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

Protoplasma DOI 10.1007/s00709-014-0631-2

ORIGINAL ARTICLE

Sporoderm and tapetum development in Eupomatia laurina (Eupomatiaceae). An interpretation Nina I. Gabarayeva & Valentina V. Grigorjeva

Received: 13 November 2013 / Accepted: 1 March 2014 # Springer-Verlag Wien 2014

Abstract 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. Keywords Sporoderm development . Tapetum–microspore connections . Self-assembly . Micelles

Handling Editor: Peter Nick N. I. Gabarayeva (*) : V. V. Grigorjeva Komarov Botanical Institute, Popov st. 2, 197376 St. Petersburg, Russia e-mail: [email protected]

Introduction Our main concern in developmental palynology is to find and explain the common features in sporoderm ontogeny, inherent in any species. In our previous papers, we suggested a hypothesis for the explanation of spore/microspore wall development by self-assembly processes, unfolding in colloidal micelle systems of the periplasmic space, with genomic control under chemical composition and concentrations of the surfactants (Gabarayeva and Hemsley 2006; Hemsley and Gabarayeva 2007). That hypothesis regards a considerable part of the developmental events as an unfolding sequence of self-assembling micelle mesophases and their aggregates in the colloidal system of the periplasmic space. Surface active glycoproteins and lipopolysaccharides of the glycocalyx (Pettitt 1976) at increasing concentrations, and later in the development—SP precursors and monomers (phenylpropanoids, especially p-coumaric acid, and fatty acids—Gubatz et al. 1986, 1993; Herminghaus et al. 1988; Wehling et al. 1989; Wiermann and Gubatz 1992; Collinson et al. 1993; van Bergen et al. 1995; Wilmesmeier and Wiermann 1995; Kawase and Takahashi 1995; Hemsley et al. 1996a; Niester-Nyveld et al. 1997; Meuter-Gerhards et al. 1999; Wiermann et al. 2001; Van Bergen et al. 2004) and newly found SP precursors, hydroxylated α-pyrone compounds (Grienberger et al. 2010), are the necessary prerequisites for the initiation and progression of such self-assembling system; the latter always starts with spherical micelles. As the concentration of spherical micelles in the periplasmic space increases, they self-arrange first into columns, and then to cylindrical micelles; the latter correspond to tufts by Rowley (1990). The next mesophase is a layer of tightly packed cylindrical micelles (hexagonal mesophase— the glycocalyx as such), then—at higher concentration of the surfactants—laminate micelles appear (= white-lined lamellae of endexine) which are bilayers of cylindrical micelles, separated by a gap (this gaps are seen in TEM sections as white lines). These main micellar mesophases and liquid crystals exist in two forms: normal (in water-based medium) and reverse (in oil-based medium), in the latter case normal micelles are turned inside out (see Fig. 3 in Hemsley and Gabarayeva 2007). We came across this

N.I. Gabarayeva, V.V. Grigorjeva

hypothesis by the observations that all the constructive elements of the sporoderm in all species are, in essence, the same: granules, rods, lamellae, their combination and variations: the forms of these units coincide with the forms of micelle mesophaes. Among the main mesophases, many transitive mesophases (e.g. columns of spherical micelles or coin-columns) and complex aggregates (e.g. bicontinual bilabyrinth structures, ordered or disordered) exist (see Figs. 4 and 7 in Hemsley and Gabarayeva 2007). Our subsequent ontogenetic studies (see references) and experimental modelling of exine-like patterns (Gabarayeva and Grigorjeva 2013) gave evidence for our hypothesis. In this work, we trace the developmental sequence of Eupomatia sporoderm to clear up if it also correspond to micellar self-assembly system. The investigations of sporoderm ontogeny are always very helpful in respect to identification and a mode of establishment of different layers, especially if the endexine is thin and poor-discernible in the mature pollen wall. The mode of exine pattern formation in Eupomatia laurina is the main point of interest in this paper. Initially, Doyle, Van Campo and Lugardon (1975) proposed that granular structure of exine was most primitive and) interpreted structureless exines as an extreme form of granular structure. Walker and Skvarla agreed (1975), but argued that essentially structureless exines as in Eupomatia and Degeneria were still more primitive. Later molecular analysis, reviewed in Soltis et al. (2005), has shown that the earliest angiosperms are “ANITA” lines. Correspondingly, Doyle (2005, 2009) concluded that the first angiosperms had columellate exines as “ANITA” species. In spite of the fact that Magnoliales are now “demoted” from the ancestral pollen type in favour to “ANITA” complex (Amborella, Nymphaeales and Austrobaileyales—Doyle 2005), they retain their important place in phylogenetic and evolutionary aspects. The meaning of granular exine structure will be discussed below. Some taxonomic data on Eupomatia are included in the papers Renner (1999) and Souquet et al. (2003). No ontogenetic studies were undertaken on the two representatives of the genus (E. laurina and E. bennettii). Pollen and pollination in the Eupomatiaceae were studies with LM by Hotchkiss (1958), who also reviewed a number of earlier works. This author described pollen grains of E. laurina as zonisulculate and shown that several types of tetrads were common for Eupomatia. Woodland and Garlick (1982) studied pollen of both species with SEM and TEM and described the aperture type as zonizonasulculate (a band-like, encircling aperture at the equator), whereas Sampson (2000) regarded the aperture type as zonizonasulcate.

Material and methods The fixed material for this study was put at our disposal from the courtesy of A. Pozhidaev. Flower buds of E. laurina R. Br.

were collected from the greenhouses of the Komarov Botanical Institute, St. Petersburg. Stamens were fixed in 3 % glutaraldehyde with addition of 2.5 % sucrose in 0.1 M phosphate buffer at pH 7.4, 20 °C, 24 h. After post-fixation in 2 % osmium tetroxide (20 °C, 4 h) and acetone dehydration, the samples were embedded in a mixture of Epon and Araldite. Ultrathin sections were contrasted with a saturated solution of uranyl acetate in ethanol and 0.2 % lead citrate. Sections were examined with a Hitachi H-600 transmission electron microscope (TEM). Mature pollen grains for SEM were mounted on stubs, coated with gold/palladium and examined with Jeol JSM-6390 instrument. To clear up a class of chemical composition of the endexinous constructive units—osmiophilic globules—the treatment with enzyme lipase (Sigma Chemical Co., Type I— Wheat germ) were applied (Dashek et al. 1971; Belitser et al. 1982). For better exposure of the second lamellated layer of endexine, a special staining for endexine with potassium permanganate was used (Weber and Ulrich 2010). To receive the proof that both suggested layers belong to endexine (are sporopolleninous), acetolysis was undertaken with dry mature flowers, pollen was embedded into agar agar cubics, dehydrated, embedded into resin and sectioned for studying with TEM.

