Development of heterocolpate pollen in Myosotis scorpioides L.(Cynoglosseae,Boraginaceae)

Structure of mature pollen of Myosotis scorpioides and sporoderm development were examined using light, scanning and transmission electron microscopy. Pollen grains of M. scorpioides are heterocolpate with three colpori alternating with three pseudocolpi (pseudoapertures). Functional pseudoapertures take part in harmomegathy but not in pollen germination. Formation of colpori and pseudocolpi starts simultaneously. Structural difference between the aperture and pseudoaperture becomes clearly noticeable at the early free microspore stage when the endexine is initiated: at the colpori (at the area of ora) the endexine is composed of only a thin loose layer, at the pseudocolpi, the endexine is homogeneous and thick. Later, on the late free microspore stage, the difference appears in the structure of the intine: at the area of ora, the intine is three times thicker comparing with the pseudoapertures and mesocolpia. Besides zonal pseudoapertures, the triangular poroid areas are present at both poles of M. scorpioides pollen. Their structure and developmental pathway are similar to the zonal pseudoapertures. Pseudocolpi and polar pseudoapertures should be considered as the structures originated de novo specialised for harmomegathy.     

A new look at the acetolysis method

The acetolysis method intreduced byGunnar Erdtman is still a very welcome and highly successful technique in palynology. However, acetolysis destroys all pollen material with the exception of sporopollenin that forms the outer pollen wall, the exine. Modern palynology in its application to plant systematics and phylogeny must consider all sporoderm characters, not only those of the exine. The neglect of the intine may distort some principal palynological aspects. This is illustrated by cases of total breakdown or gross modification of thin exine structures (e.g. inBeilschmiedia, Strelitzia) and by the clarification of apertures (e.g.,Polyalthia, Fissistigma, Calluna). In our view the investigation of both acetolysed and non-acetolysed pollen is obligatory for a well balanced view of pollen structure and function.     

Pollen morphology and pollen-wall proteins (localization and enzymatic activity) inSesamothamnus lugardii (Pedaliaceae)

The pollen grains ofSesamothamnus lugardii Stapf (Pedaliaceae of subdesert regions of SE tropical Africa) are associated in acalymmate tetrads (cross wall cohesion), with a tectate and perforate exine and 8–12 colpi. The pollen wall consists of an ectexine with a complete, perforate and ample tectum, columellated infratectum and clearly interrupted and fragmented foot layer. The endexine is built of scanty lamellae and granules. The intine is bistratificate, with a homogeneous, fibrillate layer (endintine or intine-2) and a heterogeneous, more lax and channeled layer (exintine or intine-1). Test for glycoprotein is particularly positive in the homogeneous internal intine and channels of external intine. On the other hand acid phosphatase has been localized in the exine and channeled external intine layers. These observations confirm the general interpretation of the distribution of wall compounds.     

Pectin distribution pattern in the apertural intine of Euphorbia peplus L. (Euphorbiaceae) pollen

The apertural inner layer (intine) of Euphorbia L. pollen grains has a characteristic but original structure that has paired thickenings, one on either side of the colpus. To determine the nature and role of this intine layer, pollen grains of Euphorbia peplus L. were germinated in vivo and in vitro. The germination process involves wall changes that facilitate formation of the pollen tube and its subsequent growth. In the thickenings of the intine of E. peplus, the unesterified pectin epitopes are more densely localised in the inner part of the middle intine. No such epitopes are located in the intine portion adjacent to the plasma membrane (cellulosic endintine). Unesterified pectin epitopes are also localised in the outer part of the intine but are restricted to the centre of the aperture, around and in the pore. The de-esterification of pectins is very advanced at the time of dehiscence and pollen germination. The stratification of the aperture intine may take the following pathway at the time of germination: the thin outer zone of the intine in the pore region becomes disorganised and undergoes dissolution with liberation of unesterified and esterified pectins; the middle intine thickenings undergo an important elastic modification, but without liberation of unesterified pectins; the cellulosic inner intine is the progenitor of the pollen tube wall. This special intine of E. peplus is an adaptation to the hydration process preceding germination, increasing intine and pollen grain wall elasticity.     

