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.     

PATTERN DETERMINATION OF THE EXINE IN CAESALPINIA JAPONICA (LEGUMINOSAE: CAESALPINIOIDEAE)

Exine development in Caesalpinia japonica Sieb. et Zucc. (Leguminosae) was studied by a combination of transmission electron microscopy (TEM) and field emission scanning electron microscopy (SEM) using a freeze-fracture method, with special attention to the initial process of exine pattern formation. The present study confirmed that the exine pattern is determined by the plasma membrane of microspores enclosed in the callose wall at the early tetrad stage. The plasma membrane, exclusive of the future apertures, invaginates and takes the form of a reticulate pattern. The reticulate pattern corresponds to the mature exine ornamentation. Protectum is the first to be laid down on the reticulate patterned plasma membrane. Probacules are initiated under the protectum and elongate basally on protruding sites of the plasma membrane. Primexine matrix is formed in coincidence with the probacules. After the protectum and probacules are completed within the callose wall, the invaginating plasma membrane becomes smooth. After the dissolution of the callose wall, endexine is organized by the accumulation of lamellated structures, and a foot layer is formed by the deposition of nonlamellated components on the developing endexine.     

EXINE INITIATION AND SUBSTRUCTURE IN POLLEN OF CAESALPINIA JAPONICA (LEGUMINOSAE: CAESALPINIOIDEAE)

Exine development in pollen of Caesalpinia japonica was studied using high resolution scanning electron microscopy, with attention to the initial developmental process of protectum formation and composition. The protectum is originated on the protuberant sites of the invaginated plasma membrane during the early tetrad stage. The present study shows that the initial protectum is composed of irregularly oriented fibrous threads. The fibrous threads accumulate and form a network on the plasma membrane. Granules 10–20 nm in diameter gradually aggregate within the network of fibrous threads during the tetrad stage. Subsequently the fibrous threads are almost masked by the granules. The developing protectum has a coarse texture within the callosic tetrad envelope. At the free microspore stage the granular protectum becomes homogeneous. The present study suggests that the protectum consists of an association of fibrous threads and granules. The fibrous threads may function as receptors and/or the skeleton of the developing exine.     

Exine Development in Illicium Religiosum Sieb. et Zucc. (Illiciaceae)

The exine development in Illicium was investigated using transmission electron and field emission scanning electron microscopy. The protectum and procolumellae appear on protruding sites of the microspore cytoplasm in the early tetrad stage. The protectum takes the form of a reticulate pattern with perforations within the callosic wall. After dissolution of the callosic wall, the central part of muri rises to form tectal ridges. The developing tectum, shows an echinate appearance in sectional view and has perforations at both sides around each lumen. There are two kinds of columellae; those forming continuous rings around each lumen and others which are individual rods standing beneath the tectum. The present developmental study in Illicium showed that the initial simple reticulate pattern formed within the callosic wall develops into the complex reticulate exine pattern of the differentiating tectum during the free microspore stage. The tectum has an angular shape with perforations and is supported by the two kinds of columellae.     

Microsporogenesis and exine substructure in Uraria crinita (Fabaceae)

This paper intends to elucidate the anther wall development, pollen wall development and exine substructure of Uraria crinita (L.) Desv. ex DC. (Fabaceae). The undifferentiated anther is ovoid-shaped and tetrasporangiated. The anther wall development is basic type, which is comprised of an epidermis, an endothecium layer, two middle layers and a tapetum. Anther-tapetum is glandular type and the cells are uniseriate and uninucleate. Pollen grains are tricolporate and 2-celled at the time of shedding. Before protectum development begins, a glycocalyx layer is inserted against the callose, and the plasma membrane is invaginated, exclusive of the future apertures. Subsequently, the probacula are elongate under the protectum and arise basally from the plasma membrane. The foot layer and endexine formation are concomitant with the callosic wall dissolution. The foot layer is thin and interrupted, but the endexine is thick and continuous. The intine is initially in the vacuoled stage. The substructure in the tectum, bacula and endexine is the same as a rod-shaped in side view. It composed of the loop like striate elements.     

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

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

Microsporogenesis and exine structure in Trochodendron aralioides Siebold and Zuccarini (Trochodendraceae)

This study aimed to elucidate the anther wall development, pollen wall development, and exine structure of Trochodendron aralioides Siebold and Zuccarini, a tree with primitive vessels but long considered to lack vessel elements in its wood. The anther wall is the basic type: epidermis, endothecium layer, three middle layers, and tapetum. The anther tapetum is glandular and cells are uniseriate. Microspore mother cells undergo meiosis with simultaneous cytokinesis to produce tetrahedral tetrads enclosed within a callose wall. Before development of the protectum, primexine is inserted against the callose, and the plasma membrane is invaginated. Then, the probacula are elongated under the protectum and arise basally from the plasma membrane. The foot layer formation is concomitant with callose wall dissolution. The foot layer is thick, and the endexine is thin. The foot layer and the endexine are both continuous. The intine is initially formed in the vacuolated microspore stage. Hollow Ubisch bodies are observed on the inner surface of the tapetum in free microspore stage. Pollen grains are tricolporate and 2-celled at the time of shedding. The numerous anthers of a single flower are at different development stages in both protandrous and protogynous individuals.     

