Pollen wall development in flowering plants

The outer pollen wall, or exine, is more structurally complex than any other plant cell wall, comprising several distinct layers, each with its own organizational pattern. Since elucidation of the basic events of pollen wall ontogeny using electron microscopy in the 1970s, knowledge of their developmental genetics has increased enormously. However, self-assembly processes that are not under direct genetic control also play an important role in pollen wall patterning. This review integrates ultrastructural and developmental findings with recent models for self-assembly in an attempt to understand the origins of the morphological complexity and diversity that underpin the science of palynology.     

Development of the echinate pollen wall inFarfugium japonicum (Compositae: Senecioneae)

Development of the echinate pollen grains inFarfugium (Compositae: Senecioneae) has been studied by a combination of transmission electron microscopy and field emission scanning electron microscopy with a freeze fractured method. The inner surface of the callose wall surrounding each microspore does not possess an echinate pattern before primexine deposition begins. The primexine formation coincides with the initiation of spines. The freeze fractured primexine shows probacula which form transverse rods. The developing exine has an inner spongy substructure. The endexine is formed by the accumulation of the electron dense lamellae with white lines after the dissolution of the callose wall. In the present study, it is confirmed that the developmental process of pollen formation revealed in the field emission scanning electron microscope is consistent with the results obtained using the transmission electron microscope.     

Purification of maize pollen exines and analysis of associated proteins

Zea mays (maize) pollen exines have been purified with the use of differential centrifugation and sucrose gradients, followed by mild detergent and high salt treatment. The final exine fraction is highly purified from other organelles and subcellular structures as assayed by transmission electron microscopy. Using mature maize pollen as the starting material, 0.2 to 0.3% of the total pollen protein remained associated with the exine fraction throughout the purification. Seven abundant sodium dodecyl sulfate-extractable proteins are detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the final fraction. Amino acid analysis reveals that one of the proteins contains a substantial amount of hydroxyproline, a characteristic of some primary cell wall proteins. The amino acid composition of the 25-kD protein strongly implies that it is an arabinogalactan protein. When exines are purified from earlier pollen developmental stages, a subset of the proteins found in the mature pollen exine is seen.     

An integral insight into pollen wall development: involvement of physical processes in exine ontogeny in Calycanthus floridus L., with an experimental approach

We aimed to understand the underlying mechanisms of development in the sporopollenin-containing part of the pollen wall, the exine, one of the most complex cell walls in plants. Our hypothesis is that distinct physical processes, phase separation and micellar self-assembly, underpinexine development by taking the molecular building blocks, determined and synthesised by the genome, through several phase transitions. To test this hypothesis, we traced each stage of microspore development in Calycanthus floridus with transmission electron microscopy and then generated in vitro experimental simulations corresponding to every developmental stage. The sequence of structures observed within the periplasmic space around developing microspores starts with spherical units, which are rearranged into columns to then form rod-like units (the young columellae) and, finally, white line centred endexine lamellae. Phase separation precedes each developmental stage. The set of experimental simulations, obtained as self-assembled micellar mesophases formed at the interface between lipid and water compartments, was the same: spherical micelles; columns of spherical micelles; cylindrical micelles; and laminate micelles, separated by gaps, resembling white-lined lamellae. Thus, patterns simulating structures observed at the main stages of exine development in C. floridus were obtained from in vitro experiments, and hence purely physicochemical processes can construct exine-like patterns. This highlights the important part played by physical processes that are not under direct genomic control and share influence on the emerging ultrastructure with the genome during exine development. These findings suggest that a new approach to ontogenetic studies, including a consideration of physical factors, is required for a better understanding of developmental processes.     

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.     

Pollen morphology of Zehneria s.l. (Cucurbitaceae)

A growing body of experimental data obtained from sporoderm ontogenetic studies led to the appearance of the ‘micellar’ hypothesis. The hypothesis is that the sequence of sporoderm developmental events represents the sequence of self-assembling micellar mesophases, initiated by genomically given physico-chemical parameters, which are then picked up by physico-chemical self-assembly. However, besides morphological evidence, the best proof of this hypothesis would be an experimental modelling of sporoderm-like patterns. The main idea of this study is to remove the influence of the genome, selecting substances and their concentrations for simulations to replace it, and then to trace what ‘pure’ self-assembly is capable of constructing. Our aim in this study was to simulate mainly young structures in sporoderm development, i.e. the glycocalyx and the primexine. Several polysaccharide gels (as a callose substitute) and surfactants (as glycocalyx and sporopollenin monomer substitutes) were mixed at different concentrations and combinations, thermally set and left to condense. A number of patterns were obtained in colloidal solutions in the course of condensation, simulating structures at different stages of exine development. Their structures were observed and analysed with transmission electron microscopy (TEM). Our first experiments on the modelling of biological patterns in vitro have shown encouraging results.     

