Microsporogenesis inPinus sylvestris L. VIII. Tapetal and late pollen grain development

This last portion of our developmental study ofPinus sylvestris L. pollen grains extends from just prior to the first microspore mitosis to the microsporangial dehiscence preparatory to pollen shedding. In nine years of collecting each day the duration of the above period was 7 to 11 days. Tapetal cells extended into the loculus and embraced microspores during the initial part of the above period. Thereafter tapetal cells receded, became parallel to parietal cells and so imbricated that there appeared to be two or three layers of tapetal cells. Tapetal cells were present up to the day before pollen shedding, but only rER and some mitochondria appeared to be in good condition at that time. A callosic layer (outer intine) was initiated under the endexine before microspore mitosis. After the first mitosis the first prothallial cell migrated to the proximal wall and was covered on the side next to the pollen cytoplasm by a thin wall joining the thick outer intine. There are plasmodesmata between pollen cytoplasm and the prothallial cell. After the second mitosis the second prothallial cell became enveloped by the outer intine. The inner intine appears after formation of the two prothallial cells but before the third mitosis. During this two-prothallial cell period before the third mitosis, plastids had large and complex fibrillar assemblies shown to be modified starch grains. After the third mitosis plastids of the pollen cytoplasm contained starch and the generative cell (antheridial initial), the product of that mitosis, is enveloped by the inner intine. On the day of pollen shedding cells are removed from the microsporangial wall by what appears to be focal autolysis. The tapetal and endothecial cells for 10–15 µm on each side of the dehiscence slit are completely removed. One or more epidermal cells are lysed, but both a thin cuticle and the very thin sporopollenin-containing peritapetal membrane remain attached to the undamaged epidermal cells bordering the dehiscence slit. Our study terminates on the day of pollen shedding with mature pollen still within the open microsporangium. At that time there is no longer a clear morphological distinction between the outer and inner intine but, judging by stain reactions, there is a chemical difference. The exine of shed pollen grains was found to be covered by small spinules on the inner surface of alveoli. These had the same spacing as the Sporopollenin Acceptor Particles (SAPs) associated with exine initiation and growth.     

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.     

Ontogeny of the exine in pollen of Aristea (Iridaceae)

The ontogeny of the pollen wall was studied in four species of Aristea , from the vacuolated stage of the microspores, to observe the possible formation of an endexine. At this stage, the ectexine is completely formed (tectum, columellae, and structurally homogeneous foot layer), but its maturation is incomplete and variable depending on the species. In all cases, there are one or several tripartite lamellae with a white line under the foot layer, in the apertural and extra-apertural regions. In A. major , and A. pauciflora , the exintine is not yet present, whereas in A. macrocarpa and A. glauca , it has started to initiate. In mature pollen of the four species, the tripartite endexine lamellae of the vacuolated stage disappear and there is no trace of endexine. The tripartite intine is completely formed. Maturation of exine is complete and it appears homogeneous and of medium electron density, except in A. glauca , which has particularly fragile exine, where it remains incomplete with a granular and highly electron dense appearance, which contrasts with the usually mature exine. Despite the very clear presence of endexine lamellae at the vacuolated stage, it is thus very difficult to conclude that endexine exists in pollen of the genus Aristea .     

Some characteristics of pollen wall cytochemistry and ultrastructure in Japanese larch (Larix leptolepis Gord.)

Summary The mature pollen of Larix leptolepis Gord. (Conifer) contains five different cell types, and the plasma membrane of the vegetative cell is continuous and organized. The pollen wall is composed of two morphologically and cytochemically distinct domains: the exine and the intine. In the multilayered exine, the ektexine appears granular and the endexine, lamellar. The intine is thick and bilayered with a microfibrillar structure occupying its inner portion. Cytochemical reactions of the exine and the intine are similar to those found in angiosperms. Pollen wall involvement in the male female recognition system is discussed with respecl to the angiosperms.     

