Ultrastructural study of Saffron pollen

Abstract

Pollen of Saffron (Crocus sativus L.), a sterile autotriploid plant was studied using scanning and transmission electron microscopy. The pollen of mature anthers had grains not containing pores or furrows, a discontinuous thin exine and a very thick intine crossed by tubules containing granular electron-dense material. Electron transparent material and lipid bodies were abundant in the cytoplasm which also contained a large number of vesicles. A high percentage of pollen grains showed anomalies.     

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.     

Multilamellar bodies linked to two active plasmalemma regions in the pollen grains of Sarcocapnos pulcherrima

The presence of visible multilamellar bodies in the cytoplasm of pollen grains of at least seven species of the family Papaveraceae has led us to study the behaviour of these bodies during pollen-grain ontogeny and in growing pollen tubes of Sarcocapnos pulcherrima C. Morales & R. Garcia germinated in vitro. Our transmission-electron-microscope (TEM) studies in pollen grains show that the multilamellar bodies may be classified as: 1) small, isolated and placed in the region of apertures in the cytoplasm; and 2) large, in clusters and in contact with the active plasmalemma apertures only when tubules are being formed in the apertural intine. Similar types of multilamellar bodies to those observed in the pollen apertures can be seen near the apex of the growing pollen tube (small and isolated) and in contact with the apex plasmalemma (large and clustered). Our results support the hypothesis that the multilamellar bodies are functionally linked to moments when the cytoplasmic membrane is very active. We have also linked the multilamellar bodies to Golgi vesicles as they both react positively to acid-phosphatase (AP) staining and also to the plasmalemma by the thiocarbohydrazide-silver proteinate-staining (TCH-Sp) electron-contrasting technique.     

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.     

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.     

Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana

Using the cryo-fixation/freeze-substitution method, we studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during microspore and pollen development, and pollen maturation of Arabidopsis thaliana. In young microspores forming exine (pollen outer cell wall), vacuoles looked like those of somatic cells. In microspores during the formation of intine (inner cell wall), a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic type vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs were considered to be storage vacuoles. In pollen grains from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the cytoplasm. The autolysis of pollen grains is the first finding in this study, which may contribute to the loss of ability of pollen germination after anthesis.     

Immunocytochemical localization of Cry j 1, the major allergen of<Emphasis Type="Italic"> Cryptomeria japonica</Emphasis> (Taxodiaceae) in<Emphasis Type="Italic"> Cupressus arizonica</Emphasis> and<Emphasis Type="Italic"> Cupressus sempervirens</Emphasis> (Cupressaceae) pollen grains

Immunocytochemical localization of cross-reactive antigens to Cry j 1, the major allergen of Cryptomeria japonica, in the pollen grains of Cupressus arizonica and Cupressus sempervirens, was conducted using transmission electron microscopy. Mature and activated pollen grains were fixed for immunocytochemistry using conventional and freezing protocols. Sections containing the samples were incubated with anti-Cry j 1 monoclonal antibody (mAb) (KW-S91). Cross-reactive antigens to Cry j 1 were detected in the mature pollen grains of Cupressus arizonica and Cupressus sempervirens, and abundant gold particles were seen in the orbicules and wall, and in the Golgi, nucleus, and inclusions in reserve materials. After 5 min of activation, labelling noticeably decreased. From 15 min to 48 h, the cytoplasm exhibits a new labelling pattern, with gold markers being associated with protein storage vacuoles. The decrease in cross-reactive antigens during the first 5 min of activation could be due to a rapid release of proteins recognized by Cry j 1 mAb during pollen attachment in the pollination droplet. Our results show that the content of allergenic proteins is unstable, displaying variation relative to the progress of germination in Cupressus sempervirens and Cupressus arizonica pollen grains. The ability to do so may be viewed as an adaptive strategy of Cupressaceae pollen grains to maximize their biosynthetic efficiency.     

The ultrastructure of polymorphic pollen grains of Canna indica L.

Summary Our investigations on Canna indica L. indicate that the pollen of this species is polymorphic: there are two types of pollen — a larger type and a comparatively smaller type. Transmission electron microscopy (TEM) revealed the presence of small vacuoles containing tannic substances in the generative cell (GC) of the larger grains: the GC of the mature grain contained a higher quantity of tannins than the GC of the immature grain. Mitochondria, lipid bodies, rough endoplasmic reticulum (RER) and microtubular bundles were present in the cytoplasm of the GC. Numerous mitochondria, lipid bodies and plastids were also present in the vegetative cell (VC), with the mitochondria clustered around the vegetative nucleus. The plastids were observed to be associated with the RER cisterns. During the maturation process, the number of starch grains contained in the plastids decreased.     

