Ultrastructural demonstration of a glycoproteinic surface coat in allergenic pollen grains by combined cetylpyridinium chloride precipitation and silver proteinate staining

In allergenic birch pollen grains, highly watersoluble surface substances were precipitated by the cationic detergent cetylpyridinium chloride (CPC) during aqueous fixation. After processing the pollen for electron microscopy, ultrathin sections of pollen grains were subjected to the periodic acid - thiocarbohydrazide - silver proteinate (PA-TCH-SP) procedure according to Thiery (1967) for the detection of vicinal glycol groups. It was found that the material precipitated by CPC on the surface and within the exine cavities of the pollen wall strongly reacted with the PA-TCH-SP reagent thus indicating the presence of polysaccharides on the surface of birch pollen grains. In samples which had not been treated with the cationic detergent, PA-TCH-SP reactivity was reduced to thin linings on the surface and within the exine cavities. In both cases the exine proper did not stain whereas the intine showed moderate staining. Within the aperture region of the intine, PA-TCH-SP reactivity is preferably associated with fibrillar or reticular structures. The results are discussed with special reference to biochemical findings on allergenic birch pollen proteins.     

Localization of the Major Allergen Bet V 1 in Betula Pollen Using Monoclonal Antibody Labelling

The major allergen Bet ver 1 of Betula pendula (= B. verrucosa) pollen grains has been localized by gold labelling with monoclonal antibodies. The allergen is located predominantly in the starch grains and to a slight extent in the exine and intine. The possibility that environmental factors might influence the liberation of allergenic compounds present in birch pollen grains is discussed.     

Immunoelectron-microscopic localization of diffusible Birch-pollen antigens in ultrathin sections using the protein-A/gold technique

Summary Pollen grains from Betula pendula were fixed in a mixture of p-formaldehyde and cetylpyridinium chloride (CPC) for the precipitation of soluble pollen glycoproteins. After dehydration and embedding at low temperatures in the water-soluble resin, Lowicryl K4M, ultrathin sections of the pollen grains were incubated using specific antibodies against birch-pollen extract and protein-A/gold complexes. Antigen activity was found in the CPC-precipitated surface material and within the exine (bacular cavities) and the cytoplasm (except for starch grains and lipidic droplets). There was no labelling within the intine. The region of the germinal aperture also showed a very low degree of antigen activity. The control sections were almost completely free of background staining.     

Detection and release of allergenic proteins in Parietaria judaica pollen grains

Summary. Rapid diffusion of allergenic proteins in isotonic media has been demonstrated for different pollen grains. Upon contact with stigmatic secretion or with the mucosa of sensitive individuals, pollen grains absorb water and release soluble low-molecular-weight proteins, these proteins enter in the secretory pathway in order to arrive at the cell surface. In this study we located allergenic proteins in mature and hydrated-activated pollen grains of Parietaria judaica L. (Urticaceae) and studied the diffusion of these proteins during the first 20 min of the hydration and activation processes. A combination of transmission electron microscopy and immunocytochemical methods was used to locate these proteins in mature pollen and in pollen grains after different periods of hydration and activation processes. Activated proteins reacting with antibodies in human serum from allergic patients were found in the cytoplasm, wall, and exudates from the pollen grains. The allergenic component of these pollen grains changes according to the pollen state; the presence of these proteins in the exine, the cytoplasm, and especially in the intine and in the material exuded from the pollen grains, is significant in the hydrated-activated studied times, whereas this presence is not significant in mature pollen grains. The rapid activation and release of allergenic proteins of P. judaica pollen appears to be the main cause of the allergenic activity of these pollen grains. Correspondence and reprints: Department of Plant Biology, Faculty of Biology, University of León, Campus de Vegazana, 24071 León, Spain.     

Immunoelectron-microscopic localization of diffusible birch-pollen antigens in ultrathin sections using the protein-A/gold technique

Pollen grains from Betula pendula were fixed in a mixture of p-formaldehyde and cetylpyridinium chloride (CPC) for the precipitation of soluble pollen glycoproteins. After dehydration and embedding at low temperatures in the water-soluble resin, Lowicryl K4M, ultrathin sections of the pollen grains were incubated using specific antibodies against birch-pollen extract and protein-A/gold complexes. Antigen activity was found in the CPC-precipitated surface material and within the exine (bacular cavities) and the cytoplasm (except for starch grains and lipidic droplets). There was no labelling within the intine. The region of the germinal aperture also showed a very low degree of antigen activity. The control sections were almost completely free of background staining.     