The Apertural Wall in Pollen of Linum grandiflorum

The apertural wall in tricolpate pollen of Linum grandiflorumwas investigated in order to understand its functioning duringdesiccation and rchydration. Whole and sectioned pollen grainswere studied with light or electron microscopy and by cytochemicalmeans. The areas of the apertures were examined in fresh drypollen, in grains moistened on agar gel or removed from compatiblestigmas, and in pollen from mature undehisced anthers The intine was found to consist of an inner ß-glucanlayer and an outer pectic layer. At the apertures the pecticlayer is thickened and overlaid by a ß-glucan layer.The pectinaceous intine stains red with basic fuchsin. The presenceof a third wall layer, the medine, was not confirmed. The aperturalintine thickenings possess considerable imbibitional capacityand at rehydration they appear as swollen lenticular bodies A procedure is described for obtaining intact exine free grains(EFG's) and whole, separated exines of L. grandiflorum. Invariably,the released EFG's consisted of protoplasts encased in the cellulosicintine. In most grains the outer intine remained attached tothe separated exine In L. grandiflorum the outer wall of the aperture expands whilethe protoplast and endintine are still infolded. Apparently,the exintine becomes detached from the endintine during desiccationand re-attaches at rehydration. It is suggested that the transientdetachment controls the influx of water into the vegetativecell Except for morph-specific exine processes no differences instructure of the aperture wall or its functioning at rehydrationwere observed between pin and thrum grains Pollen wallM, apertures, exintine, exine free grains, rehydration, desiccation, Linum grandiflorum     

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.     

BcMF9, a novel polygalacturonase gene, is required for both Brassica campestris intine and exine formation

Background and Aims

The polygalacturonase (PG) gene family has been found to be enriched in pollen of several species; however, little is currently known about the function of the PG gene in pollen development. To investigate the exact role that the PG gene has played in pollen development and about this family in general, one putative PG gene, Brassica campestris Male Fertility 9 (BcMF9), was isolated from Chinese cabbage (Brassica campestris ssp. chinensis, syn. B. rapa ssp. chinensis) and characterized.

Methods

RT-PCR, northern blotting and in situ hybridization were used to analyse the expression pattern of BcMF9, and antisense RNA technology was applied to study the function of this gene.

Key Results

BcMF9 is expressed in particular in the tapetum and microspore during the late stages of pollen development. Antisense RNA transgenic plants that displayed decreased expression of BcMF9 showed pollen morphological defects that resulted in reduced pollen germination efficiency. Transmission electron microscopy revealed that the homogeneous pectic exintine layer of pollen facing the exterior was over-developed and predominantly occupied the intine, reversing the normal proportional distribution of the internal endintine layer and the external exintine in transgenic pollen. Inhibition of BcMF9 also resulted in break-up of the previously formed tectum and baculae from the beginning of the binucleate stage, as a result of premature degradation of tapetum.

Conclusions

Several lines of evidence, including patterns of BcMF9 expression and phenotypic defects, suggest a sporophytic role in exine patterning, and a gametophytic mode of action of BcMF9 in intine formation. BcMF9 might act as a co-ordinator in the late stages of tapetum degeneration, and subsequently in the regulation of wall material secretion and, in turn, exine formation. BcMF9 might also play a role in intine formation, possibly via regulation of the dynamic metabolism of pectin.Key words: Brassica campestris, Chinese cabbage, exine, intine, PG, pollen wall, polygalacturonase, BcMF9     

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.     

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.     

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.     

Structure of the apertural sporoderm of pollen grains inEuphorbia andChamaesyce (Euphorbiaceae)

The tricolporate pollen grains of 38 Mediterranean and Macaronesian species ofEuphorbia L. andChamaesyce S. F. Gray have a special apertural sporoderm not found in the other taxa of theEuphorbiaceae. At the apertural margo the ectexine is thinner because of shorter columellae and the thin, fragmented or even absent foot-layer. Ectexinous granules, mixed with endexinous material, are present near the ora. The endexine is homogeneous and thickened under the colpi (at the end and at the proximity of the end of colpus). Around the ora, the endexine is granulate and lamellar with irregular cavities. The apertural intine presents a characteristic structure with thickenings running along both sides of the colpi. The arrangement and structure of these intinous thickenings depend on the distance from the ora. This special morphology of the intine is present in all taxa studied here. The genusEuphorbia is considered to be the most evolved taxon of this family. The characteristic apertural sporoderm may be an adaptative modification to different physiological conditions, so it may present an apertural mechanism which is more adapted to harmomegathic changes and thus facilitate the germination and the formation of the pollen tube.     