Palynological study of Cupressus sempervirens L. var. pyramidalis and var. horizontalis

ABSTRACT

Cupressus sempervirens L. var. pyramidalis and C. sempervirens var. horizontalis were examined for their pollen morphometry and ultrastructure to verify whether, in addition to phenotype, pollen could also show characteristics useful to discriminate the two infraspecific taxa. C. sempervirens var. pyramidalis compared to C. sempervirens var. horizontalis has mainly larger, subcircular pollen with a higher percentage of apolar and germinating grains, and a wall with thicker intine; the cytoplasm has a greater number of callose-containing vesicles. C. sempervirens var. horizontalis has pollen distributed in five classes of shape. In addition it is smaller and shows a higher percentage of ruptured grains, and a lower percentage of germination in vitro. The cytoplasm contains vesicles rich in callose and also a higher amount of osmophilic bodies. Pollen grains in both varietas contain only one cell at dispersal. The reported differences in pollen morphometry, structure and biology, together with the phenotypic characteristics of the tree, support the validity of two infraspecific taxa.     

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.     

Localization of the major allergen Bet v I in birch pollen by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to localize the major allergen, Bet v I in birch pollen. Pollen grains of Belula pendula were collected from catkins that had begun to release their pollen, freeze dried, and stored at 4°C. The pollen material was cryosectioned and stained with indirect immunofluorescence using the monoclonal antibody Bv 10. The observed localization of Bet v I was dependent on the treatment of the grains. When they had been prefixed with gaseous formaldehyde followed by acetone Bet v I was found in the cytoplasm. When the prefixation was omitted, a minor portion of Bet v I also appeared in the exine in the aperture regions. The allergen was not observed in the intine or the Zwischenkörper. Our suggestion is that the normal route for excretion of Bet v I is via the apertures on contact between pollen and the stigmatic surface of the pistil.     

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.     

Hermodactylus tuberosus L. (Iridaceae) pollen organisation before and after anther dehiscence

ABSTRACT

The morphology, cytology and viability of Hermodactylus tuberosus L. (Iridaceae) pollen were examined from the first mitosis until maturation and after anther opening. During maturation, the pollen coat becomes modified, and the vegetative cell cytoplasm accumulates several types of reserve substances. In the vegetative cell cytoplasm, starch is quickly utilised whereas lipid inclusions of different dimensions, shape and composition occur during pollen maturation. Pollen from opened anthers have a thin pollen coat; the cytoplasm has mostly lipid reserves, and many small vesicles and vacuoles. It is similar in size or larger than pollen located inside the anther, and its viability does not decrease until one day after anther dehiscence. Large osmiophilic bodies, different from those of the vegetative cell cytoplasm, are present in the generative cell cytoplasm starting from the first stage of pollen development. The poorly developed pollen coat in pollen from opened anthers suggests that it plays a minor role in attracting insects for pollination. The size and structural and ultrastructural features of mature pollen indicate that it does not undergo dehydration and possesses sufficient vigour for immediate germination.     

Effects of ovular secretions on pollen in Pseudotsuga menziesii (Pinaceae)

Effects of ovular secretions on pollen grains were examined in Pseudotsuga menziesii. The exine is cast off in the micropylar canal. A membranelike structure covers parts of pollen grains and appears to protect them. The outer intine consists of fibrous materials, but it also shows a thicker filamentous appearance in some ovules during pollen elongation. The inner intine is electron-dense. Its fibrous nature is occasionally visible. Dissolution of the outer intine varies in amount and manner in ovules from different trees. The plasma membrane near the pollen wall alternatively appears normal and distorted. These different morphologies of the outer intine and of the plasma membrane are considered to result from secretions from the ovule. The outer intine may contain electron-dense globules that are formed in the tube cell and traverse the inner intine. Pollen tube formation appears to be triggered by a secretion from the ovule. Cross-pollinated grains are less distorted compared with self-pollinated grains.     

Lipid transfer proteins in Parietaria judaica L. pollen grains: immunocytochemical localization and function

Parietariajudaica L. (Urticaceae) pollen is considered one of the most common causes of allergic respiratory symptoms in the Mediterranean area. The localization of lipid transfer proteins (LTPs) in P. judaica mature and hydrated-activated pollen grains was investigated applying a combination of transmission electron microscopy (TEM) with immunocytochemical methods. Our results show that the content of LTPs in P. judaica pollen grains changes during the process of hydration. The localization of judaica LTPs in the cytoplasm and in the lipid bodies associated with vacuoles demonstrated that LTPs represent primarily intracellular proteins. On the other hand, exposure of the pollen grains to germination medium induced the release of LTPs from the pollen grain. Thus, LTPs are cytoplasmic proteins that are secreted to become available for pollen-stigma interactions and probably induce the IgE antibody responses in allergic patients.     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