Results Premeiotic stage Young microspore mother cells (MMC) acquire roundish form (Fig. 1a, b) and are connected with each other via cytomictic channels (Fig. 1b, asterisk). MMCs gradually synthesize and augment their callose envelope. The cytoplasm density differs considerably in MMCs and in secretory parietal tapetum: the tapetal cytoplasm is packed with ribosomes in comparison with relatively transparent MMC cytoplasm (Fig. 1a). MMCs contain many small vacuoles, mitochondria, dictyosomes and lipid globules. Fig. 1 Premeiotic microspore mother cell (MMC) stage and meiosis I stages. a Microspre mother cell (MMC), nested into tapetum (Ta). The callose layer (Ca) is in the process of formation. b Neighbouring MMCs, united by a cytomictic channel (asterisk). c Metaphase-I with highly compact chromosomes (MCh) lacking nuclear envelope. d and inset A general view of MMC at early telophase-I stage, showing cytoplasmic organelles, inclusions and two telophase nuclei (a crust section). Note fibrilate structures, surrounding MMC (arrowheads). An arrow points to a specific structure, shown at high magnification in the inset: this is tipical cylindrical micelle. Mitochondria (Mi) surround nuclei. e Late telophase-I of meiosis. A partial cleavage furrowing takes place (asterisk), resulting in the formation of two semicells, connected by an isthmus. f A border of MMC, shown in d, at higher magnification. The plasma membrane lack the glycocalyx (arrow). Note fibrils (arrowhead) on the surface of callose (Ca).Ca callose, D dictyosome, ER endoplasmic reticulum, LG lipid globule, MCh metaphase chromosome, N nucleus, Ta tapetum, V vacuole, W wall of MMC. a–f 1 μm, d inset 0.1 μm

Sporoderm and tapetum development in Eupomatia laurina (Eupomatiaceae)



Meiosis At metaphase-I stage chromosomes are highly condensed (MCh), and the nucleus lack the envelope (Fig. 1c). All the meiocyte organelles and inclusions are accumulated in the peripheral cytoplasm. At telophase-I stage, a thick network of fibrils surrounds every MMC (Fig. 1d, arrowheads), the two nuclei are surrounded by mitochondria. A spiral structure is observed in the centre of MMC cytoplasm (Fig. 1d, arrow and inset at higher magnification—see discussion). At late telophase-I stage, a partial cleavage furrowing takes place (asterisk), resulting in the formation of two semicells, connected by an isthmus (Fig. 1e). A higher magnification (Fig. 1f) shows a “clean” plasma membrane surface lacking any signs of the glycocalyx, and a thick callose layer with fibrils on the surface. The tetrad period. Early tetrad stage After completing of meiosis, the four microspores are enveloped in thick callose jacket (Fig. 2a, b). Long fibrils cover the callose surface (Fig. 2a, arrowheads). The microspore plasma membrane is still bare; long cisternae of ER are disposed parallel to the plasmalemma (Fig. 2c). Soon afterwards, the initial glycocalyx appears inside the narrow periplasmic space, on the plasma membrane surface as a thin, non-differentiated layer (Fig. 2d). The picture changes very soon, revealing separate spherical units along the outer surface of the glycocalyx layer (Fig. 3a, arrowheads). Initially, these spherical units are disposed loosely (Fig. 3a), but soon become more prominent and tightly packed (Fig. 3b–d, arrowheads). The plasma membrane becomes wavy. Up to the end of early tetrad stage, spherical units on the surface of the glycocalyx are quite tightly packed; but, their individuality is evident (Fig. 4a–c, arrowheads). The microspore plasma membrane is progressively wavy. Plenty of ribosomes, mitochondria, plastids and RER cisternae are observed in the microspore cytoplasm.

glycocalyx units. At a low magnification, the outer layer of the glycocalyx in the periplasmic space seems unbroken (Fig. 5c, d); but, a higher magnification reveals individual spherical units of the previous stages (Fig. 5e, arrowheads) and in places where spherical units are arranged into columns (Fig. 5f, arrows). Late tetrad stage A survey of an anther loculus at the border of the tapetum and an adjacent tetrad shows callose disintegration (Fig. 6a, asterisks), the tapetum secretes pro-Ubisch bodies (Fig. 6a, arrowheads), and microspores have well discernible electron-dense primexine. Both tapetal and microspore cytoplasm are packed with ER cisternae. However, higher magnification shows that the primexine construction is still not completed (Fig. 6b–e). Under the outer electron-dense primexine layer which looks amorphous, in wide periplasmic space, new units appear: granules and elongated granules, close to columns (Fig. 6b– e, arrowheads). At this ontogenetic step, cisternae of SER prevails in the microspore cytoplasm (Fig. 6b–e). Post-tetrad period. Young free microspores At this stage, the ectexine is well developed, and it is not differentiated to any sublayers (Fig. 7a). The periplasmic space, which is filled with the glycocalyx, is currently an arena for the endexine development. Somewhat later, the osmiophilic layer, underlying the ectexine, is well discernible (Fig. 7b, c, arrows) and consists of small semi-fused granules, which appeared in the end of the tetrad period (Fig. 7b, arrowheads). The newcomers in the glycocalyx are additional large globules (Fig. 7c, arrowheads). The glycocalyx in the periplasmic space is highly ordered: many radially oriented rod-like units are observed (Fig. 7d, arrows). In aperture site, the ectexine layer is absent (Fig. 7e, arrow). Fibrils cover the surface of the microspores (Fig. 7a—arrows, d, e—arrowheads); some of them are attached to the tapetum.

Middle tetrad stage

Stage of vacuolization or the stage of “perforated” ectexine

A survey of a tetrad fragment shows osmiophilic layer on the microspore surfaces (Fig. 5a); whereas, the secretory tapetum is in a very active state (Fig. 5b). The reason for osmiophilicity is the initial sporopollenin (SP) precursor accumulation on the

This unusual stage shows the appearance of electron—translucent round cavities or cavernae inside the ectexine. A survey shows many vacuoles in the cytoplasm (Fig. 8a) which gradually fuse to one large central vacuole (Fig. 8b). At the margin of the aperture, the ectexine is represented by verrucae. Many osmiophilic globules are disposed in the periplasmic space, filled with the glycocalyx (Fig. 8c, arrowheads, d). Some osmiophilic substance is observed at the outer side of the plasma membrane (Fig. 8c, asterisks). Many “perforations” are seen inside the ectexine (Fig. 8d). Thick fibrils (Fig. 8d, arrows; e, arrowheads) pass from the microspore surface to the anther loculus (seen in fragments also in Fig. 8b, arrows). Many

Fig. 2

Early tetrad stage. a A border of a tetrad. Thick callose jacket (Ca) surrounds microspores. Note fibriles on the callose surface (arrowheads). b A survey of tetrahedral tetrad. c A border of microspore. The plasma membrane is naked (lack of the glycocalyx—arrow). d Slightly later stage than in c. Initial glycocalyx (G) on the plasma membrane in the narrow periplasmic space. Ca callose, ER endoplasmic reticulum, LG lipid globule, M microspore, MC microspore cytoplasm, N nucleus, Nu nucleoli, Tet tetrad, W primary wall of the tetrad. a–d 1 μm


