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.     

I. Primexine development in Passiflora racemosa Brot.: overlooked aspects of development

Developmental stages during the tetrad period were examined in detail by transmission electron microscopy with an emphasis on substructure. Our purpose was to find out whether the sequence of sporoderm developmental events provides additional evidence for our recent hypothesis on the underlying cause of exine ontogeny as a sequence of self-assembling micellar mesophases initiated by genomically given physicochemical parameters. Osmiophilic globules encrusting the surface of postmeiotic microspores and tapetal cells are temporary prepattern units which come first. The second prepattern structures are highly ordered bundles of microfilaments and microtubules which determine the position of microspore surface invaginations and clusters of the glycocalyx inside them. The first glycocalyx units are microgranules which during the middle tetrad stage rearrange into radially oriented rod-like units. The latter form lens-like clusters of the glycocalyx-1, located inside the invaginations. These clusters predestine the position of the future luminae in the exine reticulum. The second glycocalyx layer is laid down as a continuous layer over the whole microspore surface and has similar substructure, that is radial rods. Glycocalyx-2 is a framework for procolumellae which appear at the late tetrad stage. Therefore, the sequence of substructural units in the primexine is: globules, microgranules, rod-like units, and layers of radially oriented rods tightly packed in the periplasmic space. This sequence corresponds to the first three mesophases of self-assembling micelles: spherical micelles, cylindrical micelles, and layers of hexagonally packed cylindrical micelles (middle mesophase). We observed the same sequence in other species during primexine development, and the findings of this study provide new evidence for our hypothesis.     

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.     

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.     

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.     

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.     

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.     

A route for the passage of substances through the developing pteridophyte exine

Summary The developing exine ofLycopodium gnidioides is traversed from the outer to the inner surface by a series of anastomosing channels filled or lined with fibrillar glycoprotein. When living sporangia are incubated in colloidal iron, particles of ferric iron can be detected in the exine channels, the intine and the spore cytoplasm, and some iron is retained by the surface coatings on the spore. Although there is some diffuse iron staining of the exine between the channels, the main concentration of particles is associated with these structures. This, together with the fact that the proximal lasurae of the spore are closed during development, is taken as evidence that the iron has passed from the locular fluid to the surface of the protoplast principally by way of the exine channels. Results obtained from fixation in a glutaraldehyde-lanthanum nitrate mixture support this interpretation. While the exine channels are in existence, therefore, the spore protoplast is in open communication with the locular environment. The study provides no evidence to suggest that the iron which entered the spore cytoplasm did so by endocytosis. It is possible that iron altered the permeability of the plasma membrane by damaging its structure; entry of iron to the cytoplasm being effected through the damaged membrane.     

POLLEN GRAIN DEVELOPMENT AND TAPETAL CHANGES IN STRELITZIA REGINAE (STRELITZIACEAE)

The proexine that forms within the callosic envelope before the end of the microspore tetrad period is thick (about 1 μm) and exceptionally complex. It has components equatable with tectum, columellae, and a nexine that includes lamellar zones. All these components persist in the exine although late in development they become difficult to recognize because this exine is reduced in thickness, apparently by stretching, to a maximum of 0.2 μm. Strelitzia is an example of an exine template, with receptors for sporopollenin, that is not maintained during development. The Strelitzia microspore surface changes from an exine like that on an interaperture sector to the channeled intinelike system common for the apertures of pollen grains. The exine on sterile grains gives what may be a rare view of a stabilized immature exine. The mature exine on viable pollen grains resembles this early exine only in the most impressionistic way. Tapetal cells go through at least one cycle of hyperactivity, dedifferentiation, mitosis, and then again hyperactivity before they finally decline.     

Exine formation in the pollinium ofDendrobium

Summary The position of the callose wall is related to the position of the primexine matrix that forms around the peripheral tetrads during microspore development of the compound unit, the pollinium. We report a combined freeze-fracture and freeze-substitution study of the events associated with early exine development. Stage one of exine development is deposition of protosporopollenin that is probably synthesised by the microspore and secreted to the primexine matrix where it is polymerised. Enzymes for the polymerisation of the protosporopollenin may be synthesised by the microspores and then transported, via the endoplasmic reticulum, to the plasma membrane. Stage two of exine development follows callose dissolution and deposition of tapetally derived sporopollenin. Hence exine form and exine deposition inDendrobium appear to be the result of intimate cooperation between the microspore, the plasma membrane, the callose and the tapetum.     