Investigations cytochimiques,marquage immunocytochimique et effets destructeurs des U. V. sur le pollen de Dactylis glomerata L

The elucidation of the ultrastructural cytochemistry, coupled with chemical stabilizing procedures during fixation and embedding, make a significant contribution to the understanding of pollen-exine ontogenesis. A combination of the Con-A agglutination and lanthanum precipitation methods proved particular advantageous during the intine formation stage. By using immunogold techniques, it is possible to demonstrate that only the mature exine of the atmospheric pollen grains reacted positively after sectioning, not the intine. An important difference appears when immature pollen grains are treated under the same conditions: in this case, both the microsporal cytoplasm of the future pollen grain and the immature exine react, but not the intine.

In untreated pollen grains, the exine is a biological material of exceptionally high density with a very low stainability: staining for proteins and lipids is only moderate. A degradation of exine structures by U. V. radiation of 254 nm can be easily proved and the framework obtained is comparable to that induced by some chemical attacks. When sporopollenin is degraded from filamentous sub-units of the exine, stainability increases, and the cytochemical tests for acid-mucopolysaccharids are positive. It is clear that glycocalyx units within exines are chemically bound to the sporopollenin matrix. Attention can also be profitably directed to the future investigation of the function of exine frameworks, through allergen fixation in living pollen grains.     

Techniques to preserve soluble surface components in birch pollen wall: a scanning and transmission electron microscopic study

The exine of birch pollen was examined by scanning and transmission electron microscopy in the native state and after fixation in different aqueous fixatives: glutaraldehyde + OsO4; glutaraldehyde + cetylpyridinium chloride (CPC) + OsO4; glutaraldehyde + cuprolinic blue (CB); and periodate + lysine + paraformaldehyde (PLP). The native pollen exine showed a thin (3-5-nm) border of electron-dense material lining the tectum and electron-dense material within microchannels and bacula cavities. Fixation with the addition of CPC resulted in a voluminous surface coat surrounding the pollen grain, but empty microchannels and bacula cavities. After fixation with the addition of CB, there was a thin surface coat, whereas microchannels and bacula cavities were partially filled with electron-dense material. The other fixatives led to empty microchannels and bacula cavities. There was no surface coat on the pollen grain. However, after all fixation procedures, a thin electron-dense border of the tectum remained visible. Concerning the electron-dense material filling microchannels and bacula cavities in the native pollen grain, the results obtained in the present study suggest that it is either completely lost (after conventional and PLP fixation) or, after fixation with a precipitating additive, partially (CB) or completely (CPC) solubilized and precipitated on the surface of the pollen grain as a surface coat.     

THE POLLEN WALL OF CANNA AND ITS SIMILARITY TO THE GERMINAL APERTURES OF OTHER POLLEN

The pollen wall of Canna generalis Bailey is exceptionally thick, but only a minor part of it contains detectable amounts of sporopollenin. The sporopollenin is in isolated spinules at the exine surface and in the intine near the plasma membrane. There is no sporopollenin in the > 10 μ thick channeled region between spinules and intine. We suggest that the entire pollen wall of C. generalis is similar to the thick intine and thin exine typical for germinal apertures in many pollen grain types. Considered functionally, the Canna pollen wall may offer an infinite number of sites for pollen tube initiation and would differ significantly from grains that are inaperturate in the sense of an exine lacking definite germinal apertures.     

Visualization of birch pollen allergens using IgE-containing sera from human atopic individuals in immunogold labelling experiments

Summary Pollen from birch trees (Betula pendula) was fixed in paraformaldehyde with or without the addition of 0.5% cetylpyridinium chloride, dehydrated and embedded in Lowicryl K4M in the cold. Ultrathin sections were incubated using the following sequence of antibodies and antisera: IgE-containing serum from an atopic human individual allergic to birch pollen allergens, rabbit anti-human IgE antibodies, and colloidal gold-labelled goat anti-rabbit antibodies. Controls were performed by replacing the specific human antiserum by serum from an atopic person with a similar level of IgE antibodies directed against allergens other than birch pollen allergens, or by omitting the human antiserum or the anti-IgE antibody or both. In test experiments, there was a dense specific labelling of the exine and the cytoplasmic matrix of the pollen grain. There was moderate labelling of the apertural regions (poral plugs). There was no labelling of the intine. In pollen grains fixed with the addition of cetylpyridinium chloride, an electron-dense surface coat was precipitated on the outside of the pollen wall. This surface material also remained completely unlabelled.     