Fig. 3 Early tetrad stage in progress. a Appearance of the first separate spherical units at the outer border of the glycocalyx (arrowheads). b, c Spherical units are more tightly packed in a layer (arrowheads). d Spherical units at higher magnification (arrowheads). Spherical units

are most probably spherical micelles, some of them self-assemble to cylindrical micelles. Ca callose, ER endoplasmic reticulum, G glycocalyx, MC microspore cytoplasm, Mi mitochondrion, P plastid. a–d 0.5 μm

plastids, a part of them are cup-like, cisternae of RER, mitochondria and lipid globules inhabit the microspore cytoplasm. Cavities inside the ectexine differ in size (Fig. 9a–c). Some of them look empty (Fig. 9a, c, white arrows), others are filled with some substance (Fig. 9a, c, white arrowheads). Osmiophilic globules, seen in the periplasmic space, filled

with the glycocalyx, become arranged more compact (Fig. 9a, black arrows). Thick fibrils on the surface of the ectexine persist (Fig. 9a, black arrowheads). To clear up if osmiophilic globules in the periplasmic space are lipids or more resistant structures (for instance, young SP), the treatment of sections with 1 % solution of lipase at 37ºC for 4 h


Fig. 4 The end of early tetrad stage. a–c The layer of spherical units of the glycocalyx becomes gradually more compact (tightly packed spherical micelles—arrowheads). Note that the microspore plasma membrane

is progressively wavy. Ca callose, G glycocalyx, MC microspore cytoplasm, Mi mitochondrion, P plastid, RER rough endoplasmic reticulum. a–c 0.5 μm

was undertaken. The results have shown that these osmiophilic globules are resistant to lipase (Fig. 9d, arrowheads), as well as in control (Fig. 9e, arrowheads). A confirmation of enzyme activity is the disappearance of plastoglobules (Fig. 9f, arrowheads), whereas the control shows plastids with globular inclusions (Fig. 9g, arrowheads).

The appearance of intine-I and of endexine lamellae When a channeled thickening of intine-I appears at aperture site, the ectexine is still perforated (Fig. 10a, grazing section). Large globules inside the endexine zone (zEnd) persist. In a medial section, perpendicular to the plane of



the aperture, two zones of cross-sectioned intine-I are evident (Fig. 10b). At this ontogenetic step, perforations in the ectexine are already closed (Fig. 10b). Simultaneously, the formation of the inner, lamellate layer of the endexine occurs in the periplasmic space, filled with the glycocalyx (Fig. 10c, asterisks). This inner lamellate layer is formed at the plasma membrane and, together with the outer fimbrillate layer, restricts the zone of the endexine formation in the periplasmic space (Fig. 10c, ZEnd). Globules of the outer endexine layer become more compressed (Fig. 10c, arrowheads). To clear up if compressed globules of the outer endexine layer are really sporopolleninous granules, pollen grains were acetolysed and sectioned. The results have shown that this layer is preserved after acetolysis (Fig. 10d, arrowheads), but the inner lamellate layer is not observed neither in nonaperturate (Fig. 10d) nor in apertural region (Fig. 10e)—in spite of the fact that in non-acetolysed material, higher magnification clearly shows tangential lamellae with thin central “white lines” (Fig. 10f, g, asterisks). Special staining for endexine using potassium permanganate (which should make endexine more prominent) shows “white lines” better, but restricting “black lines” are very thin (Fig. 10h, arrowheads). Late free microspore stage Later on some changes in tapetum and microspores take place. Some tapetal cells penetrate between microspores, and some microspores change their form (Fig. 11a). An interesting feature is that “white lines” appear in Ubisch bodies (Fig. 11b, arrowheads). Simultaneously the intine-I becomes much thicker (Fig. 11c). The two layers of the endexine, separated by the periplasmic space, are observed: the outer, electron-dense layer, consisting of compressed granules (Fig. 11c, white arrowhead), and the inner lamellate layer. The development of exine is completed: it includes amorphous ectexine and two layers of endexine, separated by the glycocalyx. A typical bicontinual structure—a kind of micelle formations—is observed inside the plasma membrane invagination (Fig. 11d). In aperture site the channeled intine-I is covered with dark-contrasted lamellate endexine, white lines are not discernible (Fig. 11e, arrowheads). The details of the

Fig. 5

Middle tetrad stage. a Fragment of a tetrad. The developing primexine is discernible as electron-dense outer layer (arrowheads). b A border of tapetal cell (Ta). The plasma membrane is invaginative. The cytoplasm is rich of endoplasmic reticulum (ER), its cisternae are widen and contain electron-translucent secretion. Plastids with large starch grains (S). c, d The borders of microspores. Initial SP precursor and monomer accumulation on the outer spherical glycocalyx units leads to appearance of electron-dense layer (arrowheads) in the periplasmic space. e, f Higher magnification, however, shows that this layer consists of spherical units (e arrowheads) and, in places, these units are arranged into columns (f arrows). Ca callose, G glycocalyx, LG lipid globule, M microspore, MC microspore cytoplasm, N nucleus, P plastid, PS periplasmic space, V vacuole. a, b 1 μm, c–f 0.5 μm

intine-I cytoplasmic channels, surrounded by the microspore plasma membrane and comprising ER membranes, are well seen in Fig. 11f (arrowheads), as well as tangential lamellae of the inner endexine layer (Fig. 11f, arrow). Mature pollen grains Close to maturation pollen grains pass through mitosis and contain generative cell, vegetative nucleus and multiple plastids with starch grains (amyloplasts—Fig. 12a, b). The second, fine-fibrilar intine layer appears around the whole pollen grain (Fig. 12c, d). Finally, the sporoderm consists of thick ectexine, very thin osmiophilic outer endexine layer, formed of compressed granules (Fig. 12c, arrowhead), inner lamellate endexine layer (Fig. 12c, asterisk) and fibrilar intine-II. The channeled intine-I is restricted to aperture site(Fig. 12d), where both intine layers are covered with a thin endexine layer of compressed lamellae (Fig. 12d, arrowhead). Acetolysed material shows that sporopolleninous peritapetal membrane (Fig. 12e, arrow), Ubisch bodies (Fig. 12e, arrowheads) and pollen grain walls are preserved. A fracture of a pollen grain shows a thick ectexine, separated by a gap from the endexine lamellae (Fig. 12f, arrows). SEM images show mature pollen grains from equatorial (Fig. 12g) and polar (Fig. 12h) views.