Re-evaluation of a neglected layer in pollen wall development with comments on its evolution

This study focuses on one particular layer of the pollen wall, which develops below the endexine in the free microspore stage and prior to the initiation of the intine. This membranous-granular layer (MGL) has been described by different terms in the literature and has often been interpreted either as part of the endexine, or the intine. During ontogeny, however, the granular material shows a development that is clearly distinct, both in timing and mode of formation, from the endexine as well as the intine. Its chemical composition is also characteristic; the MGL resists acetolysis. Our ontogenetic observations from four dicot and one monocot species are used to illustrate the systematically widespread occurrence of this wall layer, its ultrastructure and histochemistry, and its comparable nature throughout angiosperms.     

Zusammenhänge zwischen reduzierenden Eigenschaften und Schutzwirkung von SH-Verbindungen gegen Strahlen

Zusammenfassung Der Abbau der reduzierenden Eigenschaften der Strahlenschutzstoffe Cystein, SH-Glutathion, Thioglykolat und-Monothioglycerin in reinen Lösungen und in Gemischen mit Euglobulin unter dem Einwirken von ultraviolettem Licht und von Röntgenstrahlen wird mittels direkter Jodtitrationen (Stärke als Indikator) bis zum Erliegen der Schutzstoffeigenschaften und darüber hinaus verfolgt.Die Ergebnisse der Versuche mit Cystein, SH-Glutathion und Thioglykolat machen es wahrscheinlich, daß bei diesen drei Verbindungen ein ursächlicher Zusammenhang zwischen reduzierenden Eigenschaften und Schutzstoffeigenschaften besteht.Den Herren Prof. Dr. P. Wels, Dr. F. Markwardt, Dipl.-Chem. H. Dalibor und Apotheker H. D. Schlabitz vom Pharmakologischen Institut sowie Herrn Doz. Dr. G. Henseke vom Institut für Organische Chemie der Universität Greifswald danke ich für fördernde Diskussionen. Fräulein Gerda Rahmel danke ich für technische Assistenz.     

The role of the intine and cytoplasm in the activation and germination processes of Poaceae pollen grains

Ultrastructural modifications of the intine and cytoplasm, during the maturation, activation and germination processes are described for several Poaceae pollen grains. Allergenic and antigenic proteins were found in the non apertural intine during the times of activation and germination, using TEM immunolabelling. This fact may be related to the function of the non apertural intine during the processes of pollen activation and pollen tube formation prior to fecundation. Changes in the granular particles of the cytoplasm are described and their role in pollen wall development is suggested. The pectic‐cellulosic and callosic layers of the pollen tube were formed on the degraded intine, and a relationship between pollen tube wall development and the substances expelled from the fibrillar particles was observed. The immunolabelling of the starch granules may be in agreement with their role in the allergenic process.     

Pollen morphology of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae): Taxonomic implications

In order to find new non-molecular evidence to support the phylogenetic and taxonomic position, pollen grains of 20 populations of 16 species of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae) were investigated under SEM and TEM. The pollen grains are spherical and ovoid, nonaperturate. The pollen wall is composed of a very thin exine and a thick intine. The exine is psilate or echinate. The intine consists of two layers, i.e., a thick, channeled layer (exintine) and an inner homogenous layer (endintine). The results reveal morphological congruence between the pollen grains of species of Curcuma, which according to DNA sequence data appears to be a polyphyletic genus. However the uniform pollen morphology in Curcuma provides no evidence to divide this genus into separate taxonomic entities. Our results on pollen morphology also do not provide any additional evidence to either unite or segregate Boesenbergia albomaculata and Curcumorpha longiflora in the same genus and demonstrate that more taxonomic data on the genus Boesenbergia and its relatives are needed before a final decision can be made.     

The Structure of Pollen Wall and Its Relation to Po41en Aggregation in Cymbidium goeringii (Rchb. F.) Rchb. F.

The pollen wall of tetrads located in different positions of a mature pollinium of Cymbidium goeringii was examined with the electron microscope, and the compositions of wall materials were also tested with different histochemical methods. In all tetrads of a pollinium, the pollen wall can be distingished into an exine and an intine, but the exine may be varied greatly according to the tetrad position in a pollenium. The part of the pollen wall (the outer wall) of the external tetrads, lying close, to the tapetum, is composed of two layers, i.e. the exine, and the intine. Theexine consists of tectum, granulate ectexine and endexine, without foot layer. The intine is cellulose in nature. In the outer wall between different groups of: tetrads and in the inner wall within an individual tetrad, the structure of ectexine becomes simple and the deposition of sporopollenin is roduced The degree of reduction of ectexine nicreases from the outer to inner tetrads in several external layers of a pollinium, and even the internal tetrads have a reduced ectexine or lack of it. The present study also demonstrates that the mechanism of pollen aggregation into a pollinium is built on a combined effect of the following features: (1) connected bridges formed' by intine between two pollens within a tetrad, (2) formation of cytoplasmic channels between two pollens within a tetrad, (3) incomplete cell wall formation within a tetrad, (4) little size of tetrads and compact arrangement of mature tetrads and (5) a sticky viscin material surrounded on the outside of a pollinium.     

