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2 Palynological Laboratory, Swedish Museum of Natural History, Roslagsvägen 101, S-104 05 Stockholm, Sweden; and 3 Laboratory of Plant Systematics, Institute of Botany and Microbiology, K.U.Leuven, Kard. Mercierlaan 92, B-3001 Heverlee, Belgium
Received for publication July 22, 1999. Accepted for publication April 6, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: endocingulum • Gentianales • membranous granular layer • pollen wall development • Rondeletia • Rubiaceae • ultrastructure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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11 000 species (Robbrecht, 1988, 1994
). It is regarded as a key family for understanding the phylogeny of the Gentianales. Palynological interest in Rubiaceae has recently resulted in several mostly morphological-systematic papers (e.g., Andersson, 1993
; Pire, 1997
; Stoffelen, Robbrecht, and Smets, 1997
; De Block, 1998
).
Rondeletia odorata Jacq. is a small tree with bright orange-reddish flowers native to Cuba and Panama. The genus Rondeletia Linn. (250 species) belongs to the tribe Rondeletieae in the subfamily Cinchonoideae, where tribal relationships are blurred by recent fundamental changes in the systematics, e.g., by works dealing with the delimitation of the Cinchoneae (Andersson and Persson, 1991
), the Isertieae (Bremer and Thulin, 1998
), and the Rondeletieae (Delprete, 1996
). The pollen morphology of Rondeletia has been studied previously with light microscopy (LM) and scanning electron microscopy (SEM) (Igersheim, 1993
).
Our knowledge of pollen ultrastructure and development is surprisingly poor for such a vast family as Rubiaceae. Transmission electron microscope (TEM) images of mature pollen exines have been published for a few genera, mainly in systematic papers (Johansson, 1987
; Igersheim and Weber, 1993
; Weber and Igersheim, 1994
; Endress et al., 1996
; Tilney and van Wyk, 1997
). Abadie and Keddam-Malplanche (1975)
illustrated briefly two rubiaceous species with TEM.
Available data on pollen wall development are even more scarce. Andronova (1984)
investigated pollen development in several species with special attention to the tapetum. In their series of light microscopy studies on the floral morphology and embryology of Pavetta gardeniifolia, von Teichman, Robbertse, and van der Merwe (1982) briefly described microsporogenesis.
There is only one study of pollen wall development of Rubiaceae, namely on Mitriostigma axillare, a species with permanent tetrads (Hansson and El-Ghazaly, in press). As for the order Gentianales, we know of two similar studies, one on Catharanthus roseus in the Apocynaceae (El-Ghazaly, 1990
) and the other on the Asclepiadaceae (Dannenbaum and Schill, 1991
).
In our work on the development of pollen in Rondeletia odorata we aimed at documenting the main developmental features of the exine and intine from tetrad stage until pollen maturity. Special attention was paid to relate cell organelle content of the microspores with the ontogenetic sequence of wall formation. Our data on tapetum and orbicule development in the same species will be published later.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The approximate stage of development can be determined by the dimensions of the anthers, but light microscopic squashes give a more accurate indication of the developmental stage. For different flower bud sizes, one anther was crushed on a slide, stained with toluidine blue, and examined with LM. Anthers with microspores in tetrads were 1.5 mm long, and mature anthers were ±2.7 mm. In Rondeletia the early stages of pollen development, and hence the most significant events, proceed very rapidly; this has also been observed in Mitriostigma (Rubiaceae; T. Hansson, personal communication, Palynological Laboratory, Stockholm).
TEM/LM
Decapitated anthers were fixed in 2% glutaraldehyde in 0.05 mol/L Na-cacodylate buffer, pH 7.4 for ±24 h and postfixed in 1% OsO4 for 1 h. Anthers were blockstained with uranyl acetate for 10 min, dehydrated in a series of acetone and propylene oxide, and embedded in araldite. Ultrathin sections were stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Zeiss EM906 electron microscope at 80 kV.