An approach to the knowledge of pollen and allergen diversity through lipid transfer protein localisation in taxonomically distant pollen grains

The aim of the present study is to localise non‐specific lipid transfer proteins (nsLTPs), obtained from Rosaceae (Prunus persica (L.) Batsch) in pollen grains of taxonomically distant plants, such as Iridaceae (Aristea latifolia G.J. Lewis), Platanaceae (Platanus acerifolia (Aiton) Willd.) and Urticaceae (Parietaria judaica L.), in order to compare pollen and nsLTP diversity. A combination of transmission electron microscopy, immunocytochemical techniques, and rabbit specific antiserum against peach nsLTPs (Pru p 3) were used. Abundant labelling to Pru p 3‐like proteins was observed in the cytoplasm, walls and pollenkitt of A. latifolia pollen grains. The presence of nsLTPs associated with the pollenkitt proves that it takes part in the defence mechanism of pollen grains. The labelling was less intense in the cytoplasm and walls of P. acerifolia. Immuno‐stained gold particles were associated with the vacuoles, lipid inclusions, endoplasmic reticulum and Golgi complex. No significant labelling was found in the P. judaica pollen grains incubated with anti‐Pru p 3 polyclonal antibodies. These results indicate important variations in the nsLTPs of different pollen species. Consequently, no taxonomic relationship between pollen grains and nsLTPs could be established.     

The role of allergenic proteins Pla a 1 and Pla a 2 in the germination of Platanus acerifolia pollen grains

Platanus acerifolia (Aiton) Willdenow is a plane tree, widely grown as an ornamental tree in many cities of the United States and Western Europe, which has become an important source of airborne allergens in our cities. The aim of the present study is to immunolocalize the major allergens in the pollen grain and to examine their potential function in the fertilization process. Observations were made in mature and hydrated, activated pollen of P. acerifolia for 5, 15, 30 min and 2 h in the germination medium. Specimens were fixed using freezing protocols for transmission electron microscopy (TEM). For immunogold labelling, cryosections and resin-embedded ultrathin sections were incubated using rabbit antisera against the purified pollen allergens Pla a 1 and Pla a 2. Elution of P. acerifolia allergens took place after 5 min of pollen incubation in buffered medium. Intense labelling of Pla a 1 and Pla a 2 was detected after pollen exudates were released. In pollen grains, Pla a 1 was predominantly localized in concentric cisternae of the endoplasmic reticulum (ER), situated between the vegetative nucleus and the generative cell, and was released from pollen grains 5 min after hydration; cytoplasmic localization decreased 15 min after hydration. In pollen grains, glycoprotein Pla a 2 was abundant in association with Golgi cisternae and vesicles situated in the apertural periphery of the mature pollen grains. Pla a 2 proteins were also detected in ER and in the generative cell wall. Immunolabelling of Pla a 2 decreased 5 min after pollen hydration but was again intense after 15–30 min in germination medium, presumably as a consequence of renewed expression and glycosylation of this protein. Pla a 1 belongs to a new class of allergens related to proteinaceous invertase and pectin methyl esterase inhibitors (PII, PMEI) which could be involved in membrane protection and pectin de-esterification control during pollen hydration. Pla a 2 has an exopolygalacturonase (PG) enzymatic activity consistent with pollen-stigma adhesion mechanisms or compatibility systems. Moreover, the expression of Pla a 2 found 15–30 min after hydration might contribute to pollen-tube growth and the modification of transmitting tissue cell walls. The abundant production and elution of Pla a 1 and Pla a 2 proteins may alter the environment in which pollen tube elongation occurs, thus promoting a potential crosstalk between the pollen and the gynoecium.     

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

Summary A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.     

Changes in the composition of ionogenic groups of the wall of the lily pollen grain during germination

Changes in the composition of ionogenic groups of the polymeric matrix of the cell walls of lily (Lilium longiflorum Thunb.) pollen grains were studied during its activation at the early stages of pollen germination. In the cell walls isolated from nonactivated and activated pollen grains, four types of ionogenic groups were identified: amino groups, carboxylic groups of uronic acids, phenolic OH-groups. and groups with pK_a 7–8. During the early stages of germination, ionization constants of each type groups remained unchanged, but the quantitative composition of ionogenic groups in the intine changed. In this matrix, a decrease in the content of phenolic groups and demethylated carboxylic groups of uronic acids was detected. It is supposed that, at early stages of germination, the intine loses some part of acid pectins and some phenolic compounds.