Discussion Peculiarity of meiosis Meiosis in E. laurina is of simultaneous type, as corresponded also by other authors (Hotchkiss 1958; Kamelina 1981; Furness et al. 2002). A special character of meiosis is a partial cleavage furrowing which starts to form at the late telophase-I stage, resulting in the formation of two semicells, connected by an isthmus. The same character was observed also at the same stage in Juniperus communis (Gabarayeva et al. submitted for publication), where the division by centripetal furrowings is continued at the end of meiosis-II, bringing about the appearance of the tetrads. Such uncompleted cleavage before meiosis II was also observed in Magnolia (Farr 1916, 1918). Hotchkiss noted (1958) that Eupomatia resembled Magnolia in possessing the arrested cleavage furrow but it lacked the cell plate vestige. We did not observed the cell plate in Eupomatia meiosis either, whereas in Juniperus it presents. The formation of the tetrad partitions by the furrowing is a very primitive character, known as Magnolia-type; we observed it also in Stangeria eriopus microsporogenesis (Gabarayeva and Grigorjeva 2002). In general, our studies have shown that microsporogenesis is highly labile in basal angiosperms (Gabarayeva, 1986,



1987, 1995; Gabarayeva and Rowley 1994; Gabarayeva et al. 2003), and this opinion is in good accordance with that of other authors (Sampson 1969, 1970; Furness et al. 2002). The progress of ectexine and underlying mechanism As was shown repeatedly in developmental studies of the sporoderm, the exine occurs on the framework of the plasma membrane glycocalyx (= primexine matrix), starting with some early ontogenetic works using TEM (Rowley and Flynn 1968; Heslop-Harrison 1972; Dickinson 1976), followed by many other developmental studies (see references). The first signs of the glycocalyx in Eupomatia appear on the plasma membrane as a thin layer with uncertain structure, but very soon it becomes clear that the layer consists of spherical units which are accumulated and packed tightly. This is a familiar motive in the glycocalyx development, observed in representatives of different genera under our studies (Nymphaea colorata, Anaxagorea, Nymphaea mexicana, Nymphaea capensis, Stangeria, Illicium, Cabomba, Encephalartos, Trevesia, Persea, Acer, Chamaedorea, Swida, Symphytum, Magnolia, Juniperus— see references). Such spherical units are also recognizable in micrographs of other authors’ ontogenetic studies (Cosmos bipinnatus—Dickinson 1976a, b; Hesse 1985; Triticum aestivum—El-Ghazaly and Jensen 1987; Poinciana— Skvarla and Rowley 1987; Eucommia ulmoides—Rowley et al. 1992; Caesalpinia japonica—Takahashi 1993), but usually the authors do not pay special attention to them. In numerous developmental papers of Rowley (see the full list in Blackmore and Skvarla 2012) these spherical units were evident, but up to 2009 Rowley considered them as cross sections of rod-like, fundamental exine units—tufts (Rowley 1990). The more information is accumulated in developmental palynology, the more clear it is that rod-like tufts are really fundamental, universal units of the exines, but they are, so to speak, their second hypostasis, the second form. The first one is spherical units. In Eupomatia, at the middle tetrad stage spherical units gradually form a compact layer in the outer part of the

Fig. 6

Late tetrad stage. a A border of tapetum (Ta) and an adjacent tetrad. Callose starts to disintegrate (asterisks). Primexine (PEx) is well discernible. Microspore cytoplasm is full of cisternae of endoplasmic reticulum (ER), autolytic vacuoles (AV), and vacuoles with electrontranslucent contents (V). Tapetum secretes pro-Ubisch bodies (arrowheads). b–e Higher magnifications show that developing primexine consists of ectexine (Ect), which looks internally amorphous, and of inner zone of periplasmic space (PS) with glycocalyx (G), in which new structures appear—granules and elongated granules, close to columns (arrowheads). These are elements of the future endexine. Note close contacts of SER cisternae with the plasma membrane (e). Ca callose, D dictyosome, M microspore, MC microspore cytoplasm, SER smooth endoplasmic reticulum, Ta tapetum, V vacuole. a 1 μm, b–e 0.5 μm

periplasmic space and accumulate lipidic SP precursors and monomers, which are osmiophilic (Fig. 5c, d). This is similar to what was observed in Welwitschia mirabilis at the corresponding developmental stage (Fig. 6 in Zavada and Gabarayeva 1991), where spherical units were gradually compressed into homogeneous layer of higher osmiophilicity (Fig. 9 in Zavada and Gabarayeva 1991). Therefore, it was concluded that the developmental pattern, observed in Welwitschia mirabilis, was more similar to the basal angiosperms than to other gymnosperms studied (Zavada and Gabarayeva 1991). It is necessary to mention that the issue of early SP precursors and monomers (synthesized during the tetrad period— protosporopollenin, in terminology of Dickinson 1976a, b) is still understudied. It has been suggested that the source of the SP monomers at the middle and late tetrad stages is the microspore itself or, at least, “receptor-dependent” SP (sensu Skvarla and Rowley 1987) is of both microspore and tapetal origin (Heslop-Harrison 1976; Dickinson 1976a, 1976b; Dickinson and Potter 1976; Audran 1981; Gabarayeva 1991; Uehara and Kurita 1991; Gabarayeva and Grigorjeva 2002; Gabarayeva and Hemsley 2006; Hemsley and Gabarayeva 2007). Whereas the post-tetrad synthesis of SP precursors in tapetum was studied and confirmed (Murphy and Vance 1999; Ariizumi et al. 2004; Ariizumi and Toriyama 2011), the tetrad period is obviously underestimated, and the possibility of SP precursors’ synthesis by the microspores themselves is ignored—in spite of the fact that the tetrad period indeed is the crucial point in determination of the final pattern of the pollen/ spore exines. For example, in cycadalean plants—in Ceratozamia mexicana (Audran 1981), Stangeria eriopus (Gabarayeva and Grigorjeva 2002) and Encephalartos altensteinii (Gabarayeva and Grigorjeva 2004)—the main SP accumulation is carried out during the tetrad period. An important detail is that in Eupomatia spherical units are arranged into columns (Fig. 5e, f). This phenomenon is also recurrent, observed in the course of primexine development of other species (Brasenia—Taylor and Osborn 2006; Acer— Gabarayeva et al. 2010; Chamaedorea—Gabarayeva and Grigorjeva 2010; Swida—Gabarayeva and Grigorjeva 2011; Symphytum—Gabarayeva et al. 2011; Magnolia—Gabarayeva and Grigorjeva 2012; Juniperus—Gabarayeva et al. submitted for publication). The data represented above are well explainable by our previously suggested hypothesis (Gabarayeva and Hemsley 2006; Hemsley and Gabarayeva 2007), supported by Blackmore and coauthors (Blackmore et al. 2007, 2010): the first micellar mesophases are exactly spherical and columnsof-spherical micelles. It should be mentioned that such supramolecular formations as micelles are formed not only in the periplasmic space. These can be found in any site wherever surface active substances at a proper concentration occur, for instance in the cytoplasm, which is also a colloidal solution. In Fig. 1d (and