SEM
Fresh decapitated anthers were fixed as for TEM and dehydrated in an acetone series. Anthers were transferred to gelatine capsules filled with acetone 100% and frozen in liquid nitrogen and fractured on a TF-2 chamber. After critical point drying, the anther fragments were fixed on a stub by silver paint. Pollen in Figs. 28 and 29 was acetolyzed and sectioned using a Ames lab Cryostat freeze microtome. Pollen sections were transferred to a stub. SEM observations were made with a Jeol SM6400 and a Jeol JSM-5800 LV microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although the development of the pollen wall is a continuous process in Rondeletia, we have organized the descriptions of developmental events and cytological changes in four stages: tetrads, free microspores, young pollen grains (after mitosis), and pollen grains at dehiscence.
Tetrad stage
After meiosis, tetrads of haploid microspores randomly filled the entire space of the anther locules. The tetrads were arranged in tetrahedral and decussate units. Each tetrad was surrounded by a thick, asymmetrical callose envelope. The shape of tetrads in the callosic envelope was multiangular (Fig. 1). Callose was thickest on tetrads in the center of the locules. The thickness of the callosic partitions between the microspore units of a tetrad was variable. The edges of the callosic layer appeared porous in early tetrads (Figs. 1, 2).
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In slightly older tetrads, rod-shaped electron-dense units were radially oriented in the distal part of the plasmalemma surface coat. These rods formed the columellae (Fig. 5). Sometimes more than one developmental stage was observed in the same anther. This suggests that the early events in microspore development occur rapidly.
Late tetrads
By end of tetrad stages, all layers of the exine were obvious (Figs. 6–9). The columellae were not solid and contained distinct cavities (Figs. 6, 7). The foot layer was thin and the endexine started to develop on a white line centered lamella (Figs. 7–9). The tectum was thin and interrupted by perforation, and the apertures were distinguished by development of numerous lamellae and thick granular material (Fig. 9).
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0.20 µm) with a thin continuous coat lined the surface of the tapetal cells, including the inner tangential side, the radial and the outer tangential surfaces (Fig. 10). At the end of this stage the tapetum started to mature, tapetal organelles were found in the locule (Fig. 16), and initiation of the characteristic endothecium thickenings took place. In cases where the septum between the locules was continuous, microspores in one locule showed a different stage of development from the other locule. When the septum was interrupted, all microspores in the connected locules were synchronized in development.
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Young pollen grains
The main developmental event during this stage was the formation of the intine, the last wall layer. The young pollen grains had punctate exine and three distinct apertures (Fig. 18). The tapetum appeared to be degenerating, and tapetal organelles and lipid droplets were observed in the locule between the pollen grains (Figs. 19, 20).
The intine started to develop on the radial protrusions of the plasmalemma as a continuous, evenly thick layer between the apertures (Fig. 19). Under the apertures, however, the intine was thicker and bulged out together with part of the protoplasm (Fig. 20). The distal part of the intine, close to the radially oriented MGL, had more fibrillar structure and was more electron dense than the rest of the intine (Figs. 19, 20). None of the sections with an early intine showed two nuclei. The vegetative nucleus, however, was often excentric, but this might also be due to vacuolation. We therefore assume that initiation of the intine took place prior to first mitosis. The cytoplasm contained long profiles of rough endoplasmic reticulum parallel with the plasmalemma, many mitochondria, vacuoles of different size, and numerous dictyosomes (Figs. 19, 20). The cytoplasm was dense with vesicles that were budding from dictyosomes. These vesicles were directed toward the plasmalemma and fused with it (Figs. 21, 22). The MGL appeared slightly compact and irregular in shape (Fig. 22). The white line separating the foot layer from the endexine was still visible. The foot layer was readily distinguishable and had the same stainability as the sexine (Fig. 22). Additional sporopollenin accumulated on the exine of young pollen as well as on orbicules. The tectum appeared solid, and microchannels were presumably obscured by filling material. The columellae arcade contained fibrillar material, probably remains of the primexine (Figs. 21, 22).
