Pollen and anther ontogeny in Cabomba caroliniana (Cabombaceae, Nymphaeales)

Cabomba is a small water lily genus that is native to the New World. Studies of pollen development and associated changes in the anther yield valuable characters for considering the evolution of reproductive biology in seed plants. Here we characterized the complete ontogenetic sequence for pollen in Cabomba caroliniana. Anthers at the microspore mother cell, tetrad, free microspore, and mature pollen grain stages were studied using scanning electron, transmission electron, and light microscopy. Tetragonal and decussate tetrads both occur in C. caroliniana, indicating successive microsporogenesis. The exine is tectate-columellate, and the infratectal columellae are the first exine elements to form, followed by a continuous tectum and a thin foot layer. A lamellate endexine initiates in the early free microspore stage, but becomes compressed in mature grains. Tectal microchannels and sculptural rods also initiate during the early free microspore stage, and significant pollenkitt deposition follows, supporting the hypothesis that these elements function in entomophily. The tapetum is morphologically amoeboid, with migratory tapetal cells directly contacting developing free microspores within the anther locule. Results from this study illustrate the importance of including ontogenetic data in analyzing pollen characters and in developing evolutionary and ecological hypotheses. The new palynological data also emphasize the character plasticity that occurs in basal angiosperms.     

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.     

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.     

Development of the pollen wall in Tsuga canadensis (Pinaceae)

In discussions of exine structural types, Tsuga is often mentioned as an exception, since no infratectal layer is present in the ektexine. The present investigation documents the formation of this pollen wall type at the ultrastructural level in T. canadensis . All layers of the exine are formed during the tetrad period, when the microspores are surrounded by a callose wall. The outer layer (ektexine) is elaborated on a fibrillar microspore surface coat, while the inner layer (endexine) is elaborated on lamellated structures. The deposition of the pretectum is followed by the appearance of endexine lamellae. In the initial stages, the two layers—pretectum and endexine—appear to be separated from each other only by a dense microspore surface coat. As additional wall materials are deposited, the tectal elements become convoluted and come to rest, in places, on the now recognizable footlayer. Upon release from the tetrad, intine formation begins and continuous accumulation of sporopollenin leads to an increase in ektexine thickness. The mature pollen wall of Tsuga canadensis , with a convoluted tectum resting directly on the footlayer, is characteristic of the genus.     

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.     

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 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 Vigna vexillata I. Characterization of wall layers

Light and electron microscope observations characterized the layers that comprise Vigna vexillata L. pollen walls, and identified the timing of their development. Exine sculpturings form an unusually coarse ektexinous reticulum. The structure of the ektexine is granular; this differs from the columellate/tectate type of structure typical of most angiosperm pollen. The ektexine overlies a homogeneous-to-lamellar, electron-dense endexine, which in turn surrounds a thick, microfibrillar intine. Pollen grains are triporate and operculate, with Zwischenkörper and thickened intine underlying the apertures. The ektexine forms during the tetrad period of microspore development, the endexine and Zwischenkörper during the free microspore stage, and the intine during the bicelled (pollen) stage. Coarsely reticulate exine sculpturings and the granular structure of the patterned exine wall of the pollen grains are features that make this species suitable for detailed studies of pollen wall pattern formation.     

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.     

含笑花药发育的超微结构研究

对含笑花药发育中的超微结构变化进行观察,结果显示:(1)花粉发育中有三次液泡变化过程——第一次是小孢子母细胞在形成时内部出现了液泡,这可能与胼胝质壁的形成有关;第二次是在小孢子母细胞减数分裂之前,细胞内壁纤维素降解区域形成液泡,它的功能可能是消化原有的纤维素细胞壁;第三次是在小孢子液泡化时期,形成的大液泡将细胞核挤到边缘,产生极性。(2)含笑花粉在小孢子早期形成花粉外壁外层,花粉外壁内层在小孢子晚期形成,而花粉内壁是在二胞花粉早期形成;花粉成熟时,表面上沉积了绒毡层细胞的降解物而形成了花粉覆盖物。研究认为,含笑花粉原外壁的形成可能与母细胞胼胝质壁有关,而由绒毡层细胞提供的孢粉素物质按一定结构建成了花粉覆盖物。     

POLLEN WALL AND TAPETAL ORBICULAR WALL DEVELOPMENT IN SORGHUM BICOLOR (GRAMINEAE)