Pollen wall development in Hypecoum imberbe Sm. (Fumariaceae)

We have studied the ontogeny of the pollen wall of Hypecoum imberbe. Our initial observations with conventional transmission electron microscopy (TEM) were inconclusive due to the similarities found in the electrodensity of the foot layer and the endexine, and between this latter layer and the intine. Thus we applied the PTA acetone histochemical test in order to differentiate between the cell wall components. This method proved to be efficient in resolving the differences between the layers and even allowed us to distinguish two strata within the narrow intine layer. A thin foot layer can be distinguished only by the temporary presence of a single white line separating it from the initial deposition of the endexine. The aperture consists of two colpi with no special differentiation. The tapetum is a typical secretory type giving rise to elaioplasts and tapetosomes during development, which persist as individual organelles upon the degradation of the tapetum. As the ultrastructural organisation of the sporoderm seems to offer little protection to the gametophyte, we finally discuss how the structure of the pollen sporoderm might be related to the special morphological characteristics of a flower, its habitat, and the biology of the plant as a whole.     

Sporoderm ultrastructure and development in Aristolochia manshuriensis Komarov (Aristolochiaceae)

Pollen ontogeny and sporoderm development in Aristolochia manshuriensis were studied for elaboration of the inaperturete pollen ontogeny in Aristolochia. Despite the formation of apertures in the tetrad period, the sporoderm in A. manshuriensis becomes inaperturate at the end of the free microspore period. A similar immature exine is also detected in A. macrophylla. Variants of aperture formation in the tetrad period in A. manshuriensis or formation of a polar aperture in the free microspore period in A. clematitis are associated with types of microsporogenesis. The ectexine and endexine in A. manshuriensis are formed over a longer time and reached much greater thickness than those in A. clematitis. The endexine and intine in A. manshuriensis do not reach a mature state, similar to A. clematitis. The exine of A. manshuriensis cracks, releasing a pollen tube enveloped by the intine. This fact does not hinder the functioning of the male gametophyte of A. manshuriensis.     

Cupressus arizonica pollen wall zonation and in vitro hydration

The structure of Cupressus arizonica pollen at different degrees of hydration was examined by using cytochemical staining and light (LM) and scanning electron (SEM) microscopy. Most pollen grains are inaperturate and a minority are provided with an operculate pore enveloped by a concave annulus. Intine consists of: 1) a thin polysaccharidic outer layer, 2) a large polysaccharidic middle layer that is spongy and bordered by a mesh of large and branched fibrils, and 3) an inner cellulosic thick layer with callose concentrated on the inner side, which forms a shell around the protoplast. The protoplast is egg-shaped with PAS positive cytoplasm and prominent nucleus. Exine splits during hydration and is cast off according to three major steps: 1) the split opens like a mouth and the underlying intine is expelled by swelling like a balloon, 2) the protoplast enveloped by the inner intine is sucked in the outgrowing side, and 3) the backside of the intine gets rid of the exine shell. In water containing salts, exine is rapidly released and the middle intine may expand up to break the outer layer, with disgregation of the spongy material and release of the intine shell including the protoplast. In water lacking salts, the sporoderm hydration and breaking are negatively influenced by the population effect. Pollen when air dried after the exine release become completely flat owing to disappearance of the middle intine layer which may be restored by dipping pollen in water. The results are discussed in relation to the functional potentialities of the sporoderm.     

Ultrastructural study of the relationship between generative and vegetative cells inMagnolia ×soulangeana Soul.-Bod. pollen grains

Summary InMagnolia ×soulangeana pollen grains the generative cell (GC) does not become totally free within the vegetative cell (VC), at least until the pollen tube emergence. Due to a deviation in its detachment process from the sporoderm, the opposing ends of the VC plasmalemma do not fuse themselves when the GC moves away from the intine. Consequently, the interplasmalemmic space surrounding the GC does not become isolated but rather maintains continuity with the sporoderm through a complex formation that we have called plasmalemmic cord. The real existence of this formation was confirmed through serial sectioning showing the plasmalemmic cord to consist of the VC plasmalemma. In its initial portion it is occupied by a reasonably accentuated wall ingrowth of the inner layer of the intine (intine 3). In the remainder portion, neither of the cytochemical tests used in this work have revealed the presence of a significant amount of wall material. However, ultrathin sections of samples processed either chemically or by cryofixation showed the existence of an intricate system of tubules and vesicles, some of which are evaginations of the VC plasmalemma. The hypothesis that the plasmalemmic cord may have a role in the complex interactions between the two pollen cells is discussed.     