Microspore and tapetal development in Echinodorus cordifolìus (Alismaceae)

In the microspore tetrad period the exine begins as rods that originate from the plasma membrane. These rods are exine units that on further development become columellae as well as part of the tectum, foot layer and “transitory endexine”. The primexine matrix is very thin in the future sites of the pores. At these sites the plasma membrane and its surface coating (glycocalyx) are without exine units and adjacent to the callose envelope. The exine around the aperture margin is characterized by units of reduced height. After the exine units and primexine matrix have become ca 0.2 μm in height a fibrillar zone forms under the aperture margin. It is the exine units around the aperture that are templates for exine processes on apertures of mature pollen. Oblique sections of the early exine show that the tectum consists of the distal portions of close-packed exine units. The exine enlarges in the free microspore period but initially its substructure (tectum, columellae, foot layer and transitory endexine) is not homogeneous and unit structures are visible until after the vacuolate microspore period. There are indications of a commissural line/plane (junction plane) which separates the foot layer from the endexine during early development. Our observations of development in Echinodorus pollen extend a growing number of reports of “transitory endexines” in monocot pollen. The exine unit-structures become 0.2 μm or more in diameter and many columellae are composed of only one exine unit. Spinules become exceptionally tall, many protruding ca 0.7 μm above the level of the tectum as units only ca 0.1 μm in diameter. The outer portion of the tectum fills in around spinules and by maturity they are microechinate with their bases spread out to ca 1 μm or more. Unit structures can be seen with SEM in mature pollen following oxidation by plasma ashing and in the tapetum these units are arranged both radially, as in spinules, and parallel with the tapetal surfaces. There are clear indications of such an arrangement of units in untreated fresh pollen. Units comprising the basal part of the exine are not completely fused by sporopollenin accumulated during development. This would seem to be a characteristic feature, based on published work, of the alismacean pollen. Our use of a tracer shows, however, that there is considerable space within or between exine structure of mature Echinodorus pollen. Based upon the ca 0.1 μm size of exine-units formed early in development and exine components seen after oxidative treatment it seems that the early (primary) accumulated sporopollenin has greater resistance to oxidation than sporopollenin added, secondarily, around and between units later in development. Both primarily and secondarily accumulated sporopollenin are resistant to acetolysis but published work indicates that acetolysis alters exine material. At the microspore tetrad time and until the vacuolate stages tapetal cells are arranged as in secretory tapetums. During early microspore stages there are orbicules at the inner surface of tapetal cells. At free microspore period tapetal cells greatly elongate into the loculus and surround the microspores. By the end of the microspore vacuolate period tapetal cells release their cellular contents and microspores are for a time enveloped by tapetal organelles and translocation material.     

Three-dimensional aspects of EXINE INITIATION AND DEVELOPMENT IN LILIUM LONGIFLORUM (Liliaceae)

Pollen development in Lilium longiflorum was reinvestigated with high resolution scanning electron microscopy, with special attention to three-dimensional conformation in the exine pattern formation. At the early tetrad stage, the invaginated plasma membrane takes the form of a reticulate pattern that corresponds to the mature exine tectum. Protectum is the first exine layer to be deposited on the reticulate-patterned plasma membrane. The initial protectum consists of aggregated fibrous threads and granules. Subsequently, probacules are formed under the protectum on the plasma membrane. At the free microspore stage the developing exine becomes further enlarged and the protectum develops into mature verrucate muri. The present three-dimensional investigation conflicts with the previous studies on exine development in Lilium.     

Synthesis of hierarchical materials

A major goal of material science is to produce hierarchical materials that are ordered on all length scales, from the molecular (1-100 A) via the nano (10-100 nm) to the meso (1-100 microm). In these materials, the larger-scale properties can be controlled by choosing molecular characteristics. Methods developed to produce three-dimensional, bulk-like hierarchical structures include biomimetic methods, which use polypeptides as building blocks, and amphiphile and colloidal templating, which use amphiphilic or colloidal mesophases as templates for inorganic mesoporous materials. Designing finite mesostructures with a given geometry still remains a challenge.     

Experimental modelling of exine self-assembly

Living and fossil megaspores produced by Selaginella (Lycopsida) and its extinct ancestors form distinctive (and occasionally iridescent) exines. Ultrastructural studies of these spores have provided data that demonstrate a colloidal mode of development which in turn implies a degree of self-assembly in the construction of these exines. We present here experimental evidence in support of the theory of selaginellalean megaspore exine construction by depletion flocculation. Iridescent colloidal flocculations of polystyrene latex particles demonstrate an ultrastructural organization virtually indistinguishable from that of the biological system, and clearly demonstrate that self-assembly of complex Selaginella exines by a relatively simple construction process is plausible.     

EXINE PATTERN FORMATION BY PLASMA MEMBRANE IN BOUGAINVILLEA SPECTABILIS WILLD. (NYCTAGINACEAE)

Transmission and scanning electron microscopy of exine development in Bougainvillea spectabilis (Nyctaginaceae) confirmed that the exine pattern is initiated by invagination of the microspore plasma membrane at the early tetrad stage. Invaginated plasma membranes take the form of a reticulate pattern that corresponds to the mature exine tectum. Protectum is the first exine layer to be deposited on the reticulate-patterned plasma membrane. Subsequently, probacules elongate basally on protruding sites of the plasma membrane under the protectum and in the lumina. These sites retreat as the probacules elongate. After the dissolution of the callose wall, a foot layer forms through the accumulation of lamellated structures. Clearly, the plasma membrane serves a determinative role in the initial pattern formation of exine.     

