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.     

Esterases in pollen and stigma of Brassica

The dry type stigma of Brassica is covered with a continuous layer of cuticle. Cutinase and non-specific esterases may be involved in breakdown of this cuticle barrier during pollen-stigma interaction, but only a little is known about their nature and characteristics. We report here the presence of two distinct esterases from stigma and pollen of Brassica. A 33 kD esterase assayed using MU-butyrate substrate shows high activity in stigma papillae. A similar esterase from Tropaeolum pollen has been shown to possess active cutinase activity. The esterase activity in anther tissue is due to a 24 kD enzyme with substrate specificity toward acetate esters. Both enzymes require sulfhydryl groups for their catalytic activity. Immunogold labelling of antibodies raised against these esterases localised the proteins at the subcellular level. Antibodies for MU-butyrate hydrolase gave a positive signal in the cell walls of mature stigma papillae and in the tapetum and microspores during early stages of anther development. In the mature anther, a positive signal in the cytoplasm of pollen grains with some detectable localisation in the exine layer of the pollen wall was obtained. Similar results were obtained with acetate hydrolase antibodies. These esterases are thus spatially and temporally regulated in stigma and anther tissues.Abbreviations MU methyl umbelliferyl - pAbC anti-butyrate hydrolase polyclonal antibodies - pAbE anti-acetate hydrolase polyclonal antibodies     

ULTRASTRUCTURE OF NICOTIANA ALATA POLLEN,ITS GERMINATION AND EARLY TUBE FORMATION

Both internal anatomy and external morphology of the mature pollen grain of Nicotiana alata have been studied, together with pollen activation and tube emergence and its cytological zonation. A peculiar feature of mature pollen is the occurrence in the vegetative cell cytoplasm of a large quantity of stacked cisterns of rough endoplasmic reticulum, which then dissociate during pollen activation. Pollen tube emergence generally occurs 2 hr 30 min after hydration, while cytological zonation is complete 4 hr 30 min after hydration when the first callosic plug is formed.     

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     

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.     

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.     

Development-related changes of protein ubiquitination in pollen from male and female kiwifruit (Actinidia deliciosa)

Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pistillate flowers nonetheless develop non-functional reproductive structures of the ompposite sex. Ubiquitin is a small, highly conserved protein found in all eucaryotes: a covalent ATP-dependent attachment of ubiquitin marks proteins for degradation. In the present paper, we used immunoblotting to investigate the presence of free ubiquitin and ubiquitin conjugates during pollen development in male (androfertile) and in female (androsterile) genotypes of kiwifruit. In the male, several high molecular mass protein conjugates were present throughout development. On the contrary, such a pattern characterized only early stages of pollen from the female genotype, where conjugates progressively disamppeared, until they were detectable only in trace amounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were ampproaching the first mitosis. Free ubiquitin increased continuously during development of the male microgametophyte so that mature pollen contained considerable amounts of the ubiquitin monomer at the time of its release from the anther. By contrast, only low levels were detectable in the degenerating microspores in the pistillate flowers. In vitro experiments using labeled ubiquitin indicated that early-uninucleate microspores of the female genotype had a much higher conjugation rate than those of the male genotype at the same stage. However, after feeding α-lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progressively degenerated in the microspores of the pistillate flowers starting from the early-uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity. At anthesis, the microspores in the pistillate flowers either had no nucleus or showed only traces of chromatin. Thus, the ubiquitin system seems to play an important role in protein turnover occurring during the normal developmental pathway of the kiwifruit microgametophyte, while it was mainly involved in regressive events related to microspore degeneration in the female genotype.     

Localisation pattern of homogalacturonan and arabinogalactan proteins in developing ovules of the gymnosperm plant Larix decidua Mill

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.     

