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.     

Anther, pollen and tapetum development in safflower, Carthamus tinctorius L

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having ‘two-celled’ groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of ‘non-invasive’ tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.     

Exine development in Nymphaea colorata (Nymphaeaceae)

We show a sequence of developmental events in microspores and tapetal cells in Nymphaea colorata based upon transmission and scanning electron microscopic observations. There are parallel cytoplasmic processes and surface coatings in microspores and tapetal cells. Uptake is indicated by the passage of lanthanum as a tracer from anther locule into the microspore cytoplasm and by the condition of the cytoplasmic surface of microspores. The callose envelope is not a barrier to transfer of lanthanum. During formation of the proexine glycocalyx tiny spiral elements, components of the exine substructural units, were oriented in different directions in the surface coating of microspores and tapetum. Lipoidal globules are associated with the spiral elements. After the uniform proexine stage, three regions of different exine structure and their gradations become differentiated in the sporoderm: 1) a proximal region with thick tectum and foot layer, thin columellae and a compact layer of lamellated endexine; 2) a distal pole region with separately disposed endexine lamellae; and 3) an equatorial encircling-sulcate aperture region which consists of infratectal layer, foot layer, and endexine lamellae. Based upon the presence of structurally comparable surface coats in microspores and tapetal cells, experimental uptake of lanthanum nitrate, and the co-ordinated processes in tapetum and microspores, we conclude that there is probably a reciprocal controlling influence between the microspores and the tapetum and other sporophytic tissues.     

POLLEN WALL AND APERTURE DEVELOPMENT IN HELIANTHUS ANNUUS (COMPOSITAE: HELIANTHEAE)

Wall development of tricolpate pollen of sunflower was studied by light and by scanning and transmission electron microscopy. The wall and colpi are initiated during the tetrad stage, producing a young, spinulate, two-layered exine (ektexine and endexine) separated by a “spacer layer.” After release from the tetrads, the individual microspores round up and enlarge. The exine layers increase in thickness and complexity from sporopollenin contributed by the tapetum and microspores. During the mid-vacuolate microspore stage, the tapetum becomes plasmodial and surrounds the developing microspores. At the vacuolate pollen stage, after the wall and colpi are completely formed, the plasmodial tapetum breaks down and releases its contents into the locule. Some of the contents are presumably utilized by the pollen to make storage reserves while other components, such as lipids and proteins, fill the spaces within the pollen wall exine. Pollen wall ontogeny provides a scheme of terms for mature composite walls in general. The various events associated with microsporogenesis in sunflower are compared with those reported in other pertinent studies.     

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.     

The Structure of Pollen Wall and Its Relation to Po41en Aggregation in Cymbidium goeringii (Rchb. F.) Rchb. F.

The pollen wall of tetrads located in different positions of a mature pollinium of Cymbidium goeringii was examined with the electron microscope, and the compositions of wall materials were also tested with different histochemical methods. In all tetrads of a pollinium, the pollen wall can be distingished into an exine and an intine, but the exine may be varied greatly according to the tetrad position in a pollenium. The part of the pollen wall (the outer wall) of the external tetrads, lying close, to the tapetum, is composed of two layers, i.e. the exine, and the intine. Theexine consists of tectum, granulate ectexine and endexine, without foot layer. The intine is cellulose in nature. In the outer wall between different groups of: tetrads and in the inner wall within an individual tetrad, the structure of ectexine becomes simple and the deposition of sporopollenin is roduced The degree of reduction of ectexine nicreases from the outer to inner tetrads in several external layers of a pollinium, and even the internal tetrads have a reduced ectexine or lack of it. The present study also demonstrates that the mechanism of pollen aggregation into a pollinium is built on a combined effect of the following features: (1) connected bridges formed' by intine between two pollens within a tetrad, (2) formation of cytoplasmic channels between two pollens within a tetrad, (3) incomplete cell wall formation within a tetrad, (4) little size of tetrads and compact arrangement of mature tetrads and (5) a sticky viscin material surrounded on the outside of a 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.     

Tapetal Cell Changes and Sporoderm Development in Rhigozum trichotomum (Burch.)

Rhigozum trichotomum is a perrenial woody shrub which is indigenousto the arid regions of southern Africa. Primexine developmentis initiated while the microspores are still enclosed by callose.This is followed by the appearance of probacula which give riseto the tectum, bacula and nexine. At the time of callose dissolution,the exine pattern is well established and intine developmenthas been initiated. During the tetrad stage, the protoplastsof the tapetal cells exhibit shrinkage while conspicuous stacksof rough endoplasmic reticulum become evident in their cytoplasm.These stacks produce numerous vesicles which are associatedwith lipid globules and which migrate to the tapetal/locularwall where, it is suggested, they give rise to the pro-orbicules.The pro-orbicules become coated with an osmiophilic substance,probably sporopollenin, and are released into the thecal fluidto become intimately bound to the exine, Here they are strippedof the osmiophilic layers which appear to be incorporated intothe sporoderm. Rhigozum trichotomum (Burch.), sporoderm, pollen wall, exine, orbicules, pro-orbicules, sporopollenin, tapetum     

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.     