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     

Allergenic and antigenic proteins released in the apertural sporoderm during the activation process in grass pollen grains

A combination of transmission electron microscopy with immunocytochemical methods was used to localize antigenic and allergenic proteins during the maturation and activation processes of Poaceae pollen grains. The intine undergoes a series of modifications that play a decisive role in these processes. Allergenic and antigenic proteins were detected particularly in the intine of activated in vitro grass pollen grains. Labelling of antigenic proteins was more abundant and less specific than that of allergenic proteins. At the time of hydration, the operculum was lifted up, the intine was swollen and labelling of allergenic proteins appeared highly localized in the Zwischenkörper. No significant labelling was observed when the Zwischenkörper gelatinized. Immunolocalization of allergenic proteins in the activated Zwischenkörper indicated the presence of proteins related to activation of the pollen grains. This confirms that the intine function is involved in the processes of pollen tube formation and fertilization, and also suggests the possible mechanism activated in the pollen grains when allergenic proteins reach the mucosa of sensitive subjects.     

Exploding pollen in Montrichardia arborescens (Araceae)

Pollen grains of Montrichardia are inaperturate with psilate ornamentation. The pollen wall is formed by a thin ectexine and an extraordinarily thick intine. In living as well as in dead pollen grains contact with water leads to a rapid swelling of the intine followed by an explosive opening of the exine. Within a few seconds a thick tube is formed, which is not the pollen tube. The pollen protoplast is situated at the tip of the tube. These intine tubes are interpreted as pollen connecting tools to keep pollen grains together and adhere them to the cuticle of the hairless pollinators.     

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.     

Changes in the Plantago lanceolata mature pollen ultrastructure and Pla l 1 allergen localisation after grain hydration

Abstract

We have conducted the study of ultrastructural changes of wall and cytoplasm of Plantago lanceolata (English plantain) pollen grains during the first 10 min of hydration and activation processes, prior to germination, and localisation of Pla l 1, the major allergen of these pollen grains with immunocytochemical methods. The samples were fixed using conventional and freezing protocols for transmission electron microscopy. During the activation process, the intine is thickened in the apertural region and cytoplasm undergoes changes in the number of lipid bodies, amyloplasts, vacuoles and ribosomes. Also, we observed an association between lipid bodies, cisternae of rough endoplasmic reticulum, dictyosomes and vacuoles. An increase in the presence of allergenic particles (Pla l 1) in the exine, intine and the cytoplasm in activated pollen grains was detected, whereas this presence is not significant in mature pollen grains. The increase in the production and release of this allergen when pollen grains are activated suggest that Pla l 1 has an important role in pollen–stigma recognition and in the subsequent development of the pollen tube.     

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.     

The pollen grain wall as a site for passage of lanthanum in tomato,Lycopersicon esculentum (Solanaceae)

We studied the response of the pollen grain wall to the passage of materials from the external environment. Pollen grains of Lycopersicon esculentum, a species not previously investigated in similar studies, were used to determine whether the routes of passage through the pollen wall are a characteristic of a given species. We found lanthanum deposits on the cell wall surface, inside microchannels crossing tectum, and in the infratectum. Lanthanum was also seen as a fine precipitate homogenously scattered within the substance of the exine. In the apertural region of the intine, electron-dense deposits filled the tubulations of the intine oncus, whereas the intine matrix was free of precipitate. In the cytoplasm, precipitate was observed inside small vesicles frequently located near or in contact with the plasmalemma. Our findings were similar to those reported in other species, and were not influenced by the fixative or culture medium used to incorporate the tracer element. We noted a relation between the route of passage and the stage of pollen grain maturation. In less mature grains (midbicellular pollen), electron-dense deposits were more abundant than in more mature grains, probably because apertural regions were blocked as routes of passage.     