Ultrastruktur und systematische Bedeutung des Pollens beiBocageopsis,Ephedranthus, Malmea undUnonopsis (Annonaceae)

The four genera investigated show solitary sulcate pollen grains. The structure and sculpture of the sporoderm is very similar inBocageopsis andUnonopsis, and supports the close relationship of both genera; their flower morphology also is very similar. In contrast, the sporoderm ofEphedranthus, and especially that ofMalmea, is different in some characters and suggests more remote relationships; this is also confirmed by differences in flower morphology. Within this group of genera a distinct exine progression can be recognized from non-columellate (Malmea) to granular (Unonopsis guatterioides), further to somewhat irregularly (Bocageopsis, someUnonopsis species), and finally to very regularly columellate (Ephedranthus). The sculpture of the tectum varies from a reticulum with large lumina (Malmea) to one with small performations (Ephedranthus). Within theAnnonaceae the genusMalmea is among the most primitive in respect to pollen structure. The sulcus of the four genera is very large and runs over 1/3 of the pollen grain. It is characterized by a reduction of the exine and a bulgy thickening of the intine.

     

Wall development and its autofluorescence of sterile and fertileVicia faba L. Pollen

Summary The ultrastructural changes of the pollen wall of three types of fertile and one of sterileVicia pollen were related to the autofluorescence of the pollen wall, measured by a microspectroscopic method. Till the liberation of the microspores from the tetrad, the spectrum of the ectexine shows sometimes two maxima and has a very low intensity. After this period the endexine is formed and its spectrum has one maximum with a high intensity. The differences of the pollen wall between the sterile and fertile pollen exist of the presence of one spectral maximum during the tetrad stage, a thick endexine and the absence of the intine in the sterile pollen. The different types show much differences during the tetrad stage in the callose wall as well as the ectexine. The autofluorescence illustrates the complexity and specificity of the pollen wall development.     

Cell-wall development in freeze-fixed pollen: Intine formation of Ledebouria socialis (Hyacinthaceae)

The structure and development of the inner pectocellulosic pollen wall, the intine, was re-examined using high-pressure freezing with subsequent freeze substitution in Ledebouria socialis Roth, a monocotyledonous angiosperm. The bilayered intine is formed immediately after differentiation of the endexine. Similar to somatic cell walls, intine matrix substances originate from the Golgi apparatus and leave the cytoplasm via exocytosis. Exintine development starts with the apposition of intine matrix substances to the inner polysaccharide layer of the endexine (termed inner endexine), leading to irregular cell-wall ingrowths. Subsequently the inner endexine becomes intensely infiltrated with intine matrix substances; this process is interpreted as transformation of the inner endexine into intine. Along the aperture region, cell-wall matrix substances are unevenly deposited to such an extent that more or less radially oriented tubules filled with cytoplasm remain within the growing exintine. These tubules subsequently become cut off from the microspore cytoplasm by selective membrane fusions, leading to the incorporation of ground cytoplasm and ribosomes into the exintine. Exintine formation is completed prior to the first mitotic division of the pollen grain whereas the endintine is formed as a homogeneous thin layer after mitosis. Both transformation of the inner endexine by infiltration and passive incorporation of cytoplasm and ribosomes into the exintine by membrane fusions are novel features and are only observed in optimally freeze-fixed, freeze-substituted samples; general aspects of ultrastructure preservation in high-pressure-frozen, freeze-substituted plant cells are discussed as well. Modifications of the Golgi apparatus and post-Golgi-apparatus structures during pollen wall development are correlated with increasing and decreasing polysaccharide exocytosis, respectively. These evenls strictly coincide with the formation of morphologically and chemically different pollen wall layers and therefore seem to reflect the different deposition patterns of the predominant cell-wall polysaccharides.Abbreviations ER endoplasmic reticulum - FS freeze substitution - HPF high-pressure freezing - MS microspore(s) - PATAg periodic acid-thiocarbohydrazine-silver proteinate - PGS post-Golgi-apparatus structures - UA-Pb uranyl acetatelead I am grateful to Dr. Martin Müller (Institut für Zellbiologie, ETH-Zürich) for the kind permission to use the high-pressure freezer and the freeze-substitution unit at his laboratory. I wish to thank Prof. M. Hesse, Mag. M.G. Schlag (Institut für Botanik, Universität Wien) and Dr. I. Lichtscheidl (Institut für Pflanzenphysiologie, Universität Wien) for helpfull discussions. Thanks are also due to A. Glaser and W. Urbancik for excellent technical assistence and to the Stadtgärtnerei Zürich for providing the plant material. This work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.     

