Ontogenesis in miniature. Pollen wall development in Campanula rapunculoides.

MAIN CONCLUSION: Our findings suggest a reconsideration of pollen wall ontogeny process, entailing examination of physical factors, which enable a new understanding of exine developmental processes as self-formation. The pollen wall, the most complex cell wall in plants, is especially interesting as a model of ontogeny in miniature. By a detailed study of each developmental stage of Campanula rapunculoides pollen wall, we aimed to understand the establishment of complex pollen walls and the underlying developmental mechanisms. Other aim was to compare our current observations with studies in other species to reveal the common principles. We also tried to analyse the reasons for commonalities in ontogenies of exines in remote species. TEM, SEM, comparative methods were used in this study. The sequence of events leading to exine emergence from early tetrad stage to maturity is as follows: the appearance of spherical micelles in the periplasmic space and de-mixing of the mixture in periplasm (condensed and depleted layers); appearance of plasma membrane invaginations and columns of spherical micelles inside condensed layer; appearance of rod-like units, pro-tectum and thin foot layer; the appearance of spiral substructure of procolumellae and of dendritic outgrowths on the tops of procolumellae, of vast depleted zone in aperture sites; formation of the endexine lamellae on the base of laminate micelles; gradual twisting of dendritic outgrowths (macromolecule chains) into clubs on the tops of columellae and into spines; final sporopollenin accumulation. Our observations are consistent with the sequence of self-assembling micellar mesophases. Complex organisation of the exine is established through processes of self-assembly operating together with another physical process-phase separation. After genomic determination of the exine building substances, purely physical processes which are not under direct genomic control play an important role after genomic control of constructive substances. The comparison of the underlying mechanisms of exine development in remote species occurred to be general and similar to crystallisation. Our ontogenetic experience has shown the commonality of pollen wall ontogenies in remote species.

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.

Artificial pollen walls simulated by the tandem processes of phase separation and self-assembly in vitro.

Despite numerous attempts to elucidate the developmental mechanisms responsible for the observed diversity of pollen and spore walls, the processes involved remained obscure until the structures observed during exine development were recognized as a sequence of self-assembling micellar mesophases. To confirm this, a series of in vitro experiments was undertaken in which exine-like patterns were generated in colloidal mixtures by self-assembly, without any genomic participation. The intention was to test whether all the main types of exine structure could be simulated experimentally. Mixtures of substances, analogous to those involved in microspore development, were left undisturbed while water evaporated and self-assembly occurred. We varied the substances, their combinations and concentrations, and the physical constraints to make the experiments closer to the situation in nature. The resulting dry films were then examined using transmission electron microscopy. A variety of microstructures, simulating the full range of exine types, was obtained by micellar self-assembly. Moreover, the signs of related physicochemical process (i.e. phase separation) were also observed. Simple, energy-efficient, physical-chemical interactions, phase separation and self-assembly, are capable of generating exine-like patterns, providing evidence that these processes share control of exine formation with the well-documented program of gene expression.

Mimicking pollen and spore walls: self-assembly in action.

BACKGROUND AND AIMS: Decades of research have attempted to elucidate the underlying developmental mechanisms that give rise to the enormous diversity of pollen and spore exines. The organization of the exine starts with the establishment of an elaborate glycocalyx within which the subsequent accumulation of sporopollenin occurs. Ontogenetic studies using transmission electron microscopy of over 30 species from many different groups have shown that the sequence of structures observed during development of the exine corresponds to the sequence of self-assembling micellar mesophases (including liquid crystals) observed at increasing concentrations of surfactants. This suggested that self-assembly plays an important part in exine pattern determination. Some patterns resembling separate layers of spore and pollen grain walls have been obtained experimentally, in vitro, by self-assembly. However, to firmly establish this idea, columellate and granulate exines, the most widespread forms, needed to be simulated experimentally. METHODS: We used our original method, preparing mixtures of substances analogous to those known to occur in the periplasmic space of developing microspores, then leaving the mixtures undisturbed for specific periods of time to allow the process of self-assembly to occur. We developed our method further by using new substances analogous to those present in the periplasmic space and performing the experiments in a thin layer, more closely resembling the dimensions of the periplasmic space. KEY RESULTS: The artificial microstructures obtained from our in vitro self-assembly experiments closely resembled the main types of exines, including tectate-columellate, granulate, alveolate and structureless, and permitted comparison with both developing and mature microspore walls. Compared with the previous attempts, we managed to simulate columellate and granulate exines, including lamellate endexine. CONCLUSIONS: Our results show that simple physico-chemical interactions are able to generate patterns resembling those found in exines, supporting the idea that exine development in nature involves an interplay between the genome and self-assembly.

Assembling the thickest plant cell wall: exine development in Echinops (Asteraceae, Cynareae).