its inset at high magnification), a typical large cylindrical micelle is shown in the cytoplasm of MMC. Another example is 3-D periodic surfaces, which divide a space into two interwoven labyrinths or subvolumes, giving rise to the bicontinuous phase (see Fig. 4 in Gabarayeva and Hemsley 2006 and Fig. 4c in Hemsley and Gabarayeva 2007). In Eupomatia such structure appears in the periplasmc space, where two immiscible liquids meet (Fig. 11d). Tufts, or cylindrical micelles, are usually a scaffold for columellae. But Eupomatia is a special case. At the late tetrad stage, the outer part of the primexine looks homogenous (Fig. 6). The most intriguing events occur under this layer in the glycocalyx, which is renewing constantly in the periplasmic space from the side of the plasma membrane. In this site, where rather large osmiophilic granules, elongated granules, columns of granules and columella-like units are observed (Fig. 6b–e), something like an attempt of columellae formation takes place. The progress of endexine and underlying mechanism However, this attempt failed, and procolumellae disappear like ghosts. Instead, in early free microspores granules accumulate from the inner side of the developing exine, forming a kind of fimbrillate border (Fig. 7b, c, arrows), the latter becomes more osmiophilic than the outer homogenous layer. This new layer, composed of semifused granules, is the first endexine layer. It is striking how demonstrative the substructure of the glycocalyx, which fills up the periplasmic space, is at this stage: highly ordered radially oriented cylindrical micelles—tufts—are evident (Fig. 7d, arrows). Cytochemical method of treatment of sections with lipase (Dashek et al. 1971; Belitser et al. 1982) has shown that osmiophilic granules in the periplasmic space are not simple lipids, but contain more complex substances, probably SP monomers/precursors, because lipase did not destroy them (Fig. 9d), as it did with plastoglobules (Fig. 9f).

Fig.

7 Young free microspores. a Microspore, adjacent to tapetum detaching Ubisch bodies (Ub). Large osmiophilic globules (arrowheads) are seen in the perilasmic space (PS). Note fibrils on the microspore and tapetum surfaces (arrows). b, c Periplasmic space, filled with the glycocalyx, at higher magnification. Osmiophilic globules (arrowheads) fuse each other and form a layer (arrow in c) underlying the ectexine (the latter shown not in full). Note contacts of RER cisternae with the plasma membrane (arrow in b). d Well-developed ectexine (Ect) and highly ordered glycocalyx (G) in the periplasmic space—the arena for the endexine development. Note many radially oriented rod-like units of the glycocalyx (arrows). A thick network of fibrils cover the microspore surface (arrowheads). e Aperture site: ectexine layer is absent (arrow). Ect ectexine, LG lipid globule, MC microspore cytoplasm, Mi mitochondrion, N nucleus, RER rough endoplasmic reticulum, Ta tapetum, V vacuole. a 1 μm, b–e 0.5 μm

The processes of the endexine development continues later in the development, when the channeled intine-I is initiated. The newcomers are thin tangential lamellae which most probably appear at the plasma membrane on the base of laminate micelles (Figs. 10f–h and 11c, e, f). Initially they are very thin and do not have evident white lines (Fig. 10f, g), but later white lines becomes discernible (Fig. 11c). A test with potassium permanganate staining (which makes endexine more pronounced—Weber and Ulrich 2010) reveals central white lines rather than the whole lamellae (Fig. 10h). After ectexine establishment, the whole underlying glycocalyx is a zone of endexine formation. The process reminds of an attempt of endexine building rather than a typical endexine formation: the fimbrillate layer, consisting of semifused granules, bears a shadow of uncompleteness, and the tangentional lamellate layer is not so evident as in other species. Besides, the central part of the glycocalyx does not accumulate SP, that brings about the appearance of a gap in the mature sporoderm (Fig. 12f). An interesting similarity exists between what is observed by us in Eupomatia and by Dahl and Rowley (1965) in Degeneria. Figs. 5–7 in the latter paper show a fimbrillate layer in Degeneria, which is very similar to that of Eupomatia (Fig. 10c, d, arrowheads), and also a lamellate layer between exine and intine, separated by a gap (filled with the glycocalyx) from the fimbrillate layer—exactly as it observed in Eupomatia (Fig. 10c, f, g, asterisks). Both fimbrillate and lamellate layers of the endexine, separated by a gap, are also seen in micrographs of Woddland and Garlick (1982, Fig. 1e, f), with E. laurina and Eupomatia bennettii correspondingly. The difference in our and these authors’ interpretation is in that they have called well discernible lamellate endexine layer in their Fig. 1f (E. bennettii) as the outer intine, and the underlying intine as inner intine. Neglecting these discrepancies in layers denoting, our data in this paper, the data of Dahl and Rowley (1965) and the data of Woddland and Garlick (1982) confirm the presence of fimbrillate and lamellate layers in the periplasmic space below the amorphous ectexine—the site which we call a zone of endexine (Figs. 10c and 11c, outcoming arrows). To confirm the sporopollenin status of the fimbrillate and lamellate layers of the endexine zone we used acetolysis test on SP. After acetolysis the fimbrillate layer persists, but not the lamellate layer (Fig. 10d, e, arrowheads). At first look, this does not confirm the sporopollenin status of this layer. But it should be kept in mind, that there is a gap between fimbrillate and lamellate layers, filled with the glycocalyx, lacking SP. In result, thin lamellate layer is shelled out in the process of acetolysis and lost during material treatment. The lamellate layer of the endexine is well seen, however, in Fig. 11e



(arrowheads) and f (arrow), covering the intine-I at aperture site and playing the role of the aperture membrane. The gap between two endexine layers is well seen in SEM picture of fractured pollen grain (Fig. 12f, asterisks), where the lamellate layer of endexine is preserved (Fig. 12f, arrows). Almost literally, “fimbrillate” layer was observed in the sporoderm ontogeny of Nymphaea mexicana (Gabarayeva and El-Ghazaly 1997, Figs. 32, 33 and 35) as a fringe border of a table-cloth, but the location of the fimbrillate and lamellate layers in Eupomatia is the opposite. Localization of the constructive units is variable: in the interapertural region of the developing endexine in Trevesia burckii the outer layer of the endexine is lamellate, and the inner one—granulate; in Illicium and Swida granules and lamellae are intermixed; in Chamaedorea granules, disposed under the endexine lamellae, are arranged in strings (one of micellar form), then fuse and turn into lamellae, but in the aperture sites of Borago (Fig. 21 in. and Trevesia, where the volume of the periplasmic space is increased, the opposite process is observed—lamellae scatter into granules (Rowley et al. 1999; Gabarayeva et al. 2009a, b; Gabarayeva and Grigorjeva 2003, 2010, 2011). During Acer sporoderm development two waves of spherical-to-columns transitional micelle mesophase were observed: at early tetrad stage and at free microspore stage (Gabarayeva et al. 2010). Reiteration of the micellar mesophases is observed during the exine development in Passiflora (Gabarayeva et al. 2013a, 2013b). All these variations are not surprising if one keeps in mind the most probable underlying mechanism of the exine development: the sequence of successive micellar mesophases, unfolding in the periplasmic space. The peculiarity of this sequence is in that its mesophases, starting with critical micelle concentration and subsequent increasing of it, easily change one to another, hence mixing of mesophases and their intermediate forms as e.g. strings, columns of spherical micelles, nodulate lamellae (Acer, Fig. 8c in Gabarayeva et al.