Passage of lanthanum through the pollen grain wall of Olea europaea L. during development

Summary In order to study the behavior of the exine as a site of passage of material from the locule of the anther, lanthanum nitrate was used to locate the possible routes of communication between the Olea europaea L. pollen grain and its external environment from the period of exine consolidation until dehiscence of the anther. In all four stages of development studied, dense lanthanum deposits occupied microchannel-like orifices, whereas these dense deposits occupied the apertural regions only in the first stages of development. Lanthanum precipitate is also present in the endexine, intine and cytoplasmic vesicles of mature pollen. The transportational function of the apertures is discussed in relation to the presence of dense lanthanum deposits in some stages of pollen grain.     

Pollen wall ultrastructure and ontogeny in Heliotropium europaeum L. (Boraginaceae)

The pollen grains of Heliotropium europaeum are heterocolpate, with alternation of 3 colpori and 3 pseudocolpi. The exine is characterized by a scabrate and thick tectum, massive columellae with a granular appearance and a thick nexine. The thickening of the intine at the apertural level makes the interpretation of this zone difficult. The ontogenetic study helped to understand the ultrastructure of the exine and the apertures. The different steps are as follows. The primexine matrix is formed during the beginning of the tetrad stage; it consists of an outer thick and electron dense zone and an inner one, less dense to electrons. The tectum and the infratectum begin to form in the outer zone of the matrix, towards the middle of the tetrad stage. The infratectum consists of a network of columellae variable in thickness and oriented in different directions. The foot layer is lacking. The endexine is formed on a lamella system during the callose loss and microspore separation. The endexine becomes compact very early on its inner part. The apertures are initiated during the tetrad stage; a granulo-fibrillar oncus develops. At the free microspore stage, the oncus gets fibrillar and is bordered by endexine lamellae on its outer side and by endexine granulations on its inner one and laterally. The intine is set at the end of this stage. At the vacuolated microspore stage, the intine shows three layers: two thin, clear and homogeneous layers, one outside and the other inside, and a thick middle layer that forms the zwischenkörper, crossed by trabecula, in the apertural areas.     

Exine ultrastructure of in situ peltasperm pollen from the Rhaetian of Germany and its implications

Morphology and exine ultrastructure of pollen grains of Triassic peltasperms have been studied for the first time. Pollen grains of Antevsia zeilleri from the Rhaetian of Germany are of the Cycadopites-type and monosulcate; the sculpturing is the same in the apertural and non-apertural areas. The proximal exine includes a row of lacunae covered by a solid, thick tectum and underlined by a foot layer. Pillars are hanging from the tectum between the lacunae. The exine is thinning to a homogeneous layer in the apertural region. The latter is bordered by thicker alveolate areas of the exine, in places resembling a saccus-like ultrastructure. The endexine includes white-line-centred lamellae. The exine ultrastructure is compared with that of pollen of Permian peltasperms. Although pollen types ascribed to Permian peltasperms are completely different in their general morphology, a transformation can be hypothesized by ultrastructural data from Permian Vesicaspora into Triassic Cycadopites extracted from pollen sacs of Antevsia. Comparison with Cycadopites of non-peltaspermalean (Ginkgoalean, Cycadophyte) and unknown affinities has been accomplished. The exine ultrastructure is distinctive enough to differentiate among peltaspermalean, cycadalean and bennettitalean Cycadopites; some ultrastructural features are shared with pollen of modern Ginkgo biloba. More ultrastructural data are needed as well as numerous sections of pollen grains are necessary to reveal original unchanged ultrastructure.     

Polarity, aperture condition and germination in pollen grains ofEphedra (Gnetales)

Pollen grain polarity, aperture condition and pollen tube formation were examined inEphedra americana, E. foliata, E. rupestris, E. distachya, andE. fragilis using LM, SEM and TEM. In the characteristic oblate pollen, as seen in situ in the tetrad configuration, the polar axis is the minor one and the equatorial plane runs between the two narrow ends of the microspore. The intine is thick in fresh fixed mature pollen but we have seen no indication of regions having an exceptionally thick intine that could be considered associated with an aperture or apertures. About three minutes after transferring fresh pollen to the germinating medium the ridged exine splits and twists away from the intine and its enclosed protoplast. The shed exine spreads out and curls into a scroll-like configuration that is as distinctive as that of the pollen shape had been but now having the ridges and valleys perpendicular to the long axis. The pollen tube develops, in our experience with more than a hundred germinating pollen grains, near one of the narrow tips of the pollen grain's equatorial plane. The location of the pollen tube initiation probably is related to the position of the tube cell nucleus. The pollen tube starts to grow about one hour after the exine was shed. The pollen tube emerges close to the narrow end (equator) of the gametophyte. This end emerged first as the exine is shed and is opposite to the prothallial cells. The stout pollen tube is c. 10µm in diameter grown in vitro on agar. In our germination medium the stout tube continued to elongate for about 24 hours reaching a length of c. 100 µm. With respect to exine morphology the aperture condition could be considered as inaperturate. The pollen tube, however, is formed in a germination area near one end of the exineless gametophyte.     