MAIN CONCLUSION: The exceptionally complex exine of Echinops, representing a significant investment of energy, develops from an elaborate glycocalyx which establishes, by self-assembly, a multi-layered system of micelles upon which sporopollenin polymerizes. We report on pollen development in two species of Echinops (Asteraceae, Cynareae) studied using transmission and scanning electron microscopy with an emphasis on the organisation and development of the massive sporoderm (maximum thickness 18 µm). The major events of exine deposition during the tetrad stage follow the now familiar sequence of self-assembling micellar mesophases and the subsequent incorporation of sporopollenin, observed here as: (1) spherical units with light cores; (2) columns of spherical units with dark cores; (3) large branched macromolecules arranged in a dendritic, three-dimensional network of long alveoli; and (4) alveoli with electron-transparent cores and dense walls. Later, (5) the primexine exhibits an elongated-alveolate pattern in which the alveoli have electron-dense cores and lighter exteriors. When (6) the thick inner columellae make contact with the outer primexine, sporopollenin accumulation in the cores of the primexine alveolae establishes continuity between the inner and outer columellae. In the free microspore stage, (7) the foot layer and first lamellae of the endexine appear (8). The endexine lamellae then increase in number and massive accumulation of sporopollenin occurs on all exine elements, making individual elements such as tectal spines, more pronounced. These and earlier findings, as well as experimental simulations of exine development, show that pollen wall morphogenesis involves a subtle interplay of gene-driven biological processes and physico-chemical factors offering abundant opportunities for the generation of complex, taxon-specific patterns.

Self-assembly as the underlying mechanism for exine development in Larix decidua D. C.

MAIN CONCLUSION: Our findings suggest a new approach to pollen ontogenetic investigations, entailing consideration of physical factors, which enable a better understanding of exine developmental processes. The sporopollenin-containing part of the pollen wall-the exine-is one of the most complex cell walls in plants. By tracing each stage of microspore development in Larix decidua with TEM, we aimed to understand the underlying mechanisms of its exine establishment. Our hypothesis is that self-assembly interferes with exine development. Our specific aim is to generate experimental simulations of the exine developmental pattern. The sequence of events leading to exine development includes the appearance of spherical units in the periplasmic space, their rearrangement into radial columns, and the appearance of white-lined endexine lamellae. The final accumulation of sporopollenin proceeds in the post-tetrad period. The sequence of self-assembling micellar mesophases corresponds with that of the developmental events: spherical micelles; columns of spherical micelles; and laminate micelles separated by strata of water and visible as white-lined lamellae in TEM. Several patterns, simulating structures at different stages of exine development in Larix, were obtained from in vitro experiments. Purely physicochemical processes of self-assembly, which are not under direct genetic control, play an important role in exine development and share control with the genome. These findings suggest that a new approach to ontogenetic investigations, entailing consideration of physical factors (e.g., cell tensegrity), is required for a better understanding of developmental processes.

Pollen wall development in Impatiens glandulifera: exine substructure and underlying mechanisms.

The aim of this study was to investigate in detail the pollen wall ontogeny in Impatiens glandulifera, with emphasis on the substructure and the underlying mechanisms of development. Sporopollenin-containing pollen wall, the exine, consists of two parts, ectexine and endexine. By determining the sequence of developing substructures with TEM, we have in mind to understand in which way the exine substructure is connected with function. We have shown earlier that physical processes of self-assembly and phase separation are universally involved in ectexine development; currently, we try to clear up whether these processes participate in endexine development. The data received were compared with those on other species. The ectexine ontogeny of I. glandulifera followed the main stages observed in many other species, including the late tetrad stage named "Golden gates". It turned out that the same physico-chemical processes act in endexine development, especially expressed in aperture sites. Another peculiar phenomenon observed in exine development was the recurrency of micellar sequence at near-aperture and aperture sites where the periplasmic space is widened. It should be noted that, in the whole, the developmental substructures observed during the tetrad and early post-tetrad period are similar in species with columellate exines. Evidently, these basic physical processes proceed, reiterating again and again in different species, resulting in an enormous variety of exine structures on the base of a relatively modest number of genes. Granular and alveolar exines emerge on the base of the same basic processes but are arrested at spherical and cylindrical micelle mesophases correspondingly.

Pollen wall and tapetal development in Cymbalaria muralis: the role of physical processes, evidenced by in vitro modelling.