II. Exine development in Passiflora racemosa Brot.: post-tetrad period. Overlooked aspects of development

Developmental stages during the post-tetrad period are examined in detail with TEM and SEM, with emphasis upon substructure. Our purpose was to find out whether the sequence of sporoderm developmental events gives additional evidence for our recent hypothesis on the underlying cause of exine ontogeny as a sequence of self-assembling micellar mesophases, initiated by genomically given physico-chemical parameters. Four different layers of the endexine are developed in the post-tetrad period. The first one is a layer of white line centered lamellae which appear as a demarcation line between ect- and endexine. The second layer is sponge-like and consists of “roots” of columellae and a layer between them. The third layer consists of basally disposed radially elongated granules which appear in the aperture sites only. The fourth layer emerges in interapertural sites only and is formed as stacks of uneven lamellae. Therefore, the sequence of substructural units in primexine is the next: white-lined lamellae, a layer of honeycombed substructure, globule-to-rod-like granules, stacks of wavy lamellae. These sequences correspond to the next four mesophases of self-assembling micelles: neat (=laminate) micelles, high-concentrated emulsion of sponge-like (=foam-like) substructure, spherical-to-cylindrical micelles, and laminate micelles with fenestrated laminae. Reiteration of the micellar mesophases, participating in endexine development, is observed during the post-tetrad period.     

Development of the pollen grain wall: Further work withTradescantia bracteata

Summary Development of the pollen wall inTradescantia is examined between the tetrad stage and maturity, in the light of present controversy concerning the role of the tapetum in exine secretion, and the matter of the control of this process.The view that the exine in this plant is secreted entirely by the microspore protoplast is further substantiated, and is discussed in relation to sporopollenin production in general. An hypothesis is advanced to explain the mode of exine growth observed in all plants.Evidence is presented to support the contention that the sporophyte controls exine secretion through moieties inherited from the mother cell which are present in the spore cytoplasm.The differential phasing of exine development, observed to occur between species, is discussed in relation to general phase differences in pollen development, and the physiological condition of the whole plant.The plasticity of the intine inTradescantia is reported, and its importance considered in relation to the rigidity of the exine and the consequent disruption of that wall during the growth of a pollen grain.     

Studies on the Ontogeny of the Pollinium of Goodyera Procera

Using light, transmission and scanning electron microscopy, the development of the pollinium of Goodyera procera (Ker-Gawler) Hooker. was investigated. At the early stage, sporogenous cells inside the microsporangium were seen grouping together into small aggregates each containing few cells. After the aggregates have formed the sporogenous cells inside the aggregates (which could now be called massulae) divide to form numerous pollen mother cells. Later, the pollen mother cells undergo meiosis to form tetrads. The pattern of formation of the exine of tetrads varies according to the location of the tetrads inside the micro- sporangium. Those tetrads that are situated near the outer region of the massulae can form: exine with well developed tectum, bacula and foot layer; and the sequence of events leading to the formation of this type of well developed exine is as follows the original wall and the cyto- plasmic channels associated with the wall become surrounded by a thick layer of callose thus isolating the wall from the plasmalemma. Near the plasmalemma a layer of primexine containing callose and cellulose begins to form. Later, the primexine develops into exine and between the exine and plasmalemma a layer of intine is laid down. Similar type of exine with well developed tectum, bacula and foot layer, is also present in tetrads facing the tapetum. But in this case the original wall of the tedtrad is not retained but undergoes dissolution and in its place a new exine formed. The pattern of formation of exine in the region between tetrads is even more different. Here the original wall also undergoes dissolution but instead of forming a proper exine it only forms a thin foot layer with bulges at places. The pattern of formation of the exine in the cells inside the tetrad is even more different. Here the original wall of the cells only undergoes partial dissolution. The loose fibrils of the partially dissolved wall then become mixed with the callose layer surrounding the cell. Inside this wall-fibril/callose mixture thin sheets of exine appear, but these thin sheets of exine do not develop further into tectum or bacula. In Goodyera a quite substantial amount of callose is retained in the regions between massulae and tetrads, and we believe that it is this callose which is holding the massulae and tetrads together to form pollinium.     

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.     

STUDIES OF SEED FERN POLLEN: DEVELOPMENT OF THE EXINE IN MONOLETES (MEDULLOSALES)

Monoletes pollen extracted from the seed fern synangium Dolerotheca sclerotica Baxter illustrate four stages in the development of the sporoderm. In the first stage the grains are up to 100 μm long and possess an apparent homogeneous exine in which there is little differentiation between the nexine and sexine. Numerous nexine lamellae and the initiation of sexine expansion mark stage 2 in exine ontogeny. Further expansion of the sexine continues in the third stage until the ratio between the nexine and sexine is approximately 1:5. The final stage in maturation of the sporoderm shows an expanded alveolate sexine with some of the sporopollenin units broken and disorganized. It is at this stage of development that nexine lamellae are most prominent. The formation of sporoderm layers in the fossil grains is compared with pollen grain development in living cycads (Cycadophyta) and a model proposed to account for the apparent early formation of nexine lamellae in Monoletes. The evolution of exine components in early pollen types is discussed.