Pollen wall development in Vigna vexillata I. Characterization of wall layers

Light and electron microscope observations characterized the layers that comprise Vigna vexillata L. pollen walls, and identified the timing of their development. Exine sculpturings form an unusually coarse ektexinous reticulum. The structure of the ektexine is granular; this differs from the columellate/tectate type of structure typical of most angiosperm pollen. The ektexine overlies a homogeneous-to-lamellar, electron-dense endexine, which in turn surrounds a thick, microfibrillar intine. Pollen grains are triporate and operculate, with Zwischenkörper and thickened intine underlying the apertures. The ektexine forms during the tetrad period of microspore development, the endexine and Zwischenkörper during the free microspore stage, and the intine during the bicelled (pollen) stage. Coarsely reticulate exine sculpturings and the granular structure of the patterned exine wall of the pollen grains are features that make this species suitable for detailed studies of pollen wall pattern formation.     

Aperture types withinSapranthus andPolyalthia (Annonaceae)

Within the large palaeotropical genusPolyalthia so far five different pollen types are described (two monosulcate types, two disulcate types, and a single omniaperturate type). One of the disulcatePolyalthia types is similar to the disulcate neotropical genusSapranthus. All the newly described types differ predominantly not in exine but rather in intine characters. From this it can be concluded that seemingly inaperturate grains are disculcate in function, generally with two clearly recognizable germination zones, or omniaperturate, with the whole sporoderm capable of germination. The functions of the intine layers within the germination zones and within the nongerminating areas are very different and cooperate during the first germination steps in bursting the exine. It is difficult clearly to define the germination zones of pollen grains if the exine exhibits no modifications. Based on our and other results a more precise definition is suggested.     

Development and cytochemistry of pollen and tapetum in Mitriostigma axillare (Rubiaceae)

The development of pollen grains and tapetum in Mitriostigma axillare (Rubiaceae) was studied from anther primordium to dehiscence. Anthers were freeze-cracked and studied with SEM. Embedded anthers were sectioned and studied with LM and TEM. Cytochemistry was performed in order to distinguish the different layers of the sporoderm and to determine its chemical nature at different development stages. The pollen grains remained as tetrads by partial fusion of the exine, probably because of reduced callose septa during the stage of microspore tetrads within callose envelopes. Characteristic features of the sporoderm were an irregular foot layer, an endexine composed of amalgamated granules, a transient granular-fibrous layer beneath the endexine, and a thin intine. During maturation of the exine, the endexine became chemically different from the ectexine. All layers of the sporoderm were reduced in thickness due to stretching during the engorgement of the pollen grains prior to dehiscence. The pollen grains were colpoidorate with a reticulate to microreticulate tectum covered with a scanty surface coating. The mature pollen grains were binucleate and contained a lot of starch grains. Thick intineous onci protruded through the apertures and formed papillae. About 50% of the microspores were aborted. The tapetum was of secretory type, probably with cycles of hyperactivity and protrusions of the cells into the locular cavity. No syncytium was formed and there were neither orbicules nor tapetal membrane.     

Release of proteins from the pollen wall of Linum grandiflorum

Summary The emission of proteins from the pollen wall of Linum grandiflorum stained with Coomassie blue was followed directly in moistened grains as well as in pollen prints. Within the first minute of the grain being moistened exine-borne proteins emerged from both inter-apertural and apertural sites; subsequently, proteins of a different nature were discharged from the apertures only. In a fraction of the grains the release of intine proteins was not preceded by that of exine proteins. Pin and thrum pollen did not differ in terms of mode or site of this protein emission. The presence and emergence of exine proteins from the apertures is explained by the process of infolding of the colpal wall at desiccation and its expansion at rehydration, which causes an initial trapping and subsequent re-exposure of surface materials. This explanation may also account for the occurrence of poral sporophytic proteins in the pollens of many dictoyledons.