Funktionsmorphologie des Exkretionsorgans des Spulwurms Ascaris lumbricoides L.

Zusammenfassung Das Seitenkanalsystem von Ascaris lumbricoides wurde elektronenmikroskopisch untersucht. Beim erwachsenen Tier erstreckt sich das in den lateralen Epidermisleisten eingebettete einzellige Organ vom Nervenring bis etwa zur Körpermitte. Im 2. Körperviertel besitzt es kein durchgehendes Kanallumen und erscheint degeneriert. In allen übrigen Bereichen (mit Ausnahme des Ausführungskanals) besitzt es den gleichen Aufbau aus zwei Schichten. Die das Kanallumen begrenzende innere Schicht enthält zahlreiche extraplasmatische Räume, von denen zumindest die am weitesten innen liegenden mit dem Kanallumen kommunizieren. Die äußere Zellmembran besitzt viele Einfaltungen, von denen einige weit in das Cytoplasma hineinragen. Der Gewebeanteil der lateralen Epidermisleisten, der dem Seitenkanalsystem unmittelbar anliegt, enthält sehr viele Interzellularräume, die ein zusammenhängendes Drainage-System bilden. Zur histochemischen Lokalisation von ATP-ase-Aktivität wurden Experimente durchgeführt. Die möglichen Mechanismen der Bildung der Exkretflüssigkeit werden diskutiert unter Berücksichtigung bereits veröffentlichter physiologischer Befunde.

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Ultrastructure and function of the excretory organ of Ascaris lumbricoides L. (Nematoda)

Summary The Excretory organ (H-system) of Ascaris lumbricoides has been investigated electronmicroscopically. In adult animals this single-cell-organ embedded in the lateral lines extends from the nerve ring to approximately the middle of the body.In the second quarter of the body it lacks a continuous canal lumen, and it seems to be degenerated. In all of the other regions (except the stem leading to the excretory pore) it consists of two zones. The inner zone lining the canal lumen contains several extraplasmatic spaces; at least those placed the farthest inside communicate with the canal lumen. The outer cell membrane shows many infoldings, some of which extend deeply into the cytoplasm. The tissue of the lateral line adjacent to the canal system contains very many intercellular spaces which build a coherent intracellular rainage-system. Experiments have been performed in order to localize the ATPase activity histochemically. Possible mechanisms for the forming of the excretory fluid are discussed under consideration of physiological results already published.

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Abkürzungen Ak Ausführungskanal - Bm Basalmembran - Cp Cytoplasmaplatten - lE linke Epidermisleiste - rE rechte Epidermisleiste - Ef Einfaltungen der äußeren Zellmembran - Fb Fibrillenbündel - Go Go Golgiapparat - Hg Hüllgewebe - Is lamelläre Interzellularsubstanz - Iz Interzellularraum - Kl Kanallumen - K Kutikula - Lh Leibeshöhle - Mu Muskelzelle - Mi Mitochondrien - Ms mittlerer Gewebestreifen (= Mittelstreifen) der Epidermisleiste - Mt Mikrotubuli - N Zellkern - No Nucleolus - Ne Nervenring - eP elektronendichte Partikel - sP sphärische Partikel - P Kernpore - Q Querbalken - epR extraplasmatischer Raum - Lho Längsholm - Mf Membranfusion - äS äußere Schicht des Seitenkanalsystems - iS innere Schicht der Längsholme - Sy syncytiale Cytoplasmamasse ohne Interzellularen - V Verzweigungskanal - iZ innere Zone um einen Verzweigungskanal Inauguraldissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Freien Universität Berlin (gekürzt). Herrn Prof. Dr. G. Kümmel danke ich für die Anregung zu diesem Thema und für sein ständiges kritisches Interesse der Untersuchung, Frau C. S. Friedemann für die Anfertigung der Zeichnungen und Fräulein H. Schmidt für technische Assistenz.     

Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen ofCamellia japonica

As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically. The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose, arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall.     

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.