SPORE WALL DEVELOPMENT IN ANDREAEA (MUSCI: ANDREAEOPSIDA)

The spore wall of Andreaea rothii (Andreaeopsida) is unique among mosses studied by transmission electron microscopy. The exine of other mosses is typically initiated on trilaminar structures of near unit membrane dimensions just outside the plasma membrane. The exine of Andreaea is initiated in the absence of such structures as discrete globules within the coarsely fibrillar network of the sporocyte wall. The sequence of wall layer development, nevertheless, is essentially like that of other mosses. The intine is deposited within the exine and the perine accumulates on the surface of the exine during the latter stages of spore maturation. The mature spore is weakly trilete and inaperturate. The wall consists of three layers, the inner intine, the spongy exine consisting of loosely compacted irregular globules of sporopollenin, and an outer layer of perine. The perine differs ultrastructurally from the exine only in its greater degree of electron opacity. This ultrastructural evidence of departure from the fundamental pattern of exine development in mosses supports the taxonomic isolation of Andreaea from mosses of the Sphagnopsida and Bryopsida.     

Sporoderm in Populus and Salix

Four phenomena were observed in a study of Populus tremula and P. tremula f. gigas microspores from before microspore mitosis through mature pollen which may have general significance in the ontogeny of pollen grains: 1) The exine and orbicules (Ubisch bodies) were covered by membranes. 2) The exine and the tapetal surfaces where orbicules form were covered by a polysaccharide (PAS positive) coat until after microspore mitosis; subsequently the tapetum became plasmodial. 3) Material having the staining characteristics of the nexine 2 (endexine in the sense of Fægri) accumulated on membranes in microspores in the space between the exine and the plasma membrane. That material was almost completely gone from the wall in mature pollen. The membranes on which material had accumulated migrated through the exine. Following passage through the exine these membranes were seen as empty fusiform vesicles in micrographs of anthers prepared by commonly used methods. 4) At about microspore mitosis when the cellulosic intine begins to form, microtubules about 240 A in diameter occurred near the plasma membrane and generally parallel with it. Positive acid phosphatase reactions in tapetal cells together with the morphology of orbicules and other tapetal organelles suggest that the wall of orbicules, which is like the pollen exine, may form as a residual product of a lysosome system.

Sections of mature Salix humilis pollen were compared with Populus.     

Preparation and properties of pollen sporoplasts

Summary Methods for the removal of exine from mature, ungerminatedLilium longiflorum pollen and release of intact gametophytes (sporoplasts) have been developed. These methods rely on the low temperature solvolytic activity of 4-methylmorpholine N-oxide (MMNO), which allows partial or complete detachment of exine from intine during subsequent washing procedures. These methods are: aqueous MMNO combined with cyclohexylamine (method I), aqueous MMNO at alkaline pH (method II), and aqueous MMNO containing a high Ca²⁺ concentration with added cellulysin and macerase (method III). Sporoplasts produced by methods I and II are most frequently completely separated from exine and, as shown by histochemical tests, enveloped by the intine layer. Selected enzyme activities in method II sporoplasts are measurable but, as indicated by other tests, considerable damage to the plasma membrane accompanies this treatment. Sporoplasts produced by melhod III largely remain attached to their ruptured exine layer and retain substantial biological competence in terms of extractable enzyme activities, membrane integrity, and respiration.Abbreviations MMNO 4-methylmorpholine N-oxide - SEM scanning electron microscope - TEM transmission electron microscope     

Pollen Wall Development in Gibasis (Commelinaceae)

