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.     

Developmental origins of structural diversity in pollen walls of Compositae

Compositae exhibit some of the most complex and diverse pollen grains in flowering plants. This paper reviews the evolutionary and developmental origins of this diversity in pollen structure using recent models based on the behaviour of colloids and formation of micelles in the differentiating microspore glycocalyx and primexine. The developmental model is consistent with observations of structures recovered by pollen wall dissolution. Pollen wall diversity in Compositae is inferred to result from small changes in the glycocalyx, for example ionic concentration, which trigger the self-assembly of highly diverse structures. Whilst the fine details of exine substructure are, therefore, not under direct genetic control, it is likely that genes establish differences in the glycocalyx which define the conditions for self-assembly. Because the processes described here for Compositae can account for some of the most complex exine structures known, it is likely that they also operate in pollen walls with much simpler organisation.     

Links between morphology and function of the pollen wall: an experimental approach

The wall of pollen grains exhibits morphological variation in many features including apertures, ornamentation and thickness, but the function of these characters remains to be clarified. It has been suggested that they are involved in the accommodation of volume changes (harmomegathy). To investigate this further, we developed a protocol that induces a controlled hydration of the pollen without affecting its metabolism and we applied it to six species differing in their pollen wall morphology. The entry of water caused pollen swelling and volume increase leading to breakage of the wall and/or of the plasma membrane, such that the per cent of intact grains was negatively correlated with the level of hydration. Qualitative and quantitative differences were observed between the species. Breakage of the exine was observed only in pollen lacking apertures and with thin exine. Variation in the exine ornamentation and thickness could explain the interspecific differences observed for the rates of breakage of the plasma membrane. Our results suggest that pollen wall morphology matters for survival and maintenance of pollen integrity further to volume increase due to hydration. We propose a rationale for future studies that should allow disentangling the contribution of different pollen morphological and physiological features to harmomegathy.     

Correlations between pollen exine sculpturing and angiosperm self-incompatibility systems — a reply

The exine morphology and pore size of pollen from diverse angiosperm taxa with homomorphic gametophytic, homomorphic sporophytic, and heteromorphic self-incompatibility have been studied. The results indicate that correlations alleged to exist between the type of self-incompatibility system and the nature of the pollen wall morphology should be treated with considerable caution.     

A new look at sporoderm ontogeny in Persea americana and the hidden side of development

Background and Aims

The phenomenon of self-assembly, widespread in both the living and the non-living world, is a key mechanism in sporoderm pattern formation. Observations in developmental palynology appear in a new light if they are regarded as aspects of a sequence of micellar colloidal mesophases at genomically controlled initial parameters. The exine of Persea is reduced to ornamentaion (spines and gemmae with underlying skin-like ectexine); there is no endexine. Development of Persea exine was analysed based on the idea that ornamentation of pollen occurs largely by self-assembly.

Methods

Flower buds were collected from trees grown in greenhouses over 11 years in order to examine all the main developmental stages, including the very short tetrad period. After fixing, sections were examined using transmission electron microscopy.

Key Results and Conclusions

The locations of future spines are determined by lipid droplets in invaginations of the microspore plasma membrane. The addition of new sporopollenin monomers into these invaginations leads to the appearance of chimeric polymersomes, which, after splitting into two individual assemblies, give rise to both liquid-crystal conical ‘skeletons’ of spines and spherical micelles. After autopolymerization of sporopollenin, spines emerge around their skeletons, nested into clusters of globules. These clusters and single globules between spines appear on a base of spherical micelles. The intine also develops on the base of micellar mesophases. Colloidal chemistry helps to provide a more general understanding of the processes and explains recurrent features of pollen walls from remote taxa.     

Shape and Size Modification of Recent Pollen Grains under Acetolysis

In this paper, the shape and size modification of recent pollen grains have been studied. 63 species of recent pollen grains that belongs to 55 genera, 26 families were observed before and after the acetolysis treatment with LM. The length of polar axis and equatorial axis and the thickness of exine were measured from about 20 pollen grains for each species (the thickness of exine were only measured for acetolysed pollen grains). The significance of shape and size modification was examined with the statistical method "ttest". From this investigati0n it can be concluded that: The pollen modification in size and shape 'was related to aperture type, pollen size, wall thickness and sculpture depth. Pantoporate, spheroidal pollens and small perporlate pollens usually change little in there size and shape after acetolysis. The size of colpate pollens was generally increased, or their shape changed. The size or shape change usually got bigger as the pollen size, wall thickness and sculpture depth became bigger and bigger. Oblate pollens were apter to change their size and shape than prolate pollens. The P/E ratio of most colpate pollens became a little bigger after acetolysis.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

Pollen morphology and phylogenetic relationships in neotropical Phyllanthus (Euphorbiaceae)