美乐多（Melodorum fruticosum Lour．）花粉形态观察

利用扫描电子显微镜（SEM）和透射电子显微镜（TEM）观察了美乐多（Melodorum fruticosum）的花粉形态特征。美乐多花粉为球形或扁圆形的单粒花粉,外壁纹饰为微褶皱状,有点状凹陷,无任何萌发孔或萌发沟。花粉外壁由外壁外层包括覆盖层（连续）、覆盖下层、基足层（1～3层薄片层结构,偶断裂或扭曲至6～10层）和外壁内层（连续）组成。其中,覆盖下层,其厚度为整个花粉外壁厚度的1／2,为混合型结构,即小柱状和颗粒状同时存在,但以颗粒状为主。花粉内壁分为内壁外层和内壁内层,其厚度逐渐变薄。美乐多的花粉特征（单粒、无萌发孔或沟、覆盖层连续、基足层为薄片层结构、花粉外壁内层薄等）与紫玉盘族其他类群一致。     

Pollen and anther development in Nelumbo (Nelumbonaceae)

The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera and Nelumbo lutea. Historically, the genus has been considered to be closely related to Nymphaeales, however new systematic work has allied Nelumbo with lower eudicots, particularly Platanus. In recent years, studies of pollen development have contributed greatly to the understanding of phylogenetic relationships, but little has been known about these events in Nelumbo. In this paper, pollen and anther development are morphologically described for the first time in N. lutea. A comprehensive ontogenetic sequence is documented, including the sporogenous tissue, microspore mother cell, tetrad, free spore, and mature pollen grain stages. The deposition of a microspore mother cell coat and callose wall, the co-occurrence of both tetrahedral and tetragonal tetrads, the formation of a primexine in tetrads, and primexine persistence into the late free spore stage are shown. The majority of exine development occurs during the free spore stage with the deposition of a tectate-columellate ectexine, a lamellate endexine, and an unusual granular layer below and intermixed with the endexine lamellae. A two-layered intine forms rapidly during the earliest mature pollen stage. Major events of anther development documented include the degradation of a secretory-type tapetum during the free spore stage and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The majority of mature pollen grains are tricolpate, however less common monosulcate and diaperturate grains also develop. Co-occurring aperture types in Nelumbo have been suggested to be an important transition in angiosperm aperture number. However, aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus, which occurs in the early free spore stage. This character, as well as other details of pollen and anther ontogeny in Nelumbo, are compared to those of Nymphaeales and Platanus in an effort to provide additional insight into systematic and phylogenetic relationships. Although Nelumbo is similar to both groups in several characters, the ontogenetic sequence of the genus is different in many ways.     

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.     

Pollen morphology,ultrastructure and taphonomy of the Neuradaceae with special reference to Neurada procumbens L. and Grielum humifusum E.Mey. ex Harv. et Sond.

Pollen morphology and sporoderm ultrastructure of modern Neurada procumbens L. and Grielum humifusum E.Mey. ex Harv. et Sond. were studied using light (LM) and electron (SEM and TEM) microscopy. Additionally late Holocene pollen of the Grielum-type was studied using LM. Systematic and ecological aspects have been discussed for the family Neuradaceae. The pollen grains of the studied species are characterized by similarities in size, shape, aperture type and differences in exine sculpture (reticulate semitectate exine in Neurada and finely reticulate to foveolate in Grielum) and sporoderm ultrastructure. The cavea in the exine is situated between the ectexine and endexine which are connected near the aperture region only. A combination of the palynological characters of the Neuradaceae (semitectate exine, rather loose columellae, interrupted foot layer, the cavea in the exine) increases the pollen plasticity, allowing considerable changes of the pollen grain volume but still remains insufficient to survive sharp fluctuations in hydration level.     

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.     

Conventional and novel modes of exine patterning in members of the Araceae – the consequence of ecological paradigm shifts?

Summary. In the family Araceae, the members of all subfamilies except Aroideae follow the conventional mode of exine formation pattern, which conforms with the textbook view of sporoderm stratification and chemistry (sporopollenin ektexine formed before the endexine). Only members of the subfamily Aroideae show a quite uncommon mode of exine formation pattern, with an endexine formed prior to the nonsporopollenin, polysaccharidic outer exine layer. The intine is formed simultaneously with this non-sporopollenin layer. From the differing timetable and especially from the different origin it is concluded that this outer exine layer is not homologous to the angiosperm ektexine. The fundamental question, why members of the Aroideae lack an elaborated sporopollenin ektexine, is discussed in terms of functionality of the nonsporopollenin outer exine layer. It seems that a major change in aroid evolution took place at the point when the family phylogenetically and ecologically shifted from bisexual (most subfamilies) to unisexual flowers (Aroideae only). The hypothesis is that ephemeral spathes and the absence of sporopollenin are the consequence of an adaptive syndrome for a short pollination time window in many members of the Aroideae, with short-lived pollen, an energetically not costly pollen wall, rapid germination of pollen tube, and brief receptivity of stigma. Correspondence and reprints: Institute of Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria.     