Ultrastructural features of Aloe ciliaris pollen

Summary Both the internal anatomy and the external morphology of the mature pollen grain of Aloe ciliaris have been studied, together with the cytological changes occurring during pollen activation. In mature pollen, the generative cell (GC) and the vegetative nucleus (VN) are closely associated with each other, and both can be found in the central part of the grain. In the generative cytoplasm, some organelles and microtubular bundles are present. In the vegetative cell, dictyosomes, stacks of rough endoplasmic reticulum, mitochondria, plastids, vacuoles, ribosomes, and masses of fibrillar material have been described. During pollen activation, important changes occur in both the generative and vegetative cells (VC). In the GC, the microtubular bundles become clearly visible, and the GC and VC gradually move towards the germ pore. The RER cisterns become free from the stacks, and organelles, such as dictyosomes, become very active. The fibrillar masses gradually decrease in number, and the individual fibrils become more evident and clearer in resolution.This research was carried out in the framework of contract no. BAP-0204-I of the Biotechnology Action Programme of the Commission of the European Communities     

Spatial and temporal organization of actin during hydration,activation, and germination of pollen inPyrus communis L.: a population study

Summary Dynamics of F-actin organization during activation and germination ofPyrus communis (pear) pollen was examined using rhodaminephalloidin. Prior to activation, the rhodamine-phalloidin labelling pattern appeared as circular profiles in the peripheral cytoplasm of the vegetative cell and as coarse granules around the vegetative nucleus. In activated pollen, parallel arrays of cortical F-actin were aligned circumferentially, along the polar axis in non-apertural areas of the pollen grain, and at 45° to 90° to the polar axis beneath the apertures. Some pollen also showed fluorescent granules or fusiform bodies dispersed throughout the cytoplasm, but as the number of such pollen diminished with prolonged incubation, these are being considered as intermediate patterns. In later stages, the filaments became organized as interapertural bundles traversing the three apertures. However, prior to emergence of the pollen tube, labelling became confined to a single aperture. In germinated pollen grains, actin microfilaments are aligned more or less axially with respect to the axis of the developing pollen tube.The granular labelling pattern seen around the vegetative nucleus prior to pollen activation also became clearly filamentous with pollen activation; this filamentous pattern persisted until germination when it was replaced by cables that aligned longitudinally with respect to the emerging tube axis.The results demonstrate that the organization of actin undergoes considerable changes in the period preceding pollen germination and that microfilament polarization is achieved before pollen germination.     

Deficiency of very long chain alkanes biosynthesis causes humidity‐sensitive male sterility via affecting pollen adhesion and hydration in rice

Pollen adhesion and hydration are the earliest events of the pollen–stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect‐pollinated plants. Here, we characterized the role of OsGL1‐4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1‐4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1‐4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography–mass spectrometry analysis suggested that the humidity‐sensitive male sterility of osgl1‐4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.     

Localisation of a carbohydrate epitope recognised by human IgE in pollen of Cupressaceae

The objectives of the present study were: (1) to localise, at the subcellular level, the allergens in pollen of Cupressaceae species, using a monoclonal antibody (mAb 5E6) that is specific for carbohydrate epitopes of allergenic components of Cupressus arizonica pollen extract; (2) to determine whether the glycidic epitope recognised by mAb 5E6 was present in pollen of allergenic species taxonomically unrelated to Cupressaceae; and (3) to determine whether human IgE purified from monosensitive patients recognises the same epitope as mAb 5E6 in Cupressaceae pollen. Immunogold labelling of mAb 5E6 showed a high density of gold particles on the orbicules, supporting the hypothesis that they are important vectors of allergens. A high density was also found on the exine and in the cytoplasm, with the latter finding confirming that fragments of pollen ruptured under humid conditions can represent a vector. The glycidic epitope recognised by mAb 5E6 was detected in all of the species taxonomically unrelated to Cupressaceae, although with varying density. Human IgE recognised the same epitope as mAb 5E6. These findings are consistent with observations of diffuse allergenic cross-reactivity among various allergens. The in situ localisation of a common epitope recognised by both a monoclonal antibody and human IgE could be of importance in immunotherapy.     

The interrelationship between the accumulation of lipids,protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.To whom correspondence should be addressed     

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.     

Comparisons of diverse plant species reveal that only grasses show drastically reduced levels of ubiquitin monomer in mature pollen

Ubiquitin is a ubiquitous protein involved in targeting proteins for degradation. Maize pollen was previously reported (Callis and Bedinger 1994) to show extremely low levels of ubiquitin monomer, and developmental significance was attributed to this surprising feature of maize pollen. However, we had previously shown (Muschietti et al. 1994) that tomato pollen had high levels of ubiquitin monomer. Here we show that pollen from most plant families has high levels of ubiquitin monomer. Most grasses tested show reduced levels of ubiquitin monomer, but some maize inbred lines have higher levels of ubiquitin monomer than other inbreds. There was no correlation between the level of ubiquitin monomer and either the monocotyledonous or tri-cellular condition of grass pollen or the dehydrated condition of mature pollen. Since many aspects of pollen development (i.e., wall formation, microspore mitosis, synthesis and storage of mRNAs and proteins, carbohydrates and lipids, dehydration at maturity) are stereotypical among all plant families, the reduced level of ubiquitin monomer in pollen of many grasses cannot be crucial for any feature of normal pollen development.     