In order to provide new insights into phylogenetic relationships among the neotropical taxa of Phyllanthus , 28 illustrations are provided of the pollen grains of 22 selected species studied from 11 sections of the subgenera represented in the neotropics. Special attention has been given to subgenus Conami because of its variability in pollen morphology: of eight species illustrated, the apertures are diploporate colpi in three species and pores in five species; exine ornamentation is vermiculate in two species and pilate in the other six species. The six species in the neotropical sections Pityrocladus and Microglochidion (subgenus Emblica ) are characterized by prolate grains with an increased number of colpi (4–8). Of particular interest are species in which the pollen exine is clypeate (with exine shields); clypeate pollen grains are illustrated in two species of subgenus Xylophylla and in one species of section Cyclanthera that has unique exine shields with single central pila. The pollen of the one Brazilian phylloclade-bearing species illustrated (in section Choretropsis ) has 3-colporate grains with reticulate exine, typical for subgenus Phyllanthus , and very different from the clypeate grains of the West Indian phylloclade-bearing species in section Xylophylla . This pollen evidence clearly demonstrates homoplasy in the origin of phylloclades in Phyllanthus . Pollen morphological data suggest that the neotropical taxa of Phyllanthus have arisen following colonization from Africa (subgenus Kirganelia ) and Asia (subgenus Emblica ). © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 325–338.     

Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis

Callose (beta-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.     

Pollen wall development: the associated enzymes and metabolic pathways

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways.     

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.     

Features of ionogenic group composition in polymeric matrix of lily pollen wall

The composition of ionogenic groups and ion-exchange capacity were studied in the polymeric matrix of cell walls isolated from the pollen grain and tissues of vegetative organs (leaves and stems) of Lilium longiflorum Thunb. The ion-exchange capacity was evaluated at different pH values and ionic strength of 100 mM. In the two-layered pollen wall and the somatic cell walls four types of ionogenic groups were found: amino groups, two carboxyl groups (represented by residues of uronic and hydroxycinnamic acids), and phenolic OH-groups. The groups of all four types are present in the intine, whereas the exine contains one type of anion-exchange and two types of cation-exchange groups. The contents of each type group and their ionization constants were determined. The qualitative and quantitative compositions of structural polymers of the pollen intine and somatic cell walls are significantly different. It is suggested that hydroxycinnamic acids should be involved in cross-linking of polysaccharide chains in both the intine and somatic cell primary walls, and such cross-links play a crucial role in the structural organization and integrity of the pollen grain wall.     

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.     

Effects of high-temperature stress on microsporogenesis in heat-sensitive and heat-tolerant genotypes of Phaseolus vulgaris

High ambient temperature (32/27 °C, day/night, 12 h photoperiod) applied prior to anthesis to Phaseolus vulgaris plants results in abnormal pollen and anther development during microsporogenesis. Scanning and transmission electron microscopy were used to examine anther and pollen morphology and pollen wall architecture after heat stress was applied to two genotypes that differ with respect to yield potential under high‐temperature field conditions: one, a heat‐sensitive, Mesoamerican genotype, A55, the second, a heat‐tolerant, Andean genotype, G122. High‐temperature treatment of both genotypes was applied 1–13 d before anthesis. Under heat stress, the heat‐tolerant genotype showed anther and pollen characteristics that were generally similar to the low temperature controls. In contrast, after 9 d of heat treatment before anthesis, the anthers of the heat‐sensitive genotype were indehiscent and contained abnormal pollen. Pollen wall architecture was also affected in the 12 and 13 d treatments. In addition to the morphological changes, the heat‐sensitive genotype also experienced reduced pollen viability and reduced yield in high‐temperature experiments conducted in both the greenhouse and field.     

Ripe pollen structure and histochemistry of some gymnosperms

Some aspects of pollen cytology at dispersal were studied in 12 species of gymnosperms. The pollen grains differed in: 1. volume and cell number; 2. polarization of external structure and internal cell components; 3. wall thickness, especially of the intine, and the resulting percentage of cell volume with respect to total pollen grain volume; 4. stratification and chemical nature of the various intine layers; 5. nature and location of polysaccharide reserves; 6. morphological differences between the dry and hydrated states and phenomena related to hydration; 7. presence and site of orbicles. The various characters are compared and discussed in relation to the length of the reproductive cycle and the relations between the male gametophyte and its female counterpart.     

Fertile Arabidopsis cyp704b1 mutant,defective in sporopollenin biosynthesis,has a normal pollen coat and lipidic organelles in the tapetum

The exine acts as a protectant of the pollen from environmental stresses, and the pollen coat plays an important role in the attachment and recognition of the pollen to the stigma. The pollen coat is made of lipidic organelles in the tapetum. The pollen coat is necessary for fertility, as pollen coat-less mutants, such as those deficient in sterol biosynthesis, show severe male sterility. In contrast, the exine is made of sporopollenin precursors that are biosynthesized in the tapetum. Some mutants involved in sporopollenin biosynthesis lose the exine but show the fertile phenotype. One of these mutants, cyp704b1, was reported to lose not only the exine but also the pollen coat. To identify the cause of the fertile phenotype of the cyp704b1 mutant, the detailed structures of the tapetum tissue and pollen surface in the mutant were analyzed. As a result, the cyp704b1 mutant completely lost the normal exine but had high-electron-density granules localized where the exine should be present. Furthermore, normal lipidic organelles in the tapetum and pollen coat embedded between high-electron-density granules on the pollen surface were observed, unlike in a previous report, and the pollen coat was attached to the stigma. Therefore, the pollen coat is necessary for fertility, and the structure that functions like the exine, such as high-electron-density granules, on the pollen surface may play important roles in retaining the pollen coat in the cyp704b1 mutant.     