Pollen wall development in Sorghum bicolor is morphologically and temporally paralleled by the formation of a prominent orbicular wall on the inner tangential surface of the tapetum. In the late tetrad stage, a thin, nearly uniform primexine forms around each microspore (except at the pore site) beneath the intact callose; concurrently, small spherical bodies (pro-orbicules) appear between the undulate tapetal plasmalemma and the disappearing tapetal primary wall. Within the primexine, differentially staining loci appear, which only develop into young bacula as the callose disappears. Thus, microspore walls are devoid of a visible exine pattern when released from tetrads. Afterwards, sporopollenin accumulates simultaneously on the primexine and bacula, forming the exine, and on the pro-orbicules, forming orbicules. Channels develop in the tectum and nexine, and both layers thicken to complete the microspore exine. Channeled sporopollenin also accumulates on the orbicules. A prominent sporopollenin reticulum interconnects the individual orbicules to produce an orbicular wall; this wall persists even after the tapetal protoplasts degenerate and after anthesis. While the pollen grains become engorged with reserves, a thick intine, containing conspicuous cytoplasmic channels, forms beneath the exine. Fibrous material collects beneath the orbicular wall. The parallel development and morphological similarities between the tapetal and pollen walls are discussed.     

POLLEN MORPHOLOGY AND ULTRASTRUCTURE OF THE CABOMBACEAE: CORRELATIONS WITH POLLINATION BIOLOGY

Of all species comprising the two genera of the Cabombaceae, only Brasenia schreberi J. F. Gmel. and Cabomba caroliniana Gray have been critically investigated with regard to their pollination biology. Brasenia schreberi has been shown to be anemophilous, while C. caroliniana has an entomophilous (myophilous) pollination syndrome. In the present paper, a number of pollen and pollen-related characters, including pollen size, shape, quantity, terminal settling velocity, pollen-ovule ratios, and overall exine architecture of B. schreberi and C. caroliniana are evaluated. Pollen from both species is elliptic, monosulcate, and has a tectate-columellate sporoderm with supratectal surface ornamentation. Grains of B. schreberi are small, produced in copious amounts, and settle relatively slowly. Flowers of this species have large pollen-ovule ratios. The exine of B. schreberi pollen is scabrate, relatively thin, has a uniformly thick sexine composed of a two-zoned (homogeneous/granular) tectum and distinct columellae, and a homogeneous nexine. Pollen of C. caroliniana is relatively large, produced in small quantities, and has a rapid terminal settling velocity. Flowers exhibit small pollen-ovule ratios. Exine organization of C. caroliniana pollen is typically two times thicker than that of B. schreberi; ornamentation is striate. Nonapertural sexine regions have a thick tectum and well-defined columellae, with both sexine components traversed by a dense system of channels. The nexine is relatively thin. All of the palynological characters examined correlate well with the anemophilous and entomophilous syndromes of B. schreberi and C. caroliniana, respectively. Moreover, several other parameters of exine ultrastructure from each species exhibit positive correlations with the respective pollination mechanisms, including: tectum thickness, columellae diameter, tectum-nexine ratios, and the consistency, distribution, and total amount of pollenkitt present. Overall exine ultrastructure is also discussed from a historical perspective as well as with respect to its phylogenetic significance.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

Exine ontogeny in Borago officinalis pollen

The primexine matrix is finely granulo-fibrillar up to callose digestion; it becomes distinctly fibrillar at the free microspore stage. The columellae and the tectum are initiated at the middle tetrad stage, the foot layer and the endexine are initiated when the callose wall digestion begins. The columellae are initiated by the deposition of spiral elements around a clear central zone. This hollow aspect of columella disappears when thickening. The foot layer and the endexine are built by the expansion of plasmalemma derived components. The foot layer appears first at the poles, then at the interapertural levels and at last at the apertures while the endexine appears first at the mesoapertures, then it spreads laterally towards the interapertural levels and, at last, at the poles. The gemmae are formed at the free microspore stage over all the tectum. The thickening of the exine takes place essentially during the free microspore stage and continues during the vacuolate microspore one. Apertures are entirely formed before the complete digestion of the callose wall. The ectoapertures are determined by the lacking of the columellae; the sites of the pericolpal cavities and the mesoapertures result from the plasmalemma retraction even before the setting up of the foot layer and the endexine by which they will be delimited respectively afterwards. The endoapertures are determined by the lacking of compact endexine at their level, and merge into a continuous equatorial belt.     