The development of the one and-inline of the pollen wall aredescribed for Gibasis karwinsk yana and G. venustula. Duringthe tetrad stage the appearance of electron-opaque depositionsor tri-partite plates at discrete sites between the plasma membraneof the spore and the inward surface of the callose special wallare the first indications of exine development. The sulcus rapidlydifferentiates being composed of discrete exine granules ona thin foot layer. Probacula in non-apertural areas developin an electron-opaque granular layer situated between the plasmamembrane, which is highly convoluted, and the callose specialwall. A foot layer is formed from electron-opaque lamellae atthe plasma membrane. Exine pattern is clearly established withinthe tetrad. After release of the spores from the tetrad an intimate associationis rapidly developed between the plasma membrane of the periplasmodialtapetum and the newly-formed exine. Compacted electron-opaquematerial is found at the interface between membrane and theexine and vesicular material is added from the tapetum. Theincrease in volume that occurs in both spore and anther is accompaniedby considerable vacuolation. Intine development begins just prior to pollen grain mitosisand continues rapidly at the aperture. The thin foot layer becomesdiscontinuous. Further intine deposition takes place after mitosisand a bilayer is apparent in mature grains. The matrix of thislayer contains conspicuous electron-opaque platelets. The exineof the mature spore stains less intensely than in the youngspore and the interbacula spaces are filled with material fromthe degenerate tapetum. Gibasis karwinskyana, Gibasis venustula, Commelinaceae, exine, intine, tapetum, pollen wall, ultrastructure     

DEVELOPMENT OF WHEAT (TRITICUM AESTIVUM) POLLEN. II. HISTOCHEMICAL DIFFERENTIATION OF WALL AND UBISCH BODIES DURING DEVELOPMENT

The histochemistry of different developmental stages of the pollen wall, aperture, and Ubisch bodies of Triticum aestivum is examined with light and transmission electron microscopy. Various parts of the callosic envelope of the tetrad spores stain differentially. At the late tetrad stage, the probacules and the coat of pro-Ubisch bodies are densely stained for acidic polysaccharides, protein, and neutral polysaccharides. The protectum and the core of pro-Ubisch bodies are moderately stained. Upon release of microspores from the callosic cell envelope, the stainability for acidic polysaccharides increases in the exine and in the wall of Ubisch bodies, becoming very intense in the wall of mature pollen grains and Ubisch bodies. The stainability for neutral polysaccharides is decreased in the mature pollen wall and in the Ubisch bodies, while the stainability for protein increases. The results also indicate the probability of the presence of unsaturated lipids and the absence of free aldehydes in the pollen wall and Ubisch bodies.     

ULTRASTRUCTURE AND DEVELOPMENT OF THE NEXINE AND INTINE IN THE POLLEN WALL OF SILENE ALBA (CARYOPHYLLACEAE)

Nexine and intine development in Silene alba (Caryophyllaceae) was investigated by electron microscopy and enzyme cytochemistry. Nexine-2 forms by deposition of sporopollenin along unit membrane lamellae closely associated with the microspore plasma membrane in the late tetrad stage. After the callose wall dissolves, electron density increases along the tangentially oriented fibers of the proximal primexine, forming nexine-1. When the exine is essentially complete, the intine begins to develop. In the nearly mature microspore, acid phosphatase activity appears in the peripheral cytoplasm just prior to its extrusion into the intine of the mature pollen grain.     

Pollen wall development in Sciadopitys verticillata (Sciadopityaceae)

Pollen wall development of Sciadopitys verticillata was observed by transmission electron microscopy. The pollen of S. verticillata is non-saccate and spherical, and the exine consists of the outer thick, sculptured ectexine and the inner lamellated endexine. At the early tetrad stage, the initial ectexine and lamellae of the initial endexine begin to form on the microspore plasma membrane. The ectexine granules gradually swell. Deposition of sporopollenin materials on the ectexine granules then results it their becoming partially connected to each other. Identification of the original small ectexine granules then becomes difficult, and, finally, the ectexine appears as a homogeneous, partially discontinuous layer. The granules of the early ectexine cannot be identified. At maturity, there are four to five endexine lamellae. Recent molecular data have shown that Sciadopitys first branches off from the Cupressaceae plus Taxaceae clade, which is characterized by granular exine. Although the ectexine of Sciadopitys is similar to that of the Cupressaceae during initial development, the morphology of the ectexine is significantly different in the mature pollen. The initial stage of pollen development clearly shows the structural homology of the granular ectexine. Divergence of the exine structure occurs in the later stages.     

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.     

Pollen wall development in Cryptomeria japonica (Taxodiaceae)

Pollen wall development in Cryptomeria japonica was observed by scanning and transmission electron microscopy. The pollen of C. japonica is characterized by a non-saccate, projecting papilla. The exine of C. japonica consists of the outer granular ectexine and the inner lamellated endexine. At the tetrad stage, the initial granular layer of the pro-ectexine first forms on the microspore plasma membrane. The tripartite lamellae of the pro-endexine form under the pro-ectexine. The prosporopollenin material is deposited on the pro-ectexine and pro-endexine at the free spore stage. The ectexine granule increases its volume and the endexine lamellae thicken. The papilla protrudes during the tetrad stage. The tip of the papilla bends laterally where the exine is thinner. Exine construction in C. japonica is similar to that of Cunninghamia; however, the amount and size of the granular ectexine and lamellated endexine differ. The conspicuous papilla protrudes and bends during the tetrad period.     