Western white pine (Pinus monticola Dougl.) reproduction: I. Gametophyte development

Male and female gametophyte development are described from light and transmission electron microscope preparations of ovules from first and second year Pinus monticola Dougl. seed cones. In the first year of development, pollen tubes penetrate about one-third the distance through the nucellus. The generative cell and tube nucleus move into the pollen tube. The megagametophyte undergoes early free nuclear division. First-year seed cones and pollen tubes become dormant in mid-July. In the second year, seed cones and pollen tubes resume development in April and the pollen tubes grow to the megagametophyte by mid-June. Early in June the generative cell undergoes mitosis, forming two equal-size sperm nuclei that remain within the generative cell cytoplasm. The generative cell has many extensions and abundant mitochondria and plastids. The megagametophyte resumes free nuclear division, then cell wall formation begins in early July. Cell wall formation and megagametophyte development follow the pattern found in other Pinaceae. Three to five archegonial initials form. The primary neck cell divides, forming one tier of neck cells. Jacket cells differentiate around each central cell. The central cell enlarges and becomes vacuolate; then vacuoles decrease in size and the cell divides, forming a small ventral canal cell and a large egg. Plastids in the central cell engulf large amounts of cytoplasm and enlarge. This process continues in the egg, and the peripheral cytoplasm of the egg becomes filled with transformed plastids. Mitochondria migrate around the nucleus, forming a perinuclear zone. The wide area of egg cytoplasm between these two zones has few organelles. A modified terminology for cells involved in microgametophyte development is recommended. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Pollen maturation: Where ubiquitin is not required?

A recent paper⁽¹⁾ describing the stage-specific loss of ubiquitin (UBQ) and ubiquitinated proteins (UBQ-Ps) during pollen development has raised some interesting questions regarding our understanding of the regulation of protein turnover during cellular differentiation and the specialized development of the pollen grain. The authors, Callis and Bedinger⁽¹⁾, describe experiments in which the profiles of free and protein-conjugated ubiquitin were examined during pollen development. UBQ and UBQ-Ps were immunologically detected in extracts of microspores and maturing pollen of maize at six developmental stages. Their results remarkably demonstrate that UBQ and UBQ-Ps decline to barely detectable levels during the final stages of pollen development.     

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana

Summary Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultra-structure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyo-somes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.     

朱顶红花粉萌发过程中营养细胞质的超微结构变化

本文应用透射电镜对朱顶红成熟花粉水合、活化和萌发的动态过程中营养细胞质的结构和组成变化进行了观察。成熟花粉具质体、线粒体、内质网、高尔基体。微丝束以聚集体的形式存在。花粉活化后,细胞器的数目和结构发生显著变化：质体和线粒体的片层明显增加,内质网片层狭窄,高尔基体活跃产生小泡,脂体降解及微丝聚集体散开。花粉萌发后,细胞质中出现周质微管和被刺小泡,此期细胞器的变化不明显。微丝以纤丝状遍布整个花粉管中。     

Intracellular motility,the actin cytoskeleton and germinability in the pollen of wheat (Triticum aestivum L.)

Summary The tricellular pollen of wheat germinates rapidly on a receptive stigma without the often protracted activation period characteristic of bicellular pollens. This is associated with a high level of hydration in the mature pollen and the absence of a dormancy period. Intracellular movement of organelles continues throughout development; in the mature pollen along pathways related both to the aperture site and the distribution of the amyloplasts in the vegetative cell. The movement pathways reflect the organisation of the actin cytoskeleton, elements of which are already focused upon the germination site at the time of dispersal, a disposition only achieved during rehydration and activation in bicellular pollens. Dehydration after dispersal rapidly arrests movement, disrupts the actin cytoskeleton and leads to loss of germinability. These effects are irreversible, again in contrast to the situation found in bicellular pollens such as those of the Liliaceae, species of which have been shown to be capable of withstanding several cycles of hydration and dehydration while still retaining some capacity for germination.