The next step in the ontogenetic sequence clearly showed the spindle-shaped generative cell adjacent to the intine and surrounded with an intine-like wall (Fig. 23). Later in development the generative cell migrated toward the central vegetative nucleus. The generative nucleus was characteristically surrounded by lipid droplets (Fig. 24) during its association with the vegetative nucleus. The intine-like wall around the generative nucleus remained intact. The intine became compact and appeared more electron dense. In several sites the intine protruded between the MGL and reached the endexine (Fig. 25). Later on the MGL compressed and appeared more compact than before (Fig. 25). The cytoplasm was characterized by abundance of compound starch grains and some lipid droplets (Figs. 23, 24). The increase in lipid and starch contents of the young pollen grains was concurrent with abundance of lipid droplets in tapetal cells. Ribosomes, rER, and small mitochondria were abundant near the undulating plasmalemma. Dictyosomes and Golgi vesicles, common in early pollen grains, were absent in this stage.
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Pollen grains at dehiscence
The pollen grains were fully engorged and appeared more or less circular in outline. Mature pollen grains taken from dehisced anthers were covered by a considerable amount of lipidic material (positive staining with Sudan III), which may be classed as pollenkitt (Fig. 28). This material was released by the degenerating tapetal cells, which were compressed into a tapetal membrane, densely covered by orbicules (Fig. 28).
The pollen wall was extremely stretched due to an increase in volume compared to the previous stage. This stretching affects all layers of the wall, i.e., all layers decreased in thickness. At the final stage of pollen development the intine was very thin. On the equator the apertures were covered with darkly stained tangentially oriented tubules or lamellations with a hollow, electron-transparent center (Fig. 29 and insertion). The MGL became more widely spaced and appeared discontinuous due to outstretching of the sporoderm (Fig. 30). This is obvious on full outlines of mature pollen grains (Fig. 31).
Lipid droplets were observed, often in association with the apertures (Fig. 31). The lipid bodies were much more numerous than starch grains and they were mostly enfolded in a single rER strand (Figs. 29, 31). In some sections lipid globules were conspicuously associated with the generative nucleus/sperm cells. Moreover, we observed numerous dictyosomes, elongated mitochondria, and a large number of free ribosomes (Fig. 31). The generative cell was enclosed by the intine-like wall (Fig. 31). Several pollen grains showed elongated, spindle-shaped sperm cells; thus some grains were trinucleate at dehiscence.
Mature pollen grains (LM and SEM)
The mature pollen grains were monads, small [P (polar axis) 16–(17.7)–19 µm, E (equatorial diameter) 18–(19.3)–21 µm], oblate spheroidal (P/E 0.92 on average) in equatorial view and subtriangular in polar view. The grains were planaperturate generally with three, rarely four, compound apertures. The short and narrow ectocolpi had acute ends. There was a lolongate pore in the center of each colpus; protruding onci were not observed in unacetolyzed grains. The sexine was punctate with a smooth tectum (Figs. 32–35). The perforations were variable in shape and size. The columellae were considerably longer in the mesocolpia giving rise to the subtriangular amb (Fig. 32). The inside ornamentation of acetolyzed grains was uniformly granular except for the endocingulum, which is 2 µm wide. The surface structure of the endocingulum was more coarse with rod-like elements (Figs. 32, 33). The MGL probably corresponds with the inner granular ornamentation that is observed on acetolyzed and fractured grains (Figs. 32, 33).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Pattern initiation of early exine
During the callose period the exine is initiated as rods extending from the plasmalemma. These rods are exine units that become columellae as well as part of the tectum and foot layer. Oblique sections of the early exine show that the tectum consists of the distal portions of close-packed exine units. The pro-columellae are tubular in structure, and they subsequently develop into mature wall elements by the accumulation of sporopollenin. We assume that units of pre-exine are continuous with structures in the cytoplasm, which are cytoskeletal.