洋葱花药发育中钙离子分布特征

采用焦锑酸钾沉淀钙离子技术,对洋葱（Alliumcepa）花药发育中Ca^2＋分布进行了研究。在小孢子母细胞时期,小孢子母细胞中的钙沉淀颗粒很少,但绒毡层细胞的内切向壁已出现明显的钙沉淀颗粒。在四分体时期,四分体小孢子的胼胝质壁中出现较多的钙沉淀颗粒;绒毡层细胞内切向壁的钙沉淀颗粒消失,而在外切向壁和径向壁部位的钙沉淀颗粒增加。在小孢子早期,小孢子中也出现了钙沉淀颗粒,而绒毡层细胞内切向壁表面出现了很多絮状物,其上附有细小钙沉淀颗粒。到小孢子晚期,小孢子中出现一些小液泡,细胞质中的钙沉淀颗粒有所下降。此时绒毡层细胞已明显退化,但在绒毡层膜上仍有一些乌氏体和钙沉淀颗粒。在二胞花粉早期,营养细胞中的液泡收缩、消失,细胞质中又出现了较多的钙沉淀颗粒,在质体和其内部的淀粉粒表面上附有较多的钙沉淀颗粒。到二胞花粉晚期,花粉中的钙沉淀颗粒已明显下降,仅在花粉外壁中还有一地钙沉淀颗粒．     

华北落叶松花粉发育过程中的钙动态分布

运用焦锑酸钾沉淀法研究了华北落叶松(Larix principis-rupprechtii Mayr)小孢子发育过程中不同阶段Ca2 的分布情况.减数分裂时期,小孢子囊壁表皮和中层细胞的细胞壁及细胞间隙Ca2 分布较多,绒毡层只有外切向面的细胞膜有Ca2 分布,小孢子母细胞的各部位则很少有Ca2 ;四分体时期,包围四分小孢子的胼胝质壁上有大量的Ca2 分布,在四分孢子壁上也有较多沉淀;游离小孢子时期,钙离子在小孢子壁的分布较四分体时期有所减少,而到花粉成熟时又逐渐增多;从四分体到花粉成熟,乌氏体周围的Ca2 有增多的趋势.对四分体外壁Ca2 的大量分布与花粉壁的形成及信号物质在花粉表面贮存的关系,以及小孢子囊的外壁、绒毡层和乌氏体在Ca2 向花粉运输中所起的作用进行了讨论.     

Exine and tapetum development in Nymphaea capensis (Nymphaeaceae): a comparative study

The developmental events in microspore envelope and cytoplasm and in tapetum from premeiosis until late tetrad stage were studied in Nymphaea capensis. The exceptional feature of microspore development in this species is that post-meiosis cytokinesis is retarded until the late tetrad stage. Thus, the entire development of the exine becomes completed during the tetrad stage. As a consequence of the retarded cytokinesis, the proximal portion of the forming exine lags behind the distal one during the major part of the tetrad period, but eventually the proximal part of the exine overtakes the distal part in development. The significance of this retardation is discussed. This sequence of events differs sharply from corresponding sporoderm development in other Nymphaea species. Another important topic is the microspore surface activities during exine development. The surface coatings-glycocalyx-are very similar in microspores and in tapetum cells, but their functions are completely different; the roots for this difference are discussed. A noteworthy feature of the developing microspores is the presence of gigantic, deeply cup-like mitochondria; this property is also characteristic of the microspore cytoplasm of N colorata and N. mexicana. A functional significance of these organelles and their adaptive role is discussed.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

洋葱花粉发育过程中ATP酶的分布特征(简报)

在真核细胞中,除了线粒体和叶绿体ATPase的功能是合成ATP外,其余部位ATPase是水解ATP以获取生物能量的代谢酶,在生物体细胞内广泛存在。探索ATPase在细胞中的分布状态是研究细胞生理状态的一种重要手段。ATPase在细胞中的多少可反映出细胞当时的生活状态,这一特征已被初步用于探索小麦和水稻雄性不育的细胞生物学研究中,希望通过比较可育花药和不育花药中ATPase的分布差异寻找雄性不育的机理,发现     

白菜核雄性不育花药超微结构的研究

对白菜核雄性不育两用系的可育与不育花药进行了超微结构的比较观察。结果显示不育花药的造孢细胞核仁靠边分布:包裹小孢子母细胞的胼胝质厚薄不均匀,不完整等早期异常现象。减数分裂后,四分体细胞中常有多个细胞核。从四分体释放出的小孢子外壁的孢粉素物质不均匀沉积．呈不连续的单层异常结构。最后小孢子通过细胞质收缩方式败育。在可育花药中,绒毡层细胞在小孢子发育后期已显示出退化迹象,同时在细胞中开始积累脂类物质。但在同时期的不育花药中, 绒毡层细胞没有显示出退化的迹象,也不合成脂类物质。从时间上看,败育花药中小孢子母细胞及小孢子的异常在先,绒毡层细胞的异常在后。本研究揭示了白菜核雄性不育花药的超微结构特征, 对我们以前的光学显微镜观察结果予以补充和修正。