Merging concepts: The role of self-assembly in the development of pollen wall structure

In this article, we analyse established details of exine development from a perspective that favours the integration of self-assembly. We isolate those intervals in development in which genomic control is exercised and offer a number of scenarios, which show how self-assembly can build upon a genetic basis to give rise to the fundamental pollen exine structure. This paper is a synthesis of a new concept and a detailed review of achievements in the field of developmental palynology. It seeks to link what is known regarding development with the liquid crystal realm of colloid chemistry.     

Exine and tapetum development in Symphytum officinale (Boraginaceae). Exine substructure and its interpretation

After detailing the exine ontogeny, our purpose was to find out whether the sequence of sporoderm developmental events corresponds to self-assembling micellar mesophases, initiated by genomically determined physicochemical parameters and induced by surfactant glycoproteins at increasing concentrations. Indeed, a scaffolding of the future exine, i.e., the glycocalyx, initiates with scattered clots, which then appear as clusters of spherical and worm-like micelles, derived from surface-active glycoproteins. At the middle tetrad stage, a continuous layer of the glycocalyx emerges, consisting of parallel, tightly packed cylinder-like units, which we interpret as a layer of cylindrical micelles, the so-called middle mesophase. These units bear dark-contrasted particles, arranged in strings or columns. These sites of the glycocalyx units?Cmicelles accumulate initial sporopollenin, hence the term ??sporopollenin acceptor particles?? (SAPs). This process leads to the appearance of procolumellae at the late tetrad stage. The glycocalyx units are rooted into callose and into the microspore cytoplasm. After formation of the tectum and the foot layer, the endexine initiates as a thin layer, and the latter develops into a very thick layer in the post-tetrad period. When callose disintegrates, ??bouquets?? of SAPs become evident on the tectum, which were evidently hidden inside the callose layer; these structures self-assemble into supratectal gemmae. An unusual, ??hybrid?? type of tapetum was observed. What is observed in Symphytum exine development allows us to obtain more evidence for the hypothesis of the participation of micellar self-assembly in sporoderm development and to bring together the concepts of micelles and of SAPs.     

A glycine-rich protein that facilitates exine formation during tomato pollen development

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.     

Role of genetic control and self-assembly in gametophyte sporoderm ontogeny: Hypotheses and experiment

A review of our own and literature data on the mechanisms of sporoderm (the wall of pollen grains and spores) development is presented in terms of colloidal interactions—the so-called micellar hypothesis (Gabarayeva and Hemsley, 2006; Hemsley and Gabarayeva, 2007), which suggests the participation of self-assembly processes in development. The development of exine (sporopollenin-containing part of the sporoderm) in five plant species from remote taxa has been traced in detail and interpreted as a micellar sequence. An experimental modeling of exine-like structures carried out in vitro, in which physicochemical patterns of colloidal systems (hydrophobic interactions) were the driving force, is strong evidence for the relevance of the micellar hypothesis and the promising nature of these studies. The correlation between the role of genomic control and self-assembly in the development of complex biological walls is discussed.     

Pollen wall ontogeny in <Emphasis Type="Italic">Polemonium caeruleum</Emphasis> (Polemoniaceae) and suggested underlying mechanisms of development

By a detailed ontogenetic study of Polemonium caeruleum pollen, tracing each stage of development at high TEM resolution, we aim to understand the establishment of the pollen wall and to unravel the mechanisms underlying sporoderm development. The main steps of exine ontogeny in Polemonium caeruleum, observed in the microspore periplasmic space, are spherical units, gradually transforming into columns, then to rod-like units (procolumellae), the appearance of the initial tectum, growth of columellae in height and tectum in thickness and initial sporopollenin accumulation on them, the appearance of the endexine lamellae and of dark-contrasted particles on the tectum, the appearance of a sponge-like layer and of the intine in aperture sites, the appearance of the foot layer on the base of the sponge-like layer and of spinules on the tectum, and massive sporopollenin accumulation. This sequence of developmental events fits well to the sequence of self-assembling micellar mesophases. This gives (together with earlier findings and experimental exine simulations) strong evidence that genome and self-assembly probably share control of exine formation. It is highly probable that self-assembly is an intrinsic instrument of evolution.     