In early tetrad stages of Rondeletia microspores, when the tectum becomes evident, the exine units show a honeycomb pattern resulting from close packing and interdigitation of the units. Dickinson (1970)
showed the occurrence of such a honeycomb arrangement in Lilium. El-Ghazaly and Jensen (1985 and 1986) presented a similar pattern in Triticum in early stages of microspore development. Wodehouse (1935)
pointed out that a reticulate pattern is a common theme in nature and is formed where even shrinkage occurs within a uniform matrix.
Wall layers that deserve special attention
Columellae
The columellae in Rondeletia pollen are the first wall component to be synthesized in the fibrillar plasmalemma coat in early tetrad stage. In oblique (tangential) sections from the early free microspore stage, the columellae appear circular with an electron-lucent center. Thus in three dimensions they are hollow cylindrical and radial supportive elements. Later in development the centers become obscured by material of the same electron density as sporopollenin. Nowicke, Bittner, and Skvarla (1986)
used plasma-ashing on many species of Paeonia and found that rod-shaped substructures were evident in pollen that had not been observed before ashing. Blackmore and Claugher (1984, 1987)
used fast atom bombardment in studying the exines of Fagus and Scorzonera and found that the exines were composed of hollow tubes. Blackmore (1990)
in a developmental study of pollen of Echinops found that exine processes appeared to be hollow during early stages. In Echinodorus, El-Ghazaly and Rowley (1999)
also observed probaculae with an unstaining, hollow-appearing core zone.
Foot layer
The foot layer was hardly visible in early stages. It became more pronounced and had a similar stainability as the sexine, i.e., darker than endexine, from the vacuolate stage on. In Rondeletia the junction between the endexine and the foot layer appears as a white line. Similar structure was observed in other species and described as "junction plane" (Xi and Wang, 1989
; Rowley and Rowley, 1996
) or "commissural line" (Simpson, 1983
).
Endexine
Formation of the endexine clearly involves white-line-centered lamellae. The "lamellations" can be rodlets, but there are several reports of actually sheet-like lamellations (e.g., Stone, Sellars, and Kress, 1979
). Rowley (1996)
suggested that a tubular organization with reversible lateral cross-linking offers versatility for growth and internal transport. Researches should consider the possibility of extensive lateral cross-linking of the rodlets or tubules into sheets. At the apertures endexine components separate from each other (most conspicuously in free microspore stage) contrary to the solid appearing endexine in interapertural regions. In these apertural regions the endexine shows lamellations with white-line centers in longitudinal profile and tubular components with a low-contrast core in cross sections. In Poinciana (Leguminosae) Rowley and Skvarla (1987)
also showed that endexine components in apertural regions were tubular with a low-contrast central core. In interapertural regions, where white-lines in the endexine are commonly believed to represent lamellae, an alternate interpretation of their morphology might be fascicles of the tubular form.
Our results show that ER units at the periphery of the cytoplasm extend radially toward the developing wall and form the membranous part of the MGL beneath the endexine. We have also observed these units further extended to the endexine and even to the arcade between columellae. Because our observations were consistent in several sections and the ER units were quite obvious, we resist the idea that ER might have a role in transfer of material between microspores or pollen grains and tapetal cells.
Membranous granular layer (MGL)
Echlin and Godwin (1969)
described in Helleborus that after formation of the endexine on lamellae, deposition of sporopollenin appeared as small granules that gradually coalesce. A similar phenomenon was observed in Rondeletia where membranous tubular units intermingled with granular material accumulated on the proximal side of the endexine. A similar layer was observed in Nelumbo (Kreunen and Osborn, 1999), Nymphaea (Gabarayeva and El-Ghazaly, 1997
), and Catharanthus (El-Ghazaly, 1990
). The question of whether this layer is mainly developed from extensions of the ER or whether it belongs to the endexine, e.g., endexine II, or a layer not homologous with the endexine remains open. It is definitely not part of the intine since its granular part can resist acetolysis. The granular ornamentation of the inside of mature acetolyzed grains corresponds most likely to this granular material. A histochemical study of this layer in different species will provide useful information on the chemical nature of this layer. Such a study is in progress by the authors of this paper.