A COMPARATIVE LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF MICROSPOROGENESIS IN MALE-FERTILE AND CYTOPLASMIC MALE-STERILE SUNFLOWER (HELIANTHUS ANNUUS)

Cytoplasmic male sterility (CMS) in sunflower anthers is compared with its normal (N) line by using light and electron microscopy. Degeneration and disintegration of CMS tapetum and microspore tetrads occur after meiosis II, resulting in sterility. At the onset of meiosis, the CMS tapetum enlarges radially and shows signs of disorganization of organelles and walls. The developing CMS meiocytes and tetrads of microspores do not show these abnormalities when compared with their N counterparts. The CMS microspore tetrads remain viable until a rudimentary exine forms around each microspore. At this time, the radially enlarged tapetum disintegrates, followed by disintegration of the tetrads. In N-line microsporogenesis, a peripheral, dense tapetum is present at the tetrad stage, and as each locule enlarges, free spaces occur around the tetrads. After a rudimentary exine with associated spines and colpi is formed around each microspore, the callose holding each tetrad together dissolves, freeing the microspores for further development. Eventually the binucleate tapetum becomes plasmodial, persisting until the vacuolate pollen stage.     

Cytochemistry of pollen development in Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, cytochemical staining methods were used to follow the cytochemical modifications of microspore cytoplasm and sporoderm in Campsis radicans (L.) Seem. from tetrad stage to mature pollen. Flower buds were collected at different stages of development, and the anthers were fixed and embedded in Araldite. To make cytochemical observations under light microscope, semithin sections were cut and stained with different dyes. Cytochemical methods provided the opportunity to localize the reserve material in the microspore and pollen cytoplasm, to distinguish the different layers of the sporoderm, and to determine its chemical structure at different developmental stages. Microspore cytoplasm contains variable amounts of proteins, lipids, and insoluble carbohydrates at different stages of microsporogenesis. Sporoderm formation starts at tetrad stage by the formation of primexine and is completed at vacuolated microspore stage by the addition of sporopollenin from tapetum. During the vacuolization and enlargement of the microspores, the structure and the chemical composition of the exine are modified. The endexine becomes chemically different from the ectexine. The ectexine is composed of sporopollenin and a small amount of protein, whereas the endexine is composed of sporopollenin, proteins, and traces of polysaccharides.     

Pollen wall ultrastructure and ontogeny in Heliotropium europaeum L. (Boraginaceae)

The pollen grains of Heliotropium europaeum are heterocolpate, with alternation of 3 colpori and 3 pseudocolpi. The exine is characterized by a scabrate and thick tectum, massive columellae with a granular appearance and a thick nexine. The thickening of the intine at the apertural level makes the interpretation of this zone difficult. The ontogenetic study helped to understand the ultrastructure of the exine and the apertures. The different steps are as follows. The primexine matrix is formed during the beginning of the tetrad stage; it consists of an outer thick and electron dense zone and an inner one, less dense to electrons. The tectum and the infratectum begin to form in the outer zone of the matrix, towards the middle of the tetrad stage. The infratectum consists of a network of columellae variable in thickness and oriented in different directions. The foot layer is lacking. The endexine is formed on a lamella system during the callose loss and microspore separation. The endexine becomes compact very early on its inner part. The apertures are initiated during the tetrad stage; a granulo-fibrillar oncus develops. At the free microspore stage, the oncus gets fibrillar and is bordered by endexine lamellae on its outer side and by endexine granulations on its inner one and laterally. The intine is set at the end of this stage. At the vacuolated microspore stage, the intine shows three layers: two thin, clear and homogeneous layers, one outside and the other inside, and a thick middle layer that forms the zwischenkörper, crossed by trabecula, in the apertural areas.     

Pollen wall development in Hypecoum imberbe Sm. (Fumariaceae)

We have studied the ontogeny of the pollen wall of Hypecoum imberbe. Our initial observations with conventional transmission electron microscopy (TEM) were inconclusive due to the similarities found in the electrodensity of the foot layer and the endexine, and between this latter layer and the intine. Thus we applied the PTA acetone histochemical test in order to differentiate between the cell wall components. This method proved to be efficient in resolving the differences between the layers and even allowed us to distinguish two strata within the narrow intine layer. A thin foot layer can be distinguished only by the temporary presence of a single white line separating it from the initial deposition of the endexine. The aperture consists of two colpi with no special differentiation. The tapetum is a typical secretory type giving rise to elaioplasts and tapetosomes during development, which persist as individual organelles upon the degradation of the tapetum. As the ultrastructural organisation of the sporoderm seems to offer little protection to the gametophyte, we finally discuss how the structure of the pollen sporoderm might be related to the special morphological characteristics of a flower, its habitat, and the biology of the plant as a whole.