Exine development: the importance of looking through a colloid chemistry ``window'

Most biological construction systems operate within the colloidal dimension. In view of this, it seems reasonable to reassess what is known of the early stages of exine development in the light of a brief excursion into colloid and micelle behaviour. The results of this analysis show remarkable similarity of structures and suggest that almost all of the features seen during early pollen wall development can be easily interpreted using simple, established colloidal principles. This study of exine framework and endexine development offers the possibility that growth of the early exine progresses by successive transitory mesophases of a constrained micellar system. The self-assembling micelle mesophases will all be clearly recognized as constituents of the developing exine. They include spherical, cylindrical, continuous layers of hexagonally-packed cylindrical units and lamellar mesophases which most probably correspond to future granules, columellae, complex columellar (and alveolar) microarchitecture and ``white-line-centred' lamellae. Furthermore, the various types of micelle involved have the potential to perform the functions previously loosely assigned to the exine.     

Hydration, sporoderm breaking and germination of Cupressus arizonica pollen

I n vitro and in vivo rehydration and germination in Cupressus arizonica pollen were examined using light and scanning electron microscopy. Shed pollen has 12.6% water content, which reduced to 8.2% after dispersal, and this latter pollen survived for some months at room temperature and for years at −10 °C. Rehydration requires breaking of the sporoderm walls and depends on the composition and pH of the rehydration medium. Acidity restrains the breakage, while alkalinity promotes it. Pollen division follows exine shedding and requires the persistence of the mucilaginous layer; hence, pH values countering these outcomes prevent division. Division results in a large and a small cell separated by a callosic wall. A pollen tube develops from the innermost intine of the large cell, which is callosic, and extends into the mucilaginous middle intine. The percentage germination never exceeded 17% in all tested media. In vivo , pollen rehydrates and casts off the exine in the micropylar drop. Drop withdrawal brings pollen to the apical nucellar cells that degenerate in the meantime, and it leaves a deposit on the surface of the micropylar canal. After contaction of the nucellar cells, the pollen flattens and its mucilaginous layer shrinks and disappears. This occurs simultaneously with sealing of the micropylar canal. During this time, pollen divides asymmetrically without the callosic wall, and the larger cell develops a tube in the interface with the nucellus. Only some pollen grains accomplish adhesion to the nucellus and germinate. The in vitro and in vivo developmental stages are discussed.     

The fine structure of the pollen wall inStrelitzia reginae (Musaceae)

The pollen wall ofStrelitzia reginae (Musaceae) consists of a nearly unsculptured, very thin, highly reduced, but coherent exine, and a thick intine (with an outer, channeled layer and an inner, largely homogeneous layer). After short, incomplete acetolysis the exine covers the remaining, severely shrinked protoplast as a folded, but unaltered “skin”, while the intine has totally disappeared. After extended acetolysis only the coherent, skin-like exine remains. Thus, the term “exine-less pollen” sometimes used for similar sporoderm structures in other genera ofZingiberales is misleading and should be substituted by the term “skin-like exine”. Surprisingly, the peculiar pollen wall ultrastructure ofStrelitzia and some otherZingiberales is very similar to that of some genera of theLaurales, an example for convergent evolution within the angiosperms.     

EVOLUTION OF EXINE STRUCTURE IN THE POLLEN OF PRIMITIVE ANGIOSPERMS

Diversity in the structure of the exine in 35 families of the ranalean complex is compared through a series of representative scanning electronmicrographs, and evolutionary trends in exine structure of primitive angiosperms are outlined, along with discussion of the significance of these data for understanding the evolution of exine structure in flowering plants as a whole. In order to reduce ambiguity in the palynological literature, it is suggested that persons undertaking light microscope studies of unstained, acetolyzed pollen grains adopt the morphological terms sexine-nexine in describing pollen wall layers while restricting their use of the chemically defined terms ektexine-endexine largely to pollen studies carried out with the transmission electron microscope. This study emphasizes that a clear understanding of the palynological concept of structure versus sculpturing is a necessary prerequisite for the taxonomic/ phylogenetic use of pollen wall morphology. Finally, data from investigation of a number of ranalean families of primitive angiosperms support the conclusion that the direction of a recurrent and major evolutionary trend in exine structure of flowering plants proceeds from pollen that is tectate-imperforate to tectate-perforate pollen to semitectate pollen, and more rarely, to pollen grains that are intectate.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