Fig. 8

Stage of vacuolization, or the stage of “perforated” ectexine. a, b Surveys of microspores. a A crust section, perpendicular to the equatorial aperture; there are many large vacuoles in the cytoplasm. b One large vacuole occupies the central position. c Margin of the aperture site where ectexine is represented by verrucae. Many osmiophilic globules are disposed in the periplasmic space, filled with the glycocalyx (arrowheads). Note osmiophilic substance at the outer side of the plasma membrane (asterisks) and contacts of RER cisternae with plasmalemma. d Non-aperturate region. Many “perforations” in the ectexine. Osmiophilic globules fuse each other, forming a thin dark contrasted layer of the endexine-1. Note thick fibrils (arrows) which pass from the microspore surface to the anther loculus (seen in fragments also in b— arrows). e A surface of microspore ectexine, covered with fibrils (arrowheads). Ap aperture site, CMi cup-like mitochondrion, Ect ectexine, G glycocalyx, LG lipid globule, MC microspore cytoplasm, Mi mitochondrion, RER rough endoplasmic reticulum, V vacuole. a–e 1 μm

2010) or fenestrated lamellae (Passiflora, Fig. 8d, Gabarayeva et al. 2013b). Fenestrated ectexine—another ghost structures Unexpectedly, at the stage of vacuolization round, cavernae appear in the ectexine (Figs. 8a–d, 9a–c and 10a). Their roundish form makes us remember that the ectexine is based on spherical units-micelles (Figs. 3–5). Early accumulation of SP monomers stabilizes spherical units of this outer layer to some extent, without fixing them completely. It is known that spherical, as well as other types of micelles, exist in two forms: normal and reverse (see Fig. 3 in Hemsley and Gabarayeva 2007). Normal spherical micelles have hydrophobic (hydrocarbon) cores and hydrophilic shells. Hydrocarbon cores are osmiophilic, and in Eupomatia this is seen in Fig. 3, where normal spherical micelles are revealed as the initial glycocalyx layer. Later in the development, when spherical micelles increase in number and form, the ectexine, SP precursors and monomers accumulate on them (Figs. 4 and 5), and the ectexine layer becomes more osmiophilic and looks amorphous (Figs. 6b–e and 7a–d). One of most important properties of micelle systems from biological point of view is their ability to dissolve different substances which are little or insoluble in water, for example—to solubilize lipids (or their derivates) in aqueous systems (Mittal and Mukerjee 1977). This is exactly what most probably happens with spherical micelles, the constituents of the ectexine, in the tetrad period. However, in the free microspore period, a massive entrance of lipoid SP monomers from tapetum to anther loculus takes place. This is confirmed by recent studies (Choi et al. 2011; Lallemand et al. 2013). Moreover, Lallemand and coauthors (2013) have shown that a complex of enzymes, involved in sporopollenin biosynthesis at free microspore period—a metabolon, is localized to the endoplasmic reticulum of tapetal cells. Dobritsa and coauthors (2009) have shown that a complex of three genes is required in the model species Arabidopsis to provide an indispensable subset of fatty acid-derived components for the sporopollenin biosynthesis framework. Finding themselves in hydrophobic medium of fatty acid-derived components and phenolics, normal micelles turn inside out, protecting their hydrophilic cores from the hostile hydrophobic surroundings. Reverse micelles have hydrophilic core and are capable to dissolve water-based nutrients inside them (Mittal and Mukerjee 1977). Such solubilization of water-based nutrients from the anther loculus is most probably the reason for the appearance of electronlucent cavities inside ectexine. Similar cavities open during Passiflora exine development (inside columellae—Fig. 4a, c in Gabarayeva et al. 2013b) and disappear later in development.



In general, the phenomenon of the fenestrated exine is far not unique: such exines were observed, e.g. in representatives of Compositae (see Fig. 2o, q, r, s in Blackmore et al. 2010), but fenestrations in the exines of these species are preserved in mature pollen grains; whereas in Eupomatia, they are temporary, transient formations. The opening of such cavities can be connected with passage of water-based nutrients from the anther loculus inside the microspore cytoplasm in the case if spherical reverse micelles are capable to give over their contents gradually from the outer micelles to nearby ones, occupying inner position. Indeed, the phenomenon of intermicellar exchange of solubilizates exists; in particular, the effect of the intermicellar exchange rate in reverse micelles was studied by Bagwe and Khilar (1997 and references in). An analogous opening of channels in the exines was shown (Rowley 1976; Rowley et al. 2003). These authors have shown that the process of transfer of substances from the anther loculus to the cytoplasm proceeds not only through aperture sites, but also across the nonapertural regions of the exine. Their in vivo experiments have shown that the process of transfer of substances can be visible with light and TEM microscopes (Rowley 1976). We consider the endexinous white-lined lamellae to be based on laminate (so-called neat) micelles which can act as selective chemical filters (Ball 1994). This means that the endexine lamellae may play a role of selective filter in the process of substance transfer from the tecal fluid to the microspore cytoplasm. Surface fibrils: connective structures between tapetum and microspores? In the course of the development, fibrils are observed on the MMC callose surface (Figs. 1d, f and 2a) and then, starting from the early free microspore stage, on the microspore surface (Figs. 7a, d, e, 8b, d, e, and 9a, b). Such structures were observed in our ontogenetic study of the sporoderm in Juniperus communis (Gabarayeva et al. submitted for publication), where they were especially well discernible.

Fig. 9

Stage of “perforated” ectexine (a–c) and cytochemical reactions (d–g). a, b (grazing section). c Cavities inside ectexine differ considerably in size. Some of them look empty (white arrows); others are filled with some substance (white arrowheads). Osmiophilic globules, seen in c in the periplasmic space (PS), filled with the glycocalyx, become arranged and more compact (a, black arrows). Note that there are thick fibrils on the surface of the ectexine (a, black arrowheads). d, f Treatment of sections with 1 % solution of lipase at 37ºC for 4 h and control to it (e, g). Osmiophilic globules (e, arrowheads) in the periplasmic space are preserved after treatment (d), but disappeared from plastids (f) whereas plastids contain globular inclusions in control (g). (d, e Developing parts of sporoderm are shown only). Cmi cup-like mitochondrion, Ect ectexine, LG lipid globule, MC microspore cytoplasm, P plastid, PM plasma membrane, V vacuole. a–c, f, g 1 μm; d, e 0.5 μm