Contribution of upwind pollen sources to the characterization of Juniperus ashei phenology

Local and long-range components of Juniperus ashei pollen deposition were isolated to provide a more accurate record of local pollination activity in the Arbuckle Mountains of south central Oklahoma. An aerobiological sampler recorded airborne pollen concentrations and deposition at the sample site from mid-December 1998 to the end of January 1999. Grid-based weather data was used to model the movement, position, and elevation (air mass trajectories) across the region. While a normal concentration distribution is expected for a pollination event at a single site, "very high" concentrations (>1500 pollen grains per cubic meter) creating "peaks" in the deposition record were identified using bi-hourly sample analysis of the pollen registrations in the sampler. These occurrences happened over a 2 1/2 week period beginning January 11 and are coincident with the occurrence of southerly winds throughout the region. Modeled trajectories indicate that the air masses associated with those occurrences traveled at ground level through the J. ashei population on the Edwards Plateau, some 200 kilometers to the south in Texas, then gained altitude prior to crossing the sample site, thus introducing a long-range pollen component at the sample site. Peaks with "high" concentrations (90 to 1500 pollen grains per cubic meter) were evaluated using the same methodology. Those peaks associated with trajectories having the potential of introducing a long-range component to the pollen deposition record were removed from the aerobiological record. The resulting adjusted aerobiological record shows a more normal pollen concentration distribution, reduced hourly variability, and a marked shift in the pollination initiation date. Based on the comparison of non-adjusted and adjusted aerobiological records, contributions from upwind pollen sources account for 55% of the total pollen record.     

Pollen wall and tapetum development in Plantago major L. (Plantaginaceae): assisting self-assembly

We have attempted to elucidate the underlying mechanisms of sporoderm development and pattern determination in Plantago major through a detailed ontogenetic study, using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We aim to compare our observations and interpretation with those on other species. Our study of sporoderm development in Plantago from the early tetrad stage to mature pollen grains has shown that pure physical processes, including self-assembly, which are not under direct genetic control, play an important role and represent evidently one of the instruments of evolution. Our observations fit well with the sequence of self-assembling micellar mesophases and show reiteration of some of them, confirming our self-assembly hypothesis. Some attention was also paid to the possible role of rough and smooth endoplasmic reticulum in the cortical cytoplasm of the developing microspores. The tapetum and Ubisch bodies development are also traced. The importance of detailed ontogenetic studies for understanding the establishment of complex pollen walls in any species and for understanding mechanisms underlying sporoderm development was demonstrated. We also present a simulation, obtained in vitro experiments by self-assembly, mimicking pollen grain of Plantago major. It is clear that, in pollen wall development, biological processes and purely physical factors work in tandem.     

The Pollen Morphology and Exine Ultrastructure of Paeonia L in China

The pollen morphology of 9 species of Paeonia L. has been investigated with both light microscope and scanning electron microscope. In addition, the exine structure of pollen grains of Paeonia suffruticosa and P. lactiflora was examined by transmission electron microscope. Tricolporoidate aperture is an important character of the pollen grains of the Paeonia. The surface of the exine is characterized by reticulate, foveolate and irregularly tuberculate-foveolate sculpture under the SEM. Thin sections of the pollen of this genus shows that the layers of exine are complete i.e. a perforate rectum to semitectum, columellae and foot layers. The endexine is continuous, considerably thickened in the aperture areas and relatively thin or indistinct in the mesocolpia. Paeonia has been placed in Ranunculaceae. But since the beginning of this century many authors have suggested to separating Paeonia from Ranunculaceae. Pollen marphology supports such separation. In Ranunculaceae most pollen grains are tricolpate or have other types of aperture, and exine with spinules and perforations between them. In electron microscopy, the ektexine contains a foot layer, columellae, and perforate rectum, the columellar layer with two types of columellae; the endexine is generally thin. However, the columellar layer of Paeonia has only monomorphic columellae. Some authors considered that there is a close relationship between Paeonia and the Dilleniaceae, but these also differ in the characters of the pollen grains. In Paeonia the constriction of the colpus in equator is in some degree similar to that of Theaceae (Camellia sasanqua Thunb.), Guttiferae (Hypericum L.), Actinidiaceae and Rosaceae. But in the other respects they are quite different. In sum, the pollen morphology of Paeonia is unique. So the palynological information supports Takhtajan's view that Paeonia should be elevated to a family (Paeoniaceae) or order (Paeoniales).