Our aim was to unravel the underlying mechanisms of pollen wall development in Cymbalaria muralis. By determining the sequence of developing substructures with TEM, we intended to compare it with that of other taxa and clarify whether physical processes of self-assembly and phase separation were involved. In parallel, we tried to simulate in vitro the substructures observed in Cymbalaria muralis exine development, using colloidal mixtures, to determine whether purely physical self-assembly processes could replicate them. Exine ontogeny followed the main stages observed in many other species and was initiated by phase separation, resulting in heterogeneity of the homogeneous contents of the periplasmic space around the microspore which is filled with genome-determined substances. At every stage, phase separation and self-assembly come into force, gradually driving the substances through the sequence of mesophases: spherical micelles, columns of spherical micelles, cylindrical micelles arranged in a layer, laminate micelles. The final two of these mesophases define the structure of the columellate ectexine and lamellate endexine respectively. Structures obtained in vitro from colloidal mixtures simulated the developing exine structures. Striking columella-like surface of some abnormal tapetal cells and lamella-like structures in the anther medium confirm the conclusion that pattern generation is a feature of colloidal materials, after genomic control on material contents. Simulation experiments show the high pattern-generating capacity of colloidal interactions.

Pollen wall development in Hydrangea bretschneiderii Dippel. (Hydrangeaceae): advanced interpretation through physical input, with in vitro experimental verification.

We aimed to unravel the underlying mechanisms of pollen wall development in Hydrangea bretschneiderii. For this, we tested our hypothesis that distinct physical processes, phase separation and micellar self-assembly, underpinned exine development by taking the substances, determined by the genome, through several phase transitions. We traced each developmental stage with TEM; then, we obtained in vitro simulations corresponding to those stages. The main steps of exine ontogeny observed in the microspore periplasmic space were initiated with phase separation, resulting in the conversion of homogeneous contents to heterogeneous two-layered state of the material. After each step of phase, separation self-assembly picked up the initiative and took the substances through the sequence of micellar mesophases which were the base for all the exine structures. These mesophases are as follows: spherical micelles, transforming first into columns, and then to cylindrical micelles which turn to columellae after initial sporopollenin accumulation. The tectum appeared along the interface of the phase separated material. After the tetrad disintegration and the next phase separation, laminate mesophase appeared being the base for the endexine lamellae. Then, a new step of phase separation at aperture sites brought the appearance of a granular endexine layer; the latter became intermixed finally with lamellae. This gives, together with experimental simulation, strong evidence that the genome "shifts a part of work" on exine formation onto physical processes, and the latter are an inherent mechanism of evolution.

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 ornamentation (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.

Pollen wall ontogeny in Polemonium caeruleum (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.

Sporoderm and tapetum development in Eupomatia laurina (Eupomatiaceae). An interpretation.

For the first time, the developmental events in the course of exine structure establishment have been traced in detail with TEM in Eupomatia, with the addition of cytochemical tests. A new look at unfolding events is suggested using our recent hypothesis on self-assembling micellar mesophases. The process proved to be unusual and includes "ghost" stages. The first units observed in the periplasmic space are spherical ones (= normal spherical micelles). These accumulate, resulting in a granular layer up to middle tetrad stage. Sporopollenin precursor accumulation on these units makes the ectexine layer looking as homogenous at late tetrad stage. Simultaneously, the columns of globules are added in the periplasmic space, which reminds an attempt to form columellae; but, the process failed. Instead, a fimbrillate endexine layer of compressed globules appears. The latter augments via additional globules, appearing in the periplasmic space in the free microspore period. The endexine formation is double-stepped spatially and temporally. The second, lamellate endexine layer (laminate micelles) appears late in development, when the channeled intine-I is already established-a very unusual feature. Moreover, a "fenestrated" stage comes unexpectedly at vacuolate stage, when hitherto amorphous ectexine appears pierced by cavernae-the results of reversal of normal spherical micelles (constituents of ectexine) to reverse the ones that open their cores for the entrance of hydrophilic nutrients from tapetum and give them over to the microspore cytoplasm by exchanging their solubilizates.

Sporoderm development in Acer tataricum (Aceraceae): an interpretation.

For the first time, the developmental events in the course of complicated exine structure establishment have been traced in detail with transmission electron microscope in the representative of Acer. A new look at unfolding events is suggested using the knowledge of a boundary field, colloid science. Our purpose was to find out whether the sequence of sporoderm developmental events represents, in essence, the sequence of self-assembling micellar mesophases, initiated by genomically given physicochemical parameters and induced by surfactant glycoproteins at increasing concentration. Indeed, the first units observed in the periplasmic space are globular ones (=spherical micelles) which become arranged into rod-like units (=cylindrical micelles). Then, twisted clusters of rodlets form a layer of procolumellae (middle micellar mesophase). The tectum emerges as an untwisting and merging of distal ends of procolumellae (distal untwist of micelle clusters). In the end of tetrad period, when a hydrophilic-hydrophobic switch occurs in the periplasmic space, the contrast reversal of the columellae corresponds to the change of normal micelles to reverse ones. The initiation of the foot layer and the endexine lamellae, with their typical central "white lines", corresponds to the next-"neat"-mesophase, with its typical central gaps between layers. Aperture sites during development show all the main micellar mesophases and their transitional forms. The data received have supported our previous hypothesis.