Ultrastructure of microsporogenesis and microgametogenesis in Campsis radicans (L.) Seem. (Bignoniaceae)

In the present study, microsporogenesis, microgametogenesis and pollen wall ontogeny in Campsis radicans (L.) Seem. were studied from sporogenous cell stage to mature pollen using transmission electron microscopy. To observe the ultrastructural changes that occur in sporogenous cells, microspores and pollen through progressive developmental stages, anthers at different stages of development were fixed and embedded in Araldite. Microspore and pollen development in C. radicans follows the basic scheme in angiosperms. Microsporocytes secrete callose wall before meiotic division. Meiocytes undergo meiosis and simultaneous cytokinesis which result in the formation of tetrads mostly with a tetrahedral arrangement. After the development of free and vacuolated microspores, respectively, first mitotic division occurs and two-celled pollen grain is produced. Pollen grains are shed from the anther at two-celled stage. Pollen wall formation in C. radicans starts at tetrad stage by the formation of exine template called primexine. By the accumulation of electron dense material, produced by microspore, in the special places of the primexine, first of all protectum then columellae of exine elements are formed on the reticulate-patterned plasma membrane. After free microspore stage, exine development is completed by the addition of sporopollenin from tapetum. Formation of intine layer of pollen wall starts at the late vacuolated stage of pollen development and continue through the bicellular pollen stage.     

Pollen wall characters with emphasis upon applicability

The message for exine pattern resides ultimately in the genome, yet the information for the initial form exists in the cytoplasm or with the plasma membrane and its glycocalyx. Subsequent wall development is likely to be the result of an interplay between the genome, the cytoplasm, and the intralocular environment. The exine consists of units derived from the plasma membrane glycocalyx and enveloped in the exinous polymer sporopollenin. Growth of the exine in accomodation to cell surface expansion is modeled as involving a doubling in diameter of the units, separation of components, and incorportation of new units within the nexine but not the tectum. If the tectum is thick and does not become disjunct, its restraint upon cellular expansion may result in the crushing of bacules. Both the final shape and ornamentation of the exine may be influenced by cytological processes like oncoid plugs that limit the effect of protoplast expansion to nonapertural regions or globules in the exine arcade that can cause distention of the tectum and rupturing of bacules. Subunits of exinous units can be seen in distinctive patterns at the outer surface of the exine, in the arcade of the exine, and prior to intine formation at the inner surface of the nexine.     

Sporopollenin monomer biosynthesis in arabidopsis

Land plants have evolved aliphatic biopolymers that protect their cell surfaces against dehydration, pathogens, and chemical and physical damage. In flowering plants, a critical event during pollen maturation is the formation of the pollen surface structure. The pollen wall consists essentially of the microspore-derived intine and the sporophyte-derived exine. The major component of the exine is termed sporopollenin, a complex biopolymer. The chemical composition of sporopollenin remains poorlycharacterized because it is extremely resistant to chemical and biological degradation procedures. Recent characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated that the sporopollenin polymer consists of phenolic and fatty acid-derived constituents that are covalently coupled by ether and ester linkages. This review illuminates the outlines of a biosynthetic pathway involved in generating monomer constituents of the sporopollenin biopolymer component of the pollen wall.     

The Novel Plant Protein INAPERTURATE POLLEN1 Marks Distinct Cellular Domains and Controls Formation of Apertures in the Arabidopsis Pollen Exine

Pollen grains protect the sperm cells inside them with the help of the unique cell wall, the exine, which exhibits enormous morphological variation across plant taxa, assembling into intricate and diverse species-specific patterns. How this complex extracellular structure is faithfully deposited at precise sites and acquires precise shape within a species is not understood. Here, we describe the isolation and characterization of the novel Arabidopsis thaliana gene INAPERTURATE POLLEN1 (INP1), which is specifically involved in formation of the pollen surface apertures, which arise by restriction of exine deposition at specific sites. Loss of INP1 leads to the loss of all three apertures in Arabidopsis pollen, and INP1 protein exhibits a unique tripartite localization in developing pollen, indicative of its direct involvement in specification of aperture positions. We also show that aperture length appears to be sensitive to INP1 dosage and INP1 misexpression can affect global exine patterning. Phenotypes of some inp1 mutants indicate that Arabidopsis apertures are initiated at three nonrandom positions around the pollen equator. The identification of INP1 opens up new avenues for studies of how formation of distinct cellular domains results in the production of different extracellular morphologies.