Aperture development in spores of the moss,Trematodon longicollis Mx.

Summary Young spores of the mossTrematodon longicollis Mx. are highly polar. Immediately after meiotic cytokinesis an extensive system of microtubules associated with the single plastid develops under the entire distal face. Following exine initiation on the distal surface a microtubule system is elaborated at the site of aperture development on the proximal surface. Both plastid and nucleus move from distal to proximal pole and are attached to microtubules of the proximal system. Microtubules underlie the plasma membrane as it withdraws from the exine in the initiation of both the surrounding annulus and central aperture pore. The central pore enlarges to form a bowl-shaped concavity in which a fibrillar plug develops basipetally. The annulus expands into a fibrillar-filled protrusion surrounding the central pore. The mature aperture consists of a central pore plug covered by a thin roof of exine and separated from the surrounding annulus by exine lamellae. The aperture of the mature spore is obscured by development of the ornate exine and is not a prominent feature of the mature spore surface.     

DEVELOPMENT OF STRIATE EXINE IN IPOMOPSIS RUBURA (POLEMONIACEAE)

Exine formation in Ipomopsis rubura (L.) Wherry (Polemoniaceae) was traced with transmission and scanning electron microscopy. At the early tetrad stage, plasma membranes of microspores invaginate and form a punctate pattern within the callose wall. Protectum is then deposited, taking a punctate pattern corresponding to the plasma membrane. After dissolution of the callose wall, the punctate pattern develops into a striate sculpture by partial thickening of tectum. The mature tectum is thus composed of two layers, the inner punctate and outer striate layers. With the plasma membrane determining the initial tectum pattern within the tetrad, additional sculptural elements are later formed above this pattern during the free microspore stage.     

ONTOGENETIC DEVELOPMENT OF SPINOUS EXINE IN HIBISCUS SYRIACUS (MALVACEAE)

Pollen development in Hibiscus syriacus L. (Malvaceae) was studied with light (LM), scanning (SEM) and transmission (TEM) electron microscopes, with special attention to the formation of extremely long spines of the pollen grains. At the early tetrad stage, probacules are initiated directly on the plasma membrane and grow in coincidence with the height of primexine matrix within a callosic wall. Subsequently, a pretectum appears at the top of the probacules and then a foot layer is formed by accumulation of white line centered lamellations. Before dissolution of the callosic wall, a reticulate patterned pretectum is established around the microspores. There is not, however, any morphological indication on the initiation of the spines during the tetrad period within a callosic wall. It is after dissolution of the callosic wall that the spines of exine begin to form by the apposition of lamellated sheets. The lamellated sheets show a concentric configuration around the developing supratectal spines. The mature pollen grain is spheroidal, polycolporate, 160–170 μm in diameter, with supratectal spines 20–25 μm long. The supratectal spines of Hibiscus pollen are not homologous with the other exinous protrusions which are determined within the callosic wall during tetrad stage.     

Pollen Wall Development of Xiphidium coeruleum (Haemodoraceae) and its Systematic Implications

The development of the pollen grain wall in Xiphidium coeruleum(Haemodoraceae) was studied using TEM and cytochemical stainingtechniques. Microsporocyte ontogeny initiates with the degradationof the cellulosic cell wall and subsequent deposition of a thickcallosic cell wall. Following callose deposition, successivemeiosis occurs, resulting in a tetragonal tetrad of microspores.during meiosis, the cell walls of the tapetum break down, releasingthe syncytial periplasmodium. Irregular non-sporopollenous globularbodies are deposited in this peripheral periplasmodium, whichis rich in ER, golgi bodies, vesicles, and characteristic starchplastids. Within the microspore cytoplasm, vesicles, golgi bodies,and plastids are plentiful during the early tetrad stage. Atthis time the plasma membrane of the microspore develops characteristicevaginations. An extracellular membrane, the ‘white line’,is secreted outside the microspore plasma membrane, followedby callose wall degradation. Bead-like deposits of exine orprimexine are deposited at points along the ‘white line’simultaneously on inner and outer surfaces and opposite theoriginal plasma membrane evaginations. The bead-like exine depositscontinue to grow during the release of the microspores and developinto laterally appressed, rod-shaped ektexinous elements havinga tangentially oriented commissure, the vestige of the original‘white line’. The mature intine is two-layered,the outer exintine containing radially oriented vesicular structures,which are apparently derived from plasma membrane extensions.Exine development in Xiphidium is similar to ‘nexine 1’development in Lilium and may have evolved from an ancestraltectate-columellate condition by the loss of the sexine. Walldevelopment in members of the Zingiberales is strikingly similarto that reported here for the Haemodoraceae—evidence ofa possible relationship between the two taxa. Xiphidium coeruleum, Haemodoraceae, pollen, tapetum, development, exine