Fibrils, at different degree of safety, we discovered also in sporoderm ontogeny in Persea americana, Chamaedorea microspadix, Acer tataricum and Alsophila cetosa (see references). In Eupomatia, these connective structures are not so evident as in other species, mentioned above; the reason is that in the process of fixation, lanthanum nitrate was not added to fixatives. Connective filaments, being glycoproteinous, can be well revealed only by addition of lanthanum nitrate (or ruthenium red and alcian blue) during fixation (Morbelli and Rowley 1993). The latter authors were the first who discovered such connective structures between tapetum and megaspores of Selaginella species (Morbelli and Rowley 1993), where they were very distinct. Thick connective filaments were also observed in anther of Betula pendula with SEM (El-Ghazaly 2000; Rowley et al. 2003; Rowley and Morbelli 2009), and they were similar on their morphology to viscin threads, typical for representatives of Onagraceae and Ericaceae (Skvarla et al. 1978; Hesse 1983, 2010; Hesse et al. 2000). However, these filaments do not accumulate SP (unlike viscin threads which connect pollen grains), and they are not alike elastoviscin threads either. Connective filaments between tapetum and microspores may serve as straight communications between nutritive tapetum and nursed microspores. Filaments belong to one of micellar form; they are actually very long cylindrical micelles. Summarizing the constructive units, observed in the course of exine development in Eupomatia, we conclude that all of them—granular units of the ectexine, semifused globular units of the outer layer of the endexine, lamellate units of the inner endexine, rod-like units of the glycocalyx that fills the gap between the two endexine layers and filaments on the microspore surface—are most probably based on micelles, representing successive mesophases of the micelle sequence (spherical, cylindrical, laminate micelles and intermediate forms). The same sequence was observed in the course of all our previous ontogenetic studies (see references). Additional and most important evidence for this hypothesis is experimental modelling of exine. The pioneer works on this subject were carried out by Hemsley and his coauthors (Hemsley et al. 1996b, 1998, 2000, 2003; Griffiths and Hemsley 2001; Moore et al. 2009). These experiments resulted in appearance of spore-like particles and patterns, mimicking crystal-like megaspore wall patterns of Selaginella and Erlansonisporites and of Osmunda spore wall. These authors used polystyrene in place of SP in their mimicing experiments. Our subsequent study was aimed to mimic not only mature exines, but also younger stages of exine development, including glycocalyx. Our first results occurred encouraging (Gabarayeva and Grigorjeva 2013). Several polysaccharide gels (as callose substitudes) and surfactants (as glycocalyx



and sporopollenin monomer substitudes) were mixed at different concentrations and combinations. We received a number of patterns in the course of condensation (one of self-assembly process) which simulated exine-like structures at different stages of its development. The progress of intine and underlying mechanism The intine-I appears slightly earlier than the inner lamellate endexine (Fig. 10c, f, g)—an unusual character. Cisternae of RER secrete microfibrillate substance and gradually occur enclosed inside the intine material, but the close contact with the microspore cytoplasm is saved (Fig. 11c, arrowheads). The last layer which appears in the periplasmic space is the microfibrilate intine-II (Fig. 12a– d). Dahl and Rowley (1965) shown very similar 2 layers of intine in Degeneria: channeled and fibrillate. We have found that the same constructive mechanism of the intine was in the Magnoliaceae species (Michelia fuscata, Manglietia tenuipis, Magnolia delavayi, Liriodendron chinense and Magnolia sieboldii—see references) where this layer was the second layer of intine. We also observed the channeled intine in other basal angiosperms: in Anaxagorea, Cabomba, Persea and in Chamaedorea (see references). Among these four species, only in Anaxagorea that the channeled intine is formed alike Eupomatia, with the help of RER cisternae; in the rest of the three species, the channeled intine occurs evidently by self-assembly as cylindrical micelles. The SP-free part of the Persea sporoderm (mainly, channeled) was described as intine by Hesse and Kubitzki (1983), and Rowley and Vazanthy (1993) reported analogous channeled layers (interpreted differently) in close related species of Cinnamomum. In any case, we consider that in species, where RER cisternae do not participate directly in intine development, the underlying

Fig. 10

The appearance of intine-I and lamellae in the endexine zone. a Grazing section in the plane of channeled thickening of intine-I at aperture site. Ectexine is still perforated. Large globules inside the endexine zone (zEnd) persist. b Medial section, perpendicular to the plane of the aperture. Two zones of cross-sectioned intine-I (In I) are evident. “Perforations” are closed. c Formation of the inner, lamellate layer (asterisks) in the endexine zone of the periplasmic space (zEnd, shown by outcoming white arrows). Granules of the outer endexine layer become compressed (arrowheads). d Acetolysed pollen wall; the outer granular layer of endexine is preserved after acetolysis (arrowheads). Inner lamellate endexine layer is not seen. e Acetolysed pollen wall in aperture site; thin lamellate layer is not preserved. f Aperture margin. Tangential lamellae are well discernible, but central “white lines” are very thin (asterisks). g Aperture region. Lamellate endexinous layer (= laminate micelles—asterisks) is more prominent at higher magnification.h Staining with potassium permanganate: “white lines” are seen better, but restricting “black lines” are very thin. (c, d, f, g, h only developing parts of sporoderm are shown). Ap aperture site, Ect ectexine, N nucleus, V vacuole. a, b 1 μm; c–h 0.5 μm

cause of developmental events during the channeled intine formation is a self-assemble of cylindrical micelles. Tapetum development and function The secretory activity of tapetum is high starting from early tetrad stage (plenty of ribosomes, rich ER system). Orbicules multiply at inner and radial sides of the tapetal cells, acquiring “white lines” (Fig. 11b), similar to those seen in the endexine lamellae (Fig. 11c). This gives evidence that orbicules at this stage are still in semi-liquid condition (pro-orbicules) and that the concentration of SP precursors/monomers in pro-orbicules is the same as in the inner part of endexine zone, proper for laminate micelle formation. In Tarenna gracilipes, microspore development typical “white lines” and pro-orbicules with “white-lined tails” were observed in the periplasmic space of tapetal cells (Vinckier and Smets 2005, Fig. 5b, c). These white lines are most probably the typical laminate micelles with their water-based gap between bilayers. Similar “whitelined tails” of orbicules were observed also during sporoderm ontogeny of other species (Suarez-Cervera et al. 1995; Huysmans 1998; El-Ghazaly et al. 2001). During intine-I development, some tapetal cells leave their palisade position and invade between microspores (Fig. 11a)—a character, typical for many species with parietal secretory tapeta under our studies. The fact that there are many variations of tapetal types and that borders between tapetal types become blurred were shown and noted by many authors (e.g. Pacini and Franchi 1991; Rowley et al., 1992; Hesse and Hess 1993; Furness and Ruddal 2001; Furness 2008). Such tapetal behaviour is probably connected with the supply the microspores with SP precursors/monomers by direct contact with the microspore surface (Fig. 11a, arrows). Roaming tapetal cells were observed in almost all the species under our ontogenetic studies (see references), eroding the precise limits of tapetal types, some variations of which, with two main types, were described in a review of Pacini (2010). Furness (2008) suggested a third, intermediate type—invasive nonsyncytial. Our data show that even this intermediate type has its variations. A culmination point of such deviation of “normal” types were observed in Nymphaea colorata where tapetal cells invaded into anther loculus and then retracted to parietal position at least 3 times during sporoderm ontogeny (Rowley et al. 1992). Therefore, tapetal behaviour is a high labile character, and its typification depends on the detailed study. Several words on “misleading morphology” The conclusion (Scotland et al. 2003) that morphology in some cases play a misleading role in phylogenetic



