detail in the microspore and pollen mother cells (cf. ECHLIN, 1965), we have so far detected no trace of organelle precursors to the primexine. Since the primexine is however laid down within the compound callose wall, it seems likely that exine patterns are determined by the haploid microspore and not by the diploid parental material of the anther sac surrounding them. It must further be noted that whereas in Silene the first indication of primexine comes with the deposition of electron dense material as probacula, in Ipomoea the earliest stage has the character of a negative template into the gaps in which baculoid material is subsequently injected. Further investigations may well shew that the distinction between the two types of primexine is less clear than it now appears.
MORPHOGENESIS; THE SECONDARY EXINE
When the callose wall round the individual microspores has disappeared the spines and bacula are well developed and electron dense, there arise beneath Rev. Palaeobotan. PalynoL, 3 (1967) 181-195
191
the baculoid layer the precursors of tile thick inner layer of" sporopollenin. In the loose-textured material between the plasmalennna and the baculoid layer a stranded layer develops, the elements of which are seen in section as separate strands more or less parallel with the grain surface and composed of homogeneous material similar in density lind texture to the bacular material. Most of the strands are characterised by a very sharply defined central white line of rigidly constant width of about 4 5 1 5 0 A. [Plate liD, IIIA). ~fhere is no suggestion of continuity with the baculoid layer but an occasional continuity into the base of the spines, the separateness of which however from the secondary exine below is emphasized by the dark line commonly seen between the two in section(Plate l l l C ) a n d by the way in which acetylosed grains during sectioning in paraffin wax commonly shew spinules detached at the base and displaced from the exine. Electron dense material appears to accumulate progressively upon the strands and as they widen, the matrix is left as granular threads between them (Plate IID, IIlC). Ultimately the widening strands coalesce (Plate lllCt and finally the granular matrix threads disappear leaving an apparently homogeneous or near homogeneous mass of sporopollenin (Plate IIID). By this stage little or no trace of the "white lines" remains. From the gradation in width of the strands it seems probable that those outside arise first and that others originateprogressively within them. The "white lines" can be readily seen converging on the flanks of the pores but they are associated with less and less strand thickening as they approach the pore margin and those few "white lines" that cover the pore itself seem to lead to very little exine formation. The isolated lumps of exinous material scattered on the surface of the pore seem to be homologous with the bacula. Despite the most careful search we have thus far been unable to trace any clear origin in the cell protoplast or plasmalemma of the white-cored strands, nor it must be confessed, can we define precisely the solid shape of the strand or the "white lines". The lines are visible over such considerable lengths of strand and in so many strands in each section that one is inclined to conjecture a lamellar or tape form for them. They seem to behave as centres of condensation of strand material and their uniformity and dimensions recall those of the central layer of a unit membrane. Whatever view we take of their precise nature it seems to us that they are part of a mechanism of exine formation later in time than that leading to formation of the baculoid layer and essentially different in character. It is for these reasons that it seems correct to consider the one as "primary exine" and the other as "secondary exine". These are precisely the criteria suggested recently by ERr)mMAN (1966) as a basis for morphogenetic (as distinct from topographic) classification of strata in the pollen grain wall. Indeed he arrives at a separation of the tectum plus bacula plus foot layer on the one hand from the subjacent endexine on the other on the basis that "(ils) aient des origines diff~rentes en apparaissant des moments diff~rents." It seems thus that his use of "ectine" and "endine" is intended exactly as we employ "primary exine" and "secondary exine". 192
Rev. Palaeobotan. Palynol., 3 (1967) 181-195
It remains to enquire how general is the phenomenon of a banded secondary exine and to what extent the "white lines" have been observed in relation to it. Both are clearly displayed in unpublished electron micrographs made in this department of pollen of Humulus lupulus L., Helleborous foetidus L. and Tilia platyphyllos ScoP., in each instance fixed in glutaraldehyde and osmium tetroxide and stained with potassium permanganate or lead citrate. A structure strongly suggestive of the same organisation was described by LARSON and LEWIS(1961) in the grains of Parkinsonia aculeata where the aperture membranes are said to have "laminae which have thicknesses between 300 and 700 A", "composed of paired layered fibrils with diameters of approximately 50A. The paired fibrils are rigidly oriented along the long axis of the lamina and may represent longitudinal sections through tubular structures or cross-sections throughevenly spaced parallel sheets." LARSON and SKVARLA (1961) illustrated a banded or lamellated endexine in Krameria lanceolata. Later LARSONet al. (1962)figured a lamellated and discontinuous exine below the foot layer in Ricinus communis L., and similarly placed "lamellated endexine" in Saintpaulia ionantha and Ambrosia artemisioides. Again in 1963, LARSONand LEWISfigured in Parkinsonia aculeata the same structures as "microfibrils of sporopollenin in lamellae of the endexinous aperture membrane" and they are clearly beneath a baculoid layer. A secondary exine of this kind may be widespread, but almost certainly is absent in some species, such as Scilla nutans (R. Angold, unpublished). In view of the fact that bacula commonly are of the dimensions of the strands in the secondary exine and that structures such as the spines in Ipomoea sometimes seem to shew a faintly subcellular texture like that of the mature secondary exine, it might be expected that "white lines" might also appear in the early stages of the primary exine. No sign of this has been seen or apparently reported, although ROWLEY (1962a,b) described a bundle or stranded state in tectum, bacula and foot layer of the primary wall of Poa annua microspores, and described within the bundles "windows of low density having an axial separation of 50--80 A and a diameter up to 50 A". The primary exine in Poa annua becomes progressively homogeneous in texture just as the secondary exine in Ipomoea is seen to do.
GENERALCONSIDERATIONS Our presentation of the evidence of the manner of exine formation in lpornoea has been deliberately restricted in scope and we have purposely omitted large areas of observation and interpretation where the data cannot yet be fitted to a precise developmental sequence. We thus omit reference to the abundant organelles (some of an unusual kind) and complex cellular organisation in the pollen mother cells and microspores (see ECnLIN, 1965), and we omit consideration of the intine and its relationship to the pores. Finally we have not reported upon Rev. Palaeobotan. Palynol., 3 (1967) 181-195
193
tile tapetum, although our preparattons have been such as generally to include sections of it. l'his layer, so strongly cv>m..plementary to the devcloping sporogenom; material pursues a developmental course totally out of phase with that of the pollen forming cells. It divides mitotically at an early stage and enters a condition o1 apparent autolysis during the meiotic tetrad t\~rmation of the microspores: the cellulose walls thin and disappear and the protoplasts retract by a process which appears to involve the dischalge of large vesicles at the f~ee cell su~ face. In/pomoea there appears to be no Ubisch body or plaque formation, nor have we seen any evidence for a particulate transpoit of tapetal matelial to the exines of the young microspores, either in the form of the fine fiblillar th:eads, described by ROWLEY (1962a) in Poa anmea, or in larger solid bodies. The whole subject of tapetum-microspore relationships is clearly one of great complexity and importance, to the study of which HESLOP-HARRISON(1963) has given an impressive primary consideration. We would only add the suggestion that features of exine formation may well relate to the juxtaposition of the tapetal contents in a late phase of senescent autolysis and breakdown, with the microspore surface in an early phase of ontogenetic metabolic drift with the strongest anabolic tendencies. If the earliest "'template" of the primexine is indeed cellulose, it is laid down when presumably isolated from the disorganising tapetal material by the callose wall. So far as the tectum continues to interpose such a barrier the cellulose will remain, but in those pollen grains where the tectum is perforate it is reasonable to expect the cellulose to hydrolyse under the influence of the tapetal cellulases, so producing the interbacular voids which either remain empty or are injected by the tapetal material generally if mysteriously referred to as "tryphine". The sporopollenin by contrast appears not to be susceptible to hydrolysis in the tapetal autolysis (and is indeed synthesised in plaques and l~lbish bodies at this time), so that when the callose wall disappears from the young microspores the sporopollenin already present in the exine as bacula, tectum, etc., is not attacked and indeed continues active growth, possibly by condensation from soluble precursors derived from the disorganising tapetum. Such a situation calls strongly for elucidation of the biochemistry of the sporopollenin and the role of its possible organisers including the "white lines" to which we have drawn attention.
ACK NOWLEDGEMENTS
We wish to thank the Department of Scientific and Industrial Research (now the Science Research Council) for providing the A.E.I. electron microscope and ancillary equipment used in the research, and the Cambridge Instrument Company for their kindness in allowing us to use their newly developed"Stereoscan" electron microscope for pictures of the surface of the mature grain. We are indebted to Mr. R. D. H. C. Whybrow and Miss R. Andrew for help with paraffin-wax 194
Rev. Palaeobotan. Palynol., 3 (1967) 181-195
sectioning and preparation, but most particularly to Mr. Paul Curtis who has been responsible for the photographic processing throughout.
REFERENCES
BEER, R., 1911. Studies in spore development. Ann. Botany (London), 25: 199-214. ECHLIS, P., 1965. An apparent helical arrangement of ribosomes in developing pollen mother cells of lpomoeapurpurea (L). ROTH. J. Cell Biol., 24: 150-153. ERDTMAN, G., 1966. A p r o p o s de la stratification de l'exine. Pollen Spores, 8: 5-7. HESLOP-HARRISON,J., 1962. Origin of exine. Nature, 195: 1069-1071. HESLOP-HARRISON, J., 1963. Ultrastructural aspects of differentiation in sporogenous tissue. Cell Differentiation--Symp. Soc. Exptl. Biol., 17:315-340. HESLOP-HARRISON, J., 1966. Cytoplasmic connections between angiosperm meiocytes. Ann. Botany (London), 30: 221-230. LARSON,D. A. and LEWIS, C. W., 1961. Fine structure ofParkinsonia aculeata, I. The pollen wall. Ann. J. Botany, 48: 934-943. LARSOr~,D. A. and LEWIS, C. W., 1963. Pollen wall development in Parkinsonia aculeata. Grana Palynologica, 3: 21-27. LARSON,D. A. and SKVARLA,J. J., 1961. The morphology and fine structure of pollen of Polygala alba NtrrT. and P. incarnata L. Pollen Spores, 3: 21-32. LARSON, D. A., SKVARLA,J. J. and LEwis JR., C. W., 1962. An electron microscope study of exine stratification and fine structure. Pollen Spores, 4: 233-246. ROWLEY, J. R., 1962a. Stranded arrangement of sporopollenin in the exine of microspores of Poa annua. Science, 137: 526-528. ROWLEY, J. R., 1962b. Non-homogeneous sporopollenin in microspores of Poa annua L. Grana Palynologica, 3: 3-19.
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