Immunocytochemical localization of the allergenic proteins in the pollen of Cryptomeria japonica

Applying an immunocytochemical method, a localization of the protein Cry j I in the Cryptomeria japonica pollen, which is the major allergen responsible for Japanese cedar pollinosis, is investigated with the monoclonal and polyclonal antibodies produced from the protein. The protein that reacts to the polyclonal antibody localizes on the sexine, nexine, between nexine and intine layers, orbicles, cell wall of a generative cell, Golgi body and Golgi vesicles. The allergenic protein contained in the exine and orbicles of Japanese cedar pollen can diffuse or dissolve easily from there into the mucus covering of the eye and nose, causing a response in less than 1 min after exposure. Since the orbicles have a diameter of about 430 nm, they can pass easily through the pores of most protective masks to reach the sensitive tissues of the patient. The proteins react to the monoclonal antibodies (J1BO1 and J1BO7) and localize on the Golgi body, sexine, nexine and orbicles (but not between the nexine and intine layers), and on the generative cell wall. In the young pollen grain, numerous allergenic protein particles contained in the orbicles and sexine layer, but there is only a small amount of the protein between the nexine and intine layers, since the intine layer is not yet complete at this stage. More will be accumulated there during developmental maturation. The allergenic protein is also found on the tapetal materials remaining in the young anther. Since the materials forming the exine layer and orbicles come from tapetal tissue, it is assumed that some of the allergenic protein is produced in the tapetum and localized in the orbicles and pollen wall during maturation, and that the rest of the allergenic protein is produced in the Golgi body in the mature pollen grain.     

Pollen structure, tetrad cohesion and pollen-connecting threads in Pseuduvaria (Annonaceae)

The structure of the pollen of 42 species of Pseuduvaria (Annonaceae) is described. The pollen is consistently inaperturate, isopolar and radially symmetrical. Four basic patterns of exine sculpturing are identified: rugulate, verrucate, scabrate and psilate. The exine stratification of one representative species, P. macrocarpa , is shown to be entirely ectexinal. The ectexine consists of a discontinuous outer tectal layer, a columellar infratectal layer, and an inner lamellar foliated foot layer; the intine is very thin and fibrillar. The pollen is invariably released as acalymmate tetrads, in which the tectum is absent from the proximal walls. The individual pollen grains within the tetrads are connected by crosswall cohesion, involving both exine and intine; this form of cohesion has not hitherto been reported in the Annonaceae. In addition, pollen grains of neighbouring tetrads are connected in two different ways, viz. short exine connections and non-sporopollenin pollen-connecting threads. Neither of these cohesion mechanisms has previously been reported for the genus. The function of the various forms of cohesion between pollen grains and tetrads in Pseuduvaria is discussed as a mechanism to enhance the efficiency of pollination by enabling the fertilization of multiple ovules following a single pollinator visit.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 143 , 69−78.     

Immunocytochemical localization of allergenic proteins from mature to activated Zygophyllum fabago L. (Zygophyllaceae) pollen grains

Zygophyllum fabago L. (Zygophyllaceae) can be found in the Middle East, in North Africa and in the arid zones of the Mediterranean region. It easily establishes itself in new regions, and is considered an invasive plant. They undergo ambophilous pollination, as there is a relationship between this type of pollination and its allergenic incidence. A combination of transmission electron microscopy with immunocytochemical methods was used to localize allergenic proteins during hydration and activation processes. Germination was induced in vitro for 1,2,4,6, and 30 min. The activated proteins reacting with antibodies present in human sera from allergenic patients are found in the cytoplasm, intine, exine and exudates from the pollen grains. The activation time plays an important role on the labelling intensity. Labelling of allergenic proteins was abundant at 1 and 2 min of activation, and decreased at 4 and 6 min. The rapid activation and release of the allergenic proteins appears to be the main cause of allergenic activity of Z. fabago pollen grains.     

云南松花粉形态研究

在云南松(Pinus yunnanensis Fr.)小孢子发生发育过程中,花粉母细胞、四分孢子及花粉粒均见有粘连现象。花粉气囊的形态、大小变化复杂多样。除一般具两个正常气囊的花粉粒外,还观察到气囊不发育、具一个气囊、二个异形气囊、三个气囊和四个气囊的花粉粒。成熟花粉壁从外至内可分为外壁外层、外壁内层、内壁外层和内壁内层,它们的构成成分及形态均有明显差别。贮存后花粉的内壁结构发生了明显变化。