analysis, and that it is easy to regard morphology (including pollen) as obsolete in comparison with molecular data as a source of phylogenetic information, together with exceptions, given by Doyle and Le Thomas (2012), find its explanation, if one keeps in mind that self-assembly evidently interferes into exine development. Self-assembly processes are not-linear ones, this means that, in spite of genome-ordering role, the consequence of developmental process (mature structures) may occur, to put it metaphorically, “unexpected” for genome itself. Hemsley et al. (1998) admitted that phylogenetic analysis based on selfassembled structures may differ in its conclusions from that based on DNA sequences. However, the data of developmental palynology can be useful for phylogenetics in that it shows through which stages the future mature structure has passed. Our experimental modelling of exinelike patterns (Gabarayeva and Grigorjeva 2013) has shown that when the influence of genome is excluded, granular pattern is the one occurring most easily and frequently. On the other hand, spherical micelles which are the first mesophase in the sequence of micellar self-assembly, are the base for granules and, at the same time, they are an elementary building units for several transitive structures (columns of granules, semi-fused granules in columns) on the way to rod-like units (= cylindrical micelles), the base for columellae. Under some conditions (fall in surfactant concentration), cylindrical micelles easily dissipate into spherical ones, so a transit between both forms is labile. Taking into consideration the fact that before appearing in any columellae, the system in the periplasmic space inevitably had to pass through the stage of spherical unitsmicelles, there is no any “wall” between these two constructive blocks of the exine. Granular and columellar structures form an ontogenetic and potentially evolutionary continuum. Self-assembly processes are not restricted to sporoderm domain, they are extended through living and lifeless nature, so nothing is defended from their spastic and unpredictable character.

Fig. 11

Late free microspore stage. a A survey. Some tapetal cells (Ta) penetrate between microspores (M). b An inner side of a tapetal cell with Ubisch bodies, exhibiting “white lines” (arrowheads). c The two layers of endexine, separated by the glycocalyx (G), are observed: the outer electron-dense layer of compressed granules (white arrowhead) and the inner lamellate layer (zone of endexine shown with outcoming arrows). Cisternae of RER secrete microfibrilate substance, gradually “sealing” themselves inside the intine material (black arrowheads). d Typical bicontinual structure—a kind of micelle formations (asterisk, and restricted by arrows)—appeared inside an invagination of the plasma membrane (PM). Lipoid globule (arrowhead) in the periplasmic space. e Aperture site with intine-I and dark contrasted lamellate layer of endexine (arrowheads). f A part of sporoderm shows the cytoplasmic channels (arrowheads) of intine-I (In I) and the inner lamellate endexine layer (arrow). AL anther loculus, Ect ectexine, In I intine-I, MC microspore cytoplasm, zEnd zone of endexine. a 2 μm; b–f 0.5 μm

Conclusions 1. The exine development in E. laurina is unusual: an attempt of the columellae and of the typical endexine is observed rather than their conventional formation. 2. The first—ectexine—layer is formed on the base of the glycocalyx spherical units which are evidently the first micellar mesophase—normal spherical micelles. The latter start to accumulate sporopollenin, revealing themselves as granules. The progress of this process results in the ectexine which looks internally amorphous up to the late tetrad stage. 3. However, spherical units-micelles, being constuctive elements of the ectexine, “remind of themselves” in the free microspore period, when the ectexine become “perforated”—the result of micelle inversion. Reverse micelles open their cores for the entrance of hydrophilic nutrients from tapetum. The perforated phase continues during intine-I formation and most probably promote the supply of the microspore cytoplasm with nutrients by capacity of reverse spherical micelles to exchange their solubilizates. 4. At the late tetrad stage, the occurrence of columns of granules resembles the corresponding stage in many species, where this process leads to the appearance of the infratectal columellae. But in Eupomatia, this attempt fails. 5. Instead, a thin fimbrillate endexine layer of compressed globules appears. The endexine development is two-stepped, both temporally and spatially. The second endexine layer in the form of stacked very thin lamellae with poor-discernible white lines (based on laminate micelles) appears after the intine-I formation, what is an extraordinary feature. Lamellate layer is spatially separated from the first, outer fimbrillate endexine layer by a gap. The whole endexine development resembles an attempt of endexine formation rather than a typical process. 6. The first intine layer is channeled and appears under the equatorial aperture site only. It occurs in the process of RER cisternae activity. The second intine layer is finefibrillate and wraps the whole pollen grain. 7. Fibrils (filaments), observed on the callose surface of MMC and on the surface of microspores in free microspore period, may carry out a direct contact between microspores and tapetum. 8. Such meaningful characters as an attempt of columellae formation (failed), an attempt of outer and inner endexine formation (semi-realized), and the striking appearance and subsequent disappearance of holes in the ectexine are only revealed in the course of detailed study of complete development sequence. This emphasizes once again the necessity of ontogenetic studies for the deep understanding of constructive mechanisms, and the importance of ontogenetic data for phylogenetic analysis.



Fig. 12

Mature pollen grain stage. a, b Apertural regions. The appearance of the second intine layer (In II). Pollen grains contain generative cell (a, GC), vegetative nucleus (VN) and multiple plastids (P) with starch grains (amyloplasts). c Non-aperture region. Sporoderm consists of thick ectexine (Ect), very thin osmiophilic outer endexine layer (arrowhead), inner lamellate endexine layer (asterisk) and intine-II. d Aperture site. Channeled intine-I (In I) and fine-fibrilar intine-II are covered with very thin endexine of compressed lamellae (arrowhead). e Acetolysed pollen grain and adjacent tapetum. Sporopolleninous peritapetal membrane (arrow), Ubisch bodies (arrowheads), and pollen grain wall (PGW) are preserved. f Fracture of a pollen grain, SEM (from the courtesy of Andrew Pozhidaev). Thick ectexine (Ect) is separated by a gap (asterisks) from the endexine lamellae (arrows). g, h SEM images of mature zonizonasulcate pollen grains: equatorial (g, aperture marked by asterisk), and polar (h) views. a, b, d–f 1 μm; c 0.5 μm, g, h 10 μm

Acknowledgements This work was supported by RFBR grant No. 1404-00737. We cordially thankful to Dr. Andrew Pozhidaev for giving us the material for this study. Conflict of interest The authors declare that they have no conflict of interest.

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Interpretation of an aneurysm.

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The Book of Job: A grief and human development interpretation.

The development and interpretation of the summating potential response.

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Alzheimer's disease neuropathology. Current status of interpretation of lesion development.

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