Intine
The intine, like other primary plant cell walls, develops generally after mitosis (Robards, 1970) and thus in pollen grain stage. In Rondeletia, however, intine formation might be initiated before mitosis as was also observed by Heslop-Harrison (1968)
in Lilium and Silene, and in Dioscorea dumetorum (P. Schols, personal communication, Laboratory of Plant Systematics, K. U. Leuven). Although the exact timing of intine initiation might be doubtful, the mode of deposition is not. The Golgi vesicles are clearly deposited onto the plasmalemma and are spilled out by exocytosis into the periplasmic space to form the intine, which is found pressed against the exine. This is common in other families and has been observed by Echlin and Godwin (1969)
in Helleborus foetidus, in other Ranunculaceae by Roland (1971)
, in Platanus by Suarez-Cervera and Seoane-Camba (1986)
, etc. In Rondeletia the irregular, sometimes branched ingrowths of the plasmalemma into the developing intine are very pronounced in late free microspore stage and immediately after first mitosis (Figs. 17, 19–22).
Apertures
We did not observe parallel endoplasmic reticulum profiles in future aperture sites as was reported for Lilium (Dickinson, 1970
), Helleborus (Echlin and Godwin, 1968
) and Securidaca (Coetzee and Robbertse, 1985
). From early tetrad stage, however, the plasmalemma surface coat is much thinner on three equally distributed areas believed to be the destined apertures. The intine is characteristically thicker under the apertures before engorgement of the grains. Prior to dehiscence the intine becomes very much stretched and of equal thickness all around the profile of the grain (see below). In mature pollen grains we showed that the colpus membrane is of endexinous nature (Fig. 27) and that in cross sections the pore is covered with tubular endexine components with a low-contrast core (Fig. 29).
Cytology
The dynamics of the cytoplasm components during pollen ontogeny is astonishing. Cell organelles are formed according to the demands of the developmental processes. Next to this variation in time there is also variation in space. Comparison of sections of different angles in the same developmental stage showed that the cell organelles are not evenly distributed in the cytosol. The peripheral cytoplasm at the late vacuolate stage is dense with Golgi vesicles and dictyosomes, and the vesicles fuse with the plasmalemma (Figs. 21, 22). As stated above, the distal face of the intine is formed by material transported in Golgi vesicles that through exocytosis is released into the periplasmic space. We believe that there is also membrane flow from the dictyosomes to the plasmalemma. During the exocytosis process the membranes of the vesicles are incorporated into the plasmalemma, increasing considerably its surface area. This membrane flow explains how the plasmalemma retains its surface area during formation of the radial membrane fragments in the proximal part of the intine. Membrane flow and differentiation from the ER through the Golgi apparatus to the plasmalemma have been long known (e.g., Morré et al., 1971
; Gabarayeva, 1987
; see also reviews by Steer, 1991
and Sitte, 1998
).
In Rondeletia, the vegetative cell of the maturing pollen grain synthesizes the two intracellular lipid structures found in pollen: oil bodies and intracellular membranes (Fig. 24). In mature pollen grains of Rondeletia, both starch grains and lipid bodies are found as storage products. Compound starch grains appear first, i.e., at the late free microspore stage. After the first pollen mitosis, oil body accumulation begins and starch contents of the cytoplasm decrease. At anther dehiscence lipids are the main storage product. The lipid body accumulation in Rondeletia is preceded by a transient accumulation of starch as demonstrated in many other plant species (e.g., Reznickova, 1978
; Wetzel and Jensen, 1992
; Clément et al., 1994
; Hess, 1995
).
The ER is considered to be a site of lipid body formation in both animal (Zaczek and Keenan, 1990
; Bozhkov and Dlubovskaya, 1992
) and plant cells (Grabski, de Feijter, and Schindler, 1993
; Lacey and Hills, 1996
). In late free microspore stages and young pollen grains, we observed a clear proliferation of an extensive network of ER membranes. These ER units are generally associated with maturation and expansion of the cytoplasm of the pollen vegetative cell (Jensen, Fisher, and Ashton, 1968
; Charzynska, Murgia, and Cresti, 1989
). An extensive network of ER during the young pollen grain stage was also observed in Ledebouria (Hess, 1995
) and in Brassica (Piffanelli, Ross, and Murphy, 1998
). In mature pollen grains of Rondeletia, the storage oil bodies are characteristically enfolded in a single rER strand. Similar pockets of ER have been described in the pollen of Gossypium hirsutum (Jensen, Fisher, and Ashton, 1968
), Impatiens walleriana (Fisher, Jensen, and Ashton, 1968
), and Tradescantia reflexa (Noguchi, 1990
). It has been suggested that the extensive network of ER membranes might protect the oil bodies from coalescencing during de- and rehydration (e.g., Piffanelli, Ross, and Murphy, 1997
). Secondly, the intracellular membrane system can also provide lipid precursors for the increase of surface area of the plasmalemma which follows germination and pollen tube growth (Piffanelli, Ross, and Murphy, 1997
). In addition to the prominant ER, there are numerous vesicles, apparently developed from the dictyosomes, that are laying beneath the surface of the plasmalemma. These membranous bodies undoubtedly contribute to the dramatic increase in surface area of the plasmalemma during maturation of the microspores and formation of the pollen grains (Piffanelli, Ross, and Murphy, 1998
).
We observed several grains with three nuclei at maturity; thus the second mitosis of the generative nucleus takes place before release of the pollen grains. This observation contradicts Wunderlich (1971)
and Puff (1994)
who stated that pollen of Rondeletia is bicellular when shed.
Volume increase at dehiscence
The pollen wall of Rondeletia becomes abnormally stretched just before dehiscence. This stretching affects the spacing between columellae and the thickness of all distinguished layers of the wall. The effect was particularly obvious on the radially oriented granular material. This material appeared irregular in shape and in its distribution at pollen maturity. The intine was no longer thick in apertural regions at dehiscence as is generally the case.
Endocingulum
On the inside of mature grains, a broad endocingulum occurs with an irregular rod-like surface structure in contrast to the granular ornamentation of the remainder of the nexine. The ultrastructural explanation of this structure is clear from Fig. 34. Next to the aperture there is a zone free of radially oriented granular material, endexine, and foot layer. The columellae are branched and interconnected in this area. To us, the inside view on the endocingulum shows the proximal ends of the columellae. The most plausible functional interpretation of the endocingulum is a role in harmomegathic (accommodate variations in the volume of the cytoplasm) processes but dehydrated grains observed in SEM contradict this hypothesis (Fig. 35).
Summary of major ontogenetic events
The main ontogenetic events in pollen wall development of Rondeletia odorata are presented schematically in Figs. 36–43. Our drawings are mainly based on TEM micrographs included in this work or on unpublished material. Our present study shows some interesting features that can be related to the dynamic processes of pollen development. We show ER cisternae extending between plasmalemma protrusions, and material of the membranous granular layer beneath the endexine (Fig. 15). The ER cisternae are also observed within the arcades between columellae (Figs. 15, 22). Our interpretation is that the early columellae with hollow cylindrical center, as well as other layers of the exine, may function as a dynamic transfer system between the cytoplasm of developing microspores and the tapetum. Heslop-Harrison (1963)
showed the presence of cisternae of the endoplasmic reticulum under the plasmalemma in connection with the conduits of columellae. Similar observations were reported by Skvarla and Larson (1966)
as part of their ontogenetic study of Zea mays. The interpretation then was that there might be a connection between sites of columellar initiation and ER cisternae. To understand the origins of this communication or transport system it would be helpful to study and understand the processes involved in the localization of initiation sites of exine layers on the plasmalemma.
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FOOTNOTES |
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4 Author for reprint requests (gamal.el-ghazaly{at}nrm.se
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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———, and ———. 1969 The ultrastructure and ontogeny of pollen in Helleborus foetidus L. III. The formation of the pollen grain wall. Journal of